“All flesh is coupled by the wave of circular rotational similarity” 1

“All flesh is coupled by the wave of
circular rotational similarity”
B. L. Pasternak
1
Ultralow doses
2
RUSSIAN ACADEMY OF MEDICAL SCIENCES
O. I. EPSTEIN
ULTRALOW DOSES
(HISTORY OF ONE RESEARCH)
This edition was recommended and approved for publication
by the Editorial Advisory Board of the Presidium
of the Russian Academy of Medical Sciences
Moscow
RAMS Publishing House
2009
3
Ultralow doses
UDC 615.015.32
LBC 52.81
E736
Re v i e we r s :
Seredenin Sergey Borisovich,
Academician of the Russian Academy of Medical Sciences
Chereshnev Valeriy Akeksandrovich,
Academician of the Russian Academy of Sciences
S c i e n c e e d i to r:
Sergeeva Svetlana Aleksandrovna,
Doctor of Biological Sciences, Professor
E736
Epstein O. I. Ultralow doses (history of one research). Moscow:
Publishing Office of the Russian Academy of Medical Sciences,
2008. 336 pages.
ISBN 9785790101014
This monograph is devoted to a systemic study of the effects of
potentiated (activated) preparations that contain an active substance in
ultralow concentrations. The activated preparations were shown to have
previously unknown modifying properties. Taking into account these
data, a new class of medical products (antibodies in ultralow doses) was
developed. A new direction of bipathic pharmacotherapy was proposed.
The first part of this monograph not only illustrates the results of our
studies, but also describes the physiological and physical aspects and
world outlook of ultralow doses. The final chapters are devoted to
experimental and clinical pharmacology of antibodies in ultralow doses.
The monograph is addressed to physicians of various specialties.
ISBN 9785790101014
© RAMS Publishing House, 2009
4
Devoted to my teachers
Table of contents
Preface
............................................................................................ 7
List of abbreviations ............................................................................ 10
Introduction ......................................................................................... 13
Chapter 1. Analysis of the experience of homeopathy ....................... 15
Chapter 2. Three types of effects of ultralow doses ........................... 24
Chapter 3. Dual organization of vital activity .................................... 45
Chapter 4. Holographic control of vital activity
by the immune system ...................................................... 62
Chapter 5. Principle of maintenance of the initial integrity .............. 77
Chapter 6. On the way to pharmacology of ultralow doses ............... 91
Chapter 7. Experimental pharmacology of products
from ultralow doses of antibodies .................................. 111
7.1.
Experimental study of antibodies to S100 protein
in ultralow doses ................................................................ 111
7.2.
Preclinical study of Impaza .................................................. 152
7.3.
Preclinical study of Anaferon and Anaferon for children ........... 160
7.4.
Preclinical study of Artrofoon ............................................... 173
7.5.
Preclinical study of Epigam .................................................. 180
7.6.
Preclinical study of Afala ..................................................... 190
7.7.
Preclinical study of Kardos ................................................... 198
7.8.
Study for antidiabetic activity of a new product
from ultralow doses of antibodies on the model
of streptozotocininduced diabetes in rats .............................. 204
Chapter 8. Clinical pharmacology of products
from ultralow doses of antibodies .................................. 210
5
Ultralow doses
8.1.
Use of medical products from antibodies to S100 protein
in the therapy for alcoholism and anxiety disorders ................. 210
8.2.
Use of Impaza in monotherapy and combined
treatment for erectile dysfunction ......................................... 223
8.3.
Clinical effectiveness and mechanisms
for action of Anaferon ......................................................... 241
8.4.
Artrofoon as a promising drug for pathogenetic
therapy of chronic arthropathies ........................................... 252
8.5.
Epigam in the therapy for gastric ulcer
and duodenal ulcer ............................................................. 259
8.6.
Afala in the therapy for benign prostate hyperplasia ................ 262
8.7.
Clinical pharmacology of Kardos .......................................... 265
Conclusion ........................................................................................ 275
References ........................................................................................ 277
6
Preface
This book tells you about the history of a 10year study, which was per
formed at the interface between immunology, pharmacology, pathophysiology,
and problem of ultralow doses and resulted in the development of a new class
of medical products.
Ultralow doses were used in alternative academic medicine (homeopathy)
over two centuries. Hence, this problem received little attention of scientists. At
the beginning of the 1980s, advancedtechnology studies in Russia and other
countries showed that ultralow doses have biological activity. The scientists sho
wed a cautious attitude toward these results, although they were not associated with
homeopathic doctrine. Moreover, such studies did not attract much attention of
those investigators who was interested in them (primarily, of pharmacologists).
Due to certain reasons, a systemic pharmacological study of ultralow
doses was first performed in Russia. The author of this book, O. I. Epstein, was
an initiator of such investigations in 1995. Now O. I. Epstein is the Doctor of
Medical Sciences and Professor. He was awarded the prize of the Government
of the Russian Federation in the field of science and technology. During that
period, a young physician was enthusiastic in homeopathy. He was the head of
a small pharmaceutical company, who searched for a specific direction of ac
tivity. Famous Russian specialists (investigators and clinicians) became interested
in the idea of O. I. Epstein, which determined the success of a scientific search.
The scientific way of O. I. Epstein extends from orthodox homeopathy to phar
macology (i.e., immunopharmacology). Success attended him, since the direc
tion of many researches was selected intuitively. Initially, the goal of his study
was to develop new indications for the use of homeopathic remedies. If some
God of Science protected O. I. Epstein, it was the God of Immunology.
The study may be divided into three stages. Stage I may be designated as
“nonhomeopathy”. The author assumed that ultralow doses and homeopathy
are not identical to each other. It was proposed that the phenomenon of ho
meopathy is associated with hypersensitivity of the organism to ultralow doses.
Hence, the effects of ultralow doses were explained by immunological mecha
nisms.
7
Ultralow doses
In stage II, an unusual application was found for ultralow doses. It was
shown that ultralow dose of a certain medical product modifies activity of the
original substance. Therefore, ultralow doses of modern pharmaceutical prod
ucts may be used to potentiate their effects and to reduce toxicity. Combined
treatment with the medical product in normal dose and ultralow dose received
the name “bipathy” (O. I. Epstein). Bipathy holds much promise for pharmaco
logy. The researches could focus their attention on study of bipathy.
However, the direction of investigations sharply changed. In stage III,
these researches were in close contact with immunology. To confirm the phe
nomenon of bipathy, O. I. Epstein in collaboration with M. B. Shtark (Acade
mician of the Russian Academy of Medical Sciences) and high colleagues stud
ied the effects of antibodies in ultralow doses on neurobiological models. They
showed that antibodies in ultralow and normal doses have various effects. An
tibodies in ultralow doses did not inhibit, but modified the activity of a specif
ic antigenic molecule. The discovery of “proantigenic” effects of antibodies in
ultralow doses resulted in the development of new effective and safe drugs for
the therapy of various diseases.
As an immunologist, I know that very low doses (or sometimes nano
quantities) of immunogenic proteins, peptides, and polysaccharides may induce
a strong physiological and pathophysiological effect. It mainly concerns antibod
ies, allergens, and other immunologically active molecules.
This monograph illustrates the results of advancedtechnology experi
ments and clinical studies. However, the data that extremely low concentrations
of medical products (from the viewpoint of molecular biology) exhibit the ac
tivity seem to be unexpected and paradoxical. At the modern of stage of science
development, the mechanism for action of ultralow doses can be described hy
pothetically. The author gives his opinion on this problem. Sometimes, the un
usual effects of ultralow doses are explained by uncommon events. As a pioneer
in this field, O. I. Epstein can do it.
Numerous preclinical and clinical trials were performed in leading insti
tutions of the Russian Academy of Medical Sciences and Russian Ministry of
Health and Social Development. The results of these studies indicate that ul
tralow doses have a reproducible effect, which may be evaluated and used in
evidencebased medicine. However, the author notes that the exception is ho
meopathic therapy. “An individual (similar) prescription of medical products is
similar to art. The methodology of homeopathy is not associated with general
pathophysiological approaches in pharmacology”. It is really true.
O. I. Epstein is so infatuated with his hypotheses that sometimes he passes
from a strongly scientific presentation to the emotional, speculative, or even
philosophical conclusions. It is not necessarily that strong evidence exists for
these conclusions. O. I. Epstein believes that there are no two medicines (allo
8
Preface
pathic medicine and homeopathic medicine). At a particular finestructure level,
the effects of normal and homeopathic doses in an organism are mediated by
similar mechanisms. This level includes the distant intermolecular relationships,
which are unique for each individual. O. I. Epstein assumes that the preserva
tion of individuality is an evolutionary purpose of vital activity of the organism.
This theory is close to the principles of immunology. M. Bernet, one of the
founders of modern immunology, believed that a major role of the immune sys
tem is regulation of genetic integrity in an organism.
This monograph integrates the author’s notion of distant interactions in
an organism with general principles of physiology. A lot of surprising and, some
times, doubtful facts will be of interest to the reader. The monograph is writ
ten in a vigorous and interesting style, which facilitates the understanding of
complex biological problems. O. I. Epstein not only tells us about new medi
cal products, which have high therapeutic effectiveness and hold promise for the
treatment of various diseases. He wants the reader to form an opinion of new
drugs in ultralow doses (particularly of those from antibodies).
R. M. Khaitov
Academician of the
Russian Academy of Medical Sciences
and Russian Academy of Sciences
9
Ultralow doses
List of abbreviations
AWS — alcohol withdrawal syndrome
BP — blood pressure
AID50 — aerogenic infective dose
AntiS100 — antiserum to brainspecific protein S100
AFC — antibodyforming cells
APC — antigenpresenting cells
ACE — angiotensinconverting enzyme
ARA — American Rheumatology Association
AT — angiotensin
AT1 — type 1 angiotensin II receptor
ABIRβ — antibodies to insulin receptor betasubunit
ATP — adenosine triphosphate
ATPase — adenosine triphosphatase
ADC — analogtodigital converter
EPSP — evoked postsynaptic potential
GABA — gammaaminobutyric acid
DTHR — delayedtype hypersensitivity reaction
GCD — glucocorticoid drugs
GCSF — granulocyte colonystimulating factor
GMP — guanosine 3,5monophosphate
GAD — generalized anxiety disorder
BPH — benign prostate hyperplasia
CI 95% — 95% confidence interval
DNA — deoxyribonucleic acid
LPTP — longterm posttetanic potentiation
NK — natural killer cells
GIT — gastrointestinal tract
CHD — coronary heart disease
IL — interleukin
EIA — enzyme immunoassay
IFN — interferon
CIA — collageninduced arthritis
LD — lethal dose
10
List of abbreviations
LV — left ventricle
LC — latency
LPS — lipopolysaccharide
IIEF — International index of erectile function
ICD — International classification of diseases
MTD — maximum tolerable dose
ISIAH — inherited stressinduced arterial hypertension
NAID — nonsteroid antiinflammatory drugs
AE — adverse event
OA — osteoarthritis
ARVI — acute respiratory viral infections
HTP — hydroxytryptophan
AP — action potential
EPM — elevated plusmaze
RP — resting potential
CGM — complete growth medium
PSA — prostatespecific antigen
RA — rheumatoid arthritis
LBTR — lymphocyte blast transformation reaction
RNA — ribonucleic acid
RSV — respiratory syncytial virus
SBP — systolic blood pressure
DM — diabetes mellitus
DBPM — daily (24h) blood pressure monitoring
ULD — ultralow doses
ULDH — ultralow doses of haloperidol
ULDP — ultralow doses of phenazepam
ALS — average lifespan
MADD — mixed anxiety and depression disorder
TRUSE — transrectal ultrasound examination
TS — testosterone
UA — urogenic reactive arthritis
USE ultrasound examination
CAAR — conditioned active avoidance reflex
CPAR — conditioned passive avoidance reflex
LVEF — left ventricular ejection fraction
PHA — phytohemagglutinin
PDE5 — type 5 phosphodiesterase
PI — phagocytic index
TNFβ — tumor necrosis factorβ
PBS — phosphatebuffered saline
PN — phagocytic number
11
Ultralow doses
CHF — chronic heart failure
cAMP — cyclic adenosine 3',5'monophosphate
cGMP — cyclic guanosine 3',5'monophosphate
CNS — central nervous system
CP — cyclophosphane
HR — heart rate
SE — sheep erythrocytes
ED — erectile dysfunction
ECG — electrocardiogram
5HT — serotonin receptors
ACR20 — 20% improvement by American College of Rheumatology criteria
ARA — American Rheumatology Association
AS100 — antiserum to brainspecific protein S100
AUC — trapezoid method for estimation of the area
under the concentrationtime curve
C — centesimal dilution
D — decimal dilution
EGF — epidermal growth factor
eNOS — endothelial NO synthase
FDA — USA Food and Drug Administration
HAMA — Hamilton anxiety scale
Ig — immunoglobulin
IPSS — International questionnaire for symptoms
of prostate diseases (International Prostate Symptom Score)
ITT — analysis of the results for patients included in the trial
(intention to treat analysis)
MHC — major histocompatibility complex
NMMA — NGmonomethylLarginine
NOS — NO synthase
NYHA — New York Heart Association
QoL — qualityoflife index (IPSS questionnaire)
STAI — Spielberger scale (StateTrait Anxiety Inventory)
STAIS and STAIT — state and trait anxiety by the Spielberger scale
VEGF — vascular endothelial growth factor
WOMAC — index for the severity of osteoarthritis
(Western Ontario and Mc Master Universities Osteoarthritis Index)
12
Introduction
T
he author of this book, as well as his colleagues, succeeded in an intriguing
scientific path from homeopathy to immunopharmacology, and from
traditional homeopathic remedies to hightechnology, safe, and effective medical
products. These drugs were developed on the basis of a newly discovered
phenomenon of ultralow doses of antibodies.
Several preparations from ultralow doses of antibodies, including Ana
feron, Impaza, and Proproten100, are well known. However, many physicians
and specialists do not have enough information on the mechanism for action
of these products. The phenomenon of antibodies in ultralow doses was opened
at the boundary of the following three medical disciplines: pharmacology,
immunology, and homeopathy. Therefore, this monograph includes some data
on homeopathy and immunology.
Experimental and clinical trials allowed us to obtain new data, which are
not consistent with the common notions about vital functions of the organism.
The interpretation of these facts requires other approaches and new knowledge.
The pragmatic purpose of this monograph is to develop the notion of a new
pharmacological direction.
During the preparation of this manuscript, the author should become
familiar with previously unknown areas of knowledge. It was necessary for him
to learn the notions and terminology that exist in each field of science. Medical
science consists of several special directions. A fruitful professional dialog does
not necessarily occur between experimenters and clinicians. We would like this
book to be available not only for representatives of theoretical medicine
(pharmacologists, physiologists, and immunologists), but also for physicians. To
simplify the understanding of some facts that require special biological
knowledge, they are given in a popular scientific form (history of one research).
Fundamentally, the monograph is divided into two sections. The first six
chapters are devoted to the general problem of ultralow doses. The final two
chapters show some data on experimental and clinical effectiveness of new
products. It may be of interest to various specialists, including neurologists
(Tenoten), infectious disease physicians and podiatrists (Tenoten for children
13
Ultralow doses
and Anaferon for children), urologists (Afala and Impaza), rheumatologists
(Artrofoon), narcologists (Proproten100 and Anar), and cardiologists (Kardos).
The author is grateful to Professor S. A. Sergeeva, Yu. L. Dugina, I. A.
Kheifets, and all colleagues from the Science Department of the “Materia
Medica Holding” ResearchandProduction Company for their help in the
preparation of this monograph.
14
C h a p t e r
1
Analysis of the
experience of homeopathy
T
he author of this monograph, a recent graduate of the Medical Institute,
received the book “Homeopathy” (G. Kohler) as a gift from his father in
1989. It was a rare book in the period of commodity deficit. A period of 1 year
was required to overcome a skeptical attitude of the Soviet physician toward this
“superficial” discipline. The book was opened and read. This moment may be
considered as the first successful step in a study described here. As differentiated
from various manuals on homeopathy, the general part in the book of G. Kohler
was written in downtoearth language. Otherwise, the familiarity and further
fascination with homeopathy could not occur.
After ten years of neglect of homeopathy in our country, thousands of
physicians were able to learn this discipline by visiting a variety of quasilegal
training courses. However, only some of them became the practitioners. It was
very difficult to learn homeopathy without assistance of a teacher. The author
of this monograph was successful. A twist of fate introduced him to a famous
physician T. D. Popova, who headed the Kiev school of homeopathy. She was
a bright person. O. I. Epstein had the possibility to collaborate with Tat’yana
Dem’yanovna and to look for the reception of patients. It helped him to learn
the basic principles of homeopathy.
Homeopathy is inseparably linked with the name of S. F. Hahnemann. In
1976, he published the manuscript on a new therapeutic direction. He described
the method for preparation of medical products in ultralow doses, principles of
prescription, and results of clinical trials. S. Hahnemann is one of the pioneers
15
Ultralow doses
in clinical trials with medical products. There are ambiguous data on pharma
ceutical studies in old time. The first comparative analyses of therapeutic agents
were performed only in the 18th century. However, R. Virchow believed that the
“farther of experimental pharmacology” is S. Hahnemann. Before Hahnemann,
there was no integral and intelligent approach to study of medical products. He
received a fine education and learned the ancient languages. In previous
manuscripts, Hahnemann could learn the “like cures like” principle. The deve
lopment of a new rational scientific method for medicinal treatment of various
diseases was associated with activity of this unique man. Hahnemann decided
to test the effect of cinchona bark with himself to confirm the reliability of
published data. This substance caused fever, which was typical of malaria. The
scientist concluded that a medicine for the therapy of some disease can induce
a similar state in healthy individuals.
S. Hahnemann tested the effects of various medical products (mainly of
herbs and minerals) on healthy volunteers. Clearly the trial did not meet mo
dern requirements and was performed with a small number of people (relatives,
friends, pupils, and colleagues). The results confirmed the hypothesis of Hah
nemann. The reactions induced by some substance in healthy volunteers may
be considered as an indication for therapy of similar disorders with the same
agent. The Hahnemann’s Law of Similars was formulated for the first time in
the journal of Hufeland. “One should apply in the disease to be healed, parti
cularly if chronic, that remedy which is able to stimulate another artificially pro
duced disease, as similar as possible; and the former will be healed – similia
similibus*”.
It should be emphasized that the Law of Similars was discovered by S. Hah
nemann with normal doses**. Some patients were characterized by severe drug
induced exacerbation. Hence, Hahnemann decided to reduce the dose of re
medies. Due to pedantry, the scientist developed a method for dilution of the
original substance. He empirically showed that it is necessary to combine
(exactly to combine!) a repeated dilution of the initial solution and tenfold
mechanical shaking. Hahnemann proposed to use the centesimal (C) scale of
dilution. At each stage of preparation, the initial amount of medical product is
diluted by 100 times. In the followup period, homeopaths introduced the so
called “decimal dilution” (D, successive tenfold dilution of the original substan
ce). For example, dilution C30 means that the original substance was diluted
30 times. Moreover, the concentration of this substance was reduced by 100
times at each dilution. D6 means that the concentration of the original
* Similia similibus (lat.) likes with likes.
** The term “homeopathy” originates from the Greek word “homo” (similar). It mainly
designates the principle of “remedy prescription”, but not the dose.
16
Chapter 1. Analysis of the experience of homeopathy
substance decreased by 10 times at each of six dilutions (Fig. 1.1). In the SI
system, high dilutions by the method of Hahnemann correspond to 10—n M and
100—n M. Formally, the dilutions of more than 10—24 M (D24) and 100—12 M
(C12) are submolar. They do not contain molecules of the original substance.
This fact did not disturb the founder of homeopathy. Hahnemann did not know
about the unit of “mole”, which was proposed by the physician Amedeo
Avogadro (17761856). Hence, Hahnemann operated with submolar doses.
In the 18th century, drug manufacturers were in conflict with Hahne
mann. It was related to commercial reasons, but not to the absence of molecules
of the original substance in homeopathic remedies. Hahnemann prepared these
homeopathic remedies by himself and, therefore, deprived the manufacturers of
earnings.
S. Hahnemann and his followers studied the effects of various prepara
tions on healthy volunteers. They showed that treatment with these agents in
submolar concentrations is followed by druginduced exacerbation. Even at high
dilution of one or another substance, there were two or three individuals
(respondents) with hypersensitivity to the prescribed remedy.
It should be emphasized that the respondents had similar personal
characteristics, appearance, behavioral habits, and dietary predilections. They
were predisposed to certain diseases, including inherited disorders. For example,
the subjects reacting to ultralow doses of arsenic mainly appeared as lean, fine
boned, and lightskinned individuals. They usually had a geographic tongue,
drank a considerable amount of fluid in small sips, and felt comfortable in warm
climate. These subjects differed in pedantry, anxious mood, and predisposition
1/100
1/100
1/100
1/100
1/100
↓ ⏐
↓ ⏐
↓ ⏐
↓ ⏐
↓
(Н2О)
(Н2О)
(Н2О)
(Н2О)
(Н2О)
1:1
1:102
1:104
1:106
1:108
1:1010
Basic
solution
1С
2С
3С
4С
5С
⏐
Alcohol
solution
Molecular
dilution
Homeopathic
potency
Increase in the potency of remedies
Fig. 1.1. Scheme for the preparation of homeopathic remedies (V. G. Zilov et al.,
2000).
17
Ultralow doses
to psoriasis, bronchial asthma, and other diseases with common clinical symp
toms. A complex description of the effects of any substance in ultralow dose and
characteristics of hypersensitive patients received the name “pathogenesis of
homeopathic remedy” (in this case, arsenic pathogenesis). It includes the phar
macodynamic properties of a substance and markers of individual sensitivity,
which should be taken into account in the prescription of this remedy*. S.
Hahnemann demonstrated that the maximum individualization of therapy is
necessary for clinical practice. The major therapeutic principle appeared as
follows: “cure only with a similar drug”. The similarity was considered not only
as the prescription of a medical product for certain symptoms (indications), but
also as the use of a specific remedy in patients with high individual sensitivity.
Hundreds of medical products are extensively used in homeopathy. There
are 2000 homeopathic remedies. Homeopathy was born 200 years ago. There
fore, homeopathic remedies are prepared from available raw materials (plants,
minerals and, more rarely, biological substances). The major advantage of
homeopathic remedies is that their effects were studied in details. Undoubtedly,
the arsenal of homeopathy will always include wellknown “ancient” prepara
tions. Some modern pharmaceutical (allopathic) products, including aspirin,
nitroglycerine, insulin, and prednisolone, are sometimes given in ultralow doses.
The quintessence of homoeopathy is the evaluation of sensitivity of each patient
to one of the homeopathic remedies. In this case, the treatment will produce
a therapeutic effect. Hahnemann developed the integral therapeutic discipline
of homeopathy, which differs in allsufficiency. Potentially, a skilled homeopath
may affect any disease state. The success of therapy strongly depends on the
“appropriateness” of medical treatment, but not on the nosological form or
severity of disease. A skilled homeopath is a general practitioner and good spe
cialist in propaedeutic. The prescriptions of a homeopath are based on history
taking, thorough examination, power of observation, and evaluation of patient
characteristics.
By the 20th century, homeopathy was widely distributed in Europe, North
and South America, India, and Russia. Pharmaceutical chemistry, experimental
pharmacology, and molecular biology became the specific areas of knowledge
at a later period. They determine the type of modern medicine. Due to several
reasons, homeopathy was separated from modern science. Symbiotic relation
ships between academic medicine and homeopathy developed in the followup
period. Traditionally, a European patient knows the problems that require him
*
18
The term “drug pathogenesis” is very close to the modern notion of constitution in
psychiatry and pediatrics. Hahnemann did not use the term “constitution” or
“constitutional type”. They were introduced into the homeopathic lexicon by followers
of Hahnemann.
Chapter 1. Analysis of the experience of homeopathy
to consult with a homeopath. Homeopathic therapy also develops in Russia.
After longterm neglect, this method was officially approved in Russia in 1995.
Let’s consider the main principles of the Hahnemann theory.
Principle of similarity. First of all, it should be remembered that the prin
ciple of similarity is not a prerogative of ultralow doses. The first prescriptions by
Hahnemann were made in “normal” doses. This principle suggests that the remedy
should be prescribed for a “similar” clinical manifestation. For example, arsenic
causes fever in healthy volunteers. By contrast, arsenic in homeopathic doses may
be used to cure fever in patients. However, arsenicinduced fever has several specific
features. Fever is observed in a certain time (midnight) and accompanied by
typical chill, which spreads in an upward direction of the back. Arsenic in
homeopathic doses will be therapeutically effective in patients with this type of
fever. Moreover, the effect will occur only in patients that are susceptible to
ultralow concentrations of arsenic (primarily in lean and fineboned pedants).
These features of symptoms and constitutional characteristics serve as a
marker for individual sensitivity. Modern pharmacology also postulates the
necessity of individual pharmacotherapy. Much attention is paid to the search
for genetic and phenotypic criteria of individual sensitivity.
It should be noted that each patient may have various markers. These data
indicate that individual sensitivity (at least, phenotypic sensitivity) to a substance
is determined by several markers, but not by one marker. The experience of
homeopathy shows that one sign (e.g., geographic tongue) may serve as one of
the markers for a large group of medical products. Only a specific combination
of various markers is a reliable criterion for individual sensitivity to the remedy*.
Trial with healthy volunteers. The trial with healthy volunteers was of con
siderable significance. This approach allowed S. Hahnemann and his followers
to perform a simple, rapid, and safe evaluation of indications for a large number
of medical products. The following two facts are of importance for our study.
First, medical products in ultralow concentrations cause exacerbation in
a small number of patients. Hence, this state is associated with the reaction of
hypersensitivity. Several types of immediate and delayedtype hypersensitivity
were studied and described in immunology. However, little is known about their
mechanisms. All these reactions are nonspecific. For example, anaphylactic shock
may be induced by a variety of substances. By contrast, the symptoms of drug
induced exacerbation in homeopathy are always specific for a certain substance.
And second, arsenic in toxic doses causes hyperthermia in all subjects.
Low doses of arsenic may produce the same clinical symptoms of fever in
individual volunteers, which is related to hypersensitivity. Let’s consider fever as
*
In the future, this observation allowed the author to develop a new concept of seman
tically organized constellations.
19
Ultralow doses
a protective response. In this case, the substance in high doses serves as a
“pathogen” for all individuals. By contrast, the same substance in low doses has
a pathogenic effect only on several subjects. A similar protective response should
be mediated by similar mechanisms. However, the substance in high dose
triggers these mechanisms in all subjects. It remains unclear why the substance
in low dose affects only sensitive individuals*.
Preparation of ultralow doses. It is known that highly diluted solutions do
not have biological activity. Otherwise, biological activity of these solutions is
extremely unstable. Moreover, the Hahnemann’s method of mechanical shaking
may be substituted for another external influence (electromagnetic or ultrasound
exposure). However, ultradiluted solutions do not exhibit activity without suc
cessive dilutions of the original substance in combination with external treat
ment. It is surprising that Hahnemann could combine various procedures into
a common system: preparation of medical products, study of pharmacological
activity, and principle of prescription (similarity).
The physical mechanisms for memory retention of the original substance
in highly diluted solutions remain unknown. Several scientists have cast doubt
on the use of ultralow doses and effectiveness of homeopathy. Modern studies
were performed 200 years after the discovery of homeopathy. They illustrated
that ultradiluted solutions (method of Hahnemann) have fine biological activity.
The technological process of S. Hahnemann received the name “poten
tiation”. The prepared remedies were designated as potentiated or dynamic
substances. Hahnemann proposed that these agents are characterized by the
release of an “active basis” or “vital force”. A clinical effect of potentiated pro
ducts was not observed without maximum individualization. Hahnemann sug
gested that potentiated products are ineffective without the principle of similarity
(allornone law). Various properties of potentiated agents were revealed in
modern molecular and cellular studies. Hahnemann had no technical
possibilities to determine the general properties of ultralow doses, but proposed
an approach to their use (homeopathy).
The primacy of the principle of similarity took root in the mind of Hah
nemann followers. Until the present time, homeopaths know little about other
(nonhomeopathic) variants for the use of medical products in ultralow doses.
The term “homeopathic dose” has been commonly accepted. It is more
preferable to tell about the individual (homeopathic) prescription of a medical
product in ultralow or low dose, but not about the dose. In our opinion, the
term “homeopathic remedy” means that the indications for treatment with a
*
20
The mechanisms of individual sensitivity are a major problem, which underlies the
hypothesis on dual (holographic) organization of vital functions in an organism. This
problem is discussed in the next chapters.
Chapter 1. Analysis of the experience of homeopathy
certain drug were evaluated in trials with healthy volunteers. However, the
indications for modern pharmaceutics are estimated by other methods.
The terms “low” and “ultralow” dose are also indefinite. Some authors
believe that the doses of up to 10—12 M original substance are low (molar) do
ses. The doses of not more than 10—24 M are ultralow doses. However, activity
of the potentiated agent depends little on the presence or absence of several mo
lecules of the original substance (see below). By contrast, this activity is deter
mined by the technology of preparation. The method of potentiation supplies
biological activity to ultradiluted solutions. Hence, these solutions can be used
according to the doctrine of homeopathy. It is more appropriate to use the term
“potentiated” or “activated” substance, but not the homeopathic, low, or ultra
low dose. The scale of preparation (C or D) and number of dilution cycles
(usually 6, 12, 30, 200, or 1000) should be designated.
The term “activated” preparation seems to be preferable. First, this term in
dicates than biological activity of the ultradiluted solution is related to external
treatment. Second, the term “potentiation” has another meaning in modern
pharmacology. And third, we showed that medical products in ultralow doses
have a potentiating effect under certain conditions. These features may introduce
terminological difficulties. However, the preparation of ultralow doses should
retain its historical name of “potentiation” or “dynamization” (S. Hahnemann).
Besides a rational analysis of homeopathy experience, it is necessary
to consider the subjective feelings of homeopathic physicians. At a certain stage
of professional activity, any homeopath notices that nearly all (even rarely
observed) physiological signs are manifested in the description or pathogenesis
of homeopathic remedies. For example, you see that one of the guests asks you
to prepare tea. Then he gulps down boiling water. Another guest asks you to
close the window since he cannot hear a noise (the yardkeeper cleans asphalt
with a scraper). Under these conditions a homeopath will remember the
pathogenesis of Lycopodium (club moss) and Asarum (snakeroot), respectively.
When studying the temperamental characteristics of patients sensitive to
one or another homeopathic remedy, you can see that they have a common
feature (“stem” or “algorithm”). It is difficult to explain this algorithm. How
ever, a homeopath cannot select the effective medical product without under
standing this algorithm.
Many homeopaths know that the psychological type of patients sensitive to
some medical product is progressively transformed into the psychological type of
another remedy. This feature contributes to the mosaic pattern, which resembles
a change in the properties of chemical elements in the periodic table of D. I.
Mendeleev. Psychological portraits of sensitive patients are similar in the
pathogenesis of several preparations (mineral potassium sulfate and Pulsatilla of the
family Ranunculaceae; calcium carbonate and belladonna; etc.). The remedies with
21
Ultralow doses
similar “psychological characteristics” have the same therapeutic effect. These data
suggest the systemic nature of homeopathy, which is difficult to verbalize.
This impression becomes stronger in learning the other basic principles of
homeopathy. Using the terminology of the 18th century, homeopaths classify all
pathological processes into the following three groups (depending on general char
acteristics): syphilis (destructive processes), sycosis (productive processes, including
cough), and psora (subacute areactive states). Each type of pathological processes
is characterized by a certain group of preferable homeopathic remedies. Other
principles of homeopathy are also unusual. For example, some activated
preparations are tropic for the rightsided or leftsided disease. Caustic (lime) and
Lachesis (bushmaster snake venom) are prescribed for the therapy of rightsided
and leftsided hemiplegia, respectively. The rules of C. Hering also seem
uncommon. C. Hering (18001880) is the farther of American homeopathy. He
described the spatial and temporal response of patients to homeopathic remedies.
C. Hering revealed (1998) that “the patient will be cured and the symp
toms will permanently disappear when they develop in the following direction:
from within outwards, from above downwards, and from later symptoms to
earlier symptoms (i.e. in the reverse order of their coming)”. The use of homeo
pathic remedies is rarely followed by druginduced exacerbation. When the
dynamics of druginduced exacerbation is consistent with the rules of C. Hering,
it may be considered as a prognostically favorable process. Under these condi
tions, the prescribed remedy is not withdrawn by a homeopath.
Homeopathic practitioners know that each patient is sensitive to several
homeopathic remedies (hierarchy of individual sensitivity). The higher is the
sensitivity to the remedy, the greater is the effectiveness of this remedy.
Summarizing the above we conclude that vital activity of the organism is
based not only on wellknown physiological and chemical processes, but also
on spatial and temporal laws of harmony. They are closely related to individual
characteristics of each patient. Even though the homeopathic knowledge seems
archaic, it is worthy of notice. This knowledge was obtained in a directed
clinical study of medical products. Hence, these data are objective. Moreover,
separate observations illustrate the existence of various clinical, phenotypic,
psychological, and topic criteria for individual sensitivity of an organism.
Most important in Chapt
er 1
Chapter
Homeopathy is a drug therapy based on the principle of similarity.
The principle of similarity suggests maximum individualization in the prescrip
tion of medical products, which involves a propaedeutic approach of S. Hah
nemann.
22
Chapter 1. Analysis of the experience of homeopathy
Homeopathy suggests the use of activated agents that are prepared by the meth
od of potentiation (successive dilutions of the original substance and rhythmic
mechanical shaking).
Activity of ultradiluted solutions depends on the technology of poten
tiation, but not on the concentration of the original substance. This fact is
proved, but does not have a physical explanation.
Ideally low doses in homeopathy are associated with the method of preparation.
Over two centuries, potentiated (activated) preparations were believed to produce
only the clinical effect. It was postulated that these drugs are ineffective with
out individualization of therapy.
Recent experiments showed that ultralow doses produce a fine molecularand
cellular effect, which is not directly related to the principle of similarity. Acti
vated preparations have a small effect that is insufficient for therapeutic activi
ty. Taking into account these data, we assumed that homeopathic therapy is
based on increasing the strength of ultralow doses by the mechanism of hyper
ergia.
After studying the ultralow doses of remedies with healthy volunteers, S.
Hahnemann revealed the existence of individual sensitivity to certain medical
products (complex of clinical, psychological, psychological, and topic criteria).
23
Ultralow doses
C h a p t e r
2
Three types of effects
of ultralow doses
T
he first professional experience of homeopathy showed that it is not
necessary to follow the principle of similarity in achieving a therapeutic
effect of remedies in ultralow doses. For example, a group of preparations exists
that are a priori tropic for the liver (Lycopodium, Chelidonium, and Carduus
marianus). Even without individualization of therapy, these medical products
improve the state of most patients with hepatobiliary disorders. Moreover, travel
sickness (acute paroxysmal state) may be treated with several homeopathic
remedies that reduce the symptoms of autonomic disorders. Individualization of
therapy is not required under these conditions.
The “Materia Medica Holding” company was established in 1992. In the
initial period, this company manufactured the socalled “complex homeopathic
remedies”. The pharmaceutical formulation consisted of three or four homeo
pathic drugs (granules and tablets). Other Russian and foreign complexons of
wellknown homeopathic components from plants and minerals, as well as
organotropic preparations of animal (embryonic) tissues or organs, appeared on
the pharmaceutical market. The effects of potentiated remedies and tissue
preparations are related to their tropism for a certain pathological condition and
organ, respectively.
A complex preparation Agri (homeopathic Antigrippin) was the brand of
the “Materia Medica Holding” company in the 1990s. Surprisingly, this drug
had a preventive effect on influenza and chill. These facts seem to contradict
the principle of similarity.
24
Chapter 2. Three types of effects of ultralow doses
Such “discrepancies” with the homeopathic doctrine did not alter our
relation to the basic principles of homeopathy. These data stimulated us to
search for new indications for ultralow doses.
Experimental and clinical trials of potentiated products were
conducted at the “Materia Medica Holding” company beginning from 1995.
Several discoveries were made. The first steps of a largescale scientific research
were devoted to study the effect of combined treatment with a medical product
in normal dose and activated form of the same remedy. The phenomenon of
isopathy was well known. It suggests that the symptoms of poisoning with a
certain substance are treated by the potentiated form of this substance. Due to
high risk of chemical attacks of the German army during World War II, English
volunteers were exposed to skin burns with mustard gas. These burns were then
treated with potentiated mustard gas. Complications of corticosteroid therapy,
including ItsenkoCushing syndrome, are homeopathically treated with Cortex
(activated prednisolone). However, isopathy was not subjected to a complex
clinical study. Moreover, there are no experimental data on this problem.
Similarly to the preventive effect of Agri, we decided to test ultralow doses
of a certain substance for protective activity during intoxication that was induced
by this substance in high (toxic or subtoxic) doses. As differentiated from the
isopathic method, ultralow dose (“antidote”) was administered in combination,
but not after treatment with the same substance in toxic doses (poison). We
believed that this approach does not contradict the homeopathic doctrine.
Administration of the substance in toxic doses served as a model to induce the
symptoms typical of treatment with the activated form.
The principle of our experiments was quite paradoxical. We tried to
introduce a “drop” (activated substance) into the “sea” (toxic dose of the
substance). Combined treatment with ultralow doses was performed in various
regimens. Ultralow doses were administered simultaneously or before treatment
with the toxic dose (onetoten minutes). Various routes of treatment with ultra
low dose were also analyzed. For example, the potentiated preparation was
mixed with a toxic dose of the same substance. The mixture was administered
perorally through a probe. Otherwise, the original substance was administered
parenterally, while the activated preparation was given perorally.
Combined treatment with the substance in ultralow (homeopathic) and
normal (therapeutic or toxic) doses was performed simultaneously or in a small
interval. This approach received the name bipathic* treatment (O. I. Epstein,
1996, 1997). Prednisolone was the first medical product for combined treatment.
It was the first step from homeopathy to immunopharmacology. Therefore, this
study should be described in details.
* Bipathic administration, allopathy + homeopathy.
25
Ultralow doses
Bipathic administration of prednisolone was performed at the Laboratory of
Biophysics (Kiev Institute of Otorhinolaryngologist) headed by A.F. Karas’. The animals
simultaneously received prednisolone in “normal” and potentiated doses (dilution C30). In
the latter case, the conventional concentration of prednisolone was 10—60 wt % (10—60 M).
Series I was performed on 30 rats. The effect of bipathic treatment with pred
nisolone was studied on animals with experimental acute inflammation. This state was
induced by injection of 0.05 mg 1% carrageenan into the hindlimb of rats. Activated
prednisolone was administered through a probe to group 1 animals with experimental
inflammation. Group 2 animals received prednisolone in a total dose of 20 mg per rat.
Group 3 animals were subjected to combined treatment with both drugs. The standard and
potentiated forms of prednisolone were administered twice (1 h before and 2 h after
carrageenan injection). The control groups consisted of intact animals and untreated rats
with carrageenaninduced inflammation.
We showed that administration of prednisolone alone or in combination with
activated prednisolone is followed by the reduction of inflammatory edema of the paw.
Potentiated prednisolone had no antiinflammatory activity and did not potentiate the
antiinflammatory effect. However, bipathic administration of prednisolone was
accompanied by several positive effects.
Migration of peritoneal macrophages significantly decreased during inflammation.
Normal doses of prednisolone did not improve macrophage migration. However,
macrophage migration rapidly returned to the control level after combined treatment with
study drugs.
An electron microscopic and morphological study showed that administration of 20
mg prednisolone is followed by moderate destructive changes in the liver and thymus of
animals. However, the structure of these organs was not impaired after combined
administration of prednisolone and activated prednisolone in the same doses.
Carrageenaninduced inflammation is accompanied by the increase in energy
consumption due to activation of the protective response. Prednisolone has no effect on
blood ATP concentration. Bipathic administration of prednisolone is followed by a sharp
decrease in ATP content, which reduces energy supply to the inflammatory process.
Combined administration of prednisolone in the toxic and ultralow dose improves enzyme
activity in blood cells, activates alkaline phosphatase in neutrophils, and has a normalizing
effect on the activities of ATPase, 5nucleotidase, and lactate dehydrogenase. These
changes illustrate the restoration of energy supply to cells.
As compared to “standard” prednisolone, bipathic administration was followed by
a greater increase in biosynthetic activity of rat peripheral blood lymphocytes. This
conclusion was derived from the increase in RNA level.
Series II was designed to evaluate whether potentiated prednisolone (10—60 wt %)
may prevent the side effect of chronic treatment with prednisolone in normal doses for 2
weeks. Prednisolone in a daily dose of 50 mg/kg body weight was administered through a
probe. This dose of prednisolone produced a strong antiinflammatory effect on the model
of carrageenininduced inflammation. The potentiated substance had a variety of protective
effects under these conditions (Table 2.1; V.G. Zilov et al., 2000).
These data indicate that the activated agent prevents metabolic disorders
in lymphocytes and neutrophils and, probably, has a normalizing effect on
membrane processes after treatment with normal doses of prednisolone.
A potentiated form of prednisolone also prevented the development of de
structive changes in the liver, adrenal glands, and lymph nodes and gastric
26
Chapter 2. Three types of effects of ultralow doses
Table 2.1. Protective effects of potentiated prednisolone
Prednisolone
Prednisolone +
potentiated form
Significant increase in the number
of stab granulocytes
Slight increase in the number of stab
granulocytes; significant decrease
in the absolute number of monocytes
Decrease in leukocyte count compared
to the control
Leukocyte count does not differ
from the control
No effect on peroxide chemiluminescence
Activation of peroxidation in blood plasma
Twofold decrease in adenosine triphosphate
(ATP) content in the blood
Increase in blood ATP concentration
above normal
Twofold decrease in alkaline phosphatase
level in neutrophilic leukocytes
Slight decrease in alkaline phosphatase
level in neutrophils
Increase in lactate dehydrogenase activity
in neutrophils
Lactate dehydrogenase activity in neutrophils
practically does not differ from normal
Decrease in ATPase activity in lymphocytes
(nearly by 2 times)
No changes in lymphocyte ATPase
activity
mucosal erosion, which is typical of longterm treatment with this drug.
Activated prednisolone had a normalizing effect on synthetic activity of lym
phocytes, which was suppressed after longterm administration of prednisolone.
It was manifested in an increase in the amount of chromatin proteinuncoupled
DNA. Hence, functional activity of lymphocytes returned to normal under the
influence of activated prednisolone.
We conclude that during combined (bipathic) treatment, potentiated
prednisolone has protective (adaptive) activity and abolishes the effect of
prednisolone in the toxic dose*.
The next series of experiments was performed in collaboration with
Professor Tamara Mikhailovna Vorob’eva (Head of the Laboratory of Neuro
physiology and Immunology, Ukrainian Institute of Neurology and
Psychiatry). When the protective effect of activated prednisolone was
established, the author of this monograph (professional psychiatrist) decided
to develop a new drug for the therapy of alcohol abuse and opium addiction.
We asked Tamara Mikhailovna to perform an experimental study with po
tentiated ethanol and morphine. The animals were subjected to chronic
intoxication with these substances. These experiments involved the standard
neurophysiological behavioral tests, biochemical assays, and immunological
*
In some experiments, a potentiated form of prednisolone tended to increase the anti
inflammatory activity of prednisolone. Initially, this fact received little attention.
27
Ultralow doses
methods. Electrical activity of the brain and selfstimulation of the “pleasure
center” in the lateral hypothalamus were studied*.
I would like to briefly describe the results of “bipathic” treatment with
ethanol. It was shown that ultralow dose of test substance modifies the effect
of the same substance in normal dose. Activated ethanol had a strong protective
(adaptive) effect against alcohol in the toxic dose. It was manifested in the “re
gulation” of animal behavior (e.g., conditioned responses) and normalization of
several parameters (electrical activity of the brain, structure of sleep,
neurotransmitter balance, and blood alcohol level). It should be emphasized that
the animals could stimulate the “pleasure center” via a stereotaxic electrode by
pressing the lever. However, they refused to perform selfstimulation. T. M. Vo
rob’eva believed that this unusual effect reflects a wellbalanced emotional state.
Hence, pathological alcohol addiction was reduced in these animals. Further
clinical trials with ultralow doses of alcohol showed that they have antiabsti
nence properties. An antialcohol drug AntiE (activated alcohol) was approved by
the Russian Ministry of Health. This drug was manufactured by the “Materia
Medica Holding” ResearchandProduction Company beginning from 1998.
Experiments of T. M. Vorob’eva and further clinical trials showed that
potentiated morphine has a wide range of protective activity during intoxication
with morphine or opium surrogates. However, this drug was not introduced into
clinical practice.
Our studies revealed the phenomenon of bipathy in 1996 (200year
anniversary of homeopathy). Followers of Hahnemann performed the effective,
but extremely conservative studies for 2 centuries. Homeopaths believed that the
principle of similarity is absolutely essential for the effectiveness of ultralow
doses. Initially, we shared this opinion. The results of experimental studies in the
mid1990s showed that activated preparations have biological activity. However,
this fact received insufficient attention of homeopaths. There was a great
discrepancy between homeopathic physicians and experimental biologists. On
the one hand, homeopaths could not ignore the homeopathic doctrine. On the
other hand, biologists knew a little about homeopathy and, therefore, could not
introduce the phenomenon of homeopathy into the area of rational scientific
knowledge. At the same time, studies of low and ultralow doses were conducted
for a long time. At the beginning of the 1920s, a famous Russian pharmacologist
N. P. Kravkov (1924) showed that blood flow variations in the rabbit ear can be
induced by vasoconstricting and vasodilating agents in low concentration (up to
10—32 wt %). Further experiments of A. N. Kudrin (1991) revealed that
*
28
The results of this experiment and further studies are described in the monograph
“Informational and ontological models of adaptation” (O. I. Epstein et al., 1997) and
joint articles.
Chapter 2. Three types of effects of ultralow doses
administration of epinephrine in a concentration of 10—16 wt % has a similar
effect on blood flow in frog mesenteric vessels. The effectiveness of phosphatase
in a concentration of 10—16 wt % was demonstrated by A. M. Kuzin in 1947.
The results of these experiments were published only in 1997. At the beginning
of the 1950s, A. Gay and J. Boiron showed that sodium chloride at dilution C27
modifies the dielectric constant of water. On the basis of these data, one vessel
with potentiated sodium chloride in the submolar concentration was correctly
selected from 100 vessels (99 vessels with placebo). In 1941, W. Boyd
demonstrated that activated mercury chloride in a concentration 10—6 wt %
accelerates enzymatic hydrolysis of starch.
The effects of substances in ultralow concentrations on biological objected
(primarily on plants) were described by various authors, including G. N.
ShanginBerezovskii (1982, 1986) and L. Kolisko (1953).
In the 1980s, hightechnology studies with ultradiluted solutions were de
voted to the evaluation of their biological activity. A group of investigators under
the direction of Professor E. B. Burlakova (N. E. Emanuel’ Institute of Bio
chemical Physics, 1986) showed that antioxidants at submolar dilution (10—15
M) have a stronger effect on electrical activity of the isolated snail neuron than
those in the physiological concentration (10—3 M). Further experiments of
E. B. Burlakova et al. (1986, 1990) revealed that ultralow doses have various
biological effects.
The results of a wellknown study by E. Danevas and J. Benveniste (1988)
were published in Nature. They showed that high and low doses have a similar
effect. Until the present time, this experiment is one of the most “academic”
researches in the field of ultralow doses. The authors revealed that degranulation
of basophils with surface immunoglobulin E (IgE) may be induced by antiIgE
at concentrations of 10—210—120 M. Treatment with the substance in these
concentrations was followed by successive peaks of degranulation in 4060%
basophils. Molecules of antiIgE were absent in several dilutions, which
exceeded the Avogadro constant. However, the authors hypothesized that this
method for preparation of homeopathic dilutions (thorough shaking of the
solution) provides transmission of biological information due to the arrangement
of water molecules. Basophil degranulation was also observed in the presence
of other substances at high and low dilutions, including the specific allergen
(basophils from allergic patients) and peroxidase (basophils from peroxidase
immunized rabbits). J. Benveniste et al. evaluated the degree of basophil
degranulation in the presence of phospholipase A2 from bee venom or pig
pancreas, sodium ionophore monensin (up to 90% degranulation at an
equivalent concentration of 10—30 M), and calcium ionophores A23187 and
ionomycin (10—38 M). The specific effect of high dilutions was confirmed by
experiments with ionophores. Degranulation of basophils decreased after the
29
Ultralow doses
removal of the corresponding ion from the extracellular medium (E. Danevas
et al., 1988).
These results were reproduced in six laboratories of four universities (Paris
South University, Toronto University, Jewish University, and Milan University)
and published in Nature. However, an Editorial Article (J. Maddox, 1988) has
cast doubt on the reliability of these data. In the followup period, a similar
experiment was repeated under strict conditions. The results of this study were
published in the Journal of the French Academy of Sciences (J. Benveniste, 1991).
To bridge the gap between modern biology, medicine, and homeopathy, it
was necessary to follow simple steps. The phenomenological (narrow) view of
ultralow doses should be changed to a detailed systemic evaluation of their activity.
Except for several researches in the 1980s and mid1990s, a rational study of
ultralow doses was terra incognita. Until recent times, homeopathic remedies should
meet simple requirements of Medical Regulatory Authorities in various countries.
Hence, even worldwide leaders in the production of homeopathic remedies do
not have the experience of highlevel experimental studies with ultralow doses.
In discussing the results of studies with bipathic (combined) admi
nistration of ethanol and morphine, T. M. Vorob’eva supposed that the pheno
menon of bipathy is related to biological properties of ultralow doses. These
properties should not be associated with the homeopathic doctrine. She
proposed to perform a detailed study of ultralow doses. Neurophysiological
studies of morphine in ultralow doses were performed in 1996.
A largescale study showed that the systemic effect of activated morphine
is qualitatively similar to that of normaldose morphine. Similarly to morphine
in normal doses, potentiated morphine decreased the pain threshold, facilitated
the acquisition of conditioned reactions in animals, and modulated the
emotional state (model of selfstimulation). However, morphine in ultralow
doses did not cause euphoria and addiction. These data could break the taboo
of homeopathy, which was associated with the principle of similarity. Morphine
in ultralow doses had a strong effect on experimental animals without
individualization of treatment.
Tamara Mikhailovna Vorob’eva showed that ultralow doses have a specific
biological activity, which does not depend on individual treatment. It became
clear that this activity is a general property of ultralow doses. Similarly to
homeopathy, bipathy should be considered as a particular approach to the use
of potentiated drugs. Morphine and other substances in ultralow doses have little
effect, which limits the therapeutic use of these drugs. Hence, a particular
application (homeopathy and bipathy) is of greater importance than a general
application. The indication for use, but not the dose, serves as a “watershed”
between allopathy and homeopathy. Trials of homeopathic remedies are
performed on healthy volunteers. Homeopathic remedies can cause allergic
30
Chapter 2. Three types of effects of ultralow doses
reactions in some of these individuals. They serve as a clinical criterion for the
use of this remedy. Modern pharmaceutical products are tested on patients. The
hyperergic reaction to these pharmaceutics is considered as a side effect. The
indications for use of “standard” pharmaceutics are based on their general
(physiological) properties.
Previous studies of cyclophosphane, phenazepam, and haloperidol showed
that combined treatment with a medical product and potentiated substance
holds much promise. Combined administration of cyclophosphane and ultralow
dose of this drug to experimental animals with melanoma, lung cancer, and
carcinosarcoma was followed by an increase in antitumor activity of the
cytostatic. The antimetastatic effect of cyclophosphane increased most
significantly after bipathic administration (E. N. Amosova, 2003).
To understand the mechanisms of bipathy we evaluated whether this
phenomenon is the prerogative of a living organism, or ultralow dose may
modify the effect of “high” dose in simple physicochemical systems. In vitro
experiments were performed to answer this question. The first study was con
ducted under the direction of Professor A. V. Zakharov and Senior Researcher
V. G. Shtyrlin (Candidate of Chemical Sciences) at the Kazan State University.
A nuclear magnetic resonance study was performed to estimate the effect
of potentiation on the kinetics of ATP hydrolysis with citrate buffer at
physiological pH. The rate of hydrolysis was measured after addition of
potentiated ATP or potentiated buffer (Table 2.2).
The rate of hydrolysis decreased slightly after addition of any component
in a bipathic form.
Our further studies were performed in collaboration with Professor S. I.
Petrov (Institute of Oil and Gas). Potentiated preparations of lithium chloride
and mercury nitrate were shown to modulate electroconductivity of a simple
Table 2.2. Effect of potentiated substances on hydrolysis
№
Type of sample
Hydrolysis rate, K (sec–1)
1
Reference
(3.35+0.07)×10—5
2
Reference
(3.45±0.12)×10—5
3
Bipathic
(with potentiated ATP solution)
(3.02±0.05)×10—5
4
Bipathic
(with potentiated buffer solution)
(2.60±007)×10—5
Note. (1, 2) Reactions with two various reference preparations; (3) addition of 10 vol % potentiated
homeopathic solution of ATP at dilution C30; (4) addition of 10 vol % potentiated homeopathic solution
of citrate buffer at dilution C30. K, hydrolysis rate constant for ATP in aqueous solutions of the reference
and potentiated sample with citrate buffer at pH 6.7 and T378K.
31
Ultralow doses
electrochemical system, which contained these electrolytes in normal
concentrations (S. I. Petrov et al., 2003). Professor M. A. Myagkova et al.
(2003) revealed that activated antibodies in ultralow doses have a modulatory
effect on the antibodyantigen binding constant in EIA. Similar results were
obtained in further experiments of E. A. Dukhanina.
The effect of potentiated antibodies on “standard” antibodies was studied
by means of EIA (E. A. Dukhanina).
A reaction mixture consisted of antigen (sorbed in plate wells), standard antibodies
(diluted in phosphatebuffered saline, PBS), and potentiated dilutions. The control systems
were composed of potentiated water and PBS instead of potentiated dilutions and standard
antibodies, respectively. The optical density in wells with potentiated dilutions was much
higher than that in wells with standard antibodies and water. The average optical density
Ast+dilut was estimated in four independent experiments with 1012 samples of potentiated
dilutions. This parameter varied from 0.276±0 to 0.643±0.024. The average optical density
Ast+water varied from 0.210±0.046 to 0.531±0.026. These values were not the sum of optical
densities for the reference substance and potentiated dilutions. For a quantitative study of
the effect, the relative optical density was calculated as follows: (Ast+dilutOAst+water)×100%/
Ast+water. The average value was 23.2±7.2%. These data suggest that potentiated dilutions
have a direct effect on the antigenantibody interaction. Standard antibodies and sorbed
antigen in various concentrations were used for a detailed study of this phenomenon. The
concentration of standard antibodies varied from 0.3 to 100 ng/ml. The content of standard
antibodies varied from 5.7±3.0 (at 0.3 ng/ml) to 22.7±3.9% (at 100 ng/ml). Hence, decreasing
the concentration of standard antibodies was accompanied by the reduction of effect.
We discovered the phenomenon of bipathy. During combined (bipathic)
administration of ultralow dose and normal dose, the activated preparation
always modifies the effect of the original substance. A potentiated form in vivo
and in vitro modifies the effect of the original substance. It should be
emphasized that the potentiated preparation modifies not only biological, but
also fine physicochemical properties of the original substance.
As regards the toxic and subtoxic dose* of a medical product, its
potentiated form has a strong protective (adaptive) effect. Sometimes the
potentiated preparation “strengthens” a therapeutic effect of the original
substance (e.g., antimetastatic activity of cyclophosphane). Under other
conditions the potentiated preparation has no effect on pharmacological activity
of the original substance (e.g., antiinflammatory effect of prednisolone).
Bipathy could become a major direction of activity in the “Materia
Medica Holding” ResearchandProduction Company. Moreover, potentiation
of wellknown pharmaceutics and correction of their toxicity are the urgent
problems. These approaches are developed by the world’s leading pharma
*
32
The potentiated substance can produce a complex polymodal effect on toxic doses (see
Chapter 5).
Chapter 2. Three types of effects of ultralow doses
ceutical companies. Unexpectedly, our study gained a new direction. We met a
famous scientist and one of the leading specialists in neuroimmunology M. B.
Shtark (Academician of the Russian Academy of Medical Sciences). Mark
Borisovich thoroughly examined the results of our experiments. He proposed
further studies on simple biological models to formally confirm the presence of
bipathy. In the opinion of M. B. Shtark, the socalled longterm posttetanic
potentiation (LTPTP) in surviving brain slices serves as an adequate model.
Ultrathin sections of the animal brain can retain functional activity for a long
time in a special nutrient medium. LTPTP is a wellknown electrophysiological
phenomenon. This phenomenon has been extensively used in neurobiology for
many years. Neurotropic activity of various drugs may be evaluated from their
effect on LTPTP. The phenomenon of bipathy was studied on the model of
LTPTP with antibodies to S100 protein from nervous tissue*.
In immunology, any molecule that causes the formation of complemen
tary antibodies is designated as an antigen. Hence, we shall use the term
“antibodies to S100 antigen”.
The description of our experiment may seem complicated to the reader
of this book (i.e., general practitioner). Let’s consider only the main results.
Antibodies to S100 antigen (antiS100) in normal doses have an inhibitory
effect on LTPTP. Under certain technical conditions, the potentiated substance
completely abolished a physiological effect of antiS100 in “normal” doses. A
major advantage of our experiment is the uniqueness of results. AntiS100 in
normal doses inhibited the electrophysiological reaction (LTPTP—). However,
this reaction returned to normal after combined treatment with potentiated anti
S100 (LTPTP+; O. I. Epstein et al., 1999).
This experiment was performed by M. A. Starostina, N. A. Borisov, and
N. S. Sorokina under the supervision of M. B. Shtark (Novosibirsk Institute of
Molecular Biology and Biophysics, Siberian Division of the Russian Academy
of Medical Sciences). These data are of particular importance for our scientific
research. A complete description of this experiment is presented below (ac
cording to the monograph of V. G. Zilov et al., 2000).
The study was performed on surviving slices with an artificial medium, which retains
physiological activity for a long time. It was based on a wellknown electrophysiological
phenomenon of LTPTP. During tetanic electrical stimulation in one of the regions of rat
hippocampal slice, the evoked postsynaptic potential is recorded in another region with a
special electrode. This potential persists for a long time (from 40 min to several hours) and
has specific characteristics. Synaptic effectiveness (transmission) is evaluated by recording
of the potential.
*
An unusual name of this protein is associated with the procedure of isolation. In one
of the stages, this protein is dissolved in 100% saturated solution of ammonium sulfate.
33
Ultralow doses
The development of LTPTP is a calciumdependent process. S100 protein is a
calciumbinding protein, which has an important role in synaptic processes. The antiserum
to S100 protein inhibits these processes, including LTPTP.
However, the synaptic effect was abolished after bipathic (combined) administration
of antiserum in normal dose and potentiated form C6 (10—12 wt %). A homeopathic dose
of the substance (i.e., immunological preparation) blocked the action of an effective dose.
It is important that this experiment involved an electrophysiological method, which
allowed us to repeat the measurements under standard conditions.
This series was performed to compare the effects of antibodies to
neurospecific protein S100 and potentiated sample of the same antibodies. The
influence of combined (“bipathic”) treatment with these antibodies was studied
on the model of LTPTP in the hippocampus of animals.
Experiments were performed on hippocampal slices from Wistar rats
weighing 180299 g. Transverse hippocampal sections (400 m in width) were
placed in a temperaturecontrolled chamber at 3537oC (Fig. 2.1). Flow
Yamamoto medium (Fig. 2.2) was aerated by carbogen (95% O2 and 5% CO2).
1
3
4
10
8
7
5
2
9
5
6
Fig. 2.1. Scheme of an experimental chamber to study LTPTP in surviving
hippocampal slices.
The incubation medium is delivered from reservoir 1, passes successively through polyvinyl tubes 2,
tap 3 (regulation and maintenance of the flow rate at 250 ml/min), and dropper 4 (prevention of air
bubble formation in the system), and enters compartment 6 (heatcapacity fluid). The constant
temperature of this fluid was maintained using thermostat 5. The incubation medium was delivered
through tube spirals in compartment 6, heated to a certain temperature, and entered chamber 7 (5
10 ml in volume). The medium outflowed from chamber 7 through waterjet pump 8. Incubation
chamber 7 was closed with cap 10. The fluid in chamber 7 was heated and aerated with carbogen,
which contributed to water evaporation and carbon dioxide exchange between the medium and air.
Cap 10 had the only hole above chamber 9 for the release of water vapor and CO2 that moistened
the abovechamber space and prevented a change in pH.
34
Chapter 2. Three types of effects of ultralow doses
Fig. 2.2. General view of an experimental device to study LTPTP in surviving
hippocampal slices: chamber with a flow system, micromanipulators that carry
cathode followers, preamplifiers, and stimulating and reference electrodes. Position
of electrodes is shown in Fig. 2.3.
Evoked postsynaptic potentials (EPSP) were recorded after 4060min
incubation. A stimulatory electrolytically sharpened bipolar wolfram electrode
was introduced into the zone of mossy fibers. A reference glass electrode (tip
thickness 34 m, resistance 25 mO) was filled with 2.5 M NaCl and placed in
CA3 region (initial segments of apical dendrites; Fig. 2.3).
Testing was performed with single rectangular pulses (duration 200 msec)
delivered at intervals of not less than 5 min. The amplitude of test stimuli varied
СА1
СА2
!!!!!!!!!!
СА3
!!!!! !
!
!!
!
СА4
DF
2
1
Fig. 2.3. Scheme for the position of a reference (1) and stimulating electrode (2)
to study the dynamics of LTPTP in surviving hippocampal slices. CA14, fields of
the Ammon’s horn (hippocampus); DF, dentate fascia.
35
Ultralow doses
from 10 to 30 V. EPSP were recorded on a 12digit analogtodigital converter
(Digidata, Axon Instruments Inc.). The results were analyzed on a computer
with pClamp6 (Axon Instruments Inc.) and Microcal Origin softwares.
To induce LTPTP, the amplitude of a test stimulus was selected so that
the response corresponded to 50% of the maximum value. Tetanization was
produced by three consecutive series of stimulation at 200 Hz. The length of
each series was 1 sec. Stimulation was applied at 2sec intervals. The procedure
of tetanization was repeated after 10 min. EPSP were recorded for at least 40
min after the first tetanization, which allowed us to make a conclusion about
the induction or absence of LTPTP. A significant increase in the amplitude of
EPSP (by 1.52 times), which persisted for at least 20 min after the second
tetanization, served as a criterion for the induction of potentiation.
The effect of antibodies to S100 protein was studied as follows.
Tetanization was induced in one or two slices of each series. Further
experiments with slices of this series were performed only after the induction of
LTPTP. All slices were maintained in the incubation medium after addition of
a specified amount of antibodies or reference solutions. The initial latency of
effect was considered to be 20 min (according to D. Levis and T. Teyler). The
effect of antiserum to neurospecific protein S100 on the induction of LTPTP
in rat hippocampal slices was described previously (D. Levis et al., 1986). Then
the period of incubation was selected experimentally. After study of each
dilution, the chamber was repeatedly washed with distilled water and ethyl
alcohol and completely dried with compressed air.
The indication of LTPTP is characterized by a significant increase in the
amplitude of EPSP in synapses of mossy fibers in the hippocampal dentate
fascia in response to the test stimulus after tetanization (Fig. 2.4).
Twentyminute incubation with antiserum to neurospecific protein S100
(antiS100, final dilution 1:50) completely inhibited the induction of LTPTP
mV
5
1
0
2
0
5
10
15
20
Time, μsec
Fig. 2.4. Dynamics of EPSP in CA3 region of the Ammon’s horn during extracellular
recording. Amplitude of the test stimulus is 20 V. (1) Before and (2) 10 min after
tetanization.
36
Chapter 2. Three types of effects of ultralow doses
(Fig. 2.5), which is consistent with the results of previous experiments. High
dilution of antiserum in our experiments is related to differences in the titer of
antibodies at various laboratories.
Nonimmune rabbit antiserum at the same dilution had no effect on
LTPTP induction in rat hippocampal slices (Fig. 2.6). Incubation of slices with
ethanol at a concentration present in potentiated preparations (similar dilution)
did not impair the induction of LTPTP in slices (Fig. 2.7).
To study the combined effect of test samples, antiS100 and its
potentiated form were added simultaneously to the incubation medium. This
treatment completely blocked the induction of LTPTP in slices. It should be
emphasized that combined administration of these substances did not abolish
mV
0.6
0.5
0.4
0.3
0.2
0.1
0
2
1
3
4
5
6
7
Fig. 2.5. Effect of antiS100 on the induction of LTPTP in hippocampal slices.
Ordinate, amplitude of EPSP. (1) Before treatment with antiS100; (25) over 20
min after treatment with antiS100 (57min interval); and (6, 7) 7 and 12 min after
the second tetanization, respectively. Amplitude of the test stimulus is 30 V.
1.0
0.8
0.6
0.4
0.2
0.0
1
2
3
4
5
6
Fig. 2.6. Induction of LTPTP in the presence of nonimmune rabbit serum. Dilution
1:50. Ordinate: average amplitude of EPSP. (1) After 10min incubation in non
immune serum; (2, 3) 5 and 10 min the first tetanization, respectively; and (46)
over 20 min after the second tetanization. Amplitude of the test stimulus is 12 V.
37
Ultralow doses
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1
2
3
4
5
Fig. 2.7. Induction of LTPTP after addition of 40 ml 40% ethanol. Volume of an
experimental chamber is 10 m. Ordinate: average amplitude of EPSP. (1, 2) 20min
incubation in Yamamoto medium after addition of ethanol; (3) 10 min after the
first tetanization; and (4, 5) 10 and 30 min after the second tetanization,
respectively. Amplitude of the test stimulus is 15 V.
the effect of antiS100. Similar results were obtained after 10min preincubation
of the slice with potentiated antiS100, further addition of antiS100, and 20
min incubation in a solution of both substances.
The effect of antiS100 was abolished after 20min preincubation of slices
in a solution of potentiated antiS100 (concentration 1012) and 20min
incubation in a solution of native and potentiated antiserum. Hence, the
induction of LTPTP in slices was similar to that in control samples not exposed
to antibodies (Figs. 2.8 and 2.9).
It could be suggested that the effect of native antiS100 is abolished due to long
term incubation of the slice with an ethanolcontaining solution of the potentiated form,
which results in modulation of the membrane state and/or impairment of antibodyantigen
binding. The next series was performed to test this hypothesis. Preincubation was performed
in an ethanol solution, whose concentration did not differ from that in potentiated anti
S100. Other manipulations were similar to those in the previous series. Under these
conditions, antiS100 retained the ability to block the induction of LTPTP.
These data show that nonimmune serum and 40% ethanol did not prevent the
induction of LTPTP. The inhibition of LTPTP was observed only in antiS100 solutions.
Preincubation with potentiated antiS100 for 20 min abolished the inhibition of LTPTP by
antiS100. This procedure did not prevent a normal reaction of the hippocampal CA3
region, which had a potentiating effect on synaptic effectiveness.
A study of the model of LTPTP provides strong evidence for the
phenomenon of bipathy. The experiment had unexpected consequences. In
discussing the results of this research, we hypothesized that activated antibodies
exhibit inexplicable “proantigenic” activity. T. M. Vorob’eva and M. B. Shtark
did not exclude this possibility. However, it was necessary to confirm our
hypothesis. Studies with potentiated antibodies to various neurotropic
38
Chapter 2. Three types of effects of ultralow doses
a
mV
1.0
0.8
0.6
Fig. 2.8. “Bipathic effect”.
(a) Induction of LTPTP in the
presence of antiS100 (final dilution
1:50): (13) incubation in Yamamoto
medium with antiS100 for 20 min,
interstimulus interval 57 min; (46)
10 min after the first tetanization, 3
4 min intervals; and (7, 8) 10 and 25
min after the second tetanization,
respectively. Amplitude of the test
stimulus is 12 V.
0.4
0.2
0.0
1
2
3
4
5
6
7
8
b
mV
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1
2
3
4
5
6
7
8
9
10
c
mV
2.0
1.5
1.0
0.5
0.0
5
10
15
20
11
(b) Induction of LTPTP in the
presence of potentiated antiS100 at
a concentration of 10–12 (40 mmol):
(13) incubation in Yamamoto
medium with potentiated antiS100
(1012) for 20 min, interstimulus
interval 57 min; (46) over 10 min
after the first tetanization, 34 min
intervals; and (711) over 30 min
after the second tetanization, 57
min intervals. Amplitude of the test
stimulus is 20 V.
(c) Induction of LTPTP in the pre
sence of antiS100 at a concentra
tion of 10—12 (40 mmol) and dilution
1:50: (13) incubation in Yamamoto
medium with potentiated antiS100
at a concentration of 10–12 for 20
min, interstimulus interval 10 min;
(46) incubation with potentiated
antiS100 for 20 min, 57min
intervals; (710) over 10 min after
first tetanization, 23min intervals;
and (1121) over 40 min after the
second
tetanization,
35min
intervals. Amplitude of the test
stimulus is 10 V.
substances, including morphine, delta sleepinducing peptide, histamine, and
serotonin, were performed in Kharkov and Novosibirsk. These investigations
supported our hypothesis. Under various conditions, potentiated antibodies and
antigen had “codirectional” activity. It became obvious that this is a new
immunological phenomenon. In standard immunological reactions, binding of
antibodies to the complementary antigen is followed by the inhibition of its
39
Ultralow doses
V, mV
0.7
0.6
0.4
0.2
0.0
1
2
3
4
5
6
7
Fig. 2.9. “Bipathic effect: induction of LTPTP under “bipathic” conditions: (1, 2)
20min preincubation with potentiated antiS100 (10 –12); (3, 4) incubation with
native antiS100; (5) after the first tetanization; and (6, 7) 5 and 10 min after the
second tetanization, respectively.
activity. By contrast, antibodies in ultralow doses modify the activity of this
antigen.
We asked T. M. Vorob’eva to perform the next series of experiments.
Several pairs of potentiated antibodies and antigen (S100 protein and
antibodies to S100 protein; morphine and antibodies to morphine; delta sleep
inducing peptide and antibodies to delta sleepinducing peptide; etc.) were
studied on the model of behavior and brain selfstimulation. It was shown that
psychotropic activity of antibodies in ultralow doses is higher than that of
antigen in ultralow doses.
It should be emphasized that during this period the author of this
monograph was not familiar with immunology. Similarly to the phenomenon
of bipathy, we assumed that there is a mediator between activated antibodies
and antigens (endogenous molecules, i.e., S100 protein) in the organism. I.
P. Ashmarin and I. S. Freidlin hypothesized that the socalled natural
antibodies have regulatory functions (I. P. Ashmarin et al., 1989). This
hypothesis developed the theory of a famous immunologist Pierre Grabar
about the physiological role of autoantibodies (P. N. Grabar, 1969). Under
normal conditions, nearly all molecules in the organism have “predetermined”
antibodies in very low physiological concentrations. These antibodies exhibit
affinity for the corresponding antigens and stabilize, but not inhibit their
activity. We suggested that natural antibodies serve as a target for activated
antibodies in the organism. The effect of antibodies in ultralow doses in
mediated by a change in physiological functions of predetermined antibodies
(“regulation of regulator”).
Scientific collaboration between the Institute of Molecular Biology and
Biophysics (Siberian Division of the Russian Academy of Medical Sciences),
40
Chapter 2. Three types of effects of ultralow doses
Ukrainian Institute of Neurology and Psychiatry, and “Materia Medica
Holding” ResearchandProduction Company resulted in the development of
three medical products with activated antibodies of a new class (Proprotein100,
Anar, and Tenoten). An antialcohol drug Proprotein100 contains antibodies to
S100 at dilution C1000. This is the first drug of a new pharmacological class.
Tenoten and Tenoten for children were synthesized from activated antibodies to
S100 protein in other doses. They have a wide range of pharmacological
activity. Anar is a drug for the therapy of opium withdrawal syndrome, which
contains ultralow doses of antibodies to morphine.
The discovery of a new immunological phenomenon in 1998 was
accidental. However, this discovery was associated with the results of previous
studies with ultralow doses. The phenomenon of bipathy was historically
preceded by isopathy. However, it was impossible to foresee new properties of
potentiated antibodies. Before 1998, there was only one historic “junction”
between ultralow doses and immunology. J. Benveniste showed that antiIgE
antiserum causes degranulation of basophils (E. Danevas et al., 1099). It could
be suggested that treatment with antiIgE in ultralow and normal dose causes
the same physiological phenomenon. However, new properties of activated
antibodies were not revealed due to technical reasons. These studies should be
performed on another experimental model (i.e., monospecific antibodies).
Moreover, the phenomenon of bipathy should form the basis for a new
ideology.
After “accidental contact” with immunology, we studied the basic
principles of this relatively young and rapidly developing area of biology and
medicine. It was unexpected that homeopathy and immunology have common
roots. Biologically, homeopathy is based on individual sensitivity. The immune
system maintains the individual and genetically determined integrity in an
organism. The knowledge of homeopathy and results of studying the biological
activity of ultralow doses provide new insight into physiological functions of the
immune system (see Chapter 4).
It became evident that preparations of antibodies should be
developed only by qualified pharmacologists. Rational scientists express skep
ticism about ultralow doses. We are grateful to E. D. Gol’dberg (Academician
of the Russian Academy of Medical Sciences) and A. M. Dygai (Academician
of the Russian Academy of Medical Sciences) from the Institute of Phar
macology (Tomsk Institute of Pharmacology, Siberian Division of the Russian
Academy of Medical Sciences). Despite dogmatic statements, our collaborative
studies of ultralow doses were initiated 9 years ago. The Tomsk Institute of
Pharmacology is equipped with specialized laboratories for a variety of pharma
cological investigations. The collaborative study yielded highvalue results. A
group of medical products from activated antibodies was developed.
41
Ultralow doses
Besides the study of potentiated substances, Tomsk specialists made a
contribution to the introduction and popularization of a new class of medical
products. Some remedies with ultralow doses were developed in collaboration with
the Tomsk Institute of Pharmacology and extensively used in clinical practice. They
include antibodies to the following endogenous molecules (antigens): IFNγ
(Anaferon and Anaferon for children), tumor necrosis factorβ (TNFβ, Artrofoon),
endothelial NO synthase (Impaza), prostatespecific antigen (Afala), etc. During our
collaboration with Tomsk scientists, the basic mechanisms for action of antibodies
in homeopathic doses were evaluated. For example, our experiments showed that
potentiated antibodies to the endogenous regulator modify its expression. Moreover,
potentiated antibodies have a modulatory effect on functional and metabolic
processes that are associated with this regulator (O. I. Epstein et al., 2004).
Collaborative studies with Tomsk scientists confirmed the fact that that the
system of natural antibodies has a role in the effect of antibodies in ultralow
doses. A clinical trial of potentiated antibodies to S100 protein and morphine
in patients with alcohol and heroin intoxication, respectively, was performed at
the Institute of Mental Health (Tomsk Research Center, Siberian Division of
the Russian Academy of Medical Sciences). Administration of drugs with
ultralow doses of antibodies improved the somatopsychic state of these patients
and had a normalizing effect on natural (predetermined) antibodies to S100
protein and opiates, respectively (T. P. Vetlugina, 2003; N. A. Bokhan et al., 2003).
Further studies showed that activated antibodies to IFNγ have a normalizing
effect on natural antibodies to this endogenous regulator. For example,
chickenpox patients have the increased level of natural antibodies (A. Caruso et
al., 1997). The amount of natural antibodies in these patients rapidly returned
to normal after treatment with antibodies to IFNγ in ultralow doses (as
compared to the control group).
Then clinical trials of new drugs had a major role in applied studies.
During clinical trials, the scientists had to overcome skepticism about substances
in ultralow doses. We thank V. I. Petrov (Academician of the Russian Academy
of Medical Sciences) and specialists from the Volgograd Medical University for
the first systemic study with a new class of medical products in clinical
pharmacology. Our collaboration with Volgograd colleagues resulted in the
development of a new drug Kardos for the therapy of chronic heart failure. This
preparation of antibodies to the angiotensin II receptor in ultralow doses was
initially considered as a hypotensive drug.
The major contribution to a detailed clinical study of potentiated drugs
was made by famous Moscow scientists E. B. Mazo (Corresponding Member
of the Russian Academy of Medical Sciences), V. F. Uchaikin (Academician of
the Russian Academy of Medical Sciences), and V. N. Yarygin (Academician
of the Russian Academy of Medical Sciences).
42
Chapter 2. Three types of effects of ultralow doses
In recent years, advanced clinical and experimental studies were headed
by Professor S. A. Sergeeva (Scientific Director of the “Materia Medica Hol
ding” ResearchandProduction Company). Svetlana Aleksandrovna built a
creative team of young scientists. They summarized and systematized the data
on pharmacology of ultralow doses.
Collaborative studies of several research institutes and “Materia Medica
Holding” ResearchandProduction Company to develop a new class of medical
products from ultralow doses of antibodies were awarded with the Prize of the
Government of the Russian Federation in 2005. Pupils of S. A. Sergeeva
received the same prize for a detailed investigation of Tenoten in 2006 (category
“Young Scientists”). These experiments were performed in collaboration with
the Institute of Neurology (Russian Academy of Medical Sciences).
A famous scientist Professor T. A. Voronina also made a contribution to
study of the mechanisms for action of activated products. Before our
collaboration, a qualified psychopharmacologist Tat’yana Aleksandrovna had the
experience of working with ultralow doses.
The author thanks K. V. Sudakov (Academician of the Russian Academy of
Medical Sciences) for attention to our theoretical problems. In 1997, Konstantin
Viktorovich supported the idea of “horizontal” and “vertical” regulation*. He
proposed a collaborative approach to ultralow doses. We were greatly influenced by
the theory of K. V. Sudakov about the holographic principle of functional systems
and quantization of vital activity. This theory is close to our notion of structural
organization of the organism. Our collaboration with K. V. Sudakov and V. G.
Zilov (Academician of the Russian Academy of Medical Sciences) yielded the
monograph “Elements of information biology and medicine” (2000).
A new class of medical products was developed during a difficult period
of transition in Russia. Despite difficulties, the collaboration between all
participants of this scientific research opened a new chapter in the history of
pharmacology.
Most important in Chapter 2
Three types of effects of ultralow doses were evaluated.
“Direct” fine molecularandcellular activity (confirmed by modern evidential
studies by E. B. Bulakova, J. Benveniste, and other scientists in the 1980s and
1990s).
The phenomenon of homeopathy was discovered by S. Hahnemann in study
ing the effect of ultralow doses on healthy volunteers (1976). According to
* This problem is discussed in Chapter 3.
43
Ultralow doses
modern notions, this phenomenon can be considered as a hyperergic reaction
of the organism to ultralow doses.
Bipathy (i.e., combined treatment with a medical product in normal dose and
its activated form) suggests that ultralow dose modifies the effect of normal dose.
The phenomenon of bipathy was demonstrated on biological and physicochem
ical systems. Clinical and experimental studies showed that potentiated antibod
ies modify, but do not inhibit the activity of a specific antigen (as differentiat
ed from antibodies in normal doses). This is the phenomenon of antibodies in
ultralow doses.
44
C h a p t e r
3
Dual organization
of vital activity
I
n the mid1990s, we knew the following three types of effects of ultralow
doses: direct activity and phenomena of homeopathy and bipathy.
Homeopathy and bipathy are particular cases of the activity of ultralow doses
(Fig. 3.1). S. Hahnemann studied the particular (i.e., hyperergic reactions to
ultralow doses). It was technically impossible to reveal and evaluate the general
properties of potentiated products. The latter fact emphasizes the role of this
scientist, who developed a new integral clinicalandtherapeutic discipline. In
the epoch of bloodletting and leeching (200 years ago), homeopathy was the
most effective therapeutic method.
Potentiated products exhibit activity with no concern for the homeopathic
doctrine. However, this fact did not facilitate an understanding of the nature of
ultralow doses. By contrast, this situation was similar to the mathematical
system of equations with unknowns. The greater was the number of questions
about ultralow doses, the stronger was the desire to evaluate the mechanism of
their action. It was at least necessary to systematize the data and to develop a
new approach to the understanding of these facts.
The direct activity of ultralow doses should be evaluated to under
stand the mechanisms of their action. What do we know about ultralow doses?
Experiments of J. Benveniste and E. B. Burlakova demonstrated that highly
diluted solutions exhibit physiological activity. This activity has the socalled
peaks or extremes. E. B. Burlakova revealed that ultralow dose retains the “split”
(reduced) activity of a substance. Hence, this substance may produce only some
45
Ultralow doses
Technologies of potentiation
↓
Ultrahigh dilutions
↓
→
• hyperergic reactions
General properties
• reduced activity;
• polymodal dosedependence
←
Homeopathy
Bipathy
• modifying properties
↓
Ultralow doses of antibodies
• normalizing effect on the sys
tem of natural antibodies
Fig. 3.1. Three types of effects of ultralow doses.
of the effects. However, the opposite results were not revealed in our
experiments and published data. Ultralow dose does not have the specific targets
that are untypical of the initial dose. Hence, the effects of ultralow and initial dose
are qualitatively similar. This conclusion was made by E. B. Burlakova and J. Ben
veniste. Experiments of T. M. Vorob’eva produced similar results for potentiated
ethanol and morphine (O. I. Epstein et al., 1996b; V. G. Zilov et al., 2000).
The effects of activated products are smaller than those of the initial dose.
Experiments with the isolated neuron from Helix pomatia showed that the effect
of activated substances cannot compete with the influence of drugs in normal
therapeutic doses (O. I. Epstein et al., 1999b). The results of this study are
described below.
Experiments were performed on the identified spontaneously active neurons of
subesophageal ganglia V2V6, PPa1, and PPa2 from 28 edible snails. The snails were in an
active state for at least 2 weeks before study. The following parameters were measured:
resting potential (RP), action potential amplitude (Vo), time derivative of the action
potential (AP), maximum rate of rise of AP (Vmax), spike discharge frequency, and
currentvoltage and inactivation characteristics of ion channels for inward and outward
currents. In some measurements with isolated neurons, calcium currents were recorded by
the voltage clamp technique. A simulating electrode was connected to the output of a
fixation block. The value of current to hold the membrane potential at a certain level was
measured with an amplifier. The signal from this amplifier was delivered to the second
input of an oscillograph.
Under control conditions, the substitution of physiological saline for nonimmune
serum or antiserum to sheep erythrocytes had no effect on electrical properties of the
membrane (Fig. 3.2).
46
Chapter 3. Dual organization of vital activity
%
100
Control
80
0.2%
60
2%
10-400
10-12
40
6%
20
12%
0
5
10
15
20
Time, min
25
30
35
Fig. 3.2. Maximum rate of rise of the action potential in giant neurons of snail
subesophageal ganglion after application of antibodies to S100 antigen at various
dilutions.
Vmax decreased sharply 20 min after application of antiserum to S100 protein (AS
100) at antibody dilutions of 0.2 and 2% (by 2228 and 3745%, respectively). Vmax
decreased by 6080% over the first 1015 min after treatment with AS100 at dilutions of
6 and 12% (Fig. 3.2).
Similar results were obtained in experiments with potentiated AS100 (pAS100) in
concentrations of 102 and 10400 wt %. Vmax decreased by 148% over 3035 min (Figs.
3.23.4).
Independently on the concentration of antibodies, the action of AS100 on inward
current channels is followed by a decrease in the strength of current and increase in the
steadystate inactivation at zero conditioning pulse. Vmax and inactivation curves were
shifted toward negative values of the membrane potential (Fig. 3.4).
The pAS100induced reduction of inward current was mainly associated with a
decrease in the maximum conductance of inward current channels, but not with an
increase in the steadystate inactivation. These changes probably contribute to an AS
100induced decrease in the amplitude of AP. Similar results were obtained in
experiments with pAS100 at various dilutions. Outward current remained unchanged
under these conditions (Fig. 3.5).
Potentiated AS100 decreased the amplitude of AP and inward current, but had little
effect on leakage current. These data indicate that AS100 at normal dilutions and
potentiated form has a depolarizing effect (Fig. 3.6).
Studying the influence of antiserum to S100 on functional activity of isolated
neurons showed that all dilutions of this substance (e.g., potentiated dilutions at
concentrations of 102 and 10400 wt %) have a similar, but quantitatively different effect.
This is manifested in membrane depolarization, decrease in the amplitude of AP, increase
in the maximum rate of rise of AP, decrease in the maximum conductance, and
inactivation of channels. A change in electrical properties of the membrane becomes less
significant with a decrease in the dilution of antiserum to S100 (V. G. Zilov et al., 2000).
47
Ultralow doses
%
105
100
1
2
3
95
90
4
85
80
5
0
10
5
15
20
25
30
35
40
45
50
Время, мин
Fig. 3.3. Amplitude of the action potential after application of potentiated antiserum
to S100 protein. (1) Nonimmune serum, dilution 1:5; (2) potentiated AS100 in a
concentration of 10 400 wt %; (3) 0.2% dilution of AS100; (4) 2% dilution of AS10;
and (5) 6% dilution of AS10.
%
105
100
1
2
95
3
90
4
85
80
5
0
5
10
15
20
25
Time, min
30
35
40
Fig. 3.4. Resting potential of giant neurons from H. pomatia after application of
potentiated and nonpotentiated antiserum to S100 protein. (1) Nonimmune
serum; (2) potentiated AS100 in a concentration of 10 400 wt %; (3) potentiated
AS100 in a concentration of 10 2 wt %; (4) 2% AS100; and (5) 6% AS100.
48
Chapter 3. Dual organization of vital activity
5x108 A
16
14
12
1
2
3
10
8
6
4
2
50 40 30 20 10
10
20
30
40
50
60
1
70
mV
2
3
4
5
6
Fig. 3.5. Currentvoltage characteristics of inward current channels in the
membrane of giant neurons from snail subesophageal ganglion after application
of physiological saline (1), antibodies to S100 antigen at 12% dilution (2), and
potentiated antiserum to S100 protein in a concentration of 10 2 wt % (3). Initial
membrane potential 43 mV.
О
10 mV
О
О
О
15 msec
PS
NS
С30
PS
10 mV
40 msec
PS
С6
Fig. 3.6. Action potential generation in snail neurons under the influence of pAS
100 in concentrations of 10 60 (C30) and 10 2 wt % (C6). PS, physiological saline
for H. pomatia; NS, nonimmune serum.
49
Ultralow doses
The activity of ultralow doses depends on the method of preparation
(scale of dilution, number of dilutions, and conditions of dilution), but not on
the content of the original substance. Hence, the preparation of ultrahigh
dilutions is standardized. Our experiments were not directed toward studying the
dose dependence of ultradiluted solutions. However, it became obvious that
such dependence exists. This fact is of considerable importance. It seemed that
the dose dependence has a polymodal (cyclic) nature. We used the standard
homeopathic dilutions C12, C30, C200, C1000 and, more rarely, C6 and C50.
The effects were more pronounced at low (C16, C12, and C30) or high
dilutions (C200 and C1000). In some experiments, ultrahigh dilutions did not
differ in activity. For example, the effectiveness of dilutions appeared as follows:
C1000>C30=C200.
Neurobiological studies confirmed a wellknown homeopathic fact that
that the maximum neurotropism is typical of high dilutions C1000 and C200.
These investigations showed that a mixture (chords) of various dilutions causes
the cumulative effect. For example, experiments with ultralow doses of anti
S100 revealed that the mixture of dilutions C12+C30+C200 has a stronger
antianxiety effect that C1000. The original substance and its ultralow doses
always produce a unidirectional systemic effect. Sometimes, the direction of local
effects of potentiated substances depended on the dose. In experiments of T. M.
Vorob’eva, activated ethanol was given to animals after 2week consumption of
alcohol in high doses. Blood alcohol concentration decreased after administra
tion of potentiated alcohol in dose C30, but increased in treatment with dose
C200. Dose C30 decreased, while dose C200 increased the contents of
dopamine and serotonin in the brain. It should be emphasized that potentiated
alcohol in doses C30 and C200 had a protective effect during chronic alcohol
intoxication (A. M. Titkova et al., 2002).
Submolar dilutions produce the dosedependent effect. Hence, the proper
ties of ultradiluted solutions are determined by the technology of preparation.
The effects of “molar” dilutions C6 and C12 with molecules of the original sub
stance are similar to those of “submolar” dilutions C30, C50, and C200 (O. I.
Epstein et al., 2004). These data indicate that the activity of ultradiluted solu
tions depends on the method of preparation (potentiation), but not on the
concentration of the original substance. A slight increase in the dose of medical
product (by several micrograms) has little effect on its properties. During
combined (bipathic) administration of the medical product and its activated
form, the latter compound in vitro and in vivo modifies the effect of the original
substance. Therefore, the phenomenon of bipathy illustrates that the activity of
ultralow doses is determined by the process of preparation.
The technology of potentiation allows us to retain some basic pro
perties of the original substance in ultradiluted solutions. Homeopathic studies
50
Chapter 3. Dual organization of vital activity
showed that the ability of ultradiluted solutions to produce a therapeutic effect
is observed for a long time (several years). The data show that potentiation
provides not only activity, but also stability of these solutions. After preparation
by the method of Hahnemann, potentiated solutions gain the unique modifying
properties. Despite the fact that activated products were used in medical practice
for 200 years, the mechanism of potentiation remains unclear. It is poorly
understood how a potentiated solution “remembers” the properties of the
original substance. Why these properties are transmitted to a solid pharmaceuti
cal form (carrier)? It remains unknown which mechanism is responsible for
activity of these preparations in the organism.
During potentiation, molecules of the solvent (water, alcohol, or water
alcohol mixtures) are probably arranged in a certain cluster order around the
dissolved substance. Physical properties of this substance are “fixed” in the spa
tial organization of clusters. They retain this information even when diluted
beyond Avogadro’s number (1024 M, no molecules of the original substance).
Some authors believe that the formation of these clusters results from
spatial reconstruction of an aqueous medium. For example, 912 water molecules
are arranged in the cubic structure and interact with each other by the principle
of charge complementarity (S. Zenin, 1999). Otherwise, these clusters consist
of fluid subsystems (e.g., proton subsystem; F. R. Chernikov, 1998). Indepen
dently on the basis of solution “memory” (water molecules or submolecular
particles), the potentiated solution is structured according to a fine organization
of the original (dissolved) substance. Solvent molecules are in constant motion.
Therefore, the potentiated solution is characterized by a dynamic structure. This
is a stable dynamic constellation of solvent particles (organized spatial structure).
The structure of any substance in normal doses determines its physico
chemical and biological properties. Surprisingly, this structure may be accurately
perceived and stored in the solution. Even without the molecule, its image and
properties are retained in dynamic constellations of molecules in the solution.
The structure of this “missed” molecule is a semantic content of constellations.
This fact is of considerable importance for the theory of ultralow doses. We
proposed the term “semantically organized molecular constellations”* (O. I. Ep
stein, 2002). The term “molecular constellations” was introduced by A. G. Gur
witsch. He proposed the concept of biological field (A. G. Gurwitsch, 1944).
The field is considered as an integral factor, which determines the direction and
order of biological processes. According to A. G. Gurwitsch, the constellations
consist of molecular ensembles that are combined by a common suprasystemic
factor. However, A. G. Gurwitsch did not consider the physical nature of re
lationships in constellations.
* Constellation (Latin name).
51
Ultralow doses
During potentiation, the memory of various substances is retained in the
solvent. It may be suggested that specific physicochemical properties of the
dissolved substance are stored in the structure (relationships), but not in the
nature of associations in constellations. Despite constant motion of molecules
in a solution, memory of the original substance is retained in constellations
(even during strong heating and cooling of potentiated solutions).
The process of potentiation is a surprising empirical discovery of Hah
nemann, which comprises the following two factors: successive dilution and
external influence. This process requires fundamental physical investigations.
Studies of the bipathic phenomenon on simple models showed that acti
vated solutions and, therefore, process of potentiation may be analyzed by stan
dard physicochemical methods*. We hope that this problem will be of interest
for physicists. However, several conclusions were made about the process of
potentiation.
The external influence** (10sec mechanical shaking by Hahnemann)
probably provides an additional energy source for structural reconstruction of
the solution. Repeated treatment has proper oscillation characteristics.
Hypothetically, the mechanisms of resonance contribute to the selection and
storage of a typical vibration spectrum in the solution. A wellknown study by
J. Benveniste (see Chapter 2) showed that 10 sec of shaking (according to
Hahnemann) is the minimum time required for activation of a potentiated
solution***.
Repeated removal and further transfer of only one drop from the solution
into the intact solution probably contribute to the preservation of other “basic”
oscillation parameters in the previous dilution of solvent. Repeated dilution may
be considered as a filtration of “information noise”. This fact probably deter
mines an increase in the effect with increasing the number of dilutions (e.g., in
experiments with neurotropic agents).
*
There is some controversy as to whether the potentiated solution has particular physical
properties. We can remember a humorous poem of S. Ya. Marshak:
The matrix of a song and its essence
Cannot be made out of thin air by the author.
Even God could not create anything
In the absence of raw material.
** Little is known about other types of external influences (electromagnetic and ultrasound
generators).
*** Moreover, some technological principles were demonstrated by J. Benveniste et al.
They illustrate an important role of potentiation in the activation of ultradiluted
solutions. These authors showed that potentiation can occur in water, ethanol, and
propanol, but not in dimethylsulfoxide. Various substances in usual doses differ in
thermal resistance. However, the activity of potentiated products decreases in the same
temperature range (from 70 to 80oC).
52
Chapter 3. Dual organization of vital activity
Oscillation parameters of the electromagnetic spectrum and acoustic range
are typical of any molecule. Theoretically, they may be stored in newly formed
constellations and determine the properties of these constellations. The
semantically organized constellations comprise not only solvent molecules, but
also atomic and subatomic particles and specific wave characteristics. The
“wave” constituent of constellations probably serves as a basis for distant transfer
of information about the original (dissolved) substance in a structured solution.
These data indicate that constellations may be considered as a wave structure.
The term “semantically organized constellations” suggests the essential or
ontological interpretation of the notion “information”. Information about the dis
solved substance is an essence or substantive content of constellations. This es
sence combines the relationships between solution molecules into a single
whole, “couples” the specific wave characteristics, and serves as a complex
spatial structure. The notion of semantically organized constellations is of
particular importance for physics and philosophy. From the standpoint of phy
sics, we postulate the existence of distant intermolecular (information) rela
tionships of unknown nature. Philosophically, semantic constellations are a
particular case when the “spirit” organizes the matter.
The question arises: whether the “coupled” oscillation parameters of
molecules may form a basis for distant transfer of information about the original
substance in constellations? Let’s look at some published data.
It is a wellknown fact that all biological objects have weak electromag
netic radiation. Theoretically, weak electromagnetic field may transfer informa
tion about some physiological parameters of the object and affect other biolo
gical structures. Some authors believe that the effect of electromagnetic field on
an organism is realized via membranebound acceptors with a specific frequency
of coherent oscillations. Due to resonance with these structures, electromagnetic
impulses provide synchronization of oscillations in membrane acceptors. These
receptors mediate the influence of electromagnetic impulses.
A. G. Gurwitsch (1944), V. P. Kaznacheev (1981), Yu. V. Tszyankan’
chzhen’ (1993) and other investigators attempted to confirm the possibility for
distant transfer of information between biological objects. These studies have two
features. First, not all information about the biological object, but only some
general (modal) characteristics are distantly transferred under various conditions.
And second, the biological objects serve as an inductor of information when
their vital functions are strongly suppressed or activated under the influence of
some factors. In experiments of A. G. Gurwitsch and Yu. V. Tszyankan’chzhen’
these factors were presented by degradation mitogenetic radiation and strong
growth of young plants, respectively. In studies of V. P. Kaznacheev, the tissue
culture was suppressed by viruses, mercuric chloride, and radiation. This culture
was then connected to the intact culture through an optical channel. A cyto
53
Ultralow doses
pathic effect was sometimes observed in the intact culture (suppression of vital
activity). The inhibition or activation of vital functions in the early period after
exposure to a strong external factor was probably followed by synchronization of
physiological processes (e.g., mitotic activity). Synchronous rhythms of vital
functions were accompanied by synchronization of weak electromagnetic radiation
from biological objects. Electromagnetic field was characterized by modulation and
served as a carrier of some general properties of the biological system in space.
These data suggest that electromagnetic field and, probably, other fields
contribute to a distant effect on the biological system. It should be emphasized
that the potentiated product causes a specific response, which differs from other
types of distant interaction. Our study and other researches showed that ultralow
doses have a reproducible effect, which may be used in practice (as
differentiated from the majority of distant interactions).
The quintessence of our study is the notion of semantically
organized constellation as a unique spatial structure.
It became clear that the principle of similarity is not an exclusive
principle. Ultralow doses can produce at least two types of effects. These results
were absolutely unexpected. It was very difficult to explain a variety of properties
of potentiated products. The most surprising discovery was that ultralow doses
produce a specific physiological response in each individual. This response may
be investigated by modern molecular and cellular methods. It was unclear why
the substance in ultralow dose causes “drug exacerbation” only in sensitive
patients. Which mechanisms are triggered by the essence of ultralow dose to
induce a hyperergic reaction? To answer this question, we made the following
suggestion: not only memory units of the potentiated solution are a semantically
organized spatial structure. The whole organism can be also considered as a
complex spatial structure, which consists of molecular constellations. As
differentiated from inanimate nature, the spatial structure of biological systems
has dual individualandspecies organization. Due to spatial duality of the
organism, ultralow doses sometimes cause a hyperergic reaction.
However, all facts should be considered one after another. We studied the
process of potentiation and activity of potentiated products. Potentiation was
discovered 200 years ago, but received little attention for a long time. It was a
unique chance to investigate a simple physical process at the dawn of the 21st
century. In natural sciences, there were no data on the phenomenology of this
process. We hypothesized that potentiation may be considered as dematerializa
tion, since this process results in “extraction” of the original specific information
structure from any substance. It could be suggested that the reverse process also
occurs during evolution (materialization of information). Taking into account
this hypothesis, we proposed an evolutionary approach to explain the principle
of dual organization in constellations.
54
Chapter 3. Dual organization of vital activity
There is no general evolutionary theory. We hold to the idea of Logos that
life originated from one common source due to selfdevelopment. The principle
of the original matter is postulated in the Big Bang theory, which suggests
saltatory evolution of the World. We also share the idea of V. I. Vernadsky that
biotic and nonbiotic processes occurred simultaneously, and biosphere forma
tion was a onetimeonly process (V. I. Vernadsky, 1988). Whatever the cause
and mechanism of evolution, we should emphasize that the original discrete
nonvacuum matter (“preform of material existence”) appeared in the hypo
thetical absolute vacuum at a certain primary stage of life development. Self
complication and selfdevelopment of the original matter were followed by the
appearance of the World. The original form of material existence should have
the specific geometric properties and semantic content. In our opinion, it was
an essential information structure. Selfcomplication of the original information
structure resulted in the appearance of complex information structures. These
structures were spatially congruent (identical).
The evolutionary process was probably associated with competition
between at least two original substances for the spatial and temporal organization
of a vacuum (according to principles of each original substance). The logical
consequence is a tendency toward selfcomplication of the original matter and
appearance of complex spatial structures. Only these structures provide transfer
of life principles (“jurisdiction” of the original matter) to all hypostases of the
World. The evolution of a spatially simple vacuum resulted in the formation of
a threedimensional space. The “plane” information structures were “materia
lized” or hypostatized* into complex spatial structures (e.g., biological systems).
Complex information structures were formed from simple structures
(elements) during evolution. The information structures of similar complexity
were characterized by various combinations of constituent elements, which
provided them with individual architectonics. Despite such individuality, each
information structure is similar to the original matter.
Materialization of complex information structures into biological systems
(if it occurred) has several important consequences**. First, the structure of
biological systems should have a “regular” hierarchy. This hierarchy is similar
to the original matter and reflects prebiotic selfcomplication of life. And
second, any biological system should have a dual individualandspecies spatial
*
Hypostatization is the ascription of material existence to any abstract notion, property,
or idea.
** The idea that each biological species develops from an information precursor has
something in common with the concept of nomogenesis (L. S. Berg and A. A.
Lyubishchev). This seems contrary to the theory of C. Darwin. However, this concept
is consistent with the notion of natural selection. Natural selection occurs in animate
nature, but does not serve as an “engine” of evolution.
55
Ultralow doses
organization to retain the initial predetermined integrity of functions (see
Chapter 5). It is provided by specific principles of regulation.
These assumptions contribute to the revision of common notions about
the meaning of life. Evolutionarily, each living organism should reproduce itself
in the next generation and have a “normal” function. A hypothetical structured
vacuum with information structures is the ether in a threedimensional space.
In biological systems, the relationship exists between physiological processes and
vacuum. A “normal” function contributes to a “correct” reflection and memo
rization of the environment. After biological death of the individual, its expe
rience as a “regular” information structure remains in a vacuum. This “cell” of
the universe is similar to the original matter.
Primary information structures are characterized by a certain hierarchic
relationship between constituent elements (architectonics). The initial
architectonics is preserved during materialization into objects of a three
dimensional space (e.g., biological objects). This transition without an increase
in complexity of new threedimensional systems received the name horizontal
transition (similarly to the relationships between constituent elements or
molecules in biological systems that determine the general species properties).
The species similarity does not abolish the individual properties. Hence,
there are some individual relationships between elements in each biological
system.
Spatial relationships between constituent elements of each biological
system have a specific species organization (horizontal relationships). However,
some individual relations between elements of the system also exist within the
limits of general species relationships. They received the name vertical
relationships or vertical plane of biological systems (O. I. Epstein, 1996).
To facilitate the understanding of horizontal and vertical relation
ships, we proposed the “theory” of four billiard balls. Any plane passes through
three points. Let’s assume that these three points are the billiard balls. The balls
are adjacent to each other and form a triangle on the playing field (Fig. 3.7).
A shot with ball 4 is followed by scattering of these balls into different
directions (Fig. 3.8).
Fig. 3.7. “Theory” of four billiard balls: step I.
*
56
The theory of physical vacuum was very popular. However, this theory was rejected
when A. Michelson failed to confirm the existence of a vacuum by astronomical
methods.
Chapter 3. Dual organization of vital activity
Shot
Fig. 3.8. “Theory” of four billiard balls: step II (plane constellations).
The greater is the power of the shot, the longer is the distance between
balls 1, 2, and 3 after scattering. Moreover, the quality of the shot with ball 4
may be estimated from the type of scattering (regular or rotational, smooth or
sharp, and slow or fast). An experienced player can evaluate the quality of the
break shot. Conventionally, the movement of balls 1, 2, and 3 may be con
sidered as a semantically organized constellation. It reflects the quality and vari
ous parameters of the shot with ball 4 (triangle 1'2'3 in Fig. 3.8, outlined with
a dotted line).
Let’s assume that we have four, but not three balls. These are the air balls,
but not the billiard balls. They scatter in the air after playing a shot with the
fifth ball (Fig. 3.9).
According to the simplest geometrical laws, three balls will lie in the same
plane. In the majority of cases, the fourth ball with be beyond this plane. There
fore, such constellation of four air balls will appear like a pyramid (Fig. 3.10).
The “species” relations are preserved in the base and each face of this
pyramid. The face is formed by three balls (planes 1'2'3', 2'3'4', 1'3'4', and
1'2'4'). Any three balls are within certain spatial limits. The fourth ball may
be situated in an individual, but predetermined place relative to the center of
Shot
Fig. 3.9. Constellations of “air balls”.
Fig. 3.10. Principle of the spatial organization of constellations.
57
Ultralow doses
each plane. In any case, this position of the fourth ball will contribute to a “re
gular” species organization of pyramid faces. Hence, the “individual” will be
always incorporated into the “species”.
To achieve the maximum stability, each electron occupies a certain orbit
during rotation around the nucleus. Similarly to the electron, each element has
a specific position in the biological structure. This feature provides the irrever
sibility of physiological processes. Various biological theorists, including I.
Prigozhin (1986, 2006) and E. Bauer (1935), attempted to substantiate the
irreversibility of processes on the basis of thermodynamic principles. They
suggested that the organism is an open thermodynamic system with the arranged
and directed energy flow, which provides the function of biological systems
against the chaos (antientropic vital activity). We believe that the spatially
nonlinear individualandspecies organization of biological systems contributes
to the prevention of stochastic (random) processes.
Ideally, molecules of the organism may be conceived as balls (points).
Moreover, biological systems can be imagined as thermodynamic systems*. The
response to external influences will appear as molecular constellations. They are
associated with the semantic structure of a certain external factor. First of all,
we are interested in the local and systemic response to medical products. The
experience of pharmacology indicates that any medical product has a specific
activity. The specificity of response is determined by new relationships between
metabolic or functional processes, but not by the involvement of one molecule.
Any substance causes a speciesspecific response, which does not exclude the
possibility of individual (hyperergic) reactions.
What is the cause of hypersensitivity? This state may appear as follows. Any
medical product triggers a series of genetically determined molecular (ther
modynamic) events in the organism. These events are in certain relations with
each other (constellations). Similarly to constellations in the potentiated solution,
these events are spatially “coupled” with each other by a fine structure of the
medical product. Such events reflect a threedimensional image of this structure.
“Reflection” of the medical product reaches maximum at the “peak” of
a pharmacological response. In this period, the biological system is significantly
deviated from the previous state of thermodynamic equilibrium. A negative
feedback mechanism induces the secondary reconstruction of this system, which
achieves another equilibrium state**. The achievement of equilibrium in this
*
Thermodynamics is a science that deals with the inner state of macroscopic bodies in
equilibrium. According to another definition, thermodynamics is a science that deals
with the laws of interconversion and transfer of energy.
** The equilibrium state of biological systems will be designated as an integrative state
(Chapter 5).
58
Chapter 3. Dual organization of vital activity
system serves as a biological adaptation. Pharmacologically, this process reflects
a therapeutic effect of the medical product. The experience of immunology
indicates that before responding to an external stimulus, the biological system
should evaluate its major characteristics. Semantically (i.e., spatially), this factor
can be “self” or “nonself” for the system.
The organism is considered as a combination of considerable amounts of
molecular constellations. Any organism has the conditional horizontal and ver
tical planes. A paradoxical situation may occur. The semantic structure (archi
tectonics) of an external stimulus may be similar to the individual structure of
a biological system. Hence, the newly formed constellations in an individual
(vertical) plane of the organism do not reflect (!) a fine structure of the medical
product. Spatial perception is impossible under these conditions, which ne
cessitates the construction of large constellations. The local response is trans
formed into the systemic response.
The studies to understand a major problem of homeopathy (nature of
hyperergic reactions to ultralow doses) yielded the evolutionary postulate and
notions on a dual individualandspecies spatial organization of vital functions
in biological systems.
The notion of semantic constellations allows us to illustrate schema
tically not only the mechanisms of hyperergic reactions to ultralow doses (biological
basis of homeopathy), but also the protective effect of activated products. Each
medical product is biotransformed in the organism and induces a specific metabolic
and physiological response. It results in the formation of local and systemic
constellations that are coupled by semantic parameters of the medical product.
Potentiation is accompanied by the formation of semantically organized
constellations of solvent molecules in a solution. When molecular constellations
in the organism and potentiated solution consist of the same medical product,
their spatial structures are similar (congruent).
Let’s return to the abstraction with three billiard balls. Biotransformation
in an organism and potentiation in a tube are accompanied by three stages of
biotransformation or potentiation of substance A. They are shown in Fig. 3.11.
Constellations 1'2'3', 1'’2'’3'’, and 1'’’2'’’3'’’ are the successive stages
of reflection (“reading”) of the image (structure) of substance A at various
periods. If the specific response to an external stimulus is considered as a
function, the constellations appear as derivatives of this function.
After combined administration of substance A and its activated form (A’)
during stage 1'’’2'’’3'’’ (Fig. 3.12), molecular constellations of the activated
solution initiate an adaptive response to external treatment (negative feedback
mechanism) in the earlier period than the intact nonbiotransformed molecule.
The potentiated substance is characterized by greater degree of “reading”
in time. It initiates an adaptive response of the organism, which occurs before
59
Ultralow doses
Fig. 3.11. Scheme for the phase states of constellations.
↓
Substance A
Activated substance A’
↓
Biological feedback and induction of adaptive mechanisms
↓
Advanced (predetermined) “reading” of substance A
Fig. 3.12. Scheme for the protective effect of activated substances.
biotransformation of the same substance in normal dose. During combined
treatment with medical product A and its activated form, the latter “prepares”
the organism to an external influence. This process determines the protective
effect of ultralow dose.
The next chapters will show that the phenomenon of bipathy is mediated
by more complex mechanisms. Activated forms of medical products have a
modulatory effect not only on specific targets in the organism, but also on fine
characteristics of the substance in normal doses.
Most important in Chapt
er 3
Chapter
There is a large body of evidence that ultralow doses have physiological
properties (polymodal dosedependence, “splitting” of the effect, unidirection
60
Chapter 3. Dual organization of vital activity
al systemic effect of the activated and original substance, and effectiveness of
potentiated products in molar and submolar concentrations). The physical prop
erties of ultralow doses remain unknown. However, it is obvious that the activity
of ultradiluted substances results is associated with their preparation (poten
tiation).
The effects of ultralow doses are qualitatively similar to those of the orig
inal substance. These data suggest that the potentiated solution retains a fine
structure of the dissolved substance. The memory unit of an ultradiluted struc
tured solution is presented by stable relationships between solvent molecules.
These semantically organized constellations are “coupled” by parameters of the
original substance, which is absent in the solution.
Molecules, submolecular particles of the solvent, and field processes
(acoustic and electromagnetic fields) are integrated into the common spatial
structure of semantically organized constellations. Semantically organized con
stellations in potentiated solutions may appear as the wave structures that are
capable of distant interactions.
To explain the basic principle of homeopathic therapy (hyperergic reac
tions), biological systems are considered as the integral spatial structures. They
consist of molecular constellations and have a dual individualandspecies or
ganization. The dual structure of living organisms is related to evolution of bi
ological objects from the common material (essential) information source.
Semantically organized constellations have a dynamic spatial structure,
which is characterized by specific phaseandtemporal parameters. These data
allow us to explain the phenomenon of bipathy.
61
Ultralow doses
C h a p t e r
4
Holographic control
of vital activity
by the immune system
I
n the previous chapter, we attempted to explain the mechanism of hyperegic
reactions to ultralow doses. It was hypothesized that they are related to a dual
(individualandspecies) spatial structure of the organism. This approach is very
close to the notion of holography. The term “holography” is formed by two
Greek words: “holos”, whole; and “graphein”, to write. This method to obtain
a threedimensional image of the object was proposed by Dennis Gabor in 1948.
To develop the holographic image, a photographic film is exposed to the light
beam. This beam passes through a prism and is split into two beams. Whereas
the reference beam is projected directly onto a holographic film, the object
beam first reflects off the object before reaching this film. According to the law
of wave interference, these beams are combined in the plane of a holographic
film to produce the picture of dark and light bands. The hologram is trans
formed into a threedimensional image by means of a laser technique and other
methods. The following consequences of the holographic theory are important
for our research:
1) a holographic image is stable; and
2) each point and elementary unit of the hologram include the whole
image.
The basic principle of any hologram is spatial “coupling” of constituent
elements by the general suprasystemic factor (light waves in physical holo
graphy). Before separation by a prism, optical beams are spatially coupled in the
62
Chapter 4. Holographic control of vital activity by the immune system
whole beam. These relationships are preserved after separation. Hence, the
direct (reference) and reflected (object) waves can be combined into a three
dimensional image.
In essence, semantically organized constellations of molecules are similar
to holographic structures. They are combined into a single whole by
physicochemical properties of the dissolved substance*. As differentiated from
a simple process of dissolution, potentiation allows the highly diluted solutions
to retain activity and stability. Molecules of water and other solvents are linked
by bonds (hydrogen, Van der Waals, and other bonds). These bonds exist for a
short time and easily dissociate in heating. During the manufacture of homeo
pathic remedies, it was concluded that ultradiluted solutions are resistant to
heating. These data serve as indirect evidence that constellations in the solution
are linked by other distant bonds. Conventionally, they may be designated as
information bonds.
There is no consensus on the physical nature of information (torsion
fields by G. I. Shipov, 1993; Pwaves by N. D. Kolpakov, 1997; etc.). I do not
want to be an amateur. This serious physical problem should be solved by
specialists. For medicine and biology, it is important that the molecules and
molecular processes can be coupled by information parameters.
In the 1950s, a number of interesting facts were coming to light. Memory
is distributed over the whole brain, but not stored in a certain local “cell”. Some
authors believe that these data illustrate a holographic distribution of
information in the brain. Physical holography postulates that each elementary
unit reflects the whole image. According to P. J. Beurle (1956), damage to one
or several regions in the brain does not impair an integral perception. A famous
scientist K. Pribram (1975) assumed that any brain area includes the whole
image (according to the principle of holography). In experiments of N. Yu.
Belenkov, several regions of the cortex in animals were “switched off” by cold
exposure. The animals were trained in the followup period. Then the damaged
areas were returned to a normal state (N. Yu. Belenkov, 1980). It was shown that
the interference of “trained” and “nontrained” regions in the brain is followed
by the disappearance of acquired skills in animals. These data can be explained
from the viewpoint of holography.
K. V. Sudakov is one of the authors of the theory of functional systems.
He proposed that the principle of holographic interference forms the basis for
“coupling” between signaling and satisfaction of the demand. This process
determines the activity of any functional system (K. V. Sudakov, 1984, 1990,
1996a,b).
*
The horizontal and vertical planes of an organism may be conventionally designated as
analogues of the reference and object waves, respectively.
63
Ultralow doses
It is very attractive to explain the effect of ultralow doses by holographic
principles. For the extrapolation of these data to vital activity, it is necessary to
evaluate the analogues of potentiation in an organism.
Potentiation consists of the following two components: repeated dilution
and shaking. Dilution is probably followed by separation of essential information
about the properties of dissolved molecules from “information noise” in the
solution (see above). The process of dilution does not occur in an organism. The
analogues of dilution (network processes) will be discussed below. However,
autooscillations (analogue of shaking) are typical of many structures in biolo
gical objects. It remains unclear whether all these processes are accompanied by
stable structuralization of liquid media in the organism (similarly to poten
tiation). We believe that this property is typical only of complex molecules with
a threedimensional or fourdimensional spatial structure (polypeptides and
proteins). Autooscillations in the spatially complex molecule of DNA are
probably followed by structuralization of liquid media in an organism, which
results in the dual spatial organization.
Russian scientists proposed that hereditary information has a wave
(filed) nature (A. G. Gurwitsch, 1944; P. P. Garyaev, 1997). It is difficult to
imagine that inherited characteristics have a certain “linear” arrangement in
DNA (nucleotide sequence). Obviously, hereditary “memory” of DNA is
stored in a more complex form (constellations of coding and noncoding
regions in this supermolecule*). These data suggest that genetic information
is encoded in oscillation parameters of DNA regions. It remains unclear
whether this information is transmitted directly or indirectly (thought genomic
products) into the cell. It should be emphasized that genomic products
(polypeptides and proteins) have a more complex spatial structure than the
matrix of DNA. Proteins and polypeptides probably regulate the spatial
structure of vital functions. The mechanism of these events is similar to
potentiation in the manufacture of ultradiluted solutions. During
potentiation, a holographic image (i.e., constellations of solvent molecules) is
“extracted” from the molecule of the original substance. A threedimensional
and fourdimensional structure of regulatory molecules is probably organized
by the holographic principle. Even in the absence of successive dilution (one
of the stages in potentiation), autooscillations of spatially complex molecules
can result in stable structuralization of liquid media in the organism. The
minimal liquid locus is structured around the genomic product of any location
(in the cell or intercellular space). This process is related to autooscillations
and results in the “regular” spatial organization of molecular or ionic events.
*
64
Recent studies showed that the genes are united into constellations or gene networks
(V. A. Ratner et al., 1985; N. A. Kolchanov et al., 2004).
Chapter 4. Holographic control of vital activity by the immune system
It contributes to the “regular” spatial realization of “wavy” inherited characte
ristics. Hence, DNA plays a dual role. First, the individual species hereditary
information is stored in constellations of DNA regions. And second, DNA
serves as a matrix for the synthesis of proteins and/or polypeptides that have
a specific “vertical” structure and regulate the realization of hereditary infor
mation in an organism.
The primary control is genetically mediated and involves proteins and
polypeptides. Beginning from a certain evolutionary level of multicellularity, the
immune mechanisms became involved in the regulation of vital activity. It
resulted in the appearance of secondary genetic control, or immunity. The
immune regulation in an organism is directed toward epitopes that serve as the
major molecular regions of proteins, polypeptides, and polysaccharides. Epitopes
are the smallest structures that can be “recognized” by the immune system.
They are spatially distributed within the molecule. The molecule serves as a
skeleton to combine these structures.
The notions of semantic and holographic constellations are closely related
to each other. The notion “semantic constellations” is more inclusive and em
phasizes the “determination” of constellations by information parameters. The
notion “holographic constellations” refers to the spatial coupling between
molecular and submolecular elements in constellations.
Following the apologists of holography in biology, we believe that one of
the holographic properties can be extrapolated to semantic constellations. It
suggests that properties of the whole are reflected in each element of the
hologram. It becomes clear that the immune system regulates a part of the
molecule (epitope) and, therefore, has a modulatory effect on spatial integrity
of the whole molecule (O. I. Epstein, 2002a). Holographic regulation of the
molecular structure contributes to the evaluation of its “normal” function. The
immune system has a great regulatory capacity. However, the principle of
immune function is very simple. The immune system “supervises” the whole
(function of the organism) through the small (epitope).
Epitopes are spatially distributed within the molecule. They are the
smallest structures, which can be recognized by the immune system. We believe
that epitopes are submolecular semantic constellations, which retain the spatial
and temporal properties of the whole molecule.
Immunology is a relatively young science. This science was taught in
institutes of higher education for a short time. Let’s briefly describe a physio
logical role of the immune system.
Antibodies are a major factor of humoral immunity. They were discovered
by Emil von Behring and Kitazato in 1890. These scientists revealed that
immunization of mice with tetanus toxin is followed by the appearance of serum
antitoxin (protein “bodies”) in the plasma. A famous German scientist Paul
65
Ultralow doses
Ehrlich designated the protein substances in blood plasma from animals with
bacterial infection as antibodies.
Until the middle of the 20th century, immunologists were mainly engaged
in the development of new vaccines and sera. Much attention was paid to
particular problems of antiinfective immunity. A famous Australian scientist M.
F. Bernet (1963) radically altered the role of the immune system. He proposed
that the immune response is directed to differentiation of “self” and “nonself”.
Bernet believed that a major function of immunity is the maintenance of genetic
integrity during individual (ontogenetic) development of the organism. The im
mune mechanisms should be particularly specific. They can distinguish, recog
nize, and meet the foreign agent (antigen). A simplified scheme of immuno
logical specificity appears as follows: one antigen — one antibody and one
clone* of lymphocytes.
Antigens are structurally foreign substances (molecules) that can cause an
immune response in the individual organism. Usually, the immune response is
induced by highmolecularweight molecules (proteins, polypeptides, and
polysaccharides). The immune response can be triggered by a small molecule
(haptene), which is conjugated with protein (Fig. 4.1).
The immune response is induced by a small spatial region (epitope), but
not by the whole molecule. The epitope of a protein molecule usually consists
of 612 amino acids. The epitopes interacting with T cells and B cells are de
signated as the T cell epitope and B cell epitope, respectively. The B cell directly
interacts with epitopes via the surface receptor. Immunoglobulin M is located
on the cell membrane and serves as a surface receptor during the first inter
action. By contrast, the T cell cannot directly interact with the epitope. T cells
recognize the protein molecule only in a complex with histocompatibility mole
cules. This is one of the most important features, which will be discussed below.
In 1937, an electrophoretic study showed that antibodies belong to the
γglobulin fraction of blood plasma. These antibodies are now designated as
immunoglobulins (Ig). There are the following five classes of immunoglobulins:
IgM, IgG, IgA, IgE, and IgD. They have a specific domain structure (Fig. 4.2)
and consist of two easy chains and two heavy chains. Each polypeptide chain
contains one variable domain (V), which contributes to specific binding of
antibodies to the corresponding antigen. There are also three or four constant
domains (C) in the polypeptide chain. Besides binding to the antigen, antibodies
can interact with the complement or special receptors on various cells due to
the presence of constant domains. Hence, structural dualism of antibodies
determines their binding to the specific (original) antigen and involvement in
common reactions (through the C domain).
*
66
Clone is the progeny of cells from one precursor.
Chapter 4. Holographic control of vital activity by the immune system
(145) 146151
COOH
Gem
5662
1521 (22)
113119
NH2
Fig. 4.1. Structure of myoglobin in cetacean sperm (Xray structure analysis).
Shaded areas, sequences of amino acid residues that play a role of B cell epitopes. Numerals, order
numbers of amino acid residues in polypeptide (V. G. Galaktionov, 1998).
Antigenrecognizing receptors on the surface of B cells and T cells are
structurally similar. The active antigenrecognizing site of these receptors is
formed due to the interaction between variable domains (V domains).
Similarly to all protein molecules, antigenrecognizing receptors are
encoded by specific genes. The following fact is of considerable importance: as
differentiated from other somatic cells, T lymphocytes and B lymphocytes are
characterized by recombination of gene fragments that encode the light and
heavy chain of immunoglobulins. This process is mediated by a specific
mechanism. A certain degree of variability contributes to the formation of up
to 240 billion types of various antibodies, which bind at least the same number
of types of various antigens.
Antibodies may directly bind the antigen. Before the interaction with T
cell receptors, antigens of foreign viruses and bacteria are exposed to the
intracellular preparation with antigenpresenting cells (APC). Thy bind to major
histocompatibility complex (MHC) class I and II molecules and are transformed
to the surface of APC (Fig. 4.3). This complex binds to the T cell receptor. The
next important fact suggests that antigens are squeezed in specific antigen
binding clefts of MHC molecules. Therefore, they gain another spatial
configuration before the interaction with T cell receptors (Fig. 4.4).
The immune responseinducing foreign antigens may exist in liquid media
of an organism, as well as on the surface of cells. In the first case, the foreign
agent is neutralized by humoral effector antibody molecules (humoral immune
response). In the second case, foreign antigens are killed by cytotoxic T
lymphocytes (direct mechanism) or inflammatory T cells and T helper cells
(indirect mechanism). They induce a series of molecular and cellular events
67
Ultralow doses
ain
L
C
L
V
Н
С
1
Lc
h
V
Н
H
ch
ain
a
Domain
Domain
b
Antigenbinding
site
VL
Hinge
region
VH
VH
VL
CH2
CH2
SS
SS
S
S
S
S
CL
CL
CH2
CH2
Fc fragment
CH3
CH3
Fab fragment
CH4
CH4
Fig. 4.2. Scheme (a) and molecule (b) of immunoglobulin (V. G. Galaktionov, 1988;
R. M. Khaitov, 2005).
L, light chains; H, heavy chains; V, variable domain; C, constant domain. NTerminal regions of L chains
and H chains (V domain) form two antigenbinding sites. The Fab fragment and Fc fragment of the
molecule interact with a specific membrane receptor on various cells, including macrophages,
neutrophils, and mast cells.
(cellular immunity). The cellular and humoral immune responses mainly involve
T lymphocytes and B lymphocytes, respectively.
T lymphocytes received their name from maturation in the thymus. T
lymphocyte precursors migrate from the bone marrow in the thymus. B cells
originate from the bone marrow and become mature in peripheral lymphoid
structures. Historically, the name B cells is derived from the bursa of Fabricius
in birds (one of the peripheral lymphoid organs).
The development of lymphocytes has two distinctive features.
68
Chapter 4. Holographic control of vital activity by the immune system
First, during differentiation T cells interact closely with the stroma of the
thymus. They are selected for the ability to recognize MHC class I and II
molecules (positive selection) and to interact with body’s own molecules
(negative selection for autoimmune reactions). The cells that cannot interact
with MHC molecules or react with autoantigens are removed from the
population of T cells (thymocytes).
And second, the immune response is accompanied by the following
interrelated processes:
1) shift in antibody synthesis from one type (isotype) to another (prev
alence of IgM and IgG in the initial and late stages of an immune
response, respectively); and
а
TCR
TCR
T killer cell
T helper cell
α
β
α
β
CD4
CD8
peptide
α
β
α
β
Target cell
APC
MHC II
MHC I
b
CD80/86
APC
MHC II
CD28
AG
TCR
CD40
reception
Т helper cell
CD4
costimulation
costimulation
CD154
Fig. 4.3. Scheme for the recognition of a complex of antigenic peptide and MHC
class I and II molecules by the receptor and coreceptor on T lymphocyte (a).
Interaction between T helper cells and antigenpresenting cells (b).
TCR, T cell receptor; CD4 and CD8, coreceptors, MHC I and MHC II, histocompatibility complex class
I and II antigens; and AG, antigen (by R. M. Khaitov, 2005).
69
Ultralow doses
α2
α1
N
N
C
C
α3
β2—m
b
α1
Antigenbinding
cleft
N
α2
Fig. 4.4. Spatial structure of MCHA2 class I antigen (Xray structure analysis). Side
view (a) and top view (b). Arrows, regions of the antiparallel βstructure; spirals,
αspiral fragments (R. M. Khaitov, 2005).
70
Chapter 4. Holographic control of vital activity by the immune system
2) increase in the affinity of antibodies for an immune responseindu
cing antigen.
The properties of T cells and B cells are determined before their
interaction with a foreign molecule (preantigenic stage of development). The
interaction of T cells and B cells with the antigen results in cell proliferation
and differentiation into mature effector cells, which are capable of neutralizing
and killing this antigen. The degree of lymphocyte “maturity” determines their
functional capacity and direction of the effect. Special molecules (markers) or
differentiation clusters appear on the cell surface of certain function. Core
ceptors also play a role in celltocell interactions during the immune
response. They improve the interaction between lymphocyte receptors and
antigen. This process also involves costimulators that are located on the
surface of APC.
Each event of the immune response is regulated by cytokines. Cytokines
are synthesized by various cells, including lymphocytes. The majority of cyto
kines, immunoglobulins, lymphocyte receptors, MHC molecules, coreceptors,
and several adhesion molecules belong to a superfamily of immunoglobulins.
They have a common evolutionary precursor and similar domain structure.
The most extensively studied phenomenology of an antiinfective immune
response is the largescale cascade molecularandcellular events that involve
lymphocytes, accessory cells, and various classes of biologically active substan
ces. However, some problems of immunology are poorly understood. The “loca
lization” of immunological memory, fine mechanisms of tolerance and hyper
sensitivity, and progression of autoimmune reactions remain unknown.
The Nobel Prize winner M. Bernet (1963) believed that the main role of
the immune system is regulation of genetic integrity in an organism during
ontogeny. This function is based on the ability of the immune system to dis
tinguish “self” from “nonself”. Let’s compare the commonly accepted notions
of Bernet with our “spatial” postulates.
At first, it is necessary to consider the notions of “self” and “nonself”.
In our opinion the individuality of each organism is determined by its unique
spatial structure, which combines the individual and species properties. Hence,
the maintenance of “self” integrity means the preservation of an individual
spatial structure that is organized by holographic principles.
Lymphocytes are the only somatic cells in an organism. The lymphocyte
genes encoding T cell receptors and B cell receptors of the immunoglobulin
superfamily are characterized by allowed recombination. According to the germ
line theory of L. Hood, a whole set of V genes and C genes is included in the
genome and transferred between generations with no changes. The majority of
authors believe that variability of immunoglobulins is associated with random
recombination of V segments and C segments.
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Ultralow doses
However, this problem appears to be more complex. First, all
recombinations are predetermined and depend on oscillation parameters of
DNA. Second, the combination of individual gene fragments is directed toward
the construction of an encoding region with unique spatial conformation* (in
corporation of “individual” into “general”). Hence, the encoded receptors on
T cells and B cells are characterized by a “regular” individualandspecies orga
nization. This property contributes to the holographic recognition of antigen.
And third, recombinant processes are followed not only by the appearance of
T cell receptors or specific antibodies, but also by the synthesis of immunoglo
bulin molecules of certain spatial complexity. Hence, IgM and T cell receptor
can bind several antigens of the same spatial structure.
Let us digress briefly to be more specific. In the periodic table of D. I.
Mendeleev, an increase in the weight of elements is accompanied by a change
in their chemical properties. Probably, a similar process occurs in the nature.
The spatial structure of protein (polypeptide) molecules became more complex
in evolution, which resulted in an increase of the semantic content and
appearance of “semantic series”. The semantic series may be imagined as a shelf
with books of the same genre (one detective story with another detective story;
and one novel with another novel). IgM and T cell receptor recognize the
antigenic epitope consisting of a small number of amino acid residues (612).
However, the structure of each epitope holographically reflects the overall
structure of an antigenic molecule. Hence, the spatial (semantic) complexity of
this epitope should not correlate with the molecular weight. The immune system
is prepared to analyze a spatial structure of any complexity, but not one
molecule. The T cell receptor or immunoglobulin is polyreactive and recognizes
a group of endogenous or exogenous molecules with the same degree of spatial
complexity (but not a specific antigen).
Polyvalence (hypervariability) suggests the recognition of up to 240 billion
groups of epitopes. This is only one of the unique properties of immune system.
Functional activity is also regulated by the opposite mechanism (unification of
recognition), which involves MHC. MHC is a complex of linked genes. This
complex was discovered in studying the mechanisms of tissue incompatibility in
the 1920s. The main properties of MHC (polygeny and polymorphism)
determine the individuality of MHC antigens in specimens of the same species.
The MHCencoded antigens (molecules) belong to three classes. Class I and II
molecules are particularly important for immunology. They share structural
similarity and consist of four domains, which form an antigenbinding site
*
72
Such approach suggests that the individualandspecies spatial hierarchy (specificity) of
an organism is determined during the prenatal period. This problem will be discussed
below.
Chapter 4. Holographic control of vital activity by the immune system
(cleft). The antigenbinding clefts in MHC class I and II molecules have a
similar spatial structure (Fig. 4.4).
Due to the peculiar structure of class I and II MHC, they may be
considered as the standard of individuality. The antigenbinding cleft has a role
of the Procrustean bed. All antigens undergo proteolysis and degradation in the
cytoplasm of APC. They bind to MHC class I and II molecules. A complex of
antigens and molecules is translocated to the cell surface. In the cleft of MHC
class I and II molecules, an antigenic peptide is squeezed between specific an
chor sites and forms the convolution of different shape. This convolution
interacts with the T cell receptor (Fig. 4.5). The T cell receptor does not recog
nize “self” and “nonself”, but evaluates the degree of spatial differences between
“self” and “nonself”. The greater is the differences, the stronger is the immune
response*.
Hence, the immune system follows even small conformational changes in
the “self”. Spatial characteristics of “nonself” are compared with those of
“self” (MHC class I and II molecules).
The elimination of foreign agents probably serves as an “emergency” or
“immediate” response of the immune system (antiinfective immunity). Under
normal physiological conditions, the regulatory capacity of the immune system
is directed to routine regulation of vital activity. This capacity depends on the
predetermined clone of native lymphocytes and natural autoantibodies.
Physiological functions of autoantibodies were extensively studied.
As mentioned above, the hypothesis of a regulatory role of natural
antibodies was proposed by famous Russian scientists I. P. Ashmarin and I. S.
Freidlin at the end of the 1980s (I. P. Ashmarin et al., 1989).
The notion of natural antibodies is currently undergoing revision. Recent
studies showed that natural antibodies are involved in multifactor regulation of
Fig. 4.5. Specific interaction of antigenic peptides with MHC class I molecules (V.
G. Galaktionov, 1998).
*
Previous experiments showed that the immune response depends on one autosomal
dominant gene. MHC class I and II molecules are the phenotypic products of this gene.
73
Ultralow doses
natural functions (I. P. Ashmarin, 1989, 1997; M. A. Myagkova, 2001; Y.
Shoenfeld et al., 1993). The activity of these antibodies does not correlate with
autoimmune disorders. The set of natural antibodies reflects the molecular
specificity of each adult individual (I. P. Ashmarin, 1997). There are natural
antibodies to some lowmolecularweight and highmolecularweight
substances (peptides), surface membrane structures, and intranuclear
structures. The majority of circulating natural antibodies are presented by IgG
(up to 50%).
Autoantibodies to endogenous regulatory peptides play a role in their
active transport to membrane receptors. They prevent peptide molecules from
preterm proteolysis. It was reported that natural antibodies have catalytic
(enzymelike or abzyme) activity. These antibodies modulate cell proliferation,
myelinization, activation of membrane ion channels, etc. There is a growing
body of evidence on directed transport of natural antibodies through membrane
structures and bloodtissue barriers. A combination study of natural antibodies
to endogenous regulators is used for diagnostics of various diseases (A. B.
Poletaev et al., 2002).
Physicians should consider the immune system as a global regulatory
system. Regulatory activity of the immune system is highly competitive with that
of CNS. The immune system may be imagined as a brain with dispersedly
distributed lymphocytes. Similarly to the nervous system, the immune system
is involved in the regulation of functional and metabolic processes in an
organism. This system has a role in the development of any pathological
syndrome (e.g., infectious or noninfectious syndrome). Antiinfective protection
is only a small part of physiological activity of the immune system. The main
functions of the immune system are the maintenance of homeostasis and
regulation of normal physiological functions. These functions are provided by
regulating the spatial structure of own molecules (antigens). The immune system
is responsible for local and distant regulation. During local regulation,
endogenous antigenic molecules interact with detectors of immune individuality
(MHC molecules, T cell receptors, and B cell receptors). Under normal con
ditions this interaction is not followed by antigen elimination, but initiates a
series of fine coadjustment reactions.
Natural antibodies have a distant effect. The antigenic pattern of a
variable region in immunoglobulins received the name “idiotype” (Fig. 4.6). This
region may induce an immune response. Antibody 1 always induces the
production of antiidiopathic antibody 2. This process is continuous. The same
antibody 1 is induced by antibody 2. The Nobel Prize winner H. Hjдrne
designated this phenomenon as “idiotypic/antiidiotypic network”. By the
network principle of organization, natural antibodies resemble the nervous
system. The system of natural antibodies attracted our attention. Our studies
74
Chapter 4. Holographic control of vital activity by the immune system
confirmed the fact that this system has a role in the effect of antibodies in
ultralow doses (see Chapter 2).
Natural antibodies may be considered as a “top point” of humoral
regulation. First, the synthesis of antiidiotypes provides a continuous pattern
of the antibody network (independently on the halflife of immunoglobulins).
And second, the continuous “mirrorlike” synthesis of antibodies provides
genomic regulation of the spatial structure of antibody idiotypes. Autoantibodies
are characterized by low affinity. Moreover, the concentration of autoantibodies
in the blood and liquid media of an organism is low. These data suggest that
even a single contact with the molecule (antigen) is sufficient for regulatory
activity of natural antibodies. The information about antigen is stored in
constellations of antibodies and provides distant regulation (relative to antigen).
The whole network of antibodies may be considered as a large constellation,
which reflects the parameters of all antigens in an organism. Due to the dual
function of antibodies, such holographic network follows the “regular”
individuality of an organism through V domains. It contributes to the regulation
of main functions through C domains. Natural autoantibodies may be involved in
antibodyantigen interactions for antigen elimination, which is mediated by a shift
in the synthesis of idiotypes and increase in affinity. These reactions appear to be
autoimmune in relation to own molecules. It is necessary to distinguish normal
autoimmune processes that occur in each organism under normal conditions from
a
b
VH
CH3
CH2
VL
Idiotype
Immunoglobin molecule
CH1
CL
VH
VL
«Internal
image»
Antigen
Fig. 4.6. Idiotypes, antiidiotypes, and their networks (J. Pleifer, 1999). Part of the
idiotypic/antiidiotypic network (a); antigeninduced disturbance of the network (b).
75
Ultralow doses
autoimmune diseases. Besides this, the term “autoantibody” should not be
associated with a pathological condition in medical practice.
The cause of autoimmune disease is poorly understood. These disorders may
be related not only to immune deficiency or exhaustion, but also to structural
changes in endogenous molecules. According to holographic principles, the
molecule that has a major role in certain pathological conditions may undergo
deformation and change in the fine “regular” structure. This molecule becomes
“foreign” for the organism and induces immune aggression (beyond the scope of
normal autoimmune regulation). However, physiological coadjustment functions
of antibodies are not taken into account in clinical practice.
While assuming that the system of natural antibodies serves as a target for
activated antibodies in the organism, it is necessary to understand which fine
regulatory mechanisms are affected. It can be said that we “regulate the
regulator”.
Most important in Chapter 4
The semantically organized molecular constellations are holographic
structures.
Autooscillations of molecules with a complex spatial structure (similarly
to potentiation) can determine the appearance of structured liquid loci.
Hereditary information is stored in constellations of DNA (RNA) molecules.
This information is translated to liquid media through autooscillations of DNA
regions. Autooscillations of genomic products (proteins and polypeptides) pro
vide the regulation of vital activity in the structured locus (primary genetic con
trol).
The interaction with epitopes (individual regions of “heavy” molecules, includ
ing proteins, polypeptides, and sugars) contributes to secondary genetic control
of vital functions by the immune system.
A major function of the immune system is the maintenance of individu
alandspecies spatial integrity in an organism (homeostasis).
After genetic reconstruction, immune detectors (T cell receptors, B cell receptors,
and MHC molecules) gain a specific individual structure and provide the integral
spatial (holographic) evaluation of own molecules (antigens).
The immune system is as potent as the nervous system for the overall
regulation of functional and metabolic processes.
Natural autoantibodies are the components of the immune system that play a
role in humoral regulation of vital activity. Previous studies suggest that natu
ral autoantibodies serve as a target for medical products from potentiated anti
bodies.
76
C h a p t e r
5
Principle of maintenance
of the initial integrity
M
uch progress in molecular biology, biochemistry, and genetics was
achieved during the second half of the 20th century. The doublestranded
nature of DNA and new classes of regulatory molecules were discovered. The
genome was “decoded”. Advances in biology and biotechnology provided the
development of new potent drugs. The effect of medical products was explained
schematically from their influence on the chain of “gene–protein–sign”. Mo
dern notions of vital functions are based on the linear continuum (continuity)
of vital activity. This hypothesis suggests successive transformation of molecular
(biochemical) processes to other processes.
Our study of ultralow doses showed for the first time that drugs have a
distant effect on the organism. The notion of essential information was used to
retain a scientific materialistic position. An informational approach does not
abandon the general physiological principles. In older times, the laws of classical
mechanics appeared to be insufficient to explain some physical processes. It
resulted in the appearance of quantum mechanics and new notions of the space
and time. The data on ultralow doses illustrate some new principles of vital
activity. The pharmacotherapeutic effects (e.g., adaptive activity) of ultralow
doses should be analyzed using a coordinate system.
In the previous chapter, we hypothesized that inherited characte
ristics are encoded in constellationandoscillation parameters of DNA. Auto
oscillations of DNA and genomic products (peptides and proteins) result in
structuralization of the liquid medium in an organism. All processes in this me
77
Ultralow doses
dium develop in the genetically determined spatial and temporal limits. In
herited characteristics (i.e., “wave” parameters of genetic information in struc
tured loci) appear as a semantic factor, which “integrates” the molecular pro
cesses into constellations. Figuratively, functions of a structured medium have
the semantic content and holographic shape.
Based on the experience of homeopathy, we emphasized that the specific
marker of individual sensitivity serves as an indication for treatment with several
homeopathic remedies. It depends on the association of this marker with other
markers. Analogously, we believe that each endogenous molecule enters a
considerable number of constellations and becomes involved in a variety of
distant constellation relationships. These relationships extend beyond the cell or
synapse, which forms the basis for celltocell interactions and systemic regu
lation (M. A. Pal’tsev et al., 1995). “Network” constellation processes in the
nervous, immune, and endocrine systems are particularly important for systemic
regulation. Billions of cells and subcellular factors are involved in reciprocal
relationships between the nervous and immune systems. By the degree of
complexity, these relationships are comparable with the Cosmos*. However,
local physiological processes are also based on the constellation principle. An
example is the cascade of metabolic events during intracellular transduction,
which involves secondary messengers, calcium, adenylate cyclase, phospho
inositide cycle, and protein kinases.
The existence of molecular constellations that combine cellular and inter
cellular processes eliminates the distinction between such notions as “substrate”
and “function”. More than 60 years ago the author of the theory of biological
field (A. G. Gurwitsch, 1944) assumed the existence of structured processes, but
not of the substrate or function. We shall use this good term. The formation of
constellations from hereditary information endows them with the property of
memory (i.e., holographic memory of normal function in certain molecular
ensembles). Hence, constellations are the unity of structure, function, and
regulations (holographic memory) of functioning.
The molecular ensembles maintain this trinity due to constant motion
and permanent dynamic changes in constellations. Each element of con
stellations should constantly deviate from the mean value. Functions are regu
lated by the “accuracy” of this deviation (i.e., on the basis of constellation me
mory). Hence, the most important information is stored in particular network
structures (neural networks in CNS, antiidiotypic network of natural anti
bodies, etc.).
Due to interference, all molecular and submolecular constellations
of an organism are combined into the holographic sphere. The dual individual
* The term “constellations” is most appropriate for these megaensembles.
78
Chapter 5. Principle of maintenance of the initial integrity
andspecies organization of distant intermolecular relationships in this structure
contributes to the existence of horizontal (generalspecies) and vertical (indi
vidual) planes. The notion of “holographic sphere” amplifies the term “homeo
stasis” by a new biophysical (spatial) content. The evolutionary principle pos
tulates that structure of any biological system should be similar to the original
matter. All manifestations of vital activity should be characterized by the
specific, “regular”, and evolutionarily determined spatial and temporal relation
ships between elements of the biological system. This is achieved by coupling
between the horizontal and vertical plane of an organism’s holographic sphere.
Vital functions are stable, structured, and homeostatic only within the “regular”
spatial limits. To emphasize this idea, we introduced one new term into our
hypothesis. This term (initial integrity) designates the ability of biological systems
to retain a unique individualandspatial structure according to evolutionary
(original) principles of the spatial and temporal organization of vital activity*.
The integration of all constellations into a common holographic sphere
provides the specific spatial architectonics of an organism. Spatialand
hierarchic relationships are not typical of individual molecules. They are char
acteristic of intermolecular distant (information) relations that serve as a primary
element of the holographic sphere. The hierarchy of a biological system is based
on the interrelated dual individualandspecies relationships, which develop in
the prebiotic stage of evolution.
The unique architectonics of an organism is formed during embryo
genesis. The integration (interference) of maternal and paternal “wave” infor
mation is probably a longterm process, which determines embryogenesis and
terminates only during the postnatal development. Before this period, any
foreign molecule introduced into the embryo has a specific place in the
developing architectonics. New relationships of this molecule serve as a
constituent element of the whole holographic memory in an organism (acquired
tolerance). The immune system becomes mature in the postnatal period, which
is related to genetic reconstruction of lymphocytes. This system is capable of
evaluating the structure of any endogenous or exogenous molecule. The immune
system estimates the identity of this molecule to the whole holographic structure
of an organism (principle of “self” or “nonself”).
On the basis of spatialandholographic notions, we believe that “non
self” factors are hazardous for the integrity of an organism. Exogenous substan
ces also have the property of integrity, which may be “imposed” upon the orga
nism. A structurally “nonself” molecule forms the extraneous spatial constel
lation relationships and causes disintegration in the organism. Toxic doses of a
*
By convention the compliance of organism functioning with basic principles is
designated the “correct function”.
79
Ultralow doses
slightly foreign substance also induce the formation of “incorrect” relationships
in an organism. Under these conditions, the existent information relationships
will be impaired due to a strong response to the toxic dose.
The maintenance of initial integrity is a normal physiological property of
the organism. This is similar not only to the idea of homeostasis (W. Cannon,
1915), but also to the notions of genetic integrity (M. Bernet, 1963) and anti
entropic activity (I. Prigozhin, 1986, 2006; E. Bauer, 1935). A systemic or local
disturbance in the initial integrity is always accompanied by disease. Oncological
diseases are the most prominent example. Spatial and temporal laws for the
regulation of atypical cells differ from those of normal cells.
Before the specific response to an exogenous molecule, the organism
evaluates the degree of its difference from the initial integrity. This molecule
should interact with conformational regions of endogenous protein molecules
that have a complex spatial structure (hormones, enzymes, cell receptors, and
antibodies). During the interaction of exogenous molecules with the surface of
somatic cells, these molecules are recognized by wellknown cell receptors.
During the interaction with conformational sites of microsomal enzymes, low
molecularweight xenobiotics undergo oxidationreduction and/or conjugation.
An exogenous substance is not only neutralized, but also induces the formation
of constellations to “read” its structure. Antibodies and lymphocyte receptors
are responsible for the primary evaluation of integrity during the first interaction
of antigen with immune system. This is typical of natural and synthetic antigens
with any degree of spatial complexity. The recognizing site of such antibodies
and receptors is specific for this antigen*. Large molecules are hydrolyzed in the
cell. Lowmolecularweight fragments are also evaluated by the immune system.
Under these conditions, a lowmolecularweight exogenous molecule or its
fragment interacts with the conformational site of a higher molecular weight
endogenous molecule. The weight of endogenous molecules probably “amor
tizes” autooscillations of xenobiotics, which prevents the imposition of “foreign”
integrity. Enzymes, receptors, and antibodies not only transform the signal of
an exogenous molecule, but also maintain the initial integrity. This concept
explains the domain structure of molecules that belong to a superfamily of
immunoglobulins and originate from a common gene. Nonspeciesspecific
domains of complex structure and high molecular weight provide the integral
and safe evaluation of antigen by the immune system.
As mentioned above, the medical product and its potentiated form
have a qualitatively similar effect. It is not surprising since the potentiated
substance consists of constellations of solvent molecules. After the first stage of
biotransformation, this drug in normal doses causes the formation of
* Let’s remember semantic series (see Chapter 4).
80
Chapter 5. Principle of maintenance of the initial integrity
constellations in an organism. Druginduced constellations have the same spatial
structure in vivo and in vitro. They reflect spatially a fine structure of this drug.
A systemic effect of medical product will depend on the cascade of semantically
organized molecularandconstellation events, but on the linear process
(independently on drug dose).
If both forms of the drug are constellationholographic structures, what
is their target? Probably, this target is also a holographic structure with specific
wave properties. Both forms of the drug (original and potentiated substance) can
interact with this target by the resonancelike mechanism. A therapeutic effect
of any drug is related to the direct action on pathological syndrome (i.e., holo
graphic structure), but not on a specific molecular target (concept of “magic
bullet”, P. Ehrlich).
Any pathological syndrome is a defense response. Chronic diseases are
usually characterized by hereditary (constitutional) predisposition. These data
illustrate the relationship between pathological syndrome and individualand
species structure of an organism. The question arises: what mechanisms do pro
tect the organism in pathological syndrome (abnormal structure)? First of all,
these mechanisms should maintain the initial integrity. Our evolutionary pos
tulate suggests that the hypothetic information vacuum (structured vacuum) is
characterized by a constant fight between at least two original substances for the
organization of information processes in accordance with their structure. The
tendency toward a “regular” spatial and temporal organization of normal func
tions is restricted at the information level. Hence, the inertia is typical of phy
siological processes (similarly to mechanical processes). The strength of in
dividualandspecies regulation probably decreases with time, which contributes
to the accumulation of stochastic events and natural aging of an organism.
Due to the inertia of vital functions, sooner or later the organism is
characterized by a tendency to the systemic or local formation of extraneous
spatial and temporal relationships. The increased formation of “regular”
relationships in “weak points” probably has a compensatory role. The stage of
functional disorders (predisease) is followed by deformation of the holographic
sphere in an organism. Such deformation is pathological syndrome, which
probably contributes to the maintenance of “selfproperties” (initial integrity) in
a “nonself” form. There are no “pathological” molecules in the organism. The
induced pathological distant relationships between these molecules determine a
conventionally abnormal state. Pathological syndrome should be diagnosed by the
appearance of new relationships (correlations) between clinical and laboratory
parameters, but not by a change in one biochemical or functional marker.
Holographic notions of pathological syndrome give rise to a new
understanding of the therapeutic effect of pharmaceutical products. Probably,
the remedy will be effective if it has the properties of tropism for pathological
81
Ultralow doses
syndrome and integrity. Tropic activity is associated with the resonance
interaction between druginduced constellations and pathological syndrome. The
influence should be integrative to produce a therapeutic effect. Integrity is
mainly determined by the weight of medical product. This property may be
considered as the ability of substance to retain specific physicochemical activity
(integrity) during the interaction with an organism. The relationship between
medical product and pathological syndrome depends on the integrity. It should
be sufficient to disturb the dynamic equilibrium of pathological syndrome.
The notion of pathological syndrome as a spatial structure does not
contradict the physiological hypothesis of disease pathogenesis. When one or
another endogenous molecule regulates a certain physiological process under
normal conditions, it will be involved in the development of pathological syn
drome to compensate disturbances in this process. These events are related to
the formation of “pathological” relationships.
Pathological syndrome has a specific hierarchic structure. The molecules
involved in this pathological syndrome are subordinate to each other. Without
pharmacological screening, it is difficult to estimate a priori which molecule has
a “key role” in the spatial structure of pathological syndrome. This approach
is also essential to evaluate the medical product, which will exhibit the highest
tropism for one or another type of pathological condition. Some potent phar
maceutics (aspirin, phenobarbital, and Viagra) were developed empirically.
Knowing a physiological role of these drugs, it was difficult to anticipate the in
dications for use. Pharmacological screening is a longterm and expensive process.
A new notion of tropism will allow us to develop some methods to search for
pharmaceutical products (e.g., resonance frequency analysis). Such studies can be
performed with potentiated substances, but not with medical products.
The mechanism of tropism is resonance. Therefore, tropism as a property
of substance is related to its semantic analysis in the organism. A “correct”
reflection of environmental events is the major evolutionary goal of each
organism. Any physical and chemical factors have a modulatory effect on
organism function. The first exposure to “neutral” factors is followed by only
one response of the organism (reaction to novelty; T. M. Vorob’eva, 1962). All
factors that may disturb the integrity always induce an organism’s response.
Each external factor has a specific semantic (information) structure,
which is manifested in the structure of induced molecular constellations. For
example, some information is perceived through the second signaling system and
transformed into plastic memory of the brain. These engrams are
holographically distributed in the brain. Metabolism of chemical factors (e.g.,
medical products) is followed by the appearance of constellations. Distant
relationships between all elements of the external factor and constituents of a
biological system have the same physical (information) nature. In the
82
Chapter 5. Principle of maintenance of the initial integrity
coincidence (resonance) of oscillation parameters for relationships between
discrete (final and simplest) elements of any external factor and distant inter
molecular relations in an organism (information acceptors), the image of this
external factor is distributed over the holographic structure of an organism (“re
cording”). External information is refracted through the holographic sphere
(prism) and gains an individual nature. This information is stored in compliance
with a unique structure of the organism, which constitutes its evolutionary purpose.
By convention the “portion” of external information may be compared
with the grammatical sentence, which is linguistically divided into “words” and
“syllables”. On the basis of interference, these “syllables” are combined into
new words and new “phrases”. The relationship between new phrases and ori
ginal sentence is not always obvious.
A famous linguist and Nobel Prize winner Noam Chomsky proposed the
term “generative grammar”. According to Chomsky, each notion causes the for
mation of brain associations that reflect its structure. The greater is the number
of words to describe any notion, the more exact and clear is the definition of
its meaning (N. Chomsky, 1999). A semantic analysis of medical products is
based on the same principles.
The question arises: which is the way to perform a semantic
analysis of pharmaceutical products in various doses?
A study of superdiluted solutions showed that they reproduce activity of
the original substance in a reduced form (E. B. Burlakova et al., 1990). It may
be suggested that due to little effect of the activated substance, its structure is
only partially evaluated in an organism. The activated substance causes a small
physiological response, which is mediated by the resonance mechanism and
sufficient for the image analysis of ultralow doses. Clinically, the exposure to an
activated product is followed by little activating effect.
Other results are obtained when a homeopath prescribes the potentiated
remedy according to the principle of “similarity” (i.e., individual sensitivity of
patient). The image of a potentiated substance is also “read” and “recorded”
in the holographic sphere of an organism. However, the whole spatial image of
this exposure cannot be formed in an organism. The structure of an activated
substance coincides with the individual vertical plane of a holographic sphere
and, therefore, is not reflected. To perform a complete evaluation of the
potentiated substance, druginduced constellations are combined to large
constructions in the organism. These changes contribute to the hyperergic
reaction. A specific response to ultralow dose increases due to hyperergia. In the
case of tropism for pathological syndrome, the activated product has a
therapeutic effect.
The constellations induced by a therapeutic dose also resound with
pathological syndrome. Under ideal conditions, a holographic sphere of the
83
Ultralow doses
initial integrity is restored in this locus. These changes are followed by recovery
of the patient.
It is well known that the drug has individual toxicity. A prescribed medical
product in the therapeutic dose becomes toxic when druginduced “wave”
constellations directly interact (resound) with pathological syndrome. However,
these constellations are so great that pathological syndrome cannot “absorb”
them. Treatment with the toxic dose is a priori dangerous for the initial integrity
of an organism. With respect to the initial integrity, such nonspecific event as
unreactivity to high dose of any substance appears to be substantiated. The
organism does not interact with this substance to retain its initial integrity.
The toxic dose is similar to stress exposure. Hence, stress may be con
sidered as a process to maintain the initial integrity (refusal to interact spe
cifically with a strong pathogenic factor). A similar evolutionary appropriateness
is typical of the opposite event (class reactions of hyperergia). It suggests the
refusal to interact specifically with a substance whose structure is dangerous for
the initial integrity. The experience of homeopathy shows that hyperergic
reactions retain the specific nature only after treatment with ultralow doses.
Let’s consider the interaction between ultralow dose and normal dose in
the context of the phenomenon of bipathy. Our experiments showed that
sometimes this phenomenon is reproduced during combined treatment with
both doses. Under several conditions, ultralow dose should be administered
before (several tens of minutes) treatment with “high dose”. These data indicate
that modifying activity of the potentiated substance is associated not only with
a direct effect of ultralow dose on “high” dose, but also with the readiness of
an organism to treatment with the standard drug.
It will be remembered that during bipathic treatment with prednisolone,
ethanol, morphine, and cyclophosphane, ultralow dose serves as a protective
factor against the toxic dose. The specific preparation of an organism to this
exposure probably contributes to a variety of positive protective effects that were
observed under experimental conditions.
We showed that the ultradiluted substance sometimes has a potentiating
effect on the same medical product. For example, antimetastatic activity of
cyclophosphane increased after bipathic treatment. The potentiating effect of
ultralow dose is probably related not only to its direct influence on normal dose,
but also to the preparation of common oscillatory target acceptors in an organism.
The drug in normal dose is biotransformed in an organism, which results in the
“reading” of its structure. The potentiated product is a ready constellation form.
An analysis of the potentiated product occurs more rapidly than that of the normal
dose, which determines the preparation of an organism to “high” dose.
Studying the effect of antiS100 in ultralow doses on the isolated
neuronal membrane showed that their basic property is sensitizing activity. It
84
Chapter 5. Principle of maintenance of the initial integrity
was manifested in subthreshold depolarization (with no generation of the action
potential), increase in the maximum amplitude of inward current, and moderate
functional modulation of ion channels. The systemic sensitizing effect of
activated antiS100 is manifested in variations of synaptic plasticity. This
conclusion was made in experiments on the model of LTPTP. The altered
synaptic plasticity determines a wide range of properties of neuropsychotropic
drugs in ultralow doses.
Sensitization is an increase in the individual sensitivity to a certain external
stimulus under the influence of another external factor. This phenomenon was
demonstrated in some bipathic experiments. On the one hand, we revealed that
ultralow doses of a drug specifically prepare the organism to normal dose of the
same drug. On the other hand, studies of American scientists showed that
pretreatment of neurons in the tissue culture with activated glutamate protects
them from damage by glutamate in toxic doses (W. Jonas et al., 2001).
Moreover, the protective effect against glutamate in toxic doses was also
achieved after administration of cycloheximide in ultralow doses. However, other
neurotropic drugs in ultralow doses did not have a protective effect (D. Marotta
et al., 2002). Hence, sensitization with ultralow doses is a specific process. This
conclusion was confirmed by the results of our studies with morphine (T. A.
Zapora et al., 1999), caffeine and cyclosporine A (O. I. Epstein et al., 2004),
theophylline and morphine (O. I. Epstein et al., 2003), and 5F5B6 antigen
(N. A. Beregovoi et al., 1999).
Longterm observations of antibodies in ultralow doses showed that they
have an adaptive effect. It is not surprising. Neurophysiological studies revealed
that weak and subthreshold factors have a sensitizing effect, which increases the
adaptive capacity of an organism. More than 30 years ago, I. A. Arshavskii
demonstrated the existence of adaptogenic remedies. They include extracts of
Eleutherococcus, Schizandra, ginseng, Rhodiola, etc. (I. A. Arshavskii, 1976).
Adaptive activity of these remedies is related to the nonspecific activating
effect. This activity is shortlasting and mild. Therefore, adaptogens should not
be identified with the adaptive effect of antibodies in ultralow doses. Antibodies
in ultralow doses have a strong, rapid, and specific effect. The main advantages
of activated antibodies are sensitization of natural antibodies and mobilization
of predetermined normal functions (memory). Hence, activated antibodies
produce a specific effect on the corresponding antigen. It should be emphasized
that treatment with these antibodies (i.e., mild exposure) has a sparing effect.
Adaptation is of the most complicated problems in physiology.
Adaptation to a certain environmental factor is based on the structural trace of
memory (F. Z. Meerson, 1993). By the principle of dominant, this process
contributes to the integration of topographically different nervous centers into
one constellation (A. A. Ukhtomskii, 1950, 1952). The final stage of adaptation
85
Ultralow doses
(model) is encoded in this constellation. It was named “a model of the desired
future” (N. A. Bernstein, 1990) or “useful adaptive result of activity” (P. K.
Anokhin, 1975).
According to P. K. Anokhin, parameters of the useful result integrate
various elements of a biological system into dynamic, selforganizing, and self
regulating functional systems. In other words, the goal (adaptation) combines
various physiological processes into a single whole. The integration is based on
acquired “adaptable” plastic relationships and “rigid” inherited relationships (N.
P. Bekhtereva, 1977). Functional systems are structured by the holographic
principle (K. V. Sudakov, 1984, 1990, 1996a,b). Our notions about semantic
andholographic functional principles of biological systems are close to the
classical concept of adaptation. During a semantic analysis, any external factor
induces a specific response of the organism. The image of this factor integrates
some physiological processes into dynamic constructions of memory (longterm
memory). All constellations in the organism are “coupled” by wave hereditary
information. During interference of genetic and acquired information, the latter
becomes individual for this organism. The formation of an adaptive response
results from individual experience, which is “superimposed” on generalspecies
physiological processes (genetic memory).
Our notions of adaption contradict the general theory. Approximately 10
years ago, the notions of evolutionary purposes of biological systems were revised
after experiments of Professor B. I. Lyubimov. The animals (mice) were treated
simultaneously with toxic doses of morphine and potentiated morphine C200 (10
400 wt %). Control animals received morphine in the same doses (Table 5.1).
The following results were obtained for females:
257.476(244.302:271.361)
LD50=
260.8298(238.9089:284.7621) mg/kg,
198.854(194.241:195.496)
LD16=
152,3292(148.764:155,9799) mg/kg,
340.223(339.159:341.297)
LD84=
446,6136(436,1606:457,317) mg/kg;
The following results were obtained for males:
257.476(244.302:271.361)
LD50=
275.4606(192.2007:394.7883) mg/kg,
198.854(194.241:195.496)
LD16=
86
138.0255(73.4526:259.3652) mg/kg,
Table 5.1.
Toxicity of morphine after individual treatment and bipathic administration in combination with potentiated morphine (O. I.
Esptein, 1999)
Females
Dose, mg/kg
number of animals per group
Males
number of died animals
number of animals per group
number of died animals
150
72
72
0
7
72
70
0
7
175
72
72
10
24
72
70
10
29
200
72
72
12
24
72
70
12
26
250
72
72
36
32
72
70
36
30
300
72
72
48
42
72
70
48
35
87
Chapter 5. Principle of maintenance of the initial integrity
individual
bipathic
individual
bipathic
individual
bipathic
individual
bipathic
administration administration administration administration administration administration administration administration
Ultralow doses
340.223(339.159:341.297)
LD84=
549.7437(292.5553:1033.029) mg/kg.
The standard (individual) and bipathic methods of morphine treatment
are shown in the numerator and denominator, respectively.
The experiment yielded ambiguous results. On the one hand, the
potentiated product had a protective effect against morphine in toxic doses
(LD84). On the other hand, toxicity of morphine in safe doses (LD16) was elevated
after administration of the potentiated product. A polymodal adaptive response is
typical of ultralow doses (E. B. Burlakova, 1986, 1990) and normal doses (well
known experiments of L. Kh. Garkavi et al., 1998). The question arise: what is the
biological significance of a polymodal response to substance in any dose?
We believe that the main role of adaptive capacity is not so much to pro
vide environmental adaptation as to maintain the initial integrity in an orga
nism. The tendency to integrity may be considered as a major factor, which
combines biological systems into a single whole. Biophysically, it is manifested
in an attempt of the organism to achieve a harmonic “regular” holographic
state. The analogy to electron was drawn above. Each electron occupies a certain
orbit during rotation around the nucleus. Similarly to this electron, the holographic
sphere of an organism has steady integrative multiparametric functional states
(orbits). Any medical product induces “wave” constellations in an organism.
Phase characteristics of these constellations are determined by drug dose.
Depending on the dose, this direct will “direct” all physicochemical processes
in an organism at the closest functional orbit (relative to phase characteristics).
Probably, multiparametric characteristics of a certain steady sate of the
organism’s holographic sphere are the useful result of activity (according to P.
K. Anokhin). In other words, a biological system always tends to achieve the
predetermined integrative state. These data introduce new definitions into the
classical notions of adaption in medicine and biology. Integrity appears to be at
a higher evolutionary level than health. Therefore, adaptation may be achieved
through disease. From this standpoint, any pathological condition should be
considered as the lowest level of adaptation. The organism continues to function
so long as it maintains the initial integrity and fulfils a main evolutionary
purpose (correct reflection of reality). Functional inertia contributes to the
dissociation of individual and generalspecies relationships in molecular
ensembles of one or another specific locus in the organism’s holographic sphere.
To compensate this dissociation, the organism integrates molecular processes
within the framework of pathological syndrome. During drug treatment for
pathological syndrome, we propose another program to achieve the organism’s
integrity. In regard to allopathic drugs, this is related to integrative properties of
the dose (weight). As for ultralow doses of antibodies, the specified condition
88
Chapter 5. Principle of maintenance of the initial integrity
is associated with particular properties of these agents (i.e., type of natural
antibodies).
First, the amount of natural antibodies in an organism is extremely low
to perform the role of endogenous regulators. Hence, the “degree” of
constellations induced by potentiated antibodies is sufficient for the interaction
with natural antibodies. The smaller is the target, the weaker is the weapon to
“strike” this target. Despite low physiological concentration of natural anti
bodies in the organism, their constellations include the regulations for function
of all antigens. Hence, global problems (regulation of functions) can be solved
by a “small force”.
Second, natural antibodies have a unique morphofunctional dualism that
is manifested in the existence of V domains and C domains. This property
allows natural antibodies to “trigger” coadjustment regulatory (generalspecies)
reactions in an organism. It is realized through C domains during the inter
action of one or another molecule (antigen) with V domains.
Third, antibodies are combined into the antiidiopathic network. A
disturbance from the interaction of antigen with one or several molecules of
antibodies can spread over the whole network.
And fourth, a continuous transfer of information in network structures
(e.g., antiidiopathic network of antibodies) seems to be analogues to the process
of potentiation. During continuous circulation, the same spatial images
(constellations) are superimposed on their traces. Hence, each image is divided
into various temporal (phase) constituents. The antiidiopathic network not only
includes the memory of normal function of antigens, but also encodes a step
bystep program of using this memory (program for result of action, P. K.
Anokhin)*.
Activated compounds have an “accelerating” effect on reflection of the
original substance in an organism (advanced programming of properties of the
useful adaptive result, P. K. Anokhin) and reduce the latency of an adaptive
response. This has been demonstrated with the phenomenon of bipathy. These
data suggest that the effect of products from ultralow doses of antibodies is
realized via advanced regulation of functions by natural antibodies.
Most important in Chapter 5
The cell of vital activity consists of semantically organized molecular and
submolecular ensembles (constellations) that appear as a trinity of structure,
functions, and hereditary principles (memory) of functioning.
*
Antibodies at various dilutions can affect various stages of this program. Hence, the
effect of antibodies in ultralow doses depends on their potency.
89
Ultralow doses
Any organism as a sum of molecular constellations is the integral holo
graphic structure (sphere) with a unique dual (individualandspecies) architec
tonics.
The maintenance of a unique specific structure in accordance with evo
lutionary (basic) principles of the spatial and temporal organization of vital ac
tivity is a main evolutionary purpose of any biological system.
All physiological reactions of the organism (including an adaptive re
sponse) are informationally predetermined and directed to maintain the initial
integrity.
Adaptive pathological syndrome is a holographic structure, which may be
considered as an effort to retain the initial integrity (“self”) in another structural
form (“nonself”).
The medical product in any dose can interact directly with pathological
syndrome and has a therapeutic effect under certain conditions (combination
of tropism for a certain pathological condition and sufficient integrity).
The main physiological property of remedies with ultralow doses is a sen
sitizing effect on the certain structured processes.
A specific nature of antibodies indicates that they can be used as mo
dern higheffectiveness remedies in ultralow doses. As differentiated from ho
meopathy, this approach dies not require individualization of therapy. Potenti
ated antibodies have a sensitizing effect on the system of natural antibodies
and, therefore, increase their regulatory activity (“regulation of regulator”).
90
C h a p t e r
6
On the way to
pharmacology
of ultralow doses
E
xperiments with ultralow doses showed that the division of medicine into
homeopathic and allopathic medicine is arbitrary. Medicine is a unified
science. The organism specifically reacts to a substance independently on its
dose. This simple fact emphasizes that biological systems are characterized by
a fine “informationalandessential” level of organization. At this level, the
effect of medical products in “allopathic” and “homeopathic” doses is mediated
by similar mechanisms. The effects of ultralow doses are reproducible, may be
evaluated by standard methods and, therefore, hold promise for evidencebased
medicine. The exception is homeopathic therapy. The individual (“similar”)
prescription of remedies is like art. The methodology of homeopathy is not
associated with a pathophysiological approach of modern pharmacology.
However, the experience of homeopathy is worthy of attention.
Physicians can adopt a wise (holistic) attitude of homeopaths to the patient.
Moreover, the drugs should be prescribed with caution. Skilled homeopaths take
an individual approach. They usually prescribe only one remedy, follow carefully
the patient’s reaction, and do not tend to cure the disease “at any price”. A
homeopathic approach to druginduced exacerbation is of interest to clinicians.
This state is associated with drug treatment when the observed symptoms
constitute the socalled “pathogenesis” (see Chapter 1). There is no causeand
effect relationship between the remaining symptoms and drug treatment. A
91
Ultralow doses
famous physician D. V. Popov (founder of the Kiev homeopathic school)
proposed the following three groups of symptoms in exacerbation: 1) existing in
a patient and increasing during drug treatment; 2) present in the anamnesis; and
3) observed at any time in immediate relatives. According to the rules of C. He
ring, the homeopath can predict a favorable or poor prognosis of exacerbation.
The drug is not withdrawn in a favorable prognosis. However, this state requires
a decrease in the frequency of drug treatment or shortterm interruption of
therapy. Therapy is interrupted only when druginduced exacerbation concerns
vital organs. When clinical symptoms “migrate” from vital organs to other
organs (e.g., skin) during treatment with the potentiated product, this trans
formation is considered as a positive reaction (“minimal” harm) and therapy
continues.
The historical experience of homeopathy (i.e., study of medical products
in ultralow doses with healthy volunteers, S. Hahnemann) is of particular
importance for modern clinical pharmacology. This is a paradigmatic example
of simplicity and greatness.
Generally, up to one third of patients may exhibit an atypical response
to any pharmaceutical product. The incidence of side effects, including severe
complications, is high in the group of patients with atypical reactions.
Genotyping of individual drug sensitivity is not introduced into clinical
practice. We believe that standard clinical trials of medical products should
be supplemented by the introduction of a group of healthy volunteers
receiving the activated form of drug. Such approach will allow us to evaluate
the hyperergic reaction to study drug in sensitive respondents. Therefore, all
possible complications will be rapidly and safely revealed at the initial stage
of therapy. The remaining side effects of study drug are associated with its
cumulative action and may be evaluated only in normaldose trials. Besides
the evaluation of adverse events, a study of activated products with healthy
volunteers before the start of standard clinical trials will demonstrate the
phenotypic and genetic markers (correlates) for individual sensitivity of
respondents to a certain drug.
Moreover, the activated forms of pharmaceutical agents should be tested
also in patients. Before the start of clinical trials, one or another drug in ultralow
dose may be given to a patient (onetotwo times). In the case of a strong
hyperergic reaction, the respondents should be excluded from clinical trials or
receive this drug in the reduced dose.
Besides the experience of homeopathy, combined (bipathic) admi
nistration of the drug and its activated form is of particular interest. Bipathy as
a whole may be introduced into modern medicine. After the discovery of this
phenomenon, there was a review on the protective effect of heavy metal salts
in ultralow doses against the toxic concentration. Administration of the activated
92
Chapter 6. On the way to pharmacology of ultralow doses
substance before and after treatment with heavy metals was followed by the
increased elimination of a toxic agent (A. Delbancut et al., 1997). There were
no data on the general modifying activity (except for protective properties) of
ultralow doses, which occurs after combined (bipathic) administration of the
substance and its activated form. Unfortunately, beginning from 1999 the
development and introduction of products from ultralow doses of antibodies
drew us away from further studies of bipathy. However, the results of previous
experiments are sufficient to understand the importance of this phenomenon.
The activated remedy potentiates* a pharmacological effect of the original
substance, which is of particular significance for medical practice.
Experiments at the laboratory of T. A. Voronina showed that combined
treatment with the normal and ultralow dose of phenazepam (ULDP, dilution
C12+30+200) is followed by a significant increase in anxiolytic activity of this
drug (O. I. Epstein et al., 2007). Activated phenazepam was given simultane
ously or 10 min before administration of the original substance. These studies
were conducted on the model of punished drinking. Electric current was applied
to the floor of cages and spout of drinking bowls. The animals attempted to
satisfy their natural demands for water (drinking from bowls). However, they
were punished by a mild electric current. Antianxiety drugs allowed the animals
to adapt and satisfy their biological demands for water (in spite of punishment).
The stronger was the anxiolytic (antianxiety) effect of study drug, the greater was
the number of punished drinking episodes (Table 6.1).
Another experiment of T. A. Voronina was designed to study the effect of
bipathic treatment with phenazepam on animals with corazolinduced seizures
Table 6.1.
Effect of individual or combined treatment with phenazepam in the thera
peutic and ultralow dose on the number of punished drinking episodes in
rats (conflict situation)
Group
Dose
Period between
drug treatment and
recording of the effect
Control
Number of punished
drinking episodes at
0.25mA current
20
177.75±43.02
1 ml/kg
20
415.67±113.96*
ULDP
2.5 ml/kg
20
260.67±38.21
ULDP
2.5 ml/kg
30
358.33±60.75*
ULDP+phenazepam
(simultaneously)
2.5 ml/kg + 1 mg/kg
20
1279.33±82.28**
ULDP before phenazepam
2.5 ml/kg+1 mg/kg
20
1022.00±46.36**
Phenazepam
Note. *p<0.05 and **p<0.01 compared to the control.
* For this reason, we proposed the term “activated” form instead of “potentiated” form.
93
Ultralow doses
(O. I. Epstein et al., 2007c). Ultralow doses were administered simultaneously
or 10 min before treatment with phenazepam in normal dose. Activated
phenazepam had a potentiating effect not only on anxiolytic activity, but also
on the antianxiety properties of this drug. It was manifested in an increase in
the latency of seizures, decrease in the ratio of animals with seizures, and
reduction of the mortality rate. The side effects of phenazepam were not
revealed under these conditions (sedative and myorelaxant activity; Table 6.2).
Further studies at the laboratory of T. A. Voronina showed that psycho
tropic properties of a wellknown neuroleptic drug haloperidol are not observed
after bipathic treatment of experimental animals.
Combined treatment with normal dose and ultralow dose of haloperidol
(ULDH) was followed by a decrease in the cataleptogenic effect of this drug.
The degree of catalepsy decreased by 24, 46, and 33% by the 60th, 120th, and
190th minute after administration of ULDH, respectively (compared to animals
of the haloperidol group).
Administration of Cyclodol (6 mg/kg) in combination with haloperidol
had a strong antagonistic effect on cataleptogenic activity of haloperidol.
Catalepsy was completely abolished after 60, 120, and 180 min.
The cataleptogenic effect was less pronounced after combined administra
tion of Cyclodol and ULDH. It should be emphasized that the cataleptogenic
effect was lower compared to that of Cyclodol, but higher than the activity of
potentiated haloperidol (Table 6.3).
It will be remembered that the antiblastoma effect of cyclophosphane in
creases after bipathic administration (E. N. Amosova et al., 2003). Experimental
studies (O. I. Epstein et al., 1997; V. G. Zilov et al., 2000; O. I. Epstein et al.,
2002b; A. M. Titkova et al., 2002; O. G. Berchenko et al., 2003; I. F. Pavlov
et al., 2003) and some clinical observations (N. V. Aleksandrova et al., 2003)
Table 6.2.
Effect of phenazepam in the therapeutic and ultralow dose on outbred
albino rats with corazolinduced seizures
Corazol+
Corazol+
ULDP
ULDP+
before
phenazepam
phenazepam (simultaneously)
Corazol
Corazol+
phena
zepam
Corazol+
ULDP
Latency of clonic seizures
0:11:20±
0:03:13
0:22:20±
0:05:42*
0:09:40±
0:00:35
0:37:20±
0:06:48*+
0:32:00±
0:10:26*
Latency of tonic seizures
0:16:40±
0:04:56
—
0:13:20±
0:00:34
—
0:33:00±
0:09:24*
Parameter
Tonic seizures, %
100
0
100
0
40
Mortality, %
80
0
100
0
20
Note. p<0.05: *compared to corazol; +compared to phenazepam in normal dose.
94
Chapter 6. On the way to pharmacology of ultralow doses
Table 6.3.
Effect of ULDH and Cyclodol on the degree of haloperidolinduced catalepsy
in rats (Morpurgo method)
Average score per group
Group, dose
after 60 min
after 120 min after 180 min
Haloperidol, 0.7 mg/kg
1.7±0.2
2.6±0.3
2.4±0.3
Haloperidol (0.7 mg/kg) +
Cyclodol (6.0 mg/kg), simultaneously
0.0±0.0*
0.0±0.0*
0.1±0.1*
Haloperidol (0.7 mg/kg) +
ULDH (2.5 mg/kg), simultaneously
1.3±0.1*
1.4±0.2*
1.6±0.2*
Haloperidol (0.7 mg/kg) +
Cyclodol (6.0 mg/kg) +
ULDH (2.5 mg/kg), simultaneously
0.5±0.2*
0.5±0.2*
1.0±0.2*
Note. *p<0.05 compared to animals receiving haloperidol in a dose of 0.7 mg/kg
revealed the protective effect of prednisolone, morphine, and ethanol during
combined administration of the original substance and its activated form. Several
experiments were performed with detoxification of heavy metals. The results of
these researches suggest that the phenomenon of bipathy holds much promise
not only for the potentiation of pharmacological activity, but also for the
correction of toxic properties of pharmaceutical products. Unfortunately, none
of the “bipathic” remedies is used in clinical practice (as differentiated from
medical products from ultralow doses of antibodies). It should be noted that
patents for bipathy have been granted in some countries.
Bipathy suggests a specific modifying effect of activated drugs on the
activity of original substances. Previous studies on various experimental models
showed that ultralow doses of cadmium prevent the toxic effect of cadmium.
Mercury in ultralow doses had a protective effect against toxic activity of
mercury salts (A. Delbancut et al., 1997). There are some data on the protective
activity of one substance against the toxic dose of another substance. As
mentioned above, activated cycloheximide protects neurons from glutamate in
toxic doses (D. Marotta et al., 2002). Moreover, ultralow doses of some
substances potentiate the effect of an antitumor antibiotic adriamycin (N. P.
Konovalova et al., 2002; N. P. Pal’mina et al., 2002).
We proposed that ultralow doses of antibodies can be used in clinical
practice. Activated products have a wide range of experimental and clinical
effects. Therefore, this approach may be considered as a precursor of
pharmacology of ultralow doses. At the present time, potentiated antibodies are
the most studied substances of ultralow doses.
Activated antibodies are a new class of medical products that meet
the requirements of evidencebased medicine. Due to technical reasons, they
95
Ultralow doses
are formally designated as homeopathic remedies. Probably, this classification
will change after the solution of normative questions. Principally, all products
of antibodies in ultralow doses are developed according to the general principles
of modern pharmacology. They include an experimental evaluation of specific
pharmacological activity, conduction of required toxicology studies, use of a
doubleblind placebocontrolled method in clinical trials, comparison of new
agents with modern pharmaceutical products, etc.
The results of experimental and clinical studies with products from
activated antibodies are presented in the final chapters of this monograph. They
show that ultralow doses of antibodies exhibit high effectiveness, which is
particularly important for modern pharmacology. The effectiveness of most
products was highly competitive with or surpassed that of reference drugs. Even
though the effectiveness of antibodycontaining agents in ultralow doses is lower
than that of reference drugs (Impaza and sildenafil; and Artrofoon and
indomethacin), the integral effectiveness/safety criterion of study preparations
compares well with pharmaceutical products. Sometimes the effectiveness of
antibodies in ultralow doses was unexpectedly high (even for developers). For
example, a hypoglycemic effect of peroral treatment with activated antibodies
to the insulin receptor βsubunit surpassed that of parenteral insulin. Besides
this, ultralow doses of antibodies to interferonγ (IFNγ) were effective on the
model of avian influenza. Antiinflammatory activity of Artrofoon allowed the
patients to avoid the use of nonsteroid antiinflammatory drugs.
The estimated safety of new products was expected. Ultralow doses could
not be toxic a priori. All trials for acute and chronic toxicity, reproductive
toxicity, embryotoxicity, immunogenicity, immunotoxicity, etc. confirmed the
safety of activated antibodies. Moreover, ultralow doses of antibodies to IFNγ
were shown to have antimutagenic activity.
The effect of antibodies in ultralow doses can develop in the early (e.g.,
several tens of minutes for Anaferoninduced hypothermia) or late period after
treatment (chronic diseases). It may be said that activated antibodies had the
immediate and delayed therapeutic effects in acute and chronic diseases,
respectively.
A general tendency was revealed during the therapy of chronic diseases
(rheumatoid arthritis, osteoarthritis, prostate adenoma, and cardiovascular
failure) with ultralow doses of antibodies. The improvement was observed by the
end of 1month treatment with study drugs, progressively increased in the
followup period, and reached maximum after 3 or 6 months of therapy. The
effect of “allopathic” reference drugs usually occurred after 2 weeks of therapy
and was most pronounced by the 4th week. However, many patients were char
acterized by exacerbation of the disease and development of side effects. Mild
exacerbation of the underlying disease was rarely observed after treatment with
96
Chapter 6. On the way to pharmacology of ultralow doses
ultralow doses of drugs. The adverse events were not revealed under these
conditions. Otherwise, the relationship between treatment with antibody
containing products and development of undesirable effects was ambiguous.
During combination therapy with standard drugs and products from antibodies
in ultralow doses, activated antibodies had a potentiating therapeutic effect.
Sometimes the incidence of side effects of “allopathic” drugs tended to decrease
under these conditions.
The effectiveness and safety are associated with a particular effect of
antibodies in ultralow doses on pathological syndrome. It will be remembered
that pathological syndrome is considered as an adaptive response to a change
in the individualandspecies integrity of an organism. Chronic diseases often
develop in response to the uncoupling of physiological reactions due to their
inertia, which increases with age. Pathological syndrome is a spatialand
holographic structure, which allows the organism to maintain its initial integrity.
According to the general laws of matter, any pharmaceutical agent may
exhibit tropism for one or another structured process in an organism (including
the pathological process). After a “semantic” analysis, the druginduced
molecular constellations can directly interact with pathological syndrome. This
interaction is probably mediated by the mechanism of resonance. The structure
of medical product is reflected in constellations. Because of the integrity, this
drug becomes “incorporated” into the structure of pathological syndrome. The
observed changes result in a change in spatial configuration of pathological
syndrome. Until this moment, the pathological syndrome is not “noticed” by
holographic memory of an organism. This is associated with the absence of
novelty, which serves as a major prerequisite for memory stimulation. Any
external agent or internal event of unknown integrity serves as a novel factor for
the organism. A druginduced disturbance in the dynamic equilibrium of
pathological syndrome is followed by structural changes and loss of an individual
initial integrity. The pathological syndrome becomes an object of regard with
holographic memory*. After the loss of integrity, this organism programs new
multiparametric characteristics of function to achieve the initial integrity.
Clinically, the transformation of pathological syndrome may be followed not
only by its complete or partial reduction, but also by the development of
undesired events.
*
Some data of experimental immunology are analogous to this suggestion.
Administration of an antigenic molecule to newborn or adult animals (under specific
technological conditions) may inhibit the immune response to a specific antigenic
molecule (tolerance). As mentioned above, the immune system does not respond to
small molecules or haptens (see Chapter 4). After conjugation of this “nonreactive”
antigen with hapten, the immune system “notices” a modified molecule and produces
specific antibodies.
97
Ultralow doses
A distinctive property of products from ultralow doses of antibodies is that
they modulate a unique regulatory system of natural antibodies. We emphasized
that antibodies have a specific individual structure and are combined into the
antiidiopathic network. This network includes the principles of function
(memory) for all antigens (molecules) in an organism. Potentiated antibodies
have a sensitizing effect on the antiidiopathic network, which results in the
actualization of holographic memory without structural reconstruction of
pathological syndrome. Hence, they produce a sparing effect. Our studies
showed that the therapeutic effect is achieved only after modification (through
activated antibodies) of the molecule or antigen, which has a key role in the
pathogenesis of a pathological condition. Under these conditions, the therapy
with ultralow doses of antibodies may be considered as pathogenetic. Some
evidence exists for our assumption, which is unusual for pathophysiology.
Artrofoon, which consists of antibodies to TNFa in ultralow doses, has a strong
diseasemodifying effect after longterm therapy for rheumatoid arthritis and
osteoarthritis (2 years).
A pathogenetic effect of antibodies in ultralow doses is also confirmed by
the persistence of druginduced changes after cessation of therapy. As distinct
from one of the benzodiazepine drugs, the antianxiety effect of Tenoten
(ultralow doses of antibodies to S100) is observed for at least 4 weeks (N. P.
Vanchakova et al., 2007).
The following clinical observation requires special attention: the
cumulative effect of longterm therapy with ultralow doses of antibodies is not
accompanied by an increase in the dose of prescribed drug.
The experience of homeopathy indicates that the effect of ultralow doses
may depend on their dilution (potency). Further studies revealed the unusual
types of selectivity of ultralow doses. For example, mollusk neurons of different
functions exhibit a response to various dilutions of antiS100. Otherwise, anti
S100 of different potency have a modulatory effect on various systems of
intracellular kinases. Clinical trials of Artrofoon in patients with rheumatoid
arthritis yielded a surprising result. The antiinflammatory and analgetic effects
were more significant after treatment with Artrofoon in a dose of 2 tablets 4
times a day (but not 1 tablet 4 times a day). It was unexpected that the effect
depends on the volume (number) of dilutions of activated substances. Further
studies of antiS100 were performed at the laboratory of T. A. Voronina and
“Porsolt & Partners Pharmacology” research company. The effect of these
antibodies in animals was characterized by an inverted Ushaped dependence
on the volume of dilutions of activated antibodies to S100 protein (V. Castagni
et al., 2007; Fig. 6.1).
The observed dependence is important to understand the mechanism for
effect of antibodies in ultralow doses. These data will be discussed later. It
98
Chapter 6. On the way to pharmacology of ultralow doses
80
**
60
*
40
*
20
0
20
40
2.5
5.0
7.5
15
Tenoten dose, ml/kg
Fig. 6.1. Dependence of the anxiolytic effect of Tenoten on its dose (elevated plus
maze test): “Porsolt & Partners Pharmacology”. *p<0.05 and **p<0.001 compared
to the control.
should be emphasized that the notion “dose” is also applicable to activated
products. Cumulation of the therapeutic effect without increasing the dose is a
specific feature of activated products. Moreover, clinical trials showed that
patients with rheumatoid arthritis and osteochondrosis may be treated with
lower (maintaining) doses when a therapeutic effect of the antibodycontaining
product reaches a plateau.
Tolerance of an organism to medical products results from the inertia,
which is typical of physiological processes. The stage of functionaland
metabolic “reading” of any pharmaceutical product is followed by the formation
of molecular constellations. This stage is also inert. During protracted treatment,
the therapeutic effect of prescribed drug may be maintained only under
conditions of “novelty” (i.e., increase in the dose). However, this procedure can
result in the development of drug dependence. Activated solutions are the ready
constellation product. Therefore, various drugs from ultralow doses of antibodies
should not overcome the inertia during formation of constellations. Drug
tolerance does not occur under these conditions.
The inertia is typical not only of physiological processes, but also of
conventionally pathological processes. This inertia does not allow the organism
to “recover” naturally from one or another chronic disease. Not only the
structure, but also the dynamics of pathological syndrome is encoded in the
genetic memory. This information appears as the successive and pathologically
related functionalandmetabolic stages. The program of each stage is encoded
in the previous stage. The next stage does not start before the end of the
previous stage. Increasing the dose of a therapeutic drug does not necessarily
allow us to overcome the inertia of pathological process. The therapy with
ultralow doses of antibodies has great advantages in torpid diseases. As
99
Ultralow doses
mentioned earlier, potentiated antibodies have the specific phaseanddynamic
characteristics. They serve as “time derivatives” of physiological functions of
natural antibodies in the organism. Under conditions of pathological syndrome,
these antibodies are coupled with an ensemble of other endogenous molecules.
They “freeze” at the unrealized stage of pathological process.
Ultralow doses of antibodies affect natural antibodies and transform them
(in advance) into another phasic functional state. These changes also concern
the antibodyregulated antigens and result in an “imbalance” of pathological
syndrome.
Sometimes ultralow doses of antibodies directly improve the dynamics of
pathological process (up to complete recovery or remission). These antibodies
can increase the sensitivity to previous therapy, which allows reducing the dose
of standard pharmacological agents. The course of treatment with ultralow doses
of antibodies to TNFα contributes to a twofold decrease in the dose of
nonsteroid antiinflammatory drugs and cessation of therapy in patients with
rheumatoid arthritis and osteoarthritis, respectively. The following observations
serve as an example of improved tolerance after administration of potentiated
antibodies.
Ultralow doses of antibodies to the insulin receptor betasubunit
significantly increase tolerance to glucose on the model of streptozotocin
induced diabetes. These antibodies are more potent than glybenclamide, but less
effective than insulin*.
T. M. Vorob’eva studied an antialcohol drug Proproten on the model of
lateral hypothalamic selfstimulation. The results of these experiments were
surprising for experimental narcology**. All animals refused the ability to self
stimulate a “pleasure center”, which is untypical of allopathic psychotropic
drugs. As differentiated from a variety of psychopharmacological agents, the
same phenomenon was observed without increasing the dose of study drug.
The ability to maintain a specific effect without increasing the dose
is important for longterm therapy (particularly during treatment for nearly
incurable diseases, which result from low level of adaptation). This is related to
high toxicity and risk of undesired transformation of the underlying disease.
The term “level of adaptation” is extensively used in physiology. Clini
cians also know that the lower are the adaptive capacities of an organism, the
higher is the predisposition to severe destructive diseases. The most prominent
clinical example of multistage adaptation is the development of mental
disorders due to external influences (e.g., craniocerebral injury). These
* See Chapter 7.
** Ultralow doses of ethanol had a similar, but less pronounced effect on this experimental
model.
100
Chapter 6. On the way to pharmacology of ultralow doses
disturbances are transitory (Wick’s symptoms), alternate in a certain sequence,
and usually result in asthenia. In this case, asthenia serves as a general
adaptive response.
Hence, clinical transformation of pathological syndrome is a normal sign
of adaptive capacities in the organism. The pathomorphosis of disease may be
spontaneous or induced by drug therapy. A physician must evaluate whether the
prescribed drug therapy and subsequent transformation are the symptoms of a
favorable prognosis (i.e., transfer of the disease to a higher level of adaptation).
The transformation of symptoms serves as an unfavorable prognostic factor and
may be accompanied by adverse events in severe chronic diseases (low level of
adaptation). According to FDA, 100000 people in the USA annually die from
complications of pharmacotherapy. The mortality of most patients is associated
with undesirable transformation of destructive chronic diseases. There is need
to minimize “harm” under these conditions. Incredible as it may seem, a
“severe” pathological symptom should not be “cured”. It is more important to
prevent the transformation of this symptom. Sparing therapy with potentiated
antibodies is most appropriate for such patients.
Therapy with ultralow doses of antibodies complies with the following
principle: “First, do not harm”. By the mechanism, this therapy serves as an
adaptive exposure of high effectiveness and safety. We would like a physician
to known the mechanism for action of newgeneration drugs. The safety of
potentiated products is not related to the presence of low doses of the original
substance. These drugs are an activated form of the original substance, which
has specific biological properties and involves other mechanisms of
adaptation.
Thirteen preparations from ultralow doses of antibodies were
approved in the Russian Federation. However, the number of widely used
drugs of this type is twofold lower. In recent years, antibodies in normal doses
were used for the therapy of noninfectious diseases and manufactured by the
largest international pharmaceutical companies (Table 4.6). The majority of
them are monoclonal humanized antibodies. They appear to be identical to
human antibodies and, therefore, are not perceived as a foreign agent after
parenteral treatment. These drugs are mainly used in the therapy for severe
disorders, including oncological diseases (N. I. Olovnikova et al., 2007).
Previous studies showed that these drugs are effective, but not harmless. The
development of these drugs is based on published data that antibodies directly
block one or another molecular target. We believe that the primary effect is
associated with complementary binding of antibodies to antigens. The
systemic effect of antibodies in normal and low doses may be realized via
activation of natural antibodies. This assumption is confirmed by the fact that
Remikeid and Artrofoon (preparations from normal and potentiated
101
Ultralow doses
Table 6.4. Antibodycontaining products in the world
Product (company)
Rituxan (Genetech)
Herceptin (Genetech)
Avastin (Genetech)
Molecular target
Indications for use
CD20 (B lymphocytes)
NonHodgkin’s B cell lymphoma
HER2 antigen
Breast cancer
VEGF (vascular
endothelial growth factor)
Large intestine cancer
Erbitux (Merck)
Campath (Bayer)
Zevalin (Genetech)
MyloTarg (Wyeth Ayerst)
Bexxar (GlaxoSmithKline)
Remicade (Centocor)
Oncological diseases
TNFα
Humira (Abbott)
Raptiva (Genetech)
Simulect (Novartis)
Zenapax (Hoffmann
La Roche)
OrthoClone OKT3
(Qrtho Biotech)
Psoriasis, Crohn’s disease,
ankylosing spondyloarthritis, rheu
matoid arthritis, and ulcerative colitis
Autoimmune inflammatory
diseases
ReoPro (Eli Lilly)
Platelet glycoprotein
IIb/IIIa receptor
Prevention of thrombus
formation in angioplasty
(surgical treatment for CHD)
Synagis (Medimmune)
Respiratory syncytial
virus protein
Therapy of children with respiratory
syncytial virus infection
Immunoglobulin E
Atopic diseases
Xolair
(Genetech/Novartis)
antibodies to TNFα) have a similar effect. Longterm administration of both
drugs is followed by a decrease in the level of TNFα (cytokine with
proinflammatory activity).
As regards the molecular targets (antigens), all products from ultralow
doses of antibodies are classified into the following five groups:
• antibodies to cytokines and growth factors (Anaferon, Artrofoon, and
Poetam for IFNγ, TNFα, and erythropoietin, respectively);
• antibodies to brainspecific protein S100 (Proproten, Tenoten, and
Tenoten for children);
• antibodies to enzymes (Impaza, endothelial NO synthase; and Afa
la, trypsinlike protease or prostatespecific antigen);
• antibodies to receptors (Kardos, angiotensin II AT1 receptor; and
experimental product Bation, insulin receptor bsubunit); and
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Chapter 6. On the way to pharmacology of ultralow doses
•
antibodies to lowmolecularweight compounds (histamine, Prohistam
and Epigam; cholecystokinin, Cholestam; and morphine*, Anar).
Published data show that the effectiveness of medical product from
ultralow doses of antibodies does not depend on morphofunctional
characteristics of antigen, but is determined by “tropism” for one or another
pathological condition. The effects of activated antibodies are determined by the
range of physiological activity of a particular antigen. For example, brain
specific protein S100 does not have a narrow range of physiological activity.
This protein is responsible for some basic functions of the nervous systems,
including the generation and conduction of nerve impulses, synaptic plasticity,
etc. (M. B. Shtark, 1985). It is not surprising that ultralow doses of antiS100
have a wide range of neurophysiological properties (from sensitization of the
neuronal membrane to early gene expression) and psychopharmacological
activity**. By contrast, activated antibodies to histamine exhibit an expected
high specificity for ulcer disease and allergy (as expected).
The possibility to predict pharmacological activity of antibodycontaining
products from the knowledge of pharmacological properties of a specific antigen
significantly facilitates the screening for drugs. It should be emphasized that
potentiation allows us to modulate the previously “inaccessible” molecules and
extends the number of therapeutic targets in pharmacology.
For example, the mechanisms of action for Impaza (therapy of erectile
dysfunction) differ from those for drugs consisting of phosphodiesterase type 5
inhibitors (Viagra, Levitra, and Sialis). The physiological mechanism of erection
suggests NO release in the cavernous bodies during sexual stimulation. This
process contributes to the elevation of cGMP concentration, relaxation of
smooth muscles, and increase in blood supply to the penis. Type 5 phos
phodiesterase is responsible for the consumption of cGMP. The inhibition of
this enzyme is followed by an increase in cGMP concentration. Impaza has a
normalizing effect on the reduced activity of endothelial NO synthase, increases
NO level, activates guanylate cyclase, and elevates cGMP concentration in the
cavernous bodies. Hence, endothelial NO synthase is a new pharmacological
target for ultralow doses of antibodies. Moreover, ultralow doses of antibodies
have a more physiological effect on this enzyme (modification, but not
inhibition). Impaza has a mild effect, holds promise for the therapy of patients
with hypertonia and CHD, and may be used in combination with nitrates. The
technology of potentiation allowed us to reveal a variety of new pharmacological
*
Morphine is the only endogenous antigen used in our studies. However, antibodies to
morphine can interact with some of the opiatelike lowmolecularweight endogenous
molecules.
** See Chapter 7.
103
Ultralow doses
targets (besides endothelial NO synthase), including S100 protein, prostate
specific antigen, and insulin receptor. It is most important that ultralow doses
of antibodies have a specific effect.
In the previous chapters, we described various aspects of the effect
of ultralow doses. In conclusion, we would like to briefly summarize the general
principles that concern the mechanisms of action and specific effects of
activated antibodies.
Similarly to other drugs in ultralow doses, ultralow doses of antibodies have
biological activity that is related to the technology of potentiation. Potentiated
solutions of antibodies have a similar systemic effect, which does not depend on
the presence of molecules of the original substance (O. I. Epstein et al., 2004).
The technology of potentiation provides a new property of antibodies in
ultralow doses. Subthreshold sensitization contributes to the modifying effect of
these antibodies. The physical nature of ultralow doses remains unknown.
Hence, the term “sensitization” is secondary in relation to activated products.
This term does not reflect the effect of ultralow doses, but suggests the re
storation of sensitivity (reactivity) of structured processes. Recovery of reactivity
is associated with the influence of activated antibodies on “wave” genetic
memory of an organism.
We revealed two types of the doseeffect relationship for activated
products. The effect of these substances is determined by dilution (potency) and
volume of the activated solution. The potentiated product (liquid or solid
substance) may be considered as an oscillatory circuit, whose characteristics are
determined by the method of preparation (number of dilutions) and size
(volume of the activated solution). The activated product has a selective dose
dependent effect on various phases of the same physiological process with
different frequencyoscillation characteristics, which is mediated by the
mechanism of resonance. Maximum therapeutic effect is not induced by the
highest volume of potentiated solution, but occurs when the amount of this
solution is optimal for the resonance interaction with a target. Our observations
are consistent with the general notion of Ushaped dependence (E. J. Calabrese
et al., 2001). Complex mathematical models showed that an inverted Ushaped
curve for the doseeffect relationship is not typical of substances with the
standard receptor mechanism of action (as differentiated from a monotonic
doseeffect relationship). This curve is appropriate for exogenous substances that
activate nuclear receptors (similarly to natural endogenous ligands). A Ushaped
doseeffect curve does not depend on receptor affinity for the exogenous sub
stance (M. C. Kohn et al., 2002). This approach correlates with the assump
tion of T. A. Voronina that Tenoten equilibrates the systems induced by
exogenous and endogenous substances (A. V. MartyushevPoklad et al., 2003,
2004). Previous studies of various antagonists on the model of LTPTP and
104
Chapter 6. On the way to pharmacology of ultralow doses
behavioral tests showed that the GABAA and GABAB systems are involved
in the effect of Tenoten. However, the effects of activated antibodies to S100
were not completely abolished under these conditions. These data serve as
indirect evidence that antibodies in ultralow doses have a wide rage of
modulatory (normalizing) activity.
Experiments with combined (bipathic) administration of the
medical product and its ultralow dose showed that the activated substance not
only prepares one or another structured process to the interaction with study
drug, but also potentiates the effect of this drug. The nature does not like com
plexity. Obviously, both phenomena are mediated by the same mechanism. It
may be suggested that the potentiated substance affects fine conformational pro
perties of the original molecule. Any endogenous molecule has the specific con
formation at a certain moment of time. Hypothetically, the number of mole
cular conformations is infinite or sufficiently great. The association of various
molecules in physiological or pathological processes is probably followed by syn
chronization of their conformational and oscillation parameters. Exposure of the
original molecule to its activated form is followed not only by a change in the
molecular structure, but also by modulation of distant functional relationships.
There is indirect evidence that functional activity of natural anti
bodies may be modified. The use of activated antibodies in EIA is followed by
a change in affinity of natural antibodies for the antigen (O. I. Epstein et al.,
2000). Clinical trials revealed that the content of natural antibodies returns to
normal after the course of treatment with potentiated antibodies. The reduced
level of natural antibodies to IFNγ in patients with viral infections increases
on day 1 after administration of antiIFNγ antibodies in ultralow doses (A.
Caruso et al., 1997). The content of natural antibodies to S100 protein in blood
plasma of alcoholic patients returned to normal by the end of longterm
treatment with ultralow doses of antibodies to S100 protein. A change in the
activity of natural antibodies is probably followed by programming of the
adaptive multiparametric state. Within the framework of this program, the
systemic adaptive effect of activated antibodies is mediated by a variety of
negative feedback biological reactions. The main effects are illustrated by the
example of antibodies to IFNγ in ultralow doses.
First of all, activated antibodies have a specific effect on expression
of the corresponding antigen. We showed that the course of treatment with
potentiated antibodies to IFNγ is followed by the increased production of
endogenous IFNγ in experimental animals. It should be emphasized that
activated antibodies to other cytokines had little effect on IFNγ expression*.
Besides the increase in IFNγ expression, ultralow doses of
* See Chapter 7.
105
Ultralow doses
antibodies to IFNγ had an indirect modulatory effect on the production of
functionally related IL2, IL4, IL10, and IFNα/β. These properties
contribute to a wide range of antiviral and immunomodulatory activity of
Anaferon. It is most important that Anaferon optimizes the interferon status*.
The preventive effect of Anaferon in ARVI is probably related to a normalizing
action of IFNγ on various components of immunity.
The sensitizing activity of antibodies in ultralow doses serves as a
physiological basis for their use in the prevention of various diseases.
A specific effect of activated antibodies is illustrated by the example
of antiIFNγ antibodies in ultralow doses. As shown above, potentiated
antibodies to IFNγ significantly increase the production of IFNγ, have a mild
activating effect on the expression of functionally related TNFα, and do not
modulate erythropoietin secretion (O. I. Epstein et al., 2004). Moreover,
potentiated antibodies have a speciesspecific effect. Ultralow doses of antibodies
to chicken IFNγ improved the state of chickens infected with avian influenza.
However, activated antibodies to human IFNγ were ineffective under these
conditions. High specificity of ultralow doses was also observed in studying the
phenomenon of bipathy. This specificity indicates that the activity of ultralow doses
is strongly determined. It remains unclear why potentiated speciesspecific
polyclonal antibodies (e.g., rabbit antibodies) modify the corresponding natural
antibodies of a certain patient under clinical conditions. It will be recalled that anti
bodies of any organism are individualized by the V domain due to recombinations
in the genome of lymphocytes. Hence, potentiated antibodies have a primary
effect on nonspeciesspecific regions of the C domain in natural antibodies.
The specific activity of antibodies in ultralow doses is associated
with a certain direction of effect, which depends on the initial functional state.
For example, ultralow doses of antibodies to erythropoietin and GCSF increase
erythropoietic and granulocytemacrophage activity, respectively, under
conditions of cytostatic myelosuppression. By contrast, these antibodies have an
inhibitory effect on test parameters during immobilization stress (A. M. Dygai
et al., 2003, 2004). After administration of antiS in ultralow doses the
frequency of action potential generation decreases in neurons with high basal
level of spontaneous activity, but increases in neurons with spontaneous activity
of low frequency (V. V. Andrianov et al., 2003). The effect of antibodies in
ultralow doses depends on the initial state of target organs, which illustrates the
adaptive nature of antibodyinduced changes.
The remedies with ultralow doses of antibodies are manufactured at the
“Materia Medica Holding” ResearchandProduction Company. They differ in
the implementation phase. Some products, including Impaza, Proproten100,
* See Chapters 7 and 8.
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Chapter 6. On the way to pharmacology of ultralow doses
Anaferon, and Anaferon for children, are extensively used in medical practice.
Tenoten, Afala, and Artrofoon have recently appeared in pharmacies. Kardos
and Epigam will soon be ready for manufacture. Experimental and clinical trials
with ultralow doses of antibodies will be described in Chapters 7 and 8. We
would like to show the prospects of treatment with these drugs.
Tenoten and Proprotein100 include ultralow doses of antibodies to brain
specific protein S100. The dose of antiS100 in Proprotein is C1000. Tenoten
is a mixture of activated dilutions C12+30+200. Previous experiments showed
that antianxiety activity of the mixture is higher than that of dilution C1000.
Tenoten was approved for use as an anxiolytic. Tenoten is a typical daytime
anxiolytic, since it combines the incompatible properties (antianxiety and
activating effects). As differentiated from benzodiazepine drugs and plant
preparations, ultralow doses of antibodies to S100 have no sedative and
myorelaxant properties and do not cause drug dependence. The patients
reported that Tenoten has a “gentle”, mild, and progressive effect (“sudden and
imperceptible improvement of the state”). The drug provides a feeling of light
ness and naturalness. After treatment with Tenoten the capacity for work returns
to normal (but not increases). Tenoten may be prescribed in psychological
tension (driving, examination, managing, and operating activity) and stress
conditions. Tenoteninduced “lightness” was not perceived as euphoria or
overexcitation. Clinical trials revealed that Tenoten does not cause drug addic
tion. Tenoten may be extensively used as a drug of the “normal state”, which
prevents internal stress and anxiety. Tenoten has a normalizing effect on mood
and general activity. It should be emphasized that Tenoten is effective not only
in patients with borderline mental disorders, but also in practically healthy
people with a variety of situational neurotic states. Tenoten has an antiasthenic
effect and improves cognitive function, which is particularly important for
practically healthy subjects.
In the future, we plan to introduce Tenoten into psychiatry. It will be used
mainly for the therapy of patients with depressive disorders. A unique effect of
Tenoten on synaptic plasticity should be taken into account in combined
treatment with antidepressant and antipsychotic drugs. Combined administration
of Tenoten and psychotropic drugs will allow us to reduce the volume of
pharmacotherapy and to prevent side effects of various pharmaceutical products,
including neuroleptics (J. L. Dugina et al., 2006).
Previous experiments revealed that Tenoten has a neurotrophic and protec
tive effect on the model of ischemic and hemorrhagic stroke. Hence, Tenoten holds
much promise for combination therapy of these diseases. In several trials, Tenoten
was effective for Parkinsonism, Alzheimer’s disease, and disseminated sclerosis.
Tenoten for children contains a mixture of dilutions C12+C30+C50. This
product is studied at the “Materia Medica Holding” ResearchandProduction
107
Ultralow doses
Company. Tenoten for children holds promise for the therapy of children with
nervousness and memory deficit.
The composition of Proproten100 is similar to that of Tenoten.
Proproten100 was manufactured from 1999. Proproten100 is widely used as
an antialcohol drug in Russia. The effect of Proproten is associated with the
ability of activated antiS100 to “saturate” a system of positive emotional
reinforcement, which results in the decrease in alcohol abuse. Proproten does
not cause euphoria and addiction. It can be said that Proproten has a
pseudoeuphoric effect*. The patients reported that Proproten produces a “mild”
and “specific”, but not intoxicant effect. T. M. Vorob’eva studied the effect of
Proproten on the model of lateral hypothalamic selfstimulation. These ex
periments showed that Proproten induces the socalled “emotional equili
brium”, which is difficult to describe in words. In discussing the emotiotropic
effect of Proproten, it is more appropriate to say about “normalizing activity”.
Proproten decreases alcohol addiction and has the antidepressant,
antianxiety, and neurotrophicandprotective effects. Therefore, Proproten may
be used to for the prevention of alcoholism and recurrences of this disease. The
effectiveness of drug treatment for many years (e.g., in combination with
psychotherapy) will be evaluated in future studies.
Activated antibodies to IFNγ, including Anaferon for children (mixture
of dilutions C12+30+50) and Anaferon (C12+C30+C200), were manufactured
by the “Materia Medica Holding” ResearchandProduction Company for 5
years. Ultralow doses of antibodies to IFNγ were shown to be effective in viral
infections, including influenza, chickenpox, infectious mononucleosis, genital
herpes, ophthalmic herpes, adenovirus infection, respiratory syncytial virus in
fection, rotavirus infection, coronavirus infection, calicivirus infection, tickbone
encephalitis, etc. Due to high effectiveness and safety, activated antibodies to
IFNγ are extensively for the therapy and prevention of influenza and ARVI in
Russia.
Taking into account a wide range of immunomodulatory properties of
Anaferon, we believe that the future lies with administration of this preparation
in combination with other antiviral and/or antiinflammatory drugs, vaccines,
and sera. For example, clinical trials showed that the effectiveness of acyclovir
significantly increases after combined administration of this drug and Anaferon
in patients with herpes infection (A. E. Shul’zhenko et al., 2005). After over
coming the distrust of ultralow doses, Anaferon may be used in combination
therapy for infectious diseases that are resistant to any treatment (e.g., AIDS,
*
In collaboration with E. N. Krylov, we studied the possibility of treatment with anti
bodies to morphine in ultralow doses (activated neuropsychotropic drugs) as a nonnar
cotic replacement therapy in additions (E. E. Krylov, 2003a; N. N. Ivanets et al., 2002).
108
Chapter 6. On the way to pharmacology of ultralow doses
tuberculosis, and avian influenza). Promising results were obtained in study of
avian influenza. Activated antibodies to chicken IFNγ improve the survival rate
of chickens with avian influenza. We assume that Anaferon may be used in the
therapy of noninfectious diseases, whose pathogenesis involves interferon
(disseminated sclerosis, some mental disorders, etc.). The main indication for
Anaferon therapy will be preventive treatment (particularly, in children of
decreed groups).
Besides Anaferon, the preparation from ultralow doses of antibodies to
cytokines (Artrofoon) was developed at the “Materia Medica Holding” Re
searchandProduction Company. Artrofoon consists of activated antibodies to
TNFα. This drug has a strong antiinflammatory effect. At the present time,
Artrofoon is mainly used in rheumatology. It should be emphasized that
TNFα has a wide range of physiological activity. Probably, ultralow doses of
antibodies to this cytokine may be used as an antiinflammatory agent in
pulmonology, surgery, and gastroenterology. Strong evidence exists that
Artrofoon is effective in Crohn’s disease. Our experiments showed that
ultralow doses of antibodies to TNFα have antitumor activity, which is
particularly important for oncology.
Impaza has been extensively used in recent years. This product of
antibodies to NO synthase in ultralow doses is used in the therapy for erectile
dysfunction. Impaza is a drug of choice for elderly patients. Impaza may be
given to nitratereceiving patients with coronary heart disease. Impaza has a
mild hypotensive effect. This drug may be used for a long time to prevent the
development of erectile disorders. Impaza modulates the production of NO and
has a multifactor regulatory effect on intracellular processes. Therefore, Impaza
holds much promise for the therapy of endothelial dysfunction.
The other two products, Afala (activated antibodies to prostatespecific
antigen) and Kardos* (ultralow doses of antibodies to the type 1 angiotensin II
receptor), were introduced into clinical practice in recent years. Afala is used
in the therapy for adenoma and inflammation of the prostate gland. Kardos is
used in the therapy for chronic cardiovascular failure. Both drugs have high
therapeutic effectiveness. In our opinion they hold promise for the preventive
therapy. The effectiveness of drug treatment for many years will be evaluated in
further studies.
The products from ultralow doses of antibodies to histamine, Epigam and
Prohistam, were also developed at the “Materia Medica Holding” Research
andProduction Company. They may be used in the therapy for gastric ulcer and
allergic diseases, respectively. These drugs hold much promise for secondary
prevention of diseases.
* Beginning from 2008, the drug will be designated “Kardosten”.
109
Ultralow doses
Most important in Chapter 6
There are the following three directions to the use of potentiated prod
ucts: homeopathy, bipathy, and pharmacology of antibodies in ultralow doses.
The methodology of individual prescription of ultralow doses makes it
difficult to use the experience of homeopathy by untrained physicians. The bi
pathic method and treatment with potentiated antibodies do not contradict the
paradigm of modern medicine and may be widely used in the therapy for vari
ous diseases.
The major advantages of products from ultralow doses of antibodies are
a combination of effectiveness and safety, mild (adaptive) effect, cumulative
action in longterm therapy, and absence of drug tolerance.
110
C h a p t e r
7
Experimental pharmacology
of products from ultralow
doses of antibodies
7.1. Experimental study of antibodies
to S100 protein in ultralow doses
A
n experimental study showed that ultralow doses (ULD) of antibodies to
S100 protein (ULD antiS100; Proproten100, Tenoten, and Tenoten for
children) have pharmacological activity. They exhibited the anxiolytic, antiasthenic
(activating), antidepressant, GABAmimetic, antiaggressive, stressprotective,
antihypoxic, antiischemic, neuroprotective, and nootropic properties (Fig. 7.1).
ULD antiS100 did not cause side effects (sedative and myorelaxant
effects) that are typical of benzodiazepine anxiolytics. The action of ULD anti
S100 was shown to be realized via the GABAergic and serotoninergic systems.
ULD antiS100. ULD antiS100 had a modulatory effect on neuronal plasticity
and cfos gene expression.
We studied the consequences of combined treatment with ULD antiS100
and haloperidol. ULD antiS100 did not modulate the specific psychotropic
activity, but abolished the cataleptic effect of a neuroleptic drug. The anxiolytic,
antidepressant, and nootropic properties of ULD antiS100 were also revealed
in experiments on young rats. ULD antiS100 were effective in the therapy of
memory deficit and hyperactivity. ULD antiS100 had a good safety profile in
a toxicology study.
111
Ultralow doses
ANXIOLYTIC
ACTIVATING AND ANTIASTHENIC
ANTIAGGRESSIVE
Diazepam
ANTIDEPRESSANT
Diazepam
Amitriptyline
STRESSPROTECTIVE
ULD antiS100
NOOTROPIC
Piracetam
Mexidol
Nimodipine
Cavinton
and
piracetam
ANTIHYPOXIC
NEUROPROTECTIVE
ANTIISCHEMIC
Fig. 7.1. Pharmacological properties of ULD antiS100.
Anxiolytic activity of ULD antiS100
Anxiolytic activity of ULD antiS100 was studied in the Vogel conflict test
(T. A. Voronina et al., 2000; G. M. Molodavkin et al., 1995; J. R. Vogel et al.,
1971; D. Triet et al., 1985; D. J. Sanger et al., 1991), elevated plusmaze (T. A.
Voronina et al., 2000; S. Pellow et al., 1986; G. R. Dawson et al., 1995; S. E.
File, 1995), and openfield test (T. A. Voronina et al., 2000). The benzodia
zepine anxiolytic diazepam was used as a reference drug.
Anxiolytic activity of ULD antiS100 in the Vogel test of “conflict situa
tion”. A conflict situation (G. M. Molodavkin et al., 1995; T. A. Voronina et al.,
2000; J. R. Vogel et al., 1971; D. Triet et al., 1985; D. J. Sanger et al., 1991)
resulted from the opposition of drinking and defense motivations. Each episode
of drinking was punished by pain electrostimulation. The number of punished
drinking episodes was recorded for 10 min. The anxiolytic effect of study drug
was estimated from a significant increase in the number of punished drinking
episodes.
112
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Experiments were performed on adult outbred albino rats weighing 230
250 g. The animals of groups 1 (control), 2, and 3 received intragastrically 2.5
ml/kg distilled water, 2.5 ml/kg ULD antiS100, and equivalent volume of
2 mg/kg diazepam, respectively.
During single and repeated administration (5day course), the anxiolytic
effect of ULD antiS100 compared well with that of diazepam (Fig. 7.2; T. A.
Voronina et al., 2003a; T. A. Voronina et al., 2006).
ULD antiS100 had a greater anxiolytic effect on lowactivity animals
that were predisposed to anxiety and depressive behavior (S. B. Seredenin et al.,
1994). In highactivity animals, the anxiolytic activity of ULD antiS100
compared well with that of diazepam (Fig. 7.3). These data indicate that ULD
antiS100 not only produces the anxiolytic effect, but also exhibits the
antiasthenic or activating properties (as differentiated from diazepam with
anxiolytic and sedative activity).
Anxiolytic activity of ULD antiS100 in the elevated plusmaze test. The
elevated plusmaze (EPM) consisted of four arms (length 0.5 m, width 10 cm)
% of the control
200
a
b
!
% of the control
250
!
!
150
200
!
150
100
100
50
0
50
0
1
2
1
3
2
3
Fig. 7.2. Anxiolytic effect of single administration (a) and 5day course of treatment
with ULD antiS100 (b) in the Vogel conflict test. Control (1), ULD antiS100 (2),
and diazepam (3). *p<0.05 compared to the control.
a
b
% of the control
400
!
300
200
100
0
ULD
antiS100
Diazepam
ULD
antiS100
Diazepam
Fig. 7.3. Effect of ULD antiS100 on highactivity (a) and lowactivity rats (b) in
the Vogel conflict test. *p<0.05 compared to diazepamreceiving animals.
113
Ultralow doses
that were perpendicular to each other. Two opposite arms were surrounded by
a 40cm wall from three sides. The other two arms were open. This maze was
placed at a distance of 0.5 m from the floor level. The area of EPM was il
luminated by two luminescent lamps (20 W, 60 cm from the level of arms). This
method is based on the fear of open space or fall from a height (T. A. Voronina,
et al., 2000; S. Pellow et al., 1986; G. R. Dawson et al., 1995; S. E. File, 1995).
Experimental animals were placed in the central area of EPM. The fol
lowing behavioral parameters were recorded for 3 min: latency (LC) of the first
entry into the open arms, number of complete and incomplete entries, and time
spent in the open arms. Emotionality of rats was estimated from the urination
rate and number of fecal boluses in EPM.
Experiments were performed on adult outbred albino rats weighing 230
250 g. The animals of groups 1 (control), 2, and 3 were treated with 2.5 ml/kg
distilled water, 2.5 ml/kg ULD antiS100, and 2 mg/kg diazepam (2.5 ml/kg,
reference drug), respectively. Test substances were administered intragastrically
30 min before the study.
ULD antiS100 had a strong anxiolytic effect. This conclusion was deri
ved from significant changes in the number of entries into the open arms, time
spent in the open arms, number of overhangs, and rates of defecation and
urination (Fig. 7.4; T. A. Voronina et al., 2003a; T. A. Voronina, 2006). The
effect of ULD antiS100 was similar to that of diazepam. Both drugs increased
the number of entries into the open arms (by 2.1 and 2.4 times, respectively,
p<0.05), time spent in the open arms (by 5.6 and 7 times, respectively, p<0.05),
and number of overhangs (by 5 and 9 times, respectively, p<0.05). No
differences were found in the effects of ULD antiS100 and diazepam (T. A.
Voronina et al., 2003a; T. A. Voronina, 2006).
Anxiolytic activity of ULD antiS100 in the openfield test. The openfield
test is used for a study of rat behavior (locomotor activity and emotionality;
a
b
c
% of the control
1200
!
!
1000
!
600
400
control
!
800
ULD antiS100
diazepam
!
!
200
0
Fig. 7.4. Anxiolytic effect of ULD antiS100 and diazepam in the elevated plus
maze. Number of entries into the open arms (a), time spent in the open arms (b),
and number of overhangs (c). *p<0.05 compared to the control.
114
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
T. A. Voronina et al., 2000). The box had an area of 1×1 m and was surrounded
by walls (0.5 m in height). The floor was divided into squares (10×10 cm). The
holes (4 cm in diameter) were made at each corner of these squares. The open
field was illuminated by two fluorescent lamps (20 W, 0.5 m above the floor
level).
The animals were placed in the corner of this box. Horizontal activity was
studied for 3 min. The following parameters were recorded: number of crossed
squares, vertical activity (number of rearing postures), number of entries into the
center of the open field, exploratory behavior (exploration of holes in the floor),
and frequency of grooming or scratching episodes. This study was performed to
evaluate the anxiolytic and sedative effects of drugs (Bruhwyler et al., 1995).
Experiments were performed on adult outbred albino rats weighing 230
250 g. The animals of groups 1 (control), 2, and 3 were treated with 2.5 ml/kg
distilled water and equivalent volumes of ULD antiS100 and 2 mg/kg diazepam
(reference drug), respectively. Test substances were administered intragastrically
30 min before the study.
ULD antiS100 and diazepam had a strong antianxiety effect, which was
manifested in a significant increase in the number of entries into the center of
the open field (by 2.5 and 1.8 times, respectively; Fig. 7.5). As differentiated
from diazepam, ULD antiS100 had no sedative activity. Horizontal activity of
animals did not decrease after treatment with ULD antiS100 (Fig. 7.6; T. A.
Voronina et al., 2003a; T. A. Voronina et al., 2006).
Antidepressant activity of ULD antiS100
Antidepressant activity of ULD antiS100 was studied in the Porsolt’s
forced swimming test (R. D. Porsolt et al., 1978), as well as in a water tank with
Number of entries into the center of the open field
3.5
!
!
3.0
2.5
2.0
1.5
1.0
0.5
0
0
Control
ULD antiS100
Diazepam
Fig. 7.5. Anxiolytic effect of ULD antiS100 and diazepam in the openfield test.
*p<0.05 compared to the control.
115
Ultralow doses
Number of entries into the center of the open field
25
20
15
!
10
5
0
Control
ULD antiS100
Diazepam
Fig. 7.6. Effect of ULD antiS100 and diazepam on horizontal activity of rats in
the openfield test. *p<0.05 compared to the control.
wheels (G. M. Molodavkin et al., 1994; S. Nomura et al., 1982). A standard
tricyclic antidepressant amitriptyline was used as the reference drug.
Antidepressant activity of ULD antiS100 in the Porsolt’s forced swimming
test. This method is based on recording of the immobility time in rats (R. D.
Porsolt et al., 1978). The animals were placed in a water tank (diameter 40 cm,
depth 60 cm). Under these conditions, locomotor activity of rats was directed
to the avoidance of an aversive (unpleasant) situation. However, these animals
became “suspended” in water during the followup period. They remained
immobile or made slight movements to maintain the head above water. The
duration of immobility served as a criterion of depressiveness.
Experiments were performed on male outbred rats weighing 250300 g.
The animals received 2.5 ml/kg distilled water (control group), 2.5 ml/kg ULD
antiS100 (treatment group 1), and equivalent volume of 10 mg/kg amitriptyline
(reference drug, treatment group 2). Test substances were administered
intragastrically 30 min before the study.
Single and repeated treatment (5day course) with ULD antiS100 and
amitriptyline had an antidepressant effect, which was manifested in a significant
decrease in the immobility time. The activity of ULD antiS100 compared well
with that of a tricyclic antidepressant (Fig. 7.7; O. I. Epstein et al., 2003c; T. A.
Voronina et al. 2006).
Antidepressant activity of ULD antiS100 in the Nomura’s forced
swimming test in a water tank with wheels. This method is extensively used to
study the effect of antidepressant drugs (G. M. Molodavkin et al., 1994; S.
Nomura et al., 1982). The rats were placed in a water tank. The reservoir was
divided into four compartments with wheels. The animal’s snout was opposite
to a wheel in each compartment. The number of wheel revolutions was recorded
for 10 min.
Experiments were performed on male outbred rats weighing 250300 g.
The animals received 2.5 ml/kg distilled water (control group), 2.5 ml/kg ULD
antiS100 (treatment group 1), and 10 mg/kg amitriptyline in an equivalent
116
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
% of the control
a
% of the control
140
140
120
120
100
b
100
!
80
!
80
!
!
60
60
40
40
20
20
0
0
Control
ULD antiS100
Amitriptyline
Control
ULD antiS100
Amitriptyline
Fig. 7.7. Antidepressant activity of ULD antiS100 and amitriptyline in the Porsolt’s
forced swimming test. Single administration (a) and 5day course of treatment (b).
Ordinate: immobility time. *p<0.05 compared to the control.
volume of the solvent (treatment group 2). Test substances were administered
intragastrically 30 min before the study.
ULD antiS100 and amitriptyline significantly reduced the signs of a
depressive state. The number of wheel revolutions increased by 1.8 times after
treatment with both drugs (Fig. 7.8; O. I. Epstein et al., 2003c; T. A. Voronina
et al., 2006).
Studying the possible side effects of ULD antiS100
Previous experiments showed that the anxiolytic activity of ULD anti
S100 is similar to that of a benzodiazepine anxiolytic diazepam. However, the
range of pharmacological properties of benzodiazepine drugs includes the
sedative and myorelaxant effects. Further investigations were conducted to study
these effects.
% of the control
250
!
!
200
150
100
50
0
Control
ULD antiS100
Amitriptyline
Fig. 7.8. Antidepressant activity of ULD antiS100 and amitriptyline (single
treatment) in the Nomura’s forced swimming test in a water tank with wheels.
*p<0.05 compared to the control.
117
Ultralow doses
The possible myorelaxant and sedative effects of ULD antiS100 were
evaluated in the rotarod test (T. A. Voronina et al., 2000; N. W. Dunham et al.,
1957) and openfield test (T. A. Voronina et al., 2000).
Studying the possible myorelaxant effect of ULD antiS100 in the rotarod
test. The rats were placed on a rotating rod (diameter 4 cm, rotation speed 10
rpm). The number of falling animals and latencytofall from the rod were
recorded for 2 min ((T. A. Voronina et al., 2006; N. W. Dunham et al., 1957).
Experiments were performed on male outbred rats weighing 250300 g.
The animals were divided into groups. The rats received 2.5 ml/kg distilled water
(control group), 2.5 ml/kg ULD antiS100, or equivalent volume of diazepam
in doses of 1, 2, and 4 mg/kg. Test substances were administered intragastrically
30 min before the study.
ULD antiS100 had no myorelaxant activity (Fig. 7.9; T. A. Voronina et
al., 2003). Diazepam produced a dosedependent myorelaxant effect. The per
centage of animals falling from a rotating rod after administration of diazepam
in doses of 2 and 4 mg/kg was 40 and 70%, respectively.
Studying the possible sedative effect of ULD antiS100 in the openfield
test. The sedative or stimulating effect of ULD antiS100 was evaluated in the
openfield test (increase or decrease in locomotor activity and exploratory be
havior; T. A. Voronina et al., 2000a).
Experiments were performed on adult outbred albino rats weighing 250
300 g. They were divided into the control group and two treatment groups. The
animals received 2.5 ml/kg distilled water, 2.5 ml/kg ULD antiS100, or 2 mg/kg
diazepam in an equivalent volume of the solvent (reference drug). Test sub
stances were administered intragastrically 30 min before the study.
ULD antiS100 had little effect on horizontal and vertical activity and
exploratory behavior of animals (exploration of holes). Administration of
a
%
120
b
%
80
100
!
60
80
!
!
40
60
40
!
20
20
0
0
Control
ULD
antiS100
Diazepam,
2 mg/kg
Diazepam,
4 mg/kg
Control
ULD
antiS100
Diazepam,
2 mg/kg
Diazepam,
4 mg/kg
Fig. 7.9. Effect of ULD antiS100 and diazepam on muscle tone of rats in the
rotarod test. Ordinate: percentage of animals (in group). Remaining animals (a) and
falling animals (b). *p<0.05 compared to the control.
118
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
diazepam in a dose of 2 mg/kg was followed by a significant decrease in horizontal
activity of rats, which reflects the sedative effect of this drug (Table 7.1).
These data indicate that ULD antiS100 do not cause a sedative effect (as
differentiated from diazepam; T. A. Voronina et al., 2003a).
Mechanisms for the effect of ULD antiS100
Role of the GABAergic system in the anxiolytic effect of ULD antiS100.
Anxiolytic activity was studied in the Vogel conflict test (T. A. Voronina et al.,
2000; J. R. Vogel et al., 1971). Experiments were performed on male outbred
rats weighing 250300 g. The animals received intragastrically distilled water (2.5
ml/kg), ULD antiS100 (2.5 ml/kg), or reference drug diazepam (2 mg/kg, 2.5
ml/kg).
GABAA receptor blockade was induced by intraperitoneal injection of a
receptor antagonist bicuculline in a dose of 1 mg/kg (ICN Biomedicals Inc.; R.
W. Olsen et al., 1984). Picrotoxin in a dose of 1 mg/kg (ICN Biomedicals Inc.;
M. G. Corda et al., 1984) was injected intraperitoneally to cause Cl channel
blockade in the GABAbenzodiazepine receptor complex.
The anticonflict effects of ULD antiS100 and diazepam were
significantly reduced under conditions of bicucullineinduced GABAA receptor
blockade and picrotoxininduced Cl channel blockade. Hence, these subunits
of the GABAbenzodiazepine receptorchloride ionophore complex are involved
in the anxiolytic effect of test substances (Fig. 7.10; T. A. Voronina et al.,
2003b).
Role of the GABA(B)ergic system in the anxiolytic and antidepressant
effects of ULD antiS100. The role of the GABA(B)ergic system in the
anxiolytic and antidepressant effects of ULD antiS100 was studied in the Vogel
conflict test and forced swimming test (water tank with freely rotating wheels),
respectively.
Table 7.1. Studying the sedative effect of ULD antiS100 in the openfield test (M±m)
Parameter
Control
ULD antiS100
(2.5 ml/kg)
Diazepam
(2 mg/kg)
Horizontal activity
18.2±2.4
15.8±2.1
Vertical activity
8.2±3.3
5.8±2.6
6.2±1.4
Exploratory activity
11.2±3.1
8.9±1.6
8.7±1.5
0
2.4±0.7*
1.8±0.9*
Number of entries into the center
of the open field
12.5±1.8*
Note. *p<0.05 compared to the control.
119
Ultralow doses
+
x
2
3
!
!
% of the
control
150
100
50
0
1
4
5
6
7
8
9
Fig. 7.10. Effect of GABAbenzodiazepine receptor antagonists on anxiolytic activity
of ULD antiS100 and diazepam. Ordinate: number of punished drinking episodes.
Control (1); ULD antiS100 (2); diazepam (2 mg/kg, 3); bicuculline (1 mg/kg, 4);
ULD antiS100 + bicuculline (5); diazepam + bicuculline (6); picrotoxin (1 mg/kg,
7); ULD antiS100 + picrotoxin (8); and diazepam + picrotoxin (9). p<0.05: *com
pared to the control; +compared to the “diazepam + bicuculline” and “diazepam +
picrotoxin” groups; xcompared to the “ULD antiS100 + bicuculline” and “ULD anti
S100 + picrotoxin” groups.
This study was performed with the following substances (equivalent
volume of distilled water as the control):
1) ULD antiS100 (2.5 ml/kg intragastrically, 40 min before the experi
ment);
2) diazepam (Polfa; 2 mg/kg intragastrically, 40 min before the ex
periment);
3) amitriptyline (Spofa; 10 mg/kg intragastrically, 40 min before the ex
periment);
4) selective GABAB receptor agonist baclofen (ICN; 1 mg/kg intraperi
toneally; L. Gilbo et al., 2000), 30 min before the experiment and 10
min before administration of ULD antiS100, diazepam, or amitrip
tyline; and
5) GABAB receptor antagonist faclofen (ICN; 10 mg/kg intraperito
neally; L. Gilbo, 2000), 30 min before the experiment and 10 min be
fore administration of ULD antiS100, diazepam, or amitriptyline.
The number of punished drinking episodes in rats of the ULD antiS100
group (507.33±120.59) was comparable to that in diazepamreceiving animals
(Table 7.2).
A GABAB receptor agonist baclofen (1 mg/kg) diminished the anxiolytic
effect of ULD antiS100. The number of punished drinking episodes decreased
to 231.67±79.66 (Table 7.2). By contrast, a GABAB receptor antagonist
faclofen (10 mg/kg) potentiated the anticonflict effect of ULD antiS100. It was
manifested in an increase in the number of punished drinking episodes.
120
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Table 7.2.
Incidence of punished drinking episodes (conflict situation) and number of
wheel revolutions (forced swimming test) in rats after administration of ULD
antiS100 and GABA(B)ergic substances (M±m)
Group
Control (distilled water)
Number of punished
drinking episodes
Number of wheel
revolutions
288.6±39.59
61.70±28.66
ULD antiS100 (2.5 ml/kg)
507.33±52.59*
116.1±23.5*
ULD antiS100 (2.5 ml/kg) and baclofen (1 ml/kg)
231.67±39.66+
78.20±12.51+
ULD antiS100 (2.5 ml/kg) and baclofen
(10 ml/kg)
705.00±81.47*+
66.60±21.64+
Diazepam (2 mg/kg)
623.33±115.65*
Not measured
Diazepam (2 mg/kg) and baclofen (1 ml/kg)
567.00±76.67*
Not measured
Amitriptyline (10 mg/kg)
Not measured
112.40±24.86*
Amitriptyline (10 mg/kg) and baclofen (1 ml/kg)
Not measured
122.10±18.88*
Note. p<0.05: *compared to the control; +compared to the ULD antiS100 group.
However, baclofen did not modulate the anxiolytic effect of diazepam (Table
7.2; T. A. Voronina et al., 2006b; T. A. Voronina et al., 2003).
Rats of the control group tried to escape from a water tank with wheels.
The average number of wheel revolutions was 61.70±28.66. These attempts
failed due to free rotation of wheels. Sometimes the activity was resumed, but
remained unsuccessful.
After administration of ULD antiS100, the number of wheel revolutions
was 116.1±30.5. The effect of ULD antiS100 was similar to that of a standard
antidepressant amitriptyline. Amitriptyline in a dose of 15 mg/kg increased the
number of wheel revolutions to 112.40±24.86 (Table 7.2).
GABA(B)ergic substances baclofen and faclofen were equally potent in
diminishing the effect of ULD antiS100 (decrease in the number of wheel
revolutions).
Our results indicate that administration of ULD antiS100 in combination
with GABA(B)ergic substances is followed by the interaction between these
compounds. A GABAB receptor agonist baclofen diminishes, while faclofen
potentiates the anticonflict effect of ULD antiS100.
Role of the serotoninergic system in the anxiolytic and antidepressant
effects of ULD antiS100. The serotoninergic system is involved in the genesis
of anxiety and depression. Serotonin uptake inhibitors are extensively used for
the therapy of depression. Much attention is paid to the search for new methods
to modulate the serotoninergic system, including serotonin receptors (D. N.
Middlemiss et al., 2002). 5HT1A receptor agonists (buspirone, gepirone,
ipsapirone, flesinoxan, etc.) and 5HT3 receptor antagonists (ondansetron,
tropisetron, bemesetron, granisetron, etc.) have the anxiolytic effect.
121
Ultralow doses
Under experimental conditions, ULD antiS100 exhibit the anxiolytic and
antidepressant activity. Hence, a 5HT2/5HT1C receptor antagonist ketanserin
was selected for a pharmacological study of the serotoninergic system. These
receptors have a role in the development of anxiety and depression. Ketanserin
is not approved as a pharmaceutical product in the Russian Federation. This
substance is used as an analyzer. Depression is accompanied by serotonin
deficiency. Therefore, a serotonin precursor 5hydroxytryptophan (5HTP) was
also used to analyze the effect of ULD antiS100.
These experiments were designed to evaluate a possible role of the
serotoninergic system in the anxiolytic and antidepressant effects of ULD anti
S100. The study was conducted with a selective 5HT2/5HT1C receptor
antagonist ketanserin and serotonin precursor 5HTP.
Experiments were performed on male outbred albino rats weighing 200
250 g. Anxiolytic activity of ULD antiS100 was studied in the Vogel conflict
test. The antidepressant effect of test substances was analyzed in the Nomura’s
forced swimming test (water tank with wheels).
The following substances were analyzed:
1) ULD antiS100 (single intragastric dose 2.5 ml/kg, 30 min before the
experiment; distilled water as the control);
2) diazepam (Polfa; single intragastric dose 2 mg/kg, 30 min before the
experiment);
3) ketanserin (ICN; 1 mg/kg intraperitoneally, 40 min before the experi
ment and 10 min before administration of ULD antiS100); and
4) 5HTP (ICN; 50 mg/kg intraperitoneally, 30 min before the experi
ment and 10 min before administration of ULD antiS100).
In a conflict situation, the number of punished drinking episodes for
control animals was 102.20±25.06. ULD antiS100 had a strong anxiolytic
effect, which was manifested in an increase in the number of punished drinking
episodes to 324.09±40.61. The effect of ULD antiS100 was comparable to that
of diazepam in a dose of 2 mg/kg (Table 7.3).
Ketanserin exhibited anxiolytic activity and increased the number of
punished drinking episodes to 224.5±31.7. A serotonin precursor 5HTP also
increased the number of punished drinking episodes (statistically insignificant).
The anticonflict effect of ULD antiS100 became less pronounced after
treatment in combination with ketanserin or 5HTP (Table 7.3).
In the forced swimming test, control rats tried to escape with freely
rotating wheels. However, their attempts failed. Sometimes the activity was
resumed. The number of wheel revolutions for control animals was
73.00±25.19.
After administration of ULD antiS100, the number of wheel revolu
tions was 159.50±29.77. These data reflect the antidepressant effect of ULD
122
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
antiS100. Antidepressant activity of ULD antiS100 compared well with that
of a standard antidepressant amitriptyline (119.13±19.16 wheel revolutions,
Table 7.3).
Individual administration of ketanserin or 5HTP was followed by an
increase in the number of wheel revolutions, which reflects the antidepressant
effect of drugs. However, the antidepressant effect of study drugs was reduced
after administration in combination with ULD antiS100. A decrease in the
antidepressant effect was most significant after combined treatment with
ketanserin and ULD antiS100 (Table 7.3).
Our results illustrate the interaction between ULD antiS100 and
serotoninergic substances. A selective 5HT2/5HT1C receptor antagonist
ketanserin had the anticonflict and antidepressant properties. The anxiolytic and
antidepressant effects became less pronounced after combined administration of
ULD antiS100 and ketanserin.
A serotonin precursor 5HTP induces a significant increase in brain
serotonin content. 5HTP had a strong antidepressant effect and mild anxiolytic
effect. Combined administration of ULD antiS100 and 5HTP was also
accompanied by a decrease in the anxiolytic and antidepressant properties of
ULD antiS100 (statistically insignificant).
We conclude that the anxiolytic and antidepressant effects of ULD anti
S100 are modified after administration of this product in combination with
ketanserin and 5HTP. It may be suggested that the serotoninergic system has
a role in the realization of these effects (T. A. Voronina et al., 2006a; J. L. Du
gina et al., 2005c).
Table 7.3.
Results of the conflict situation test and forced swimming test in rats after
administration of ULD antiS100 and serotoninergic substances (M±m)
Number of punished
drinking episodes
(conflict situation)
Number of wheel
revolutions (forced
swimming test)
Control
102.20±15.06
73.00±25.19
ULD antiS100 (2.5 ml/kg)
324.90±40.61*
159.50±29.77*
Diazepam (2 mg/kg)
372.60±45.18*
Not measured
5HTP (50 mg/kg)
135.00±33.15
146.60±30.16*
Ketanserin (1 mg/kg)
224.5±31.7*
117.10±24.87*
Group
ULD antiS100 (2.5 ml/kg) and
5HTP (50 mg/kg)
ULD antiS100 (2.5 ml/kg) and
ketanserin (1 mg/kg)
Amitriptyline (15 mg/kg)
259.2±48.4*
102.90±44.36
168.70±40.12+
80.50±18.14+
Not measured
119.10± 19.16*
Note. p<0.05: *compared to the control; +compared to ULD antiS100.
123
Ultralow doses
Effect of ULD antiS100 on early gene cfos expression in the hypotha
lamic paraventricular nucleus. An increased expression of the early gene cfos in
the hypothalamic paraventricular nucleus serves as a criterion for the stress
response of an organism (L. W. Swanson et al., 1980; T. R. Tolle et al., 1995;
Z. Tan et al., 2002).
Experiments were performed on male Wistar rats weighing 250280 g. The
animals were divided into groups of highactivity and lowactivity specimens in
the openfield test (E. V. Koplik et al., 1995; E. V. Koplik et al., 2001). The rats
received intragastrically distilled water (2.5 ml/kg), ULD antiS100 (2.5 ml/kg),
or imipramine (12 mg/kg). Test substances were administered once or several
times (20day course). Each group consisted of active (n=5) and passive rats
(n=5). Thirty minutes after acute treatment or last administration of substances,
the animals were exposed to 1h immobilization and electrocutaneous stimu
lation (46 V, frequency 50 Hz, pulse duration 1 msec, 1618 stimulations per
1 h). The rats were killed 90 min after stress. The brain was removed and frozen
in liquid nitrogen. The slices were prepared and stained immunohistochemically
with antibodies to cFos protein. The number of fospositive cells in the
hypothalamic paraventricular nucleus and intensity of cfos gene expression were
estimated in five intact (nonstressed) active and passive rats.
Stress was followed by a significant increase in cfos gene expression in
active and passive animals (by 2025 times). These changes were particularly
pronounced in passive specimens.
The course of treatment with imipramine was accompanied by a decrease
in the number of immunoreactive cells in active and passive animals (by 1.2 and
1.5 times, respectively). The observed changes were statistically significant in
passive specimens (Fig. 7.11).
Administration of ULD antiS100 for 20 days was followed by a 1.3fold
decrease in the number of fospositive cells in passive animals (p<0.0, Fig. 7.11).
Imipramine and ULD antiS100 were equally effective in this respect.
Effect of ULD antiS100 on longterm posttetanic potentiation in surviving
hippocampal slices. The model of longterm posttetanic potentiation (LTPTP)
in surviving hippocampal slices is extensively used to study the molecular
mechanisms of synaptic plasticity and effect of substances on synaptic
transmission. The induction of LTPTP is a Ca2+dependent process. This
process involves not only Ca2+regulatory proteins, but also Ca2+binding
protein S100. The induction of LTPTP in hippocampal slices is accompanied
by an increase in the content of membranebound protein. Application of
antiserum to S100 protein inhibits the induction of LTPTP in hippocampal
slices (T. Lewis et al., 1986).
Experiments were performed on hippocampal slices from adult Wistar rats
weighing 180200 g. Transverse sections (400 m in width) were placed in a
124
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
a
25
b
25
20
20
15
15
10
10
5
5
0
+
!
!
0
Intact
Control
Imipramine
ULD antiS100
active
Intact
Control
Imipramine
ULD antiS100
passive
Fig. 7.11. Effect of single administration (a) and 20day course of treatment with
ULD antiS100 and imipramine (b) on cfos gene expression in rats after
immobilization stress and electrocutaneous stimulation. Ordinate: number of Fos
positive cells. p<0.05: *compared to the control; +compared to imipraminereceiving
animals.
temperaturecontrolled chamber at 3537oC. Flow Yamamoto medium was
aerated by carbogen (95% O2 and 5% CO2). Evoked potentials were recorded
after 4060min incubation. A stimulatory electrolytically sharpened bipolar
wolfram electrode was introduced into the zone of mossy fibers. A reference
glass electrode (tip thickness 35 m, resistance 25 mO) was filled with 2.5 M
NaCl and placed in CA3 region (initial segments of apical dendrites).
Testing was performed with single rectangular pulses (duration 200 msec)
delivered at an interval of not less than 57 min.
The amplitude of test stimuli usually varies from 10 to 30 V. Evoked
potentials were recorded on a 12digit analogtodigital converter (Digidata,
Axon Instruments Inc.). The results were analyzed with pClamp6 (Axon
Instru-ments Inc.) and Microcal Origin softwares.
To induce LTPTP, the amplitude of test stimulus was selected to
produce a halfmaximal response. Tetanization was induced by three conse
cutive series of stimulation at 200 Hz. The length of each series was 1 sec. Sti
mulation was applied at 2sec intervals. The procedure of tetanization was re
peated after 10 min.
The internal potentials were recorded for at least 40 min after the first
tetanization, which reflected the induction or absence of LTPTP. A significant
increase in the amplitude of EPSP (by 1.52 times), which persisted for 20 min
after the second tetanization, served as a criterion for the induction of
potentiation.
After study of each dilution, the chamber was repeatedly washed with
distilled water and ethyl alcohol and completely dried with compressed air.
Experiments were performed with monospecific rabbit antiserum to
neurospecific protein S100 and ULD antiS100. Nonimmune rabbit antiserum
served as the control.
125
Ultralow doses
The induction of LTPTP was characterized by a significant increase in the
amplitude of evoked potentials in response to the test stimulus after tetanization.
Twentyminute incubation with antibodies to S100 protein (final dilution 1:50)
completely inhibited the induction of LTPTP, which is consistent with published
data (T. Lewis et al., 1986). Nonimmune rabbit antiserum at the same dilution
had no effect on LTPTP induction in rat hippocampal slices.
The average amplitude of EPSP after 20min incubation in Yamamoto
medium with antiserum to S100 (concentration 1012) at the interstimulus
intervals of 10 and 57 min was 0.91.1 and 0.80.9 mV. Over 10 min after first
tetanization at 23min intervals, this parameter was 1.1 mV.
The effect of antiS100 was completely abolished after preincubation of
slices with ULD antiS100 (concentration 1012, 20 min) and subsequent
incubation in a solution of native and potentiated antisera (20 min). Hence, the
induction of LTPTP in slices was similar to that in control samples (no
treatment with antibodies). Over 40 min after the second tetanization at 35
min intervals, the average amplitude of EPSP was 1.72.0 mV.
These data show that preincubation with ULD antiS100 modifies the in
hibitory effect of antiserum to S100 protein. The inhibition of LTPTP is not
observed under these conditions. This phenomenon is probably related to a di
rect modulatory effect of ULD antiS100 on the corresponding endogenous li
gand (O. I. Epstein et al., 1999a; M. B. Shtark, 2001).
Effect of ULD antiS100 on electrical properties of neuronal membranes.
Antibodies to S100 protein cause reversible changes in the passive and active
properties of neuronal membranes from snail subesophageal ganglia (Kh. L.
Gainutdinov et al., 1999).
Moreover, ULD antiS100 have a strong effect on the membrane of giant
neurons from H. pomatia (O. I. Epstein et al., 1996b).
The snails were in an active state for at least 2 weeks before study.
Experiments were performed on the identified spontaneously active neurons of
subesophageal ganglia V2V6, PPa1, and PPa2. The resting potential, action
potential amplitude, time derivative of the action potential, maximum rate of
rise of the action potential (Vmax), spike discharge frequency, and current
voltage and inactivation characteristics of ion channels for inward and outward
current were measured on a Hitachi device. In some experiments with isolated
neurons, calcium currents were recorded by the voltage clamp technique. The
study was performed with glass microelectrodes (internal resistance 720 mΩ).
The neuronal membranes were polarized with depolarizing and hyperpolarizing
pulses through a reference electrode (up to 30 V).
The dilution of antibodies to S100 protein was 0.2, 2.6, and 12%. The
concentration of ULD antiS100 was 1012 and 10400 wt %. Nonimmune and
immune sera of sheep erythrocytes served as the control.
126
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Under control conditions, the substitution of physiological saline for
nonimmune serum or antiserum to sheep erythrocytes had little effect on
electrical properties of the membrane.
Vmax decreased sharply 20 min after application of antibodies to S100
protein at dilutions of 0.2 and 2% (by 2228 and 3745%, respectively). Vmax
decreased by 6080% over the first 1015 min after treatment with antibodies
to S100 protein at dilutions of 6 and 12%. Similar results were obtained in
experiments with ULD antiS100. Vmax decreased by 148% over 35 min.
Treatment with various dilutions of antibodies to S100 protein and poten
tiated form of these antibodies was followed by a decrease in the strength of inward
current and increase in the steadystate inactivation at zero conditioning pulse.
Currentvoltage characteristics and inactivation curves were shifted toward negative
values of the membrane potential. The antibodyinduced reduction of inward
current was mainly associated with a decrease in the maximum conductance of
inward current channels, but not with an increase in the steadystate inactivation.
These data show that ULD antiS100 have a strong effect on membrane
electrical properties of the isolated neuron. The effect of ULD antiS100 is
qualitatively similar to that of native antiserum in concentrations of 2, 6, and
12%. This is manifested in membrane depolarization, decrease in action
potential amplitude, increase in the maximum rate of rise of the action
potential, decrease in the maximum conductance, and inactivation of channels
(O. I. Epstein et al., 1999b).
Other pharmacological effects of ULD antiS100
Antiaggressive activity of ULD antiS100. Antiaggressive activity of ULD
antiS100 was studied under conditions of motivated (Yu. V. Burov et al., 1976)
and unmotivated aggression (T. A. Voronina et al., 2000).
Antiaggressive activity of ULD antiS100 in the test of unmotivated
aggression. The test of unmotivated aggression is based on studying the threshold
of aggressive response for two rats on an electrode floor at increasing the
strength of stimulating current (T. A. Voronina et al., 2000). Pairs of rats were
placed on the electrode floor of a Plexiglas chamber (27.5×27.5×40 cm).
Alternating voltage of increasing amplitude (beginning from 15 V) was applied
to the chamber floor using a special stimulator. The duration of stimulation was
3 sec. The interstimulus interval was 1 sec. If three stimulations of similar
intensity did not induce the aggressive response, voltage was increased by 1 V.
Stimulation was continued until the aggressive response to at least three pulses
of similar strength. This voltage was considered as a threshold. During the
aggressive response, both rats stand up on their hindlimbs “face to face” and
paw or bite each other.
127
Ultralow doses
Experiments were performed on 120 male outbred albino rats weighing
250300 g. Distilled water (2.5 ml/kg) was given to control animals. The
remaining rats received 2.5 ml/kg ULD antiS100 (treatment group 1) or
equivalent volume of 5 mg/kg diazepam (treatment group 2). Test substances
were administered intragastrically 40 min before the study.
Single administration and, particularly, repeated treatment with ULD
antiS100 and diazepam caused an increase in the aggressive threshold (Fig.
7.12). The activity of ULD antiS100 compared well with that of diazepam (S.
A. Sergeeva et al., 2004).
Antiaggressive activity of ULD antiS100 in the test of motivated aggression.
A study of motivated aggression is based on the analysis of aggressive behavior
in two rats that try to avoid punishment on a crowded safe bench (Yu. V. Burov
et al., 1976).
Experiments were performed on 120 male outbred rats weighing 250300 g.
Study was conducted in two stages. A conditioned response to avoid nociceptive
stimulation of the limbs was elicited on day 1. A safe bench was put in the
center of the chamber. The animals were placed in this chamber. Pulses of
alternating voltage (35 V, duration 3 sec, interpulse interval 1 sec) were applied
to the electrode floor after 5min adaptation. After successful avoidance, the rat
was permitted to remain on the bench for 30 sec. Then the rat was placed in
the home cage. The same procedure was repeated after 2 min. The response was
considered to be elicited when avoidance LC did not exceed 9 sec (100% trials).
On the next day, pairs of rats were placed in the chamber. The behavior
of animals was studied for 2 min. Control animals fought for a safe bench,
although it was sufficient for two specimens. Antiaggressive substances allowed
the rats to avoid nociceptive stimulation by getting to the bench. The efficacy
of test substances was estimated from the time of avoidance for both rats.
Threshold of aggression,
% of the control
160
!
!
!
!
120
80
40
0
Control
ULD antiS100
Diazepam
Fig. 7.12. Antiaggressive effect of ULD antiS100 and diazepam in the test of
unmotivated aggression. Single administration (light bars) and course of treatment
(dark bars). *p<0.05 compared to the control.
128
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
The animals were divided into three groups. They received distilled water
(2.5 ml/kg, control group 1), ULD antiS100 (2.5 ml/kg, group 2), or diazepam
(5 mg/kg in an equivalent volume, group 3). Test substances were administered
intragastrically 40 min before the study.
Single administration and course of treatment with ULD antiS100 and
diazepam had a strong antiaggressive effect (Fig. 7.13). These drugs caused an
increase in the time of avoidance by 3.8 and 3.3 times, respectively (S. A.
Sergeeva et al., 2004).
Antihypoxic activity of ULD antiS100. Antihypoxic activity of ULD anti
S100 was studied under conditions of acute hypobaric hypoxia. Acute hypobaric
hypoxia in mice was produced in a flowexhaust attitude chamber (M. V.
Korablev et al., 1976). Pressure was measured with an altimeter. The rate of
climb was measured with a variometer. The animals were “elevated” to a height
of 11,000 m (20 m/sec). The time of exposure was 10 min. The mice were
returned to a normal environment over the next 5 min. Alkali (3035%) was put
into the chamber to prevent hypercapnia. The lifespan and survival rate of
animals were calculated relative to the control (100%). The control and treated
mice were simultaneously placed in the chamber to study hypoxia under the
same conditions.
Experiments were performed on male outbred albino mice weighing 18
24 g. ULD antiS100 were administered on days 1 (2.5 ml/kg twice a day at
10.00 and 17.00) and 2 of study (2.5 ml/kg at 10.00; and 5 ml/kg at 15.00). Treat
ment was conducted 30 min before the study. An equivalent volume of mexidol
(100 mg/kg) was given similarly. Control animals received physiological saline.
Repeated treatment with ULD antiS100 was followed by an increase in
the lifespan of mice during hypobaric hypoxia (Fig. 7.14). Antihypoxic activity
of ULD antiS100 was highly competitive with that of mexidol.
Time of collective avoidance,
% of the control
500
!
!
400
!
!
300
200
100
0
Control
ULD antiS100
Diazepam
Fig. 7.13. Antiaggressive effect of ULD antiS100 and diazepam in the test of
motivated aggression. Single administration (light bars) and course of treatment
(dark bars). *p<0.05 compared to the control.
129
Ultralow doses
% of the control
300
!
250
!
200
150
100
50
0
Control
Mexidol
ULD antiS100
Fig. 7.14. Effect on ULD antiS100 on the lifespan of mice after acute hypobaric
hypoxia. *p<0.05 compared to the control.
Neuroprotective activity of ULD antiS100. Neuroprotective activity of ULD
antiS100 on the model of ischemic stroke. The antiischemic and antiamnesic
properties of ULD antiS100 were studied on the model of irreversible focal
ischemia due to bilateral focal photothrombosis of the prefrontal cortex in rats
(G. A. Romanova et al., 1998; B. D. Watson et al., 1985, 1998). Bilateral focal
ischemia of the prefrontal cortex in rats was induced by photochemical
thrombosis. This method was developed by B. D. Watson et al. and modified by
I. V. Viktorov. The surgery to induce photothrombosis was performed in animals
under chloral hydrate anesthesia (300 mg/kg intraperitoneally). The head of
shamoperated and treated rats was fixed using a stereotaxic device.
Amnesia is a prominent symptom of integrative dysfunction in CNS
during photothrombosis of the prefrontal cortex in rats. Integrative dysfunction
of the brain was estimated from the impairment of acquisition and performance
of a conditioned passive avoidance response (CPAR; T. A. Voronona et al.,
2000).
CPAR was elicited by the standard method. LC of transition from the
dark compartment to the light compartment was measured. On day 1 of
training, the rat was placed in the light compartment. The animal explored this
area and moved to the dark compartment after several seconds. The door of this
compartment was closed, and the rat remained in darkness for 300 sec. The
procedure was repeated after 1 h. In this trial, the rat was immediately removed
from the dark compartment. On the next day, this procedure was repeated two
times at a 1h interval. When the rat entered the dark compartment, the door
was closed. Electric current was delivered through a metal grid floor. CPAR was
considered to be elicited at LC of 300 sec (preoperative period). The animals
with a shorter period of LC were excluded from further observations.
Before the start of CPAR training, locomotor activity of rats was studied
in the automated openfield test. The number of crossed squares was measured
for 300 sec.
130
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
A standard nootropic agent piracetam (200 mg/kg) and cavinton (4 mg/kg)
were used as the reference drugs.
The rats were divided into six groups. Group 1 animals (control, n=20)
were trained in CPAR and received 0.9% NaCl. Group 2 specimens (n=20)
were shamoperated and treated with ULD antiS100. The rats of groups 36
were subjected to photothrombosis and received 0.9% NaCl (group 3, n=12),
ULD antiS100 (group 4, n=12), piracetam (group 5, n=12), and cavinton
(group 6, n=7).
In groups 36, test substances (2.5 ml/kg) were administered orally 1 h
after photothrombosis and then daily for 9 days. In the last day, these drugs were
given 40 min before testing. Control rats and shamoperated animals received
physiological saline or ULDantiS100 according to the same regimen.
Locomotor activity of all rats was studied in an automated open field on
day 9 after photothrombosis of the prefrontal cortex. A pathomorphological
study was performed immediately after CPAR testing.
The natural extinction of CPAR in intact animals was observed 9 days
after acquisition. LC decreased by 23% (G. A. Romanova et al., 2003). Irrever
sible focal ischemia of the prefrontal cortex caused amnesia, which was mani
fested in the impairment of response performance and 1.8fold decrease in the
latency of CPAR.
ULD antiS100 prevented the natural extinction of CPAR in shamope
rated animals. Amnesia due to ischemic stroke was not observed after admini
stration of ULD antiS100. LC in treated rats did not differ from that in intact
animals. Piracetam and cavinton had little antiamnesic effect (Fig. 7.15).
Administration of ULD antiS100 was followed by a significant decrease
in the area of ischemic injury (by 40%), which illustrates the neuroprotective
(antiischemic) effect of this product (Fig. 7.16).
CPAR LC, % of the control
120
!
100
80
60
40
20
0
1
2
3
4
5
6
7
Fig. 7.15. Effect of ULD antiS100 on CPAR performance in rats on day 9 after
ischemic stroke. Intact animals (baseline, 1); control (2); sham operation + ULD
antiS100 (3); photothrombosis (4); photothrombosis + ULD antiS100 (5);
photothrombosis + piracetam (6); and photothrombosis + cavinton (7). *p<0.05
compared to untreated rats with stroke.
131
Ultralow doses
a
b
Fig. 7.16. Effect of ULD antiS100 on focal ischemic injury in the prefrontal cortex
due to photothrombosis: control (a) and treated rats (b).
Neuroprotective activity of ULD antiS100 in the model of hemorrhagic
stroke. Hemorrhagic stroke was induced in adult male outbred rats weighing
200250 g (A. N. Makarenko et al., 1990; A. Jackowski et al., 1990). Soft tissues
and periosteum in the parietal and central regions of the cranium were removed
under nembutal anesthesia (40 mg/kg intramuscularly). A hole (diameter 1 mm)
was made in the left hemisphere of the cranial bone (1.51.88 mm posterior to
the bregma, 2.53.0 mm lateral to the sagittal suture). A special device (mandrel
knife) was inserted into the hole at a depth of 4 mm. The brain tissue (internal
capsule) was destructed in a stereotaxic device. The blood (0.020.03 ml) was
taken from the sublingual region and introduced into the area of injury after 23
min. A morphological study showed that this treatment causes bilateral focal
stroke in the internal capsule (diameter 2 mm, depth 3 mm). The upper
structures of the brain and neocortex remain intact under these conditions.
The dynamics of intracerebral hemorrhagic trauma was monitored for 14
days. The state and behavior of animals were examined on days 1, 3, 7, and 14
(T. A. Voronina et al., 2006b,c).
ULD antiS100 (2.5 ml/kg) or nimodipine (0.1 mg/kg) was administered in
tragastrically using a special probe. The probe was equipped with a thickened olive.
Test substances were administered once a day. The first treatment was performed
4 h after surgery. The animals were tested on days 1, 3, 7, and 14 after surgery.
Shamoperated animals of the control group were subjected to scalping
in the parietal and central regions of the cranium and skull drilling with no
damage to brain structures.
132
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
We studied the effect of substances on the survival rate of animals,
neurological deficit (I. V. Gannushkina, 1996) and cognitive dysfunction (T. A.
Voronina et al., 2000) due to hemorrhagic stroke, emotional state of rats (T. A.
Voronina et al., 2000), and change in muscle tone (T. A. Voronina et al., 2000;
N. W. Dunham et al., 1957).
Blood infusion into the internal capsule (model of hemorrhagic stroke)
caused death of some animals (50% mortality on day 14). This treatment was
also followed by neurological deficit (60% survived animals), reduction of
muscle tone (50% rats), and amnesia (2fold decrease in CPAR LC; Figs. 7.17
7.21). High anxiety of animals was manifested in a decrease in the time spent
in the open arms (A. N. Makarenko et al., 2004; S. A. Sergeeva et al., 2005).
ULD antiS100 increased the survival rate of rats to 100%, significantly
decreased the percentage of animals with neurological deficit and reduced
muscle tone, and produced an antiamnesic effect in the CPAR test (no
differences between treated and intact animals). Administration of ULD anti
S100 also had an anxiolytic effect on day 1 after stroke. It was manifested in
an increase in the time spent in the open arms (by 20 times) and elevation of
locomotor activity (by 2 times).
A reference drug nimodipine had little effect on the survival rate of rats.
The action of nimodipine on neurological deficit and muscle tone was similar
to that of ULD antiS100. Nimodipine was less potent than ULD antiS100 in
producing an antiamnesic effect in the CPAR test. The anxiolytic effect of
nimodipine was less pronounced that that of ULD antiS100.
Nootropic activity of ULD antiS100. Nootropic activity of ULD anti
S100 was evaluated from CPAR performance by intact animals and specimens
% of the control
110
2
100
90
80
3
70
!
4
60
1
50
40
1
3
7
Time, days
14
Fig. 7.17. Effect of ULD antiS100 on the survival rate of rats with hemorrhagic
stroke. Control animals (1); sham operation (2); ULD antiS100 (3); and nimodipine
(4). *p<0.05 compared to the control.
133
Ultralow doses
%
120
100
80
60
!
!
1
!
!
3
4
2
40
20
0
1
3
7
Time, days
14
Fig. 7.18. Effect of ULD antiS100 on neurological deficit in rats with hemorrhagic
stroke. Ordinate: animals with neurological deficit. Control animals (1); sham
operation (2); ULD antiS100 (3); and nimodipine (4). *p<0.05 compared to the
control.
%
80
70
4
60
1
50
40
!
30
20
!
3
!
2
!
10
0
1
3
7
Time, days
14
Fig. 7.19. Effect of ULD antiS100 on muscle tone in rats with hemorrhagic stroke.
Ordinate: animals with reduced muscle tone. Control animals (1); sham operation
(2); ULD antiS100 (3); and nimodipine (4). *p<0.05 compared to the control.
with scopolamineinduced amnesia or Alzheimer’s disease (Yu. V. Burov et al.,
1991; T. A. Voronina et al., 2006b).
Effect of ULD antiS100 on CPAR performance. The test of CPAR in
animals is based on passive avoidance of aversive events with no active
movements (T. A. Voronina et al., 2006b). CPAR was studied in a special device
(Lafaette Instruments Co.). This device consisted of the illuminated platform,
which was placed at a height and connected with the dark compartment.
Nociceptive stimulation was applied to the floor of this compartment. A hole
between the platform and compartment was closed by a movable partition wall.
After appearing on the illuminated platform, all rats were characterized by
instinctive behavior to enter the dark compartment. The animals explored this
134
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
sec
180
160
140
120
100
80
60
40
20
0
!
!
1
4
5
3
2
1
7
Time, days
14
Fig. 7.20. Effect of ULD antiS100 on CPAR performance in rats with hemorrhagic
stroke (T. A. Voronina et al., 2006b). Intact animals (1); control (hemorrhagic stroke
and distilled water, 2); sham operation (3); ULD antiS100 (4); and nimodipine (5).
*p<0.05 compared to the control.
% of intact animals
250
a !
b
200
150
100
!
!
50
0
!
+
+
1 2 3 4 5
1 2 3 4 5
Fig. 7.21. Anxiolytic effect of ULD antiS100 on rats with hemorrhagic stroke.
Anxiety in the EPM test. Time spent in the open arms (sec, a) and number of entries
into the arms (b). Intact animals (1); sham operation (2); control (stroke, 3); stroke
+ ULD antiS100 (4); and stroke + nimodipine (5). p<0.05: *compared to animals
of the stroke group; +compared to intact and shamoperated animals.
compartment for 3 min, but sometimes visited the open area. In the followup
period, a partition wall between the platform and dark compartment was closed.
Electric current was applied to the floor of this compartment for 10 sec. Hence,
the dark compartment became dangerous. CPAR acquisition was tested on the
next day. The rats were placed on the illuminated platform. LC of the first entry
to the dark compartment, as well as the total time spent in this compartment
was recorded for 3 min.
ULD antiS100 (2.5 ml/kg) or piracetam (400 mg/kg) was administered
immediately before training (1 h before study), immediately after training, and
24 h after training (1 h before performance). Diazepam in a single dose of 5
135
Ultralow doses
mg/kg was given 40 min before training. Control animals did not receive any
drug.
As differentiated from ULD antiS100 and piracetam, diazepam had an
adverse effect on the acquisition and performance of CPAR (Fig. 7.22). These
data indicate that ULD antiS100 do not cause one of the side effects, which
is typical of a standard anxiolytic agent diazepam (impairment of cognitive
function; T. A. Voronina et al., 2006c).
Antiamnesic activity of ULD antiS100 under conditions of scopolamine
induced amnesia. Antiamnesic activity of ULD antiS100 was estimated from
CPAR performance by animals with scopolamineinduced amnesia (T. A.
Voronina et al., 2006b; R. Ader et al., 1972). A muscarinic receptor antagonist
scopolamine (single dose 2 mg/kg) was injected intraperitoneally 15 min before
CPAR training to induce amnesia in rats.
ULD antiS100 in a dose of 2.5 ml/kg were administered intragastrically
before training (1 h before study), immediately after training, or 24 h after
training (1 h before performance).
The reference drug piracetam (Nootropil) in a dose of 400 mg/kg was
administered intragastrically (similarly to ULD antiS100). Control animals
received an equivalent volume of physiological saline.
ULD antiS100 reduced the symptoms of scopolamineinduced amnesia
(increase in learning ability for CPAR; Fig. 7.23). Nootropic activity of ULD
antiS100 compared well with that of piracetam (T. A. Voronina et al., 2006c).
Efficacy of ULD antiS100 in experimental Alzheimer’s disease.
Experimental Alzheimer’s disease in rats was induced by the method of Yu. V.
Burov et al. (1991) and T. A. Voronina et al. (2006b). This method is based on
the induction of cholinergic deficit, which occurs during aging and serves as a
major pathogenetic mechanism of Alzheimer’s disease. Experiments were
performed on male outbred albino rats.
% of trained animals
120
100
80
60
!
40
20
0
1
2
3
4
Fig. 7.22. Effect of single treatment with ULD antiS100 on CPAR performance in
rats. Control (1); ULD antiS100 (2); piracetam (3); and diazepam (4). Control, 100%
(posttraining level). *p<0.05 compared to the control.
136
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
% of trained animals
120
100
80
!
!
60
40
20
0
1
2
3
4
Fig. 7.23. CPAR performance in rats with scopolamineinduced amnesia after single
administration of ULD antiS100. Control (1); scopolamine (2); ULD antiS100 + sco
polamine (3); and piracetam + scopolamine (4). *p<0.05 compared to the control.
The rats were randomized into three groups of control (group 1) and trea
ted specimens (groups 2 and 3). A muscarinic receptor antagonist scopolamine
in a daily dose of 1 mg/kg was injected intraperitoneally for 20 days to induce
cholinergic deficit in treated rats. Control animals received distilled water.
On days 2130, the rats of groups 1 (passive control) and 2 (active control)
received orally distilled water. ULD antiS100 were given to group 3 rats (2.5 ml/
kg twice a day at 10.00 and 16.00). Cognitive functions were estimated from the
dynamics of CAAR acquisition after scopolamine administration (number of
conditioned responses, percent of the total number of presentations; and ratio of
rats reaching the learning criterion). Neurological status was assessed by the Mc
Grow scale. Muscle tone was measured in the rotarod test. Movement coordination
was studied in the hanging wire test. Anxiety was determined in the EPM test.
Chronic administration of scopolamine was followed by delayed learning
of CAAR, weakness, “floppy” movements, unilateral partial ptosis or ptosis
(50% rats), and hypotonia (impaired coordination of movements; Figs. 7.24
Performance of conditioned
responses, %
80
1
60
3
40
!
!
!
2
20
0
1
2
3
4
5
Training, days
6
7
Fig. 7.24. Effect of ULD antiS100 on the dynamics of CAAR performance during
experimental Alzheimer’s disease. Control (1); scopolamine (2); and ULD antiS100
+ scopolamine (3). *p<0.05 compared to the control.
137
Ultralow doses
7.26). Anxiety of animals was manifested in a decrease in the time spent in the
open arms of EPM. The rats were also characterized by a decrease in locomotor
activity (T. A. Voronina et al., 2004).
The course of treatment with ULD antiS100 and piracetam resulted in
the improvement of learning (similarly to intact animals) and decrease in the
percentage of animals with hypotonia (1.52fold decrease in muscle tone) and
partial ptosis or ptosis.
As differentiated from piracetam, ULD antiS100 had an anxiolytic effect
(5fold increase in the time spent in the open arms of EPM). Moreover,
locomotor activity of animals increased after administration of ULD antiS100.
ULD antiS100 improved the CAAR learning ability of rats with cognitive
dysfunction due to subchronic blockade of muscarinic receptors and further
depletion of the cholinergic system. The efficacy of ULD antiS100 was similar
to that of piracetam.
%
!
60
45
30
15
0
1
2
3
Fig. 7.25. Effect of ULD antiS100 on amnesia during experimental Alzheimer’s
disease: percentage of animals with the elicited response (5th day of training).
Control (1); scopolamine (2); and ULD antiS100 + scopolamine (3). *p<0.05
compared to the control.
а
%
30
б
25
20
!
15
!
10
5
0
1
2
3
1
2
3
Fig. 7.26. Effect of ULD antiS100 on amnesia during experimental Alzheimer’s
disease: no adequate response. Days 1 (a) and 2 (b). Control (1); scopolamine (2);
and ULD antiS100 + scopolamine (3). *p<0.05 compared to the control.
138
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Effects of combined treatment
with ULD antiS100 and haloperidol
The modulatory effect of ULD antiS100 on activity of haloperidol was
studied in the test of apomorphine verticalization (K. S. Raevskii et al., 2000)
and under conditions of catalepsy induced by high dose of haloperidol.
Combined administration of ULD antiS100 and haloperidol. Influence of
ULD antiS100 on the effect of haloperidol in the test of apomorphine
verticalization. Experiments were performed on male outbred albino mice
weighing 2532 g. The animals were randomly divided into four groups of 12
specimens each. Apomorphine in a dose of 2.5 mg/kg was injected
subcutaneously to group 1 mice (control). Group 2 mice were treated with
apomorphine 10 min after intraperitoneal injection of haloperidol in a dose of
0.1 mg/kg (Gedeon Richter). Group 3 mice were subjected to successive
treatment with ULD antiS100 (2.5 ml/kg intragastrically), haloperidol (5 min
after ULD antiS100 administration), and apomorphine (10 min after
haloperidol injection). Group 4 mice received ULD antiS100 (2.5 ml/kg
intragastrically for 7 days), haloperidol (5 min after the last treatment with ULD
antiS100), and apomorphine (10 min after haloperidol injection).
Immediately after apomorphine injection, the animals were placed in
cylindrical wire chambers (diameter 13 cm, height 16 cm). Vertical activity
was measured 10 min after injection of apomorphine. The measurements were
performed for 1 h at 2min intervals. The degree of verticalization was
determined by a 4point scale (number of limbs to hold on to a vertical wall;
M. Vasse et al., 1985). The following parameters were calculated: total score
of verticalization for each animal over the whole period of study; average
degree of verticalization for each group; and vertical activity (relative to the
control).
Apomorphine injection was followed by a strong stimulatory effect on
vertical activity of mice. The average score of vertical activity was 63.7±15.9
points over 60 min. A typical neuroleptic haloperidol completely abolished the
stimulatory effect of apomorphine on vertical activity of animals (Fig. 7.27).
Single administration of ULD antiS100 did not modulate the influence of
haloperidol on apomorphine verticalization.
These data indicate that single and repeated administration of ULD anti
S100 has little effect on the specific psychotropic action of haloperidol in the
therapeutic dose on apomorphine verticalization (T. A. Voronina, 2005).
Influence of ULD antiS100 on the degree of catalepsy induced by high dose
of haloperidol. Experiments were performed on male outbred albino rats
weighing 250280 g. Catalepsy was induced by intraperitoneal injection of
haloperidol in a dose of 1.0 mg/kg (ampoule solution, Gedeon Richter) 60 min
139
Ultralow doses
Points
80
60
40
20
!
!
!
2
3
4
0
1
Fig. 7.27. Effect of haloperidol and ULD antiS100 on vertical activity of
apomorphinereceiving mice. Apomorphine (1); haloperidol + apomorphine (2); ULD
antiS100 (single administration) + haloperidol + apomorphine (3); and ULD anti
S100 (course of treatment) + haloperidol + apomorphine (4). *p<0.05 compared
to the control.
before the study (T. A. Voronina et al., 2000a). ULD antiS100 in a dose of 2.5
ml/kg were injected intraperitoneally 40 min after haloperidol administration (20
min before the study). Each group consisted of 10 animals.
The degree of catalepsy (ability of animals to maintain a fixed body
posture for some time) was estimated 60, 120, and 180 min after haloperidol
injection. The placement of paws on the step (7.5 and 12.5 cm in height) was
studied in the staircase test. We evaluated the ability of rats to hold the paw on
the step for 10 sec.
The degree of catalepsy was determined by a 6point scale: 1 point, only
one of the forelimbs remains on the bottom step; 2 points, both forelimbs and
one hindlimb remain on the bottom step; 4 points, both hindlimbs remain on
the bottom step; 5 points, only one of the forelimbs remains on the tope step;
and 6 points, both forelimbs remain on the top step.
Treatment with high dose of a neuroleptic drug haloperidol caused
catalepsy in rats (maintenance of a given posture for a long time). The degree
of catalepsy in control animals was maximum (6point scale) over the whole
period of study (180 min, Table 7.4).
The degree of catalepsy in haloperidolreceiving animals significantly
decreased 60 min after administration of ULD antiS100 (by 1.33 times, Table
7.4). The effect of ULD antiS100 was less pronounced after 120 and 180 min.
However, the average score of catalepsy in these rats was much lower than in
control animals (by 1.22 and 1.07 times, respectively).
After administration of ULD antiS100, the highest degree of catalepsy
(6 points) was not observed in 60% animals.
Hence, single treatment with ULD antiS100 was followed by a
significant decrease in cataleptogenic activity of haloperidol in a dose of 1 mg/
kg (T. A. Voronina, 2005).
140
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Table 7.4.
Effect of ULD antiS100 on the degree of haloperidolinduced catalepsy
Animals without the highest
degree of catalepsy (6 points), %
Average score
Group
Haloperidol
Haloperidol +
ULD antiS100
after
60 min
after
120 min
after
180 min
after
60 min
after
120 min
after
180 min
6
6
6
0
0
0
4.50±1.58*
4.90±1.29*
5.60±0.84
60
60
20
Note. *p<0.05 compared to the haloperidol group.
Range of pharmacological properties
of ULD antiS100 in young rats
Experiments were performed on 767 outbred rats (384 males and 383
females) aging 3035 days. We studied the anxiolytic, antidepressant, and
nootropic properties of ULD antiS100, efficacy of ULD antiS100 under
experimental conditions of memory deficit and hyperactivity, and influence of
ULD antiS100 on locomotor and behavioral activity of highactivity and low
activity rat pups.
Anxiolytic activity of ULD antiS100 in young rats. Anxiolytic activity of
ULD antiS100 was studied in a conflict situation (T. A. Voronina et al., 2000c;
G. M. Molodavkin et al., 1995; J. R. Vogel, 1971; D. Triet, 1985; D. J. Sanger,
1991) and EPM test (T. A. Voronina et al., 2000c; S. Pellow et al., 1986; G. R.
Dawson et al., 1995; S. E. File, 1995). The benzodiazepine anxiolytic diazepam
was used as a reference drug.
Anxiolytic effect of ULD antiS100 on young rats in the Vogel test. Experiments
were performed as described elsewhere (T. A. Voronina et al., 2000c; J. R. Vogel,
1971). The animals (n=36, 3035 days of life) were divided into three groups. The
rats received 2.5 ml/kg distilled water (control group 1, n=12), 1.5 mg/kg diazepam
(group 2, n=12), or 2.5 ml/kg ULD antiS100 (group 3, n=12). Test substances
were administered intragastrically 30 min before the study.
The behavior of control rat pups was characterized by high anxiety in a
conflict situation. This conclusion was derived from a small number of
approaches to the drinking bowl and low incidence of punished drinking
episodes.
Anxiety of animals in a conflict situation was significantly reduced after
administration of diazepam. The number of punished drinking episodes in
diazepamreceiving rats was 2.7fold higher than in control animals (Fig. 7.28).
The anxiolytic effect of ULD antiS100 in a conflict situation was similar
to that of diazepam. It was manifested in a significant increase in the number
of punished drinking episodes compared to the control (Fig. 7.28).
141
Ultralow doses
Number of punished
drinking episodes
300
!
!
200
100
0
Control
ULD
Diazepam
antiS100
Fig. 7.28. Anxiolytic effect of ULD antiS100 on young rats in the Vogel conflict
test. *p<0.05 compared to the control.
Therefore, ULD antiS100 in a dose of 2.5 ml/kg had the anxiolytic effect
on young rats in a conflict situation. Under these conditions, anxiolytic activity
of ULD antiS100 compared well with that of diazepam in a dose of 1.5 mg/kg.
Anxiolytic and antistress effect of ULD antiS100 on young rats in the EPM
test. Experiments on young animals were performed as described for adult
specimens (T. A. Voronina et al., 2000c; S. Pellow et al., 1986; S. E. File, 1995).
The animals (n=36, 3035 days of life) were divided into the control and
treatment groups of 12 specimens each. The rats received 2.5 ml/kg distilled
water (control group), 1.5 mg/kg diazepam (treatment group 1), and 2.5 ml/kg
ULD antiS100 (treatment group 2). Distilled water and test substances were
administered intragastrically 20 min before the study.
ULD antiS100 had a normalizing effect on the EPM behavior of
animals. Administration of this product was followed by a significant increase
in the number of entries into the open arms and central area of EPM (by 5.6
and 2.1 times, respectively; Fig. 7.29).
Diazepam also had an anxiolytic effect. The number of entries into the
open arms and central area of EPM increased by 8.3 and 3.6 times, respectively,
after diazepam treatment (p<0.05, Fig. 7.29).
No significant differences were found in the anxiolytic effect of ULD
antiS100 and diazepam on young rats in EPM.
Antidepressant effect of ULD antiS100 on young rats. The antidepressant
effect of ULD antiS100 on young rats was studied in the Porsolt’s forced
swimming test (R. D. Porsolt et al., 1978), as well as in a water tank with wheels
(G. M. Molodavkin et al., 1994; S. Nomura et al., 1982).
Antidepressant effect of ULD antiS100 on young rats: Porsolt’s forced
swimming test. Experiments were performed as described elsewhere (R. D.
Porsolt et al., 1978). The animals (n=40) were divided into three groups. They
142
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
sec
80
!
60
!
40
+ +!
!
20
0
Control
ULD antiS100
Diazepam
Fig. 7.29. Anxiolytic effect of ULD antiS100 on young rats in the EPM test. Light
bars, time spent in the central area; dark bars, time spent in the open arms. p<0.05:
*compared to the control; +compared to diazepam.
received 2.5 ml/kg distilled water (n=20, control group), 10 mg/kg amitriptyline
in 2.5 ml/kg distilled water (n=10, treatment group 1), or 2.5 ml/kg ULD anti
S100 (n=10, treatment group 2). Test substances were administered
intragastrically 30 min before the study.
ULD antiS100 and amitriptyline had an antidepressant effect in the
Porsolt’s test of behavioral despair. The immobility time in treated rats was
much lower than in control animals (by 1.3 and 1.4 times, respectively; Fig.
7.30). Therefore, the antidepressant activity of ULD antiS100 compared well
with that of amitriptyline.
Antidepressant effect of ULD antiS100 on young rats in the Nomura’s forced
swimming test with rotating wheels. Experiments on young rats were performed as
described for adult specimens (G. M. Molodavkin et al., 1994; S. Nomura et al.,
1982). The animals (n=40, 3035 days of life) were divided into three groups. The
rats received 2.5 ml/kg distilled water (n=20, control group), 10 mg/kg amitriptyline
in 2.5 ml/kg distilled water (n=10, group 2), or 2.5 ml/kg ULD antiS100 (n=10,
group 3). Test substances were administered intragastrically 30 min before the study.
The number of wheel revolutions in the Nomura’s forced swimming test
with freely rotating wheels increased by 1.8 and 1.7 times after administration
Immobility time, sec
400
!
300
!
200
100
0
Control
ULD antiS100
Amitriptyline
Fig. 7.30. Antidepressant effect of ULD antiS100 on young rats in the Porsolt’s
forced swimming test. *p<0.05 compared to the control.
143
Ultralow doses
of ULD antiS100 and amitriptyline, respectively (p<0.05). Drug treatment was
also followed by an increase in the correlation coefficient between the numbers of
wheel revolutions during the first and second 5min periods of study (Fig. 7.31).
These data indicate that test substances have a strong antidepressant
effect.
Nootropic effect of ULD antiS100 on young rats. Nootropic activity of
ULD antiS100 was studied in the CPAR test under conditions of scopolamine
induced amnesia (T. A. Voronina et al., 2000b). The product was also tested in
rat pups with poor learning ability. Piracetam was used as a reference drug.
Nootropic effect of ULD antiS100 (single administration and course of
treatment) during scopolamineinduced amnesia. The device and design of
experiments were similar to those in studies with adult animals (T. A. Voronina
et al., 2000b). The parameter of DLC was calculated as follows:
ΔLC=LC2LC1,
where LC2 and LC1 are LC of the first entry into a compartment with the
electrode floor during CPAR acquisition and testing (24 h after training),
respectively. The optimal conditions of CPAR training for young rats were
selected in previous experiments (eight series of electrostimulation with 0.6 mA,
pulse duration 1 sec, interpulse interval 2 sec).
CPAR amnesia was induced by the standard method. A cholinoceptor
antagonist scopolamine in a dose of 1.4 mg/kg was injected subcutaneously
15 min before training (according to the Manual on Experimental (Preclinical)
Study of New Pharmacological Substances, 2005).
Rat pups were divided into the following groups:
• group 1 (passive control, n=27): distilled water, 2.5 ml/kg intragastri
cally, single or repeated administration (10 days, last treatment 40 min
before training); and distilled water, 2 ml/kg subcutaneously, 15 min
before training;
Number of wheel
revolutions
160
!
!
120
80
40
0
Control
ULD antiS100
Amitriptyline
Fig. 7.31. Antidepressant effect of ULD antiS100 on young rats in the Nomura’s
forced swimming test. *p<0.05 compared to the control.
144
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
•
group 2 (active control, n=27): distilled water, 2.5 ml/kg intragastri
cally, single or repeated administration (10 days, last treatment 40 min
before training); and scopolamine, 1.4 mg/kg subcutaneously, 15 min
before training;
• group 3 (n=15): 400 mg/kg piracetam in 2.5 ml/kg distilled water,
intragastrically for 10 days (last treatment 40 min before training); and
scopolamine, 1.4 mg/kg subcutaneously, 15 min before training;
• group 4 (n=12): 400 mg/kg piracetam in 2.5 ml/kg distilled water,
intragastrically, 40 min before training; and scopolamine, 1.4 mg/kg
subcutaneously, 15 min before training;
• group 5 (n=15): ULD antiS100, 2.5 ml/kg intragastrically, 40 min
before training; and scopolamine, 1.4 mg/kg subcutaneously, 15 min
before training; and
• group 6 (n=12): ULD antiS100, 2.5 ml/kg intragastrically for 10 days
(last treatment 40 min before training); and scopolamine, 1.4 mg/kg
subcutaneously, 15 min before training.
Memory processes were retained in rat pups of the passive control group.
These animals remained in the dangerous dark compartment for a longer period
than during training (by 10 times, p<0.05). Scopolamineinduced amnesia was
manifested in a decrease in LC of entry into the dark compartment (by 7.6
times, p<0.05) and reduction of DLC (by 18.2 times compared to the passive
control, p<0.05; Table 7.5).
The amnesic effect of a cholinoceptor antagonist scopolamine was less
pronounced after single administration of piracetam. This conclusion was
derived from an increase in LC of entry into the dangerous dark compartment
(by 3.4 times, p<0.05) and DLC (by 7.4 times compared to the active control,
p<0.05; Table 7.5).
Single administration of ULD antiS100 had a similar effect under
conditions of scopolamineinduced amnesia. LC of entry into the dark
compartment and DLC increased by 2.2 and 4.6 times, respectively, compared
to the active control (Table 7.5).
Treatment with ULD antiS100 and piracetam was followed by an in
crease in the number of rat pups that reached the learning criterion (Fig. 7.32).
These data show that single administration of ULD antiS100 and
piracetam has a strong antiamnesic effect on the model of scopolamineinduced
amnesia. The antiamnesic effect of single treatment with ULD antiS100 is less
pronounced than that of piracetam. Due to low effectiveness of ULD antiS100
after single treatment, further experiments were performed with repeated
administration of ULD antiS100 and piracetam (10day course).
A 10day course of treatment with ULD antiS100 and piracetam had a
greater antiamnesic effect. The effect of ULD antiS100 was most pronounced
145
Ultralow doses
Table 7.5.
Antiamnesic effect of single treatment with ULD antiS100 and piracetam
in the test for scopolamineinduced amnesia of CPAR (M±m, n=15)
24 h after training
Before training
Substance
Passive control
LC1
LC2
ΔLC=LC2–LC1
10.13±3.81
100.47±21.27
90.27±19.86
8.3±3.1
13.33±8.08+
4.97±3.30+
Active control
(amnesia + scopolamine)
ULD antiS100 + scopolamine
6.77±2.0
29.80±9.84*
23.03±9.90*
Piracetam + scopolamine
8.07±2.32
44.67±10.58*
36.6±10.9*
Note. p<0.05: *compared to the active control; +compared to the passive control.
under these conditions. It was manifested in an increase in LC of entry into the
dark compartment (by 3.4 times compared to the active control, p<0.05) and
DLC (by 8.6 times, p<0.05; Table 7.6). In animals of the piracetam group these
parameters increased by 3.8 and 8.9 times, respectively (p<0.05). It can be
concluded that the antiamnesic effect of repeated treatment with ULD anti
S100 compares well with that of piracetam.
Hence, ULD antiS100 and piracetam have a strong antiamnesic effect
in the test for scopolamineinduced amnesia. During single administration, the
antiamnesic effect of ULD antiS100 was smaller than that of piracetam. After
the course of treatment, ULD antiS100 and piracetam had a similar effect.
Effect of repeated treatment with ULD antiS100 on training of young rats
with poor learning ability. Experiments were performed by the standard method
(T. A. Voronina et al., 2006b). DLC for each animal was calculated as follows:
Trained animals, %
120
100
80
60
+
+
40
+
!
20
0
1
2
3
4
5
Fig. 7.32. Effect of ULD antiS100 on CPAR performance during scopolamine
induced amnesia. Control (1); scopolamine (2); piracetam + scopolamine (3); ULD
antiS100 (single administration) + scopolamine (4); and ULD antiS100 (course
of treatment) + scopolamine (5). p<0.05: *compared to the control; +compared
to scopolamine.
146
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Table 7.6.
Antiamnesic effects of repeated treatment with ULD antiS100 and
piracetam (10day course): CPAR of 3035dayold rat pups in the test for
scopolamineinduced amnesia (M±m, n=12)
Training
24 h after training
Substance
LC1
LC2
ΔLC=LC2–LC1
Passive control
(distilled water)
8.83±3.15
109.25±25.03
98.25±23.87
Active control (scopolamine)
9.08±2.28
14.83±3.12+
5.08±1.95+
ULD antiS100 + scopolamine
7.17±2.25
50.83±17.43*
43.50±16.88*
Piracetam + scopolamine
10.17±1.82
55.58±14.42*
45.42±14.02*
Note. p<0.05: *compared to the active control; +compared to the passive control.
ΔLC=LC2LC1,
where LC2 and LC1 are LC of the first entry into a compartment with the
electrode floor during CPAR acquisition and testing (24 h after training),
respectively.
DLC was low under specified conditions of training (five series of electro
stimulation with 0.45 mA, pulse duration 1 sec, interpulse interval 2 sec). The
animals were characterized by poor learning ability, which met the requirements
of this series. Previous experiments with scopolamineinduced amnesia showed
that the efficacy of drugs is much higher after the course of treatment. In the
present study, test substances were administration for 10 days before training.
Rat pups were randomized into three groups of 15 specimens each. The
animals received intragastrically distilled water (2.5 ml/kg, group 1), ULD anti
S100 (2.5 ml/kg, group 2), or piracetam (400 mg/kg, group 3) for 10 days (last
treatment 40 min before training).
LC of entry into the dark compartment tended to increase after piracetam
administration (no statistically significant differences from the control, Table 7.7).
LC of entry into the dark compartment and DLC significantly increased
after treatment with ULD antiS100 (by 1.8 and 4 times, respectively; Table 7.7).
The results indicate that a 10day course of treatment with ULD anti
S100 improves memory processes in rat pups with poor learning ability. ULD
antiS100 were more potent than piracetam in modulating the learning ability
of these animals.
Effect of ULD antiS100 on cognitive function, motor activity, and anxiety
of highactivity young rats with inadequate behavior: experimental model of
attention deficiency and hyperactivity. Highactivity animals with impulsive
(inadequate) behavior in the openfield test and provoking stimulation were
selected from the population of outbred rat pups (scale of Broudy and Nauta).
147
Ultralow doses
Table 7.7.
Effects of ULD antiS100 and piracetam on CPAR acquisition in young rats
with poor learning ability (M±m, n=15)
Before training
Substance
24 h after training
LC1
LC2
ΔLC
Control
10.3±1.1
15.1±2.3
4.8±1.9
ULD antiS100
7.2±1.1
26.4±4.6*
19.2±3.9*
Piracetam
9.2±2.1
19.2±4.8
10.3±5.7
Note. *p<0.05 compared to the control.
The animals were divided into three groups. Rat pups received intragastrically
2.5 ml/kg distilled water (group 1, n=49), 125 mg/kg phenibut (group 2, n=20),
or 2.5 ml/kg ULD antiS100 (group 3, n=36) for 7 days.
Each group was divided into two subgroups. Subgroup 1 rats were trained
in CPAR 15 min after the last administration of test substances. EPM anxiety
of subgroup 2 animals was studied 20 min after the last treatment.
Administration of phenibut was followed by a 2.8fold increase in LC of
entry into the dark compartment (p<0.05 compared to the control). Phenibut
also increased the number of animals not entering the dark compartment.
Under these conditions the number of transitions between the illuminated
platform and dark compartment decreased by 3 times (Table 7.8, Fig. 7.33).
Treatment with ULD antiS100 was followed by an increase in LC of
entry into the dark compartment (by 3.4 times, p<0.05), time spent on the
illuminated platform (by 2 times, p<0.05), and number of animals not entering
the dark compartment. Under these conditions the number of transitions
between the illuminated platform and dark compartment decreased by 3 times
(Table 7.8, Fig. 7.31).
These data indicate that ULD antiS100 improve CPAR learning and
decrease the hyperactivity of highactivity young rats with inadequate behavioral
reactions. ULD antiS100 had a modulatory effect on learning ability during
CPAR performance in the dark compartment (i.e., measurement of LC). This
effect was manifested in an increase in the time spent on the illuminated
platform. The number of animals not entering the dark compartment increased
under these conditions. Moreover, hyperactivity of highactivity rat pups was
shown to decrease after administration of ULD antiS100. This conclusion was
derived from a decrease in the number of transitions between the illuminated
platform and dark compartment. The effect of ULD antiS100 on learning of
highactivity rat pups was more pronounced than that of phenibut.
The EPM test showed that the behavior of selected rat pups is
characterized by high motor activity. The animals exhibited multiple entries into
148
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Table 7.8.
Effects of ULD antiS100 and phenibut on CPAR acquisition in highactivity
young rats with inadequate behavior (M±m)
Control
(n=27)
Phenibut, 125
mg/kg (n=10)
ULD antiS100,
2.5 ml/kg (n=18)
26.61±11.26
73.40±36.56*
91.33±34.14*
Time spent on the illuminated
platform, sec
51.99±20.85
73.40±36.56
102.56±32.58*
Time spent in the dark
compartment, sec
128.5±20.8
106.60±36.56
77.72±32.56
Number of transitions
2.40±0.67
0.80±0.26*
0.78±0.34*
Parameter,
24 h after training
Latency of entry into the dark
compartment, sec
Note. *p<0.05 compared to the control.
the closed arms, active movement in the closed arms, and anxiety (rare visits
to the open arms and central area, little time spent in the open arms and central
area, considerable number of fecal boluses, and high incidence of grooming
episodes).
ULD antiS100 had a normalizing effect on the EPM behavior of high
activity animals. ULD antiS100 decreased the number of entries into the
closed arms (by 3.5 times, p<0.05), but increased the number entries into the
open arms (by 5.2 times, p<0.05) and central area (by 2 times, p>0.05). The
number of movements in the open and closed arms was similar after
administration of ULD antiS100. The total number of these movements for
animals of the ULD antiS100 group was lower compared to the control (4.6
and 6.1, respectively).
Treatment with ULD antiS100 was followed by an increase in the time
spent in the central area and open arms (by 2.7 and 1.8 times, respectively,
p<0.05). Moreover, the number of fecal boluses decreased by 3.1 times (p<0.05).
The observed changes reflect a decrease in the degree of anxiety (Table 7.9).
Trained animals, %
40
!
30
20
10
0
Control
ULD antiS100
Phenibut
Fig. 7.33. Effect of ULD antiS100 on CPAR performance in highactivity young
rats with inadequate behavior. *p<0.05 compared to the control.
149
Ultralow doses
Table 7.9.
Effects of ULD antiS100 and phenibut on the behavior of highactivity rat
pups in the elevated plusmaze (M±m)
ULD antiS100,
2.5 ml/kg (n=18)
Control
(n=22)
Phenibut, 125
mg/kg (n=10)
into the closed arms
4.98±0.85
2.50±0.53*
1.44±0.33*
into the open arms
0.33±0.39
1.2±1.0
1.72±0.89*
to the central area
0.75±0.53
2.50±0.73*
1.50±0.66
on the central area
3.71±3.03
20.0±5.3*
10.06±5.21*
in the open arms
3.69±4.79
8.5±7.31
6.56±3.32
25
40
50
Grooming
2.48±0.73
0.80±0.64*
2.50±0.51
Number of boluses
1.88±0.69
0.30±0.42*
0.61±0.39*
Parameter
Number of entries
Time, sec
Animals entering the open arms, %
Note. *p<0.05 compared to the control.
These data indicate that ULD antiS100 decrease the motor activity and
have an anxiolytic effect on highanxiety rat pups.
Phenibut also had a normalizing effect on the EPM behavior of high
activity animals. This substance decreased the number of entries into the closed
arms (by 2 times, p<0.05), but increased the number of entries into the open
arms (by 3.6 times) and central area (by 3.3 times, p<0.05).
However, phenibut was less potent than ULD antiS100 in decreasing the
number of entries into the closed arms (by 3.6 and 2 times, respectively; Table
7.9). Therefore, phenibut had a smaller effect on the increased motor activity
of highactivity animals.
Phenibut also had a strong anxiolytic effect. This substance increased not
only the number of entries into the open arms and central area, but also the
time spent in the illuminated area. The incidence of grooming episodes and
number of fecal boluses decreased under these conditions (Table 7.9). Phenibut
had a greater anxiolytic effect on highactivity rat pups than ULD antiS100.
These data indicate that ULD antiS100 and phenibut have a normalizing
effect on the EPM behavior of highactivity animals. Test substances decreased
the number of entries into the closed arms, but increased the number of entries
and time spent in the open arms and central area. The number of fecal boluses
decreased under these conditions. Hence, ULD antiS100 and phenibut
produce an anxiolytic effect on highactivity rat pups.
The anxiolytic effect of phenibut was greater than that of ULD antiS100.
However, ULD antiS100 were more potent than phenibut in normalizing the
increased motor activity of highactivity animals.
150
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Safety of ULD antiS100
A complete toxicology study was performed to evaluate the safety profile,
possible side effects, target organs, and safe dosage range of ULD antiS100.
These experiments were conducted in accordance with the recommendations
given in the Manual on Experimental (Preclinical) Study of New
Pharmacological Substances and approved by the Pharmacological Committee
of the Russian Ministry of Health in 2000. The purpose of studies with ULD
antiS100 was to determine the acute toxicity (experiments on mice and rats),
chronic toxicity (6month treatment of rats and rabbits), reproductive and
allergic toxicity (experiments on rats), immunotoxicity, mutagenicity (chromo
somal aberration assay in mouse bone marrow cells), and genotoxicity (test
system for somatic mosaicism in wing cells of Drosophila melanogaster).
Our experiments demonstrated a good safety profile of ULD antiS100.
An acute toxicity study showed that this substance in the maximum permissible
dose does not cause death of animals. Drugrelated death of animals was not
observed after 6month treatment with ULD antiS100 in the highest dose. The
product had no toxic effect on organs of experimental animals. A
pathomorphological study did not reveal damage to the internal organs or local
irritation of the gastric mucosa after drug administration. ULD antiS100 did
not cause reproductive disorders in male and female rats. The embryotoxic effect
of ULD antiS100 was not detected. ULD antiS100 had no mutagenic,
allergenic, and immunotoxic properties.
Acute toxicity of ULD antiS100 was also studied on young rats. Due to
the absence of mortality, it was difficult to evaluate LD50 of ULD antiS100 for
young animals (similarly to adult specimens). Conventionally, the dose of ULD
antiS100 exceeding the maximum permissible dose (by volume, according to
the route of administration) was considered as LD50.
* * *
Experimental studies for pharmacological activity of ULD antiS100
allowed us to make the following conclusions.
Experiments on adult and young animals showed that the product of
ULD antiS100 has a wide range of psychotropic and neurotropic pharmaco
logical properties, including the anxiolytic, antidepressant, antistress, nootropic,
neuroprotective, antiischemic, and antihypoxic effects. ULD antiS100 differ
from modern neuropsychotropic drugs in a variety of properties, absence of side
effects (typical of highefficacy reference drugs), and mechanism of action.
A wide range of pharmacological properties of ULD antiS100 is related to
the improvement of general adaptive processes in CNS (Fig. 7.34). The impairment
of these processes is followed by a deficiency of endogenous stresslimiting systems
151
Ultralow doses
Modification of functional activity
of endogenous protein S100
and its ligands
GABA(A)
receptor
complex
GABAmimetic effect,
functional recovery
of the GABAergic
system
• Activation of inhibitory
processes in CNS
• Stresslimiting activity
• Neuromodulatory effect::
neuronal plasticity
growth factor for
serotoninergic neurons
reduced production of
hypothalamic corticotropin
releasing hormone
• Functional regulation of other
neurotransmitter systems
Other ligands
and regulatory targets
for S100 protein
Intracellular
calcium,
Ca 2+dependent
processes
Membrane
adenylate cyclase
Restoration
of intracellular
Ca 2+ homeostasis
Regulation
of cAMPdependent
processes
• Stabilization of neuronal impulse activity
• Stressprotecting effect on cells
• Regulation of functional plasticity
of neurons:
through the activity of calmodulin,
protein kinase C, and other
Ca 2+dependent kinases
regulation of enzyme activity for energy
metabolism and plastic metabolism
(through dephosphorylation
or phosphorylation)
functional regulation of glutamate
receptors and GABA receptors
Recovery/increase in the activity
of endogenous stresslimiting systems
• Nuclear factor NFkB
• Antiapoptotic
factor Bcl2
• Cytoskeletal proteins
(Tauproteins etc.)
Regulation of the cell cycle,
regeneration of neurons and glia,
and cytoskeletal integrity;
antiapoptotic effect
• Signal transduction from
G proteincoupled receptors
• Regulation of functional
plasticity of neurons:
through protein kinase A
through protein kinase G
functional regulation of glutamate
receptors and GABA receptors
Regulation of enzyme expression
for energy metabolism
and plastic metabolism
Recovery/increase in neuronal plasticity
(natural stressprotective mechanism in CNS)
Fig. 7.34. Possible pathways and mechanisms for pharmacological activity of ULD
antiS100.
(primarily of the GABAergic system) and chronic dysregulation of neuronal plas
ticity. Functional recovery is accompanied by a variety of pharmacological effects
of ULD antiS100. The prevalence of any mechanism for dysregulation determines
the site of action and effects of this product under various pathological conditions.
The proposed pathways and mechanisms for pharmacological activity of
ULD antiS100 are consistent with the results of experimental and clinical
studies. Moreover, they fit naturally into the existing neurobiological paradigm.
ULD antiS100 have a normalizing effect and increase the activity of
endogenous stresslimiting systems. The product regulates neuronal plasticity.
These properties determine the neuroprotective and trophic effect of ULD anti
S100. Hence, ULD antiS100 hold much promise for the pathogenetic therapy
of various neurological and mental disorders.
7.2. Preclinical study of Impaza
Impaze consists of affinity purified rabbit polyclonal antibodies to
endothelial NO synthase (eNOS) in ULD (mixture of homeopathic dilutions
C12, C30, and C200).
152
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Previous experiments showed that Impaza improves sexual behavior and
erectile function in male rats with decreased sexual activity. Moreover, Impaza
had a positive effect on sexual function in females (Fig. 7.35). Impaza activates
eNOS and increases the production of cGMP, nitrates, and nitrites in the ca
vernous bodies of male rats. The drug has no effect on hemodynamics in nor
motensive animals, but decreases blood pressure (BP) in hypertensive rats. These
data indicate that Impaza may be used in the therapy of cardiovascular disorders.
A toxicology study was performed in accordance with the recommenda
tions of the Pharmacological Committee of the Russian Ministry of Health and
Social Development. This study showed that Impaza exhibits a good safety pro
file. Combined treatment with Impaza and nitroglycerine was not accompanied
by a further decrease in BP. The data indicate that Impaza may be prescribed
for patients with CHD.
Effect of Impaza on sexual behavior of rats
Effect of Impaza on sexual behavior of male Wistar rats. Although the
development of erectile dysfunction (ED) is associated with a variety of factors, the
major pathogenetic types of this disorder have a common mechanism. It suggests
functional insufficiency of the NO synthase — NO guanylate cyclase — cGMP
cascade and, primarily, inadequate production of NO (K. E. Andersson, 2001; M.
Ushiyama et al., 2004). Previous experiments were designed to study the effect
of Impaza on sexual behavior and activity of this regulatory pathway.
The effect of Impaza on sexual behavior of Wistar rats was studied on two
models of decreased sexual activity (seasonal and agerelated suppression).
IMPAZA
Effect on sexual behavior
Males
Seasonal
suppression
Females
Mechanism of action
Effect on eNOS activity
and production of cGMP,
nitrates, and nitrites
Agerelated
suppression
Effect on hemodynamics
Normotensive rats
Hypertensive rats
Fig. 7.35. Preclinical study of Impaza.
153
Ultralow doses
Series I was performed on 4monthold animals (400450 g) in the winter
period. Series II was performed on old males (16 months old, 600700 g).
Impaza was administered intragastrically in a daily dose of 1.5 ml for 5 days
(treatment group, n=10). Control rats (n=10) received an equivalent volume of
distilled water. Sexual behavior in the open field was studied before and after
therapy (J. Bures et al., 1983).
The males were mated with 34monthold females (300400 g). The
stage of estrus in females was induced by 4fold injection of 0.05% folliculin in
a daily dose of 0.02 mg/kg. The profile of sexual activity was estimated from LC
of mounting, total number of mountings, and number of matings (T. G. Bo
rovskaya, et al., 2002).
The course of treatment with Impaza in males with seasonal suppression
of sexual activity was followed by an increase in the total number of mountings
and matings by 2 and 3 times, respectively. These parameters in control animals
increased by 25 and 35%, respectively (Fig. 7.36).
Therefore, the course of treatment with Impaza improves sexual function
and motivation (T. G. Borovskaya, et al., 2002).
Experiments on old rats showed that Impaza is effective only in animals
with preserved sexual behavior (55% specimens). The number of matings
(ejaculations) is one of the major parameters, which determines sexual moti
vation and sexual function. Under basal conditions, this parameter was low in
16monthold males of the control and treatment groups. The number of
matings (ejaculations) increased by 3.3 times after administration of Impaza for
5 days (p<0.05). By contrast, this parameter decreased in control rats. LC of
mating serves as the criterion of sexual motivation. Administration of distilled
water and Impaza had no effect on this parameter in rats. The number of
mountings reflects not only sexual function, but also sexual motivation. The
a
b
!
30
25
20
control
Impaza
15
10
!
5
0
Baseline
After
treatment
Baseline
After
treatment
Fig. 7.36. Effect of Impaza on sexual behavior of male rats with a seasonal
decrease in sexual activity. Number of mountings (a) and matings (b). *p<0.05
compared to the baseline value.
154
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
number of mountings decreased in the control group, but increased after a 5
day course of treatment with Impaza (by 1.8 times, p<0.05 compared to the
baseline value; Fig. 7.37).
The data indicate that a 5day course of Impaza improves sexual function
in male rats with a seasonal decrease in sexual function and agerelated erectile
dysfunction (T. G. Borovskaya et al., 2001; T. G. Borovskaya et al., 2002).
The effect of Impaza on the basal activity of the NOS NO cGMP
cascade in the cavernous bodies was studied on male Wistar rats aging 4 months.
The animals received intragastrically Impaza (1.5 ml, single administration or
5day course of treatment), distilled water, or reference drug sildenafil citrate
(single dose 10 mg/kg; Pfizer). They were killed 3 h after the last treatment. The
cavernous tissue was isolated. cGMP content was estimated by a direct enzyme
immunoassay with Amersham kits. The concentration of NO derivates was
measured colorimetrically with CN Biosciences kits. NOS activity was measured
colorimetrically (Oxford Biomedical Research).
Impaza improved copulative (erectile) function and had a stimulatory
effect on sexual behavior under conditions of seasonal or agerelated suppression
of reproductive function.
A biochemical study showed that the improvement of erectile function
after treatment with Impaza is accompanied by significant changes in the basal
activity of NOS, production of NO, and content of cGMP in the cavernous
tissue of male rats. Single oral administration of Impaza was followed by an
increase in the activity of endothelial NO synthase and concentration of NO
derivates in the cavernous bodies of male rats (by 2 and 1.4 times, respectively;
p<0.05). A 5day course of treatment of Impaza also produced a 4fold increase
in cGMP content in the penile cavernous tissue (Yu. P. Bel’skii et al., 2003; A.
V. MartyushevPoklad et al., 2003). A reference drug sildenafil increased only
the content of cGMP (Fig. 7.38).
a
7
6
5
4
3
2
1
0
b
!
control
Impaza
!
Baseline
After
treatment
Baseline
After
treatment
Fig. 7.37. Effect of Impaza on sexual behavior of old male rats with decreased
sexual activity. Number of mountings (a) and matings (b). *p<0.05 compared to
the baseline value.
155
Ultralow doses
% of the baseline
700
!
600
500
400
300
200
100
0
b
а
!
!
!
Single treatment
control
Course of treatment
sildenafil
Impaza
% of the baseline
140
130
120
110
100
90
80
70
60
50
!
Single treatment
control
!
Course of treatment
sildenafil
Impaza
c
% of the baseline
300
!
!
250
200
150
100
50
0
Single treatment
control
Course of treatment
sildenafil
Impaza
Fig. 7.38. Major mechanism for the effect
of Impaza: content of cGMP (a), concentra
tion of NO derivatives (b), and activity of
eNOS (c) in the cavernous tissue of male
rats. *p<0.05 compared to the control (dis
tilled water).
Hence, the most probable peripheral mechanism for Impaza action is an
increase in endothelial NO synthase activity and recovery of NO production
(i.e., normalization of endothelial function).
Effect of Impaza on sexual function in females. In addition to studying the
effect of Impaza on sexual behavior of male rats, the influence of this drug on
sexual behavior of estrous females was evaluated (T. G. Borovskaya et al., 2002).
Sexual receptivity of females (readiness for mating) is determined by the
reduced aggressiveness to males and appearance of the lordosis posture. Impaza
had no effect on the number of lordosis postures in females, but increased the
lordosis/mounting ratio (by 1.9 times, p<0.05). Moreover, Impaza increased the
number of active females (exhibiting the lordosis posture) and decreased the
number of aggressive females.
Effect of Impaza on the cardiovascular system
The vascular endothelium is a neuroendocrine organ, which maintains the
relationship between blood and tissues (G. A. Chumakova et al., 2006).
Endothelial dysfunction causes a variety of diseases, including hypertension,
156
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
CHD, and ED (D. V. Nibieridze, 2005; O. D. Ostroumova et al., 2005).
Previous experiments showed that Impaza improves endothelial function (NO
synthase — NO guanylate cyclase — cGMP cascade). Further investigations
were performed to evaluate the effect of this drug on the cardiovascular system.
The effects of Impaza on the cardiovascular system were studied in two
series with normotensive Wistar rats (series I) and hypertensive ISIAH rats
(series II).
Effect of Impaza on hemodynamic parameters in normotensive Wistar rats.
We studied the effect of test substances on the following parameters of systemic
hemodynamics: cardiac output (CO), stroke volume, mean BP (MBP), central
venous pressure, total peripheral resistance, and heart rate (HR).
The drugs to modulate sexual potency (sildenafil, vardenafil, and tadalafil;
according to the Instructions for Use) should not be administered in combi
nation with nitrates (nitrites) and hypotensive pharmaceuticals, which limits
their use in patients with CHD and arterial hypertension. It was interesting to
evaluate the effect of combined treatment with nitroglycerine and Impaza.
Experiments were performed on 100 male Wistar rats weighing 200250 g.
The animals were divided into five groups as follows: group 1, single intragastric
administration of distilled water (2 ml); group 2, single intragastric administra
tion of 10 mg/kg sildenafil (2 ml, Pfizer); group 3, single intragastric admini
stration of Impaza (2 ml); group 4, 5day course of intragastric treatment with
Impaza (2 ml); and group 5, 5day course of intragastric treatment with distilled
water (2 ml).
Single and repeated administration of Impaza had no effect on a short
term decrease in MBP induced by nitroglycerine (5 mg/kg intravenously). The
timetorecovery of MBP remained unchanged under these conditions.
Moreover, various routes of treatment with Impaza did not affect the systemic
hemodynamics in healthy rats.
Effect of Impaza on BP in ISIAH rats with inherited hypertension. ISIAH
rats were bred at the Institute of Cytology and Genetics of the Siberian
Division of the Russian Academy of Sciences (Novosibirsk). They are
characterized by inherited stressinduced arterial hypertension. Experiments
were performed on 30 male ISIAH rats aging 56 months. The animals were
divided into three groups of 10 specimens each. Group 1 rats (control) received
orally 0.5 ml distilled water for 10 days (through a glass pipette). Group 2
animals were subjected to a 10day course of treatment with Impaza in a dose
of 0.5 ml. Losartan in a dose of 10 mg/kg was administered to group 3 rats
for 5 days. BP was measured 2 h after the 5th and 10th treatment, as well as
7 days after drug withdrawal (day 17 after the start of therapy). Indirect
measurements were performed with a special tail cuff. During this procedure,
the animals were placed in a plastic chamber (B. N. Van Vliet et al., 2000).
157
Ultralow doses
Impaza significantly decreased BP in hypertensive rats (A. L. Markel’ et al.,
2002). The hypotensive effect of Impaza developed progressively. On the 10th
day of treatment, BP in Impazareceiving rats significantly differed from the
control (Figs. 7.39 and 7.40).
The results suggest that Impaza contributes to activation of endothelial
NO synthase and recovery or increase in the production of endothelial NO
(improvement of endothelial function). A possible mechanism for the effect of
Impaza is shown in Fig. 7.41. Probably, eNOS activity reaches the individual
physiological optimum after longterm administration of Impaza.
Safety profile of Impaza. A complete toxicology study was performed to
evaluate the safety profile, possible side effects, target organs, and safe dosage
range of Impaza. These experiments were conducted in accordance with the
recommendations given in the Manual on Experimental (Preclinical) Study of
New Pharmacological Substances and approved by the Pharmacological
Committee of the Russian Ministry of Health and Social Development in 2000.
The purpose of studies with Impaza was to determine the acute toxicity
(experiments on mice and rats), chronic toxicity (6month treatment of rats and
rabbits), reproductive and allergic toxicity (experiments on rats),
immunotoxicity, mutagenicity (chromosomal aberration assay in mouse bone
marrow cells), and genotoxicity (test system for somatic mosaicism in wing cells
of Drosophila melanogaster).
Impaza had a good safety profile. An acute toxicity study showed that
Impaza in the maximum permissible dose does not cause death of animals.
Drugrelated death of animals was not observed after 6month treatment with
Impaza in the highest dose. The product had no toxic effect on organs and
systems of organs in experimental animals. A pathomorphological study did not
Decrease in MBP,
% of the control
14
12
10
8
6
4
2
0
1
2
3
Fig. 7.39. Effect of the course of treatment with Impaza on systolic BP in ISIAH
rats with inherited stressinduced arterial hypertension. Effect of Impaza on day
5 of treatment (1); effect of Impaza on day 10 of treatment (2); and effect of losartan
on day 5 of treatment (3).
158
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
MBP, mm Hg
195
190
185
!
180
175
170
165
Baseline
After
2h
After
5 days
After Seven days
10 days
after
withdrawal
Time after the start of Impaza treatment
Fig. 7.40. Dynamics of systolic BP in ISIAH rats during treatment with Impaza.
*p<0.05 compared to the baseline value.
Impaza
+++
eNOS
eNOS
Vascular endothelium
NO
NO
GTP
Phosphate
Smooth
muscle cell
cGMP
Cell relaxation
Activation of guanylate cyclase
Fig. 7.41. Possible mechanism for the effect of Impaza. NO, nitric oxide; eNO,
endothelial NO synthase; GTP, guanosine triphosphate; cGMP, cyclic guanosine
monophosphate.
reveal damage to the internal organs or local irritation of the gastric mucosa
after drug administration. Impaza did not produce a damaging or embryotoxic
effect on the reproductive system in male and female rats. Impaza had no
mutagenic, allergenic, and immunotoxic properties.
The results of preclinical studies show that Impaza improves copulative
(erectile) function, increases sexual motivation, and stimulates sexual behavior
159
Ultralow doses
of rats. These effects are realized via the NOS – NO guanylate cyclase – cGMP
cascade. Impaza had a moderate hypotensive effect on animals with stress
induced arterial hypertension, which was observed after the course of longterm
treatment with this drug. Impaza had no general toxic properties and did not
produce an adverse effect on reproductive function, fertility, and development
of the offspring. Moreover, Impaza did not possess mutagenic and allergenic
properties.
7.3. Preclinical study of Anaferon
and Anaferon for children
The active substances of Anaferon and Anaferon for children are affinity
purified polyclonal antibodies to human interferonγ (IFNγ) in ULD.
Experimental studies showed that the course of treatment with oral anti
bodies to IFNγ in ULD has an antiviral effect on the model of various infec
tions. This effect is related to their ability to stimulate the production of endo
genous IFNγ and functionally related cytokines. This property also contributes
to a wide range of immunomodulatory effects of the product (Fig. 7.42).
ANAFERON
Antiviral effect
Models of viral infections
influenza
herpes
genital herpes
Immunomodulatory effect
In vivo:
humoral response (antibody production)
cellular response
(delayedtype
hypersensitivity response)
effect on phagocytosis
In vitro:
T lymphocytes
B lymphocytes
NK cells
Mechanisms of effect (ex vivo)
effect on the cytokine status
(functional activity of type
1 and 2 T helper cells)
specific effect
on IFNγ induction
Fig. 7.42. Preclinical study of specific pharmacological properties of Anaferon and
Anaferon for children.
160
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Antiviral effect of Anaferon
To study antiviral activity of Anaferon, the infectious process was induced
by aerogenic (aerosol) administration of influenza virus strain A/Aichi/2/68
(H3N2) in a dose of 50100 AID50 (aerogenic infectious dose). Anaferon in a
daily dose of 0.2 ml was administered intragastrically (through a catheter) to
outbred albino mice. Anaferon was given for 5 days before infection and 5 days
after infection to study the preventive and therapeutic effects of this drug,
respectively. Control mice of the reference group received distilled water. The
preventive and therapeutic effects of Anaferon were estimated from the
concentration of influenza viruses in the lungs of infected mice (treatment group
and control group) on days 2, 3, 4, and 5 after infection. The presence of
influenza viruses in clarified homogenates of the lungs was determined by
titration on 10dayold developing chick embryos.
The preventive and therapeutic treatment with Anaferon had an antiviral
effect. This conclusion was derived from a decrease in the concentration of
influenza viruses in the lungs of animals (by 2.7 times on day 2 of infection; and
by 4.6 times on day 4 of infection, respectively) compared to the control group
(p<0.05, Fig. 7.43; A. N. Sergeev et al., 2004).
To induce herpes virus infection, outbred albino mice received intraperi
toneal injection of herpes simplex virus type 2 (HSV2; strain MS, ATCC) in a
dose of 5 LD50.
An aqueous solution of Anaferon was administered intragastrically for 5
days before infection (daily treatment). The animals were examined for 14 days.
The survival of mice and virus concentration in the brain were estimated on days
Virus concentration,
lg (EID)
7
a
Virus concentration,
lg (EID)
7
b
*
6
6
*
5
!
5
!
4
4
2
3
4
3
5
2
Time after infection, days
control
3
4
5
Anaferon
Fig. 7.43. Efficacy of Anaferon in mice with experimental influenza: preventive (a)
and therapeutic treatment (b). *p<0.05 compared to the control.
161
Ultralow doses
6 and 9 after infection. Virus concentration in animals of the treatment group
was much lower than in control specimens. The mortality rate of control and
treated mice was 69.2 and 28.6%, respectively. The average lifespan of died animals
from the treatment group was much greater than that of control specimens (by
3.3 days). The data indicate that preventive administration of Anaferon has a
strong protective effect on mice (p<0.05). This treatment was followed by a
decrease in herpes virus concentration in the brain (by 10 times) and increase
in the average lifespan of animals (Fig. 7.44; M. A. Susloparov et al., 2004).
The therapeutic efficacy of Anaferon was studied in guinea pigs with
experimental genital herpes infection. The animals were infected with HSV2
virus (strain EC). Beginning from the first day after infection, the animals
received intragastrically distilled water (5 ml/kg, control), acyclovir (100 mg/kg),
or Anaferon (5 ml/kg) twice a day for 15 days. Group 4 guinea pigs were treated
with Anaferon for 5 days before infection and 15 days after infection. The
general state of animals, local symptoms, and viral titer in vaginal smears were
estimated over 2 months.
The therapeutic and, particularly, therapeuticandpreventive treatment
with Anaferon was followed by a significant decrease in the severity and duration
of general and local symptoms of herpes infection. Virus elimination also
decreased under these conditions (Fig. 7.45).
The antiviral effect of ULD antiIFNγ on chickens with avian influenza
virus (H5N1, strain A/Chicken/Suzdalka/Nov11/2005) was studied at the
“Vektor” State Research Center for Virology and Biotechnology (Novosibirsk)
in 20062007.
ULD antiIFNγ significantly increased the survival of chickens after
infection with avian influenza virus in LD75 (p<0.05, Table 7.10). Administra
a
Survived
animals, %
80
b
BFU/ml
*
1000000
100000
*
control
Anaferon
*
4
6
60
10000
40
1000
100
20
10
0
1
Control
Anaferon
2
9
Time after infection, days
Fig. 7.44. Efficacy of Anaferon as a prophylactic drug in systemic herpes virus
infection. Survival of mice on day 14 after infection with HSV2 (5 LD50, a); and HSV2
virus concentration in the brain of mice (b). *p<0.05 compared to the control.
162
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
а
c
% of the control
120
Days
3
100
*
80
*
2
*
60
40
1
*
0
1
2
Points
1200
3
4
20
0
1
2
3
4
b
1000
800
600
400
*
200
0
1
2
3
4
Fig. 7.45. Efficacy of Anaferon on the
model of experimental genital herpes.
Duration of mucosal damage in genital
herpes (a), total score of symptoms (b),
and titer of genital herpes virus (c).
Control (1); acyclovir (2); Anaferon (3);
and preventive and therapeutic treatment
with Anaferon.
tion of this product was followed by a significant increase in the survival time
of chickens infected with avian influenza virus in LD90. However, the effect of
ULD antiIFNγ was less pronounced than that of a reference drug Tamiflu
(Fig. 7.46).
These data indicate that the therapeutic, preventive, and therapeuticand
preventive treatment with Anaferon has an antiviral effect. Hence, the action of
this product is related to immunomodulatory activity and modulation of the key
mechanisms for antiviral protection.
Experimental models for drug efficacy in viral infections reflect functional
activity of all systems that determine the antiviral resistance of an organism
% of the baseline
80
**
60
*
40
20
0
Control
ULD of antibodies
Tamiflu
Fig. 7.46. Effect of test substances on the survival rate of chickens on day 4 after
infection with avian influenza virus in LD 90. *p=0.03 and **p=0.01 compared to
the control.
163
Ultralow doses
Table 7.10.
Effects of ULD antiIFNγ on the average lifespan of chickens after infection
with avian influenza virus in LD75
Parameter
Survived chickens, %
Average lifespan, days (n=20)
Control
(distilled water)
ULD antiIFNγ
25
55*
4.5±0.3
4.9±0.3
Note. *p=0.0528 compared to the control.
(interferon system, NK cells, and specific cellular and humoral immunity). The
effects of Anaferon on these systems were subjected to detailed analysis in
studying the immunomodulatory properties.
Studying the immunomodulatory activity of Anaferon
Immunotropic properties of Anaferon and Anaferon for children were
studied in accordance with the Manual on Experimental (Preclinical) Study of
New Pharmacological Substances (2000).
Experiments were performed on 372 CBA/CaLac mice (318 males and 54
females), 25 male F1(CBAґC57Bl/6) mice, and 36 male C57Bl/6 mice (22.5
months old, 1820 g), and 120 male and female outbred albino rats (2023 g).
Experiments on females were conducted in studying the delayed type hyper
sensitivity (DTH) reaction and phagocytosis of neutrophils. Other experiments
were conducted on male animals. In vitro experiments were performed with the
suspension of peripheral blood mononuclear cells from 10 healthy donors (22
36 years of age).
To study the immunotropic properties, Anaferon (0.2 ml) was administered
orally for 5 or 10 days. The reference of group of mice was treated with distilled
water (solvent). The control group consisted of intact animals of the same sex.
To study the effect of Anaferon on the humoral immune response, the
mice were intraperitoneally immunized with sheep erythrocytes (SE) in the
minimum dose (5×106/ml). Single immunization was performed at the
beginning of a 5day course of treatment. Experiments were conducted on
healthy and immunosuppressive CBA mice. To induce immunosuppression,
cyclophosphane (CP, Biokhimik) in a single dose of 125 mg/kg (1/2 maximum
tolerated dose, MTD) was injected intraperitoneally at the beginning of
Anaferon treatment. The total number of splenocytes (total cellularity of the
spleen [TCS], ґ106; E. D. Gol’dberg et al., 1992), relative (%) and absolute
number (×106) of antibodyproducing cells (APC) in the spleen (A. I. Cunning
ham, 1965), and antibody (AB) titer (standard hemagglutination reaction, HAR)
were measured on day 5 after immunization.
164
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
To study the effect of Anaferon on the cellular immune response (DTH
reaction), the mice were sensitized with SE (R. V. Petrov et al., 1984). A chal
lenge dose of SE was administered into the hindlimb pad (single subcutaneous
injection) after a 10day course of Anaferon treatment (5th day after sensiti
zation). An equivalent volume of physiological saline was injected to the control
limb. In a special series with the DTH reaction, some animals of the treatment
and control groups received intraperitoneal injection of a specific NO synthase
inhibitor NGmonomethylLarginine (NMMA) on days 4 and 5 after treatment
with the sensitizing dose of SE. The reaction index was estimated in each
animal (relative weight of the SEinjected to control limb, %).
The effect of Anaferon on phagocytic activity of neutrophils and
macrophages in the peritoneal exudate was analyzed after a 10day course of
treatment with this drug or distilled water. The ability of these cells to phago
cytize a 1dayold culture of St. aueus was estimated. The following parameters
were evaluated: percent of microbeengulfing neutrophils or macrophages (pha
gocytic index, PI); and average number of phagocytized staphylococci per cell
(phagocytic number, PN).
In in vitro experiments, the aqueous solution of Anaferon was added to
a complete nutrient medium (CNM, 50 ml/ml). The in vitro effect of Anaferon
on proliferative activity of T lymphocytes and B lymphocytes was studied in the
reaction of spontaneous or mitogeninduced blast transformation (LBTR).
Induced LBTR was conducted with the T cell mitogen (PHA) or B cell mitogen
(pokeweed mitogen) in the suboptimal concentration. Anaferonfree CNM
served as the control. The results were analyzed by the number of pulses per
minute and stimulation index (ratio of radioactivity levels in the presence and
absence of mitogen stimulation).
The in vitro effect of Anaferon on production of interleukin1 (IL1) or
IL2 was estimated from functional activity of IL1 and IL2 in supernatants
of 1dayold mononuclear cell cultures after incubation with the test substance
and lipopolysaccharide (LPS, induction of IL1 production) or PHA (induction
of IL2 production). Control supernatants of 1dayold mononuclear cell
cultures were incubated with LPS, PHA, or test substance. Otherwise, incuba
tion of control samples was performed in CNM. IL1 activity was determined
from comitogenic activity of IL1 (S. B. Mizel, 1980). The activity of IL2 was
evaluated from its ability to stimulate proliferation of lymphoblast cells (R. V.
Petrov et al., 1984).
The suspension of peripheral blood mononuclear cells served as a source
of NK cells to study the in vitro effect of Anaferon on cell function. Functional
activity of NK cells (i.e., ability to lyse tumor cells without presensitization) was
estimated from lysis of myeloblastic K562 cells in the cytotoxic reaction (B. B.
Fuchs radiometric assay; R. V. Petrov et al., 1984).
165
Ultralow doses
The effect of Anaferon on production of IFNγ, IL2, IL4, and IL10
by splenic lymphocytes was studied in mice after a 10day course of drug
treatment. Lymphocytes were incubated in the absence (spontaneous reaction)
or presence of PHA (induced reaction) for 24 h. The content of IFNγ, IL2,
IL4, and IL10 in culture supernatants was measured by enzyme immunoassay
with commercial kits.
Anaferon had a strong immunomodulatory effect. This drug in vivo in
creased the humoral and cellular immune response (course of oral administra
tion) and had a direct stimulatory effect on functional activity of immune cells
(A. V. MartyushevPoklad, 2003).
A 5day course of treatment with Anaferon contributed to an increase in
the humoral immune response to SE (immunization with the minimum dose
of antigen in combination with the first administration of Anaferon). It was
manifested in an increase in the percentage of APC in the spleen (by 1.7 times)
and elevation of hemagglutinin titer in blood plasma (by 1.6 times, p<0.05
compared to the control; Fig. 7.47).
The course of Anaferon treatment significantly activated the humoral
immune response to SE in mice with CPinduced immunosuppression (1/2
MTD). These mice were characterized by a significant increase in the relative
number of APC in the spleen (by 2 times) and elevation of specific
hemagglutinins in blood plasma (by 3 times) compared to control animals (SE
immunization in cytostatic disease with no drug treatment).
These data show that Anaferon increases the humoral immune response
to a complex corpuscular antigen (e.g., during immunization with the minimum
dose of antigen under conditions of cytostaticinduced immunosuppression).
Therefore, Anaferon has a positive effect on the regulatory (activation of type
Antibody titer to SE, log 2
10
+
*
8
6
4
2
0
1
2
3
4
5
Fig. 7.47. Effect of the course of oral Anaferon on the humoral immune response
to SE: production of immune antibodies. Intact animals (1); control (SE, 2); SE +
CP (3); SE + Anaferon (4); and SE + CP + Anaferon (4). p<0.05: *compared to the
control; +compared to the SE + CP group.
166
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
2 T helper cells) and/or effector components of the humoral immune response
(antibody production).
Studying the DTH reaction to sensitization with SE showed that the
course of treatment with Anaferon significantly activates the cellular immune
response (Fig. 7.48).
In control and Anaferonreceiving specimens, the effector component of
this reaction was mainly realized via NOdependent mechanisms. The activation
was abolished after in vivo suppression of NO production by a NO synthase
inhibitor NMMA. The DTH index decreased to 110.5±3.1 and 110.8±3.7%, re
spectively (p<0.05).
The course of treatment with oral Anaferon was accompanied by stimu
lation of the phagocytic immune response in intact mice. The ratio of staphy
lococcusengulfing neutrophils (phagocytic index) increased from 21.3±0.8 to
29.2±3.2% (p<0.05 compared to the control; Fig. 7.49). Phagocytic activity of
peritoneal macrophages significantly increased after administration of Anaferon.
The phagocytic index increased from 12.2±0.9 to 19.7±1.1% (p<0.05). However,
the phagocytic number remained unchanged under these conditions.
Reaction index, %
140
*
130
120
110
100
Control
Anaferon
Fig. 7.48. Effect of the course of oral Anaferon on the cellular immune response
to sheep erythrocytes: DTH reaction. *p<0.05 compared to the control.
a
%
30
b
!
25
!
20
15
10
5
0
Control
Anaferon
Control
Anaferon
Fig. 7.49. Effect of the course of Anaferon treatment on phagocytic activity
(phagocytic index) of neutrophils (a) and macrophages (b). *p<0.05 compared to
the control.
167
Ultralow doses
These data indicate that the course of oral Anaferon stimulates the pha
gocytic activity of neutrophils and macrophages (another component of the im
mune system), which is related to an increase in the ratio of active phagocytes.
These changes reflect the in vivo effect of Anaferon, which is probably
mediated by various regulatory systems of the organism. The next series showed
that Anaferon has a direct effect on immune cells under in vitro conditions.
Addition of Anaferon in combination with the T cell mitogen or B cell
mitogen to cultured MNC (induced blast transformation) was followed by an
increase in the stimulation index for T lymphocytes (from 52.9±9.3 to
88.6±10.5) and B lymphocytes (from 74.7±32.6 to 120.7±39.7). The observed
changes were statistically insignificant due to a wide scatter of data. However,
the drug had no effect on spontaneous blast transformation of lymphocytes.
Therefore, addition of Anaferon to the culture of MNC produces a moderate
comitogenic effect on T lymphocytes and B lymphocytes.
IL1 production in the culture of MNC was much higher after addition
of Anaferon and LPS (compared to cell culturing with LPS). The stimulation
index was 20.9±3.1 and 11.5±3.2, respectively. Oneday incubation of MNC
with Anaferon also stimulated the production of IL1 (vs. IL1 concentration
in supernatants of cell cultures in CNM). However, the effect of Anaferon was
less pronounced than that of the mitogen. During culturing in the presence of
mitogen, the stimulation index increased from 2.7±0.8 to 4.6±1.5.
Anaferon had little effect on in vitro production of IL2 in the culture of
peripheral blood MNC.
Addition of Anaferon to the culture of MNC was followed by a significant
increase in functional activity of NK cells. It was manifested in an increase in
the cytotoxicity index at a target/effector cell ratio of 1:25 (from 63.6±2.7 to
71.7±1.9, p<0.05; Fig. 7.50). Hence, Anaferon increases functional activity of
NK cells that play an important role in the protection from intracellular
parasites and tumor cell growth.
Cytotoxicity index
75
!
70
65
60
55
50
Control
Anaferon
Fig. 7.50. Direct in vitro effect of Anaferon on functional activity of NK cells.
*p<0.05 compared to the control.
168
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
IFNγ is one of the major inductors of NO production by effector cells
of the immune system (U. Boehm et al., 1997).
A quantitative study (enzyme immunoassay) was performed to evaluate ex
vivo production of IFNγ by splenic lymphocytes from Anaferonreceiving ani
mals. Spontaneous production of IFNγ by lymphocytes significantly increased
on days 1 and 37 after treatment with Anaferon. The observed changes were
most pronounced 3 days after Anaferon administration. IFNγ production in
this period increased to 116.34±23.71 pg/ml (p<0.001; Fig. 7.51, a, b), which
exceeded the baseline (14.03±1.31 pg/ml, more than 8 times) and control level
(17.93±1.75 pg/ml, by 6.5 times).
The increase in PHAstimulated production of IFNγ by lymphocytes was
less pronounced. The maximum level was achieved on day 7 after treatment and
exceeded the control by 9.5% (2636.22±63.41 and 2407.32±104.59 pg/ml,
respectively; p<0.05).
These data show that Anaferon significantly increases spontaneous and
mitogenstimulated ex vivo production of functionally active IFNγ (key
cytokine of type 1 T helper cells) by T lymphocytes from intact animals. IFNγ
induces and regulates the cellular immune response and serves as a component
of the interferon system (autonomic system for antiviral resistance). Oral
administration of Anaferon has a modulatory (inducing) effect on the systemic
production of endogenous IFNγ, which confirms the notion that ULD of
antibodies to endogenous regulators have modifying properties.
Spontaneous ex vivo production of IL2 by lymphocytes from treated mice
increased on days 27 after administration of Anaferon. However, statistically
significant differences were found only on days 3 and 7. This parameter increased
from 42.13±2.92 to 61.07±7.65 pg/ml and from 38.71±2.76 to 56.46±5.70 pg/ml,
respectively (p<0.05). The increase in PHAstimulated production of IFN2 by
lymphocytes was less pronounced under these conditions. The maximum level was
observed on days 2 (increase from 589.04±33.91 to 689.41±18.34 pg/ml, by 17%),
5 (increase from 532.68±22.27 to 684.37±29.89 pg/ml, by 28.6%), and 7 (increase
from 502.78±41.10 to 649.47±31.97 pg/ml, by 29.3%, p<0.05; Fig. 7.51, c).
Spontaneous production of IL4 by lymphocytes from experimental animals
significantly increased on days 7 (from 5.37±0.23 to 16.06±4.10 pg/ml, by 198%)
and 10 after administration of Anaferon (from 5.21±0.18 to 7.38±0.80 pg/ml, by
41.7%, p<0.05). Anaferon produced the opposite effect on PHAstimulated
production of IL4 in various periods. The product had a stimulatory effect on days
1 (increase from 86.18±2.99 to 121.87±13.92 pg/ml, by 41%), 2 (increase from
80.48±0.76 to 111.02±8.49 pg/ml, by 38%), and 4 (increase from 84.27±3.62 to
100.67±7.73 pg/ml, by 19%; p<0.05). By contrast, PHAstimulated production of
IL4 decreased on days 6 (from 62.74±5.75 to 52.13±5.49 pg/ml, by 17%) and 10
(from 52.63±5.77 to 38.50±3.75 pg/ml, by 26.8%; Fig. 7.51, d).
169
Ultralow doses
The course of treatment with Anaferon had no effect on spontaneous
production of IL10 by splenic lymphocytes, but caused a significant increase
in PHAstimulated production of this compound on days 15 of study (by 40.6
84.5%, p<0.05 compared to the control; Fig. 7.51, e).
a
Treatment/control
*
6
b
Treatment/control
0.14
5
0.12
4
0.10
*
0.08
3
0.06
2
0.04
1
0.02
0
*
0
1
2
3
4
5
7
10
c
Treatment/control
0.5
6
1
4
5
6
*
*
*
1.5
1.0
0.1
0.5
0
0
1
2
3
4
6
7
10
0.5
1
2
*
*
3
4
5
*
6
7
10
* *
*
0.6
5
* *
e
Treatment/control
1.0
0.8
10
2.5
*
0.2
7
d
2.0
0.3
0.4
3
Treatment/control
*
*
0.4
0.1
2
*
*
0.2
0
0.2
1
2
3
4
5
6
7
0.4
Anaferon administration, days
170
10
Fig. 7.51. Effect of the course of oral
Anaferon on spontaneous (light bars)
and PHAstimulated ex vivo production
(dark bars) of IFNγ (a, b), IL2 (c), IL4
(d), and IL10 by lymphocytes (e). Increa
se, compared to the control.
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
We conclude that the course of treatment with oral Anaferon has a
modulatory effect on the production of not only IFNγ, but also of functionally
related cytokines in type 1 T helper cells (IL2) and type 2 T helper cells (IL4
and IL10). These properties contribute to the ability of Anaferon to stimulate
the cellular and humoral immune response.
It should be emphasized that an increase in spontaneous cytokine production
mainly reflects the basal activity of producing cells. The reserve capacity of
lymphocytes to produce cellular factors is evaluated from the intensity of sti
mulated cytokine production. Type 1 T helper cells exhibited a spontaneous re
sponse to the test substance. By contrast, the reaction of type 2 T helper cells was
revealed under conditions of mitogenic stimulation (Fig. 7.52). These differences
illustrate a change in the reserve capacity, but not in the basal activity of cells.
These data indicate that Anaferon possesses the immunotropic properties,
stimulates cellular and humoral immunity, has a modulatory effect on the balance
between regulatory components of the immune system (type 1 and 2 T helper
cells), and activates the specific (antibody production and cellular cytotoxicity) and
nonspecific immune mechanisms (phagocytosis, NK cells, and interferon system).
A strong effect of Anaferon on the IFN system during therapy of acute
respiratory viral infections was confirmed in further clinical studies of this drug.
Studying the toxicity of ULD antiIFNγ
A preclinical safety study of Anaferon and Anaferon for children was
performed in accordance with the recommendations given in the Manual on
Experimental (Preclinical) Study of New Pharmacological Substances in 2000.
a
Treatment/control
7
6
5
4
3
2
1
0
1 2 3 4 5
1
IFN+IL2
IL4+IL10
6
7
10
Treatment/control
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
0.2
0.4
1 2 3
b
4
5
6
7
10
Anaferon administration, days
Fig. 7.52. Effect of the course of oral Anaferon on an increase in spontaneous
cytokine production (a) by type 1 T helper cells (light bars) and PHAstimulated
cytokine production (b) by type 2 T helper cells (dark bars).
171
Ultralow doses
The study was designed to evaluate acute toxicity, general chronic toxicity,
allergenic properties, genotoxicity, embryotoxicity, reproductive toxicity, and
teratogenicity.
Test products were classified to a group of lowhazard substances. They
had no general toxic and allergic effects (systemic or local) and did not cause
reproductive disorders. Antimutagenic properties of ULD antiIFNγ were re
vealed in the test for somatic mosaicism in wing cells of Drosophila melanogaster
(O. L. Voronova et al., 2002).
Some common effects of ULD antibodies
to endogenous regulators by the example
of antibodies to IFNγ in ULD
This experiment was designed to solve two problems. First, we studied the
specificity of the pharmacological effect produced by ULD antibodies to a
certain endogenous regulator. And second, we evaluated whether the effect of
antibodies depends on their dose.
The induction of endogenous IFNγ after oral administration of ULD
antiIFNγ was estimated ex vivo with the supernatant of splenocytes (O. I.
Epstein et al., 2004).
Experiments were performed on 342 CBA/CaLac mice weighing 1820 g.
The study was conducted with goat polyclonal antibodies to human IFNγ (IgG
fraction) and rabbit polyclonal antibodies to erythropoietin (EP) and human
tumor necrosis factorα (TNFα). ULD antibodies were obtained by the
standard homeopathic method of potentiation. ULD antiIFNγ were admini
stered in the molar (dilution C3; equivalent concentration 106 wt %; 1012 M)
or submolar dose (mixture of dilutions C12+C30+C50; equivalent concentrations
of 1024, 1060, and 10100 wt %; 1030 M). ULD antibodies to EP and TNFα
were used in a mixture of dilutions equivalent to concentrations of 1024, 1060,
and 10100 wt % (1030 M). Test substances (0.2 ml) were given orally for 10
days. Control mice received an equivalent volume of the solvent (distilled water).
The reference group consisted of intact animals.
The ability of substances to modulate IFNγ production by lymphocytes
from experimental animals was studied as described above. IFNγ concentration
in culture supernatants was measured by enzyme immunoassay with Amersham
Pharmacia Biotech kits.
Antibodies to an endogenous regulator IFNγ (oral administration) were
highly potent in inducing the production of this substance (Fig. 7.53). Anti
bodies to IFNγ in molar and submolar doses had the same effect.
Preclinical studies showed that Anaferon has the immunomodulatory and
antiviral properties that are related to the induction of IFNγ and activation of
172
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
IFNγ production,
% of the control
500
*
400
*
300
200
100
0
1
2
3
4
Fig. 7.53. Specificity and dose dependence of the pharmacological effect of ULD
antibodies to an endogenous regulator (antibodies to IFNγ): spontaneous
production of IFNγ by splenocytes after administration of ULD antiIFNγ (molar
and submolar dilutions, 12) and ULD antibodies to erythropoietin (3) and TNFα
(4). *p<0.05 compared to the control.
the key immune mechanisms. The product demonstrated a good safety profile.
Moreover, ULD antibodies to IFNγ produced a specific effect.
7.4. Preclinical study of Artrofoon
The active substances of Artrofoon are antibodies to human TNFα in
ULD. Pharmacological activity of Artrofoon was estimated in a largescale expe
rimental study (Fig. 7.54). Artrofoon not only had a strong antiinflammatory effect
on the model of experimental arthritis, but also possessed the analgetic properties.
Studying the mechanisms for action of Artrofoon revealed that this product affects
the system of proinflammatory and antiinflammatory cytokines. A good safety
ARTROFOON
Antiinflammatory
activity
Analgetic
activity
Acetic
acidinduced
writhing test
Modulation
of cytokine production
Hotplate
test
Adjuvantinduced
arthritis (immune
inflammation)
Collageninduced
arthritis (immune
inflammation)
Fig. 7.54. Preclinical study for the range of pharmacological activity of Artrofoon.
173
Ultralow doses
profile of Artrofoon was demonstrated in a toxicology study. The effect of Artrofoon
on tumor growth and dissemination was evaluated on various tumor models.
Antiinflammatory activity of Artrofoon
Experiments were performed on the following two models of immune
inflammation: adjuvantinduced arthritis and collageninduced arthritis (CIA).
Antiinflammatory activity of Artrofoon on the model of adjuvantinduced
arthritis. Experimental adjuvant arthritis is extensively used to study the
antiinflammatory effect of pharmaceutical substances in Russia (Manual on
Experimental (Preclinical) Study of New Pharmacological Substances, 2000,
2005) and other countries (A. Bendele, 1999; F. A. J. Van de Loo, 2004). In the
present study, immune inflammation was induced by subplantar injection of
complete Freund’s adjuvant (ICN) in a single dose of 0.1 ml. Experiments were
performed on male outbred albino rats weighing 180200 g. Artrofoon (2.5
ml/kg) and reference drug indomethacin (5 mg/kg) were administered 1 day
before injection of complete Freund’s adjuvant and over the whole period of
inflammation. The severity of edema was evaluated for 21 days at 2day inter
vals. The measurements were performed using a plethysmograph (evaluation of
the volume of fluid displaced by the submerged limb).
Antiinflammatory activity of Artrofoon compared well with that of
indomethacin (O. I. Epstein et al., 2001a). The effect was most pronounced on
day 10 after drug treatment. The severity of edema decreased by 43.5 and 55%,
respectively, compared to the control (p<0.05). No significant differences were
found in the effect of Artrofoon and indomethacin.
Antiinflammatory activity of Artrofoon on the model of collageninduced
arthritis. CIA serves as an experimental model of rheumatoid arthritis (RA), which
is extensively used to study the effect of antirheumatic drugs (F. Kato et al., 1996;
A. Bendele et al., 1999; A. C. Tellander et al., 2000). CIA is progressive auto
immune inflammation of the joints. CIA is a suitable model to study new antirheu
matic drugs for the early stage of disease. In the present work, CIA was studied on
male Wistar rats (D. E. Trentham et al., 1977). CIA was induced by twofold sub
cutaneous injection of rat collagen type II (100 mg) in 100 ml incomplete Freund’s
adjuvant. This solution was injected into the tail at a 7day interval. Experiments
were performed in accordance with the manual “Successful Induction of Collagen
Induced Arthritis in Rats” (Chondrex Company). Fortyfive animals of the treat
ment group received intragastrically Artrofoon (0.5 ml) for 90 days beginning from
the 8th day of study (i.e., next day after repeated injection of collagen). Distilled
water (solvent) was given to 45 control rats according to the same regimen.
The inflammatory response in rats was evaluated three times a week on
days 897 of study (i.e., days 190 of treatment with Artrofoon or distilled
174
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Points
3.0
2.5
2.0
1.5
1.0
0.5
0
control
!
!
! !
Artrofoon
!
!
!
!
1 3 6 8 10 13 15 17 20 22 24 27 29 31 34 36 38 41 43 45 48 50 52 55 57 59 62 64 66 69 71 73 76 78 80 83 85 87 90
Time after the start of therapy, days
Fig. 7.55. Effect of Artrofoon on the severity of inflammation in rats with collagen
induced arthritis. *p<0.05 compared to the control.
water). The following parameters were studied: onset of arthritis; number of
animals with arthritis; and count of arthritic joints in each rat. The degree of
damage was expressed in points (04 points for one limb; and 016 points,
overall severity for four limbs).
Artrofoon had a strong antiinflammatory effect during type II collagen
induced immune inflammation (similarly to the previous series). The course of
treatment with Artrofoon was followed by the reduction of joint inflammation
in rats with CIA. It was expressed in a decrease in the severity of joint injury
(Fig. 7.55), number of animals with arthritis, and count of arthritic joints.
Analgetic activity of Artrofoon
Nonsteroid antiinflammatory drugs (NAID), including Artrofoon,
produce the antiinflammatory and analgetic effects. Analgetic activity of
Artrofoon was studied in the acetic acidinduced writhing test and hotplate test.
Analgetic activity of Artrofoon in the acetic acidinduced writhing test.
Acetic acidinduced writhing was studied on male outbred mice weighing
2225 g. The animals received intraperitoneal injection of 0.75% acetic acid
(0.1 ml/10 g). Experimental mice were divided into three groups of 10
specimens each. Distilled water (2.5 ml/kg), Artrofoon (2.5 ml/kg), or indo
methacin (5 mk/kg) was administered intragastrically through a probe for
5 days (last treatment 1 h before injection of acetic acid). The number of
writhing episodes and LC of writhing were estimated for each animal over
15 min after acetic acid injection.
The analgetic effect of Artrofoon was manifested in a significant decrease
in the incidence of writhing episodes compared to the control (by 29%). The
efficacy of Artrofoon was comparable with that of indomethacin. Indomethacin
decreased the number of writhing episodes by 44.2% compared to the control
(p<0.05, Fig. 7.56; O. I. Epstein, 2001a).
175
Ultralow doses
Analgetic activity of Artrofoon in the hotplate test. The hotplate test was
performed on male outbred rats weighing 180200 g. The animals were divided
into three groups of 10 specimens each. The rats received intragastrically 2.5 ml/
kg distilled water (group 1), 2.5 ml/kg Artrofoon (group 2), or 5 mg/kg
indomethacin (reference drug, group 3) for 5 days (daily treatment). In
flammation of the right hindlimb was induced by subplantar injection of 0.1 ml
complete Freund’s adjuvant on day 2 of treatment with test substances. Test sub
stances were administered 2 h after the induction of inflammation. Each animal
was placed on a hot plate (64oC) 3 h and 1 or 3 days after injection of Freund’s
adjuvant. The analgetic effect was evaluated from the time of staying on a hot
plate. LC of paw licking was recorded after the placement of rats to a hot plate.
The nociceptive threshold decreased by 3.7 and 3 times on days 1 and 3
after administration of Artrofoon (p<0.05 compared to the control). The effect
of Artrofoon persisted for a longer time compared to that of indomethacin.
Indomethacin significantly decreased the nociceptive threshold in rats only on
day 1 after treatment (Fig. 7.57; O. I. Epstein, 2001a).
Number of writhing episodes
60
50
!
40
!
30
20
10
0
Control
Indomethacin
Artrofoon
Fig. 7.56. Analgetic activity of Artrofoon on the model of aceticacidinduced
writhing. *p<0.05 compared to the control.
LC, sec
50
!
!
control
indomethacin
Artrofoon
40
30
!
20
10
0
3h
1 day
3 days
Time after injection of complete Freund’s adjuvant
Fig. 7.57. Analgetic activity of Artrofoon in the hotplate test. *p<0.05 compared
to the control.
176
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
These data show that Artrofoon and typical NAID indomethacin have a
similar effect on two models for studying the analgetic properties of pharma
ceutical substances.
Immunotropic effect of Artrofoon
Immune inflammatory diseases, including RA and systemic lupus
erythematosus, are associated with immune dysregulation (C. D. Hamilton,
2005). RA is characterized by the prevalence of type 1 T helper cells and over
production of proinflammatory cytokines IL1 and TNFα. The antiinflam
matory and analgetic properties of some drugs (glucocorticosteroids, GCS; and
monoclonal antibodies to TNFα, Infliximab) are related to their influence on
the production of proinflammatory and antiinflammatory cytokines (e.g.,
inhibition of TNFα and IL1; C. D. Hamilton, 2005). TNFα can induce the
production of IL1, IL6, and IL8. Moreover, TNFα modulates the secretion
of IFNγ, IL4, IL10, and other cytokines. Hence, studying the effect of
Artrofoon (ULD antibodies to TNFα as an active substance) on the cytokine
profile of animals with immune inflammation was necessary to evaluate the
mechanism for action of this product. Prednisolone that belongs to a group of
GCS was used as a reference drug.
Experiments were performed on 200 male CBA/CalAc mice with CIA
(1820 g). Experimental animals were divided into three groups. The mice
received orally 0.2 ml distilled water (14day course, from the day before CIA
induction; control group 1), 0.2 ml Artrofoon (14day course, from the day
before CIA induction; group 2), or 53 mg/kg prednisolone (Nikomed; 11day
course, from the day before CIA induction; group 3).
CIA was induced by subplantar injection of type II collagen (single dose
100 mg) in 50 ml complete Freund’s adjuvant. This solution was injected into
the right hindlimb of mice. The concentrations of proinflammatory and
antiinflammatory cytokines (TNFα, IL1, IL6, IFNγ, IL4, and IL10) in
blood plasma and supernatants of peritoneal macrophages (TNFα, IL1, and
IL6) and lymphocytes (IFNγ, IL4, and IL10) were measured after 3 h and
1, 3, 5, 9, 13, 17, and 21 days. The overall and mean production of cytokines
was estimated by calculating the area under the concentrationtime curve.
Artrofoon had the antiinflammatory and immunomodulatory effect on
mice with CIA and immune inflammation. The activity of Artrofoon compared
well with that of a reference drug prednisolone. The inflammatory index in
Artrofoonreceiving animals decreased by 1027% on days 113 of inflammation
(p<0.05 compared to the control). Administration of prednisolone was followed
by a 3658% decrease in the inflammatory index on days 19 of inflammation
(p<0.05 compared to the control).
177
Ultralow doses
Prednisolone and Artrofoon produced the same changes in systemic
production of cytokines (blood cytokine level; Fig. 7.58).
These data show that Atrtrofoon has the antiinflammatory properties.
Similarly to prednisolone, the effect of Artrofoon is related to the inhibition of
proinflammatory cytokine production.
Safety profile of Artrofoon
The antiinflammatory and analgetic effects of Artrofoon compared well
with those of typical nonsteroid antiinflammatory drugs (indomethacin) and
glucocorticoids (prednisolone). Artrofoon reduces the symptoms of experimental
immune inflammation. Similarly to prednisolone, Artrofoon has a modulatory
effect on cytokine production. However, NAID and GCS cause some side
effects. NAID contribute to gastropathy, allergy, and elevation of BP. Treatment
with GCS is followed by gastropathy, hyperglycemia, and other disorders.
Modern pharmaceuticals that affect TNFα (biological inhibitors of TNFα,
including Infliximab, Etanercept, and Adalimumab) are potent in the therapy
of autoimmune diseases, but cause serious side effects. Druginduced immune
suppression increases the risk of tuberculosis, meningitis, fungal diseases,
histoplasmosis, and sepsis (C. D. Hamilton, 2005).
A complete toxicology study was performed to evaluate the safety profile,
possible side effects, target organs, and safe dosage range of Artrofoon. The
purpose of studies with Artrofoon was to determine the acute toxicity
% of the control
200
!
180
!
160
!
!
140
!
!
120
100
80
!
!
!
!
!
60
40
20
0
TNFα
IL1
control
IL6
Artrofoon
IFNγ
IL4
IL10
prednisolone
Fig. 7.58. Effect of Artrofoon of systemic cytokine production on days 121 of
experimental arthritis. Ordinate: average concentration of cytokines. *p<0.05
compared to the control.
178
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
(experiments on mice and rats), chronic toxicity (6month treatment of rats and
rabbits), reproductive and allergic toxicity (experiments on rats), immunoto
xicity, mutagenicity (chromosomal aberration assay in mouse bone marrow
cells), and genotoxicity (test system for somatic mosaicism in wing cells of
Drosophila melanogaster).
Artrofoon appears to have a good safety profile. An acute toxicity study
showed that this substance in the maximum permissible dose does not cause
death of animals. Drugrelated death of animals was not observed after 6month
treatment with Artrofoon in the highest dose. The product had no toxic effect
on organs and functional systems of experimental animals. A pathomorphologi
cal study did not reveal damage to the internal organs or local irritation of the
gastric mucosa after drug administration. Artrofoon did not cause reproductive
disorders in male and female rats. The embryotoxic effect of Artrofoon was not
observed. Artrofoon had no mutagenic, allergenic, and immunotoxic properties.
Despite good results of a toxicology study, it was important to evaluate the
effect of Artrofoon on the cytokine system (e.g., TNFα). TNFα holds much
promise for oncology. This cytokine possesses antiblastic activity and has a direct
cytotoxic effect on malignant cells. This effect is associated with hemorrhagic
necrosis of the tumor and activation of the immune system (S. M. Navashin et
al., 1989; S. G. Zubkova et al., 2001; R. Horssen et al., 2006). Multicenter
clinical trials confirmed the efficacy of TNFα infusion for therapy of some
tumors (R. Horssen, 2006). However, TNFα may serve as a tumorinducing
agent. The increased activity of TNFα provides favorable conditions for tumor
cell dissemination, including stimulation of angiogenesis (N. Ahmaazadeh et al.,
1990; D. Bertolini et al., 1986; F. Brennan et al., 1989; F. Brennan et al., 1997;
P. Cunha et al., 1992; J. Dayer et al., 1985; C.A. Dinareilo et al., 1986;
M. Feldman et al., 1986; C. Hawonh et al., 1991; E. Lupia et al., 1996;
K. Macnaul et al., 1992; M. Shingu et al., 1993; G. Tilz et al., 1997). A TNFα
inhibitor Infliximab increases the risk of tumor development (P. W. Szlosarek et
al., 2006; L. Biancone et al., 2005).
A special series was performed to determine the antitumor activity of
Artrofoon. It was interesting to evaluate whether Artrofoon may contribute to
tumor progression.
Experiments on tumor models (Lewis lung carcinoma and B17 mela
noma) showed that Artrofoon has no antitumor activity. The drug did not
stimulate primary tumor growth. Moreover, Artrofon had no stimulatory effect
on the development and growth of metastases. By contrast, Artrofoon exhibited
the antitumor and antimetastatic properties under conditions of tumor
transplantation with a small number of cells (E. N. Amosova et al., 2001).
This series showed that Artrofoon does not have an adverse effect on
tumor growth, but exhibits the antitumor activity.
179
Ultralow doses
Preclinical studies showed that the antiinflammatory and analgetic effects
of Artrofoon compare well with those of GCS and NAID. These properties of
Artrofoon are related to its influence on the cytokine system. Toxicity was not
observed after longterm treatment with Artrofoon in doses that exceed the
recommended human dose by more than 1000 times. Artrofoon does not have
the mutagenic, allergenic, immunotoxic, or tumorigenic properties. The results
of experimental studies were confirmed by clinical observations.
7.5. Preclinical study of Epigam
Epigam consists of affinity purified polyclonal antibodies to histamine in
ULD.
Studying the antiulcer activity of pharmaceutical products requires a stage
of investigations on animals with experimental ulcers. The existence of various
etiopathogenetic factors for ulcer disease and no general agreement concerning
the cause, pathogenetic mechanism, and course of this disorder make it difficult
to develop a general model for all manifestations of the pathological processes.
Antiulcer activity of drugs is usually studied on several models of ulcer disease
that differ in etiology and specific characteristics. The antiulcer effect of Epigam
was evaluated on several models of acute and chronic ulcers. Studying the
specific activity of an antiulcer drug suggests the evaluation of its effect on
secretory and motorevacuation function of the gastrointestinal tract (GIT).
Moreover, these experiments were designed to determine the analgetic,
spasmolytic, antiedematous, and antitumor effects of Epigam (Fig. 7.59).
Experiments were performed on 215 outbred albino rats (males, 300350
g; and females, 220240 g) and 390 outbred mice (females, 2224 g). The
animals were obtained from the Laboratory of Experimental Biological
Modeling (Institute of Pharmacology, Tomsk Research Center, Siberian Division
of the Russian Academy of Medical Sciences).
Antiulcer activity of Epigam
Antiulcer activity of Epigam on the model of acute ulcers. The effect of
Epigam on acute ulcerative lesion of the gastric mucosa was studied on animal
models for various etiologic factors of ulcer disease, including stress (neurogenic
ulcer), NAIDinduced gastric mucosal injury (indomethacin, acetylsalicylic
acid, butadiene), alcohol consumption, and acidpepsin factor (Shay ulcer).
On the model of acute ulcers, Epigam was administered intragastrically
to mice (0.3 ml) and rats (0.5 ml). The product was given once or several times
for 58 days (last treatment 1 h before the induction of ulcerative lesions).
180
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
EPIGAM
Antiulcer
activity
Antiinflammatory
activity
Acute ulcers
Antiedematous
Chronic ulcer
Antiproliferative
Effect on functional activity
of the gastrointestinal tract
Spasmolytic
Analgetic activity
Secretory
Motorevacuation
Fig. 7.59. Studying the pharmacological properties of Epigam
Control animals received distilled water (solvent) according to the same
regimen.
The animals were killed by cervical dislocation under ether anesthesia or
ether overdose. The number and area of gastric mucosal injury were estimated
macroscopically. They were differentiated into petechia (less than 1 mm), large
ulcers (more than 1 mm), and linear lesions. The average number of ulcerative
lesions in one animal and percentage of mice with ulcers were evaluated as
described elsewhere (Ya. I. Khadzhai, 1962). The Pauls index was determined
as an integral criterion for the number of lesions (F. Pauls et al., 1947). Antiulcer
activity of Epigam was estimated as the control/treatment ratio of the Pauls
index (G. V. Obolentseva et al., 1974).
Neurogenic ulcer. Stress is one of the major etiologic factors for GIT
diseases. Immobilization is a strong stress factor, which causes ulceration in GIT.
Stressinduced ulceration during immobilization is associated with nervous and
humoral changes (I. S. Zavodskaya et al., 1981). The animals were suspended
by application of dressing forceps to the skinfold of the neck. Partial immo
bilization for 22 h was followed by ulceration of the gastric mucosa (Yu. I.
Dobryakov, 1978). The bedding and food were removed from mouse cages 1 day
181
Ultralow doses
before stress. The antistress effect of test substances was estimated from the
number of gastric ulcers and weights of the adrenal glands, thymus, and spleen.
Preventive treatment with Epigam for 6 days had a strong gastroprotective
effect under conditions of neurogenic ulceration. The antiulcer activity of
Epigam was 3.52 U. Epigam decreased the number of petechia and large ulcers
by 3.5 and 5.5 times, respectively, compared to the control (p<0.05). The aver
age number of ulcers in animals of the Epigam group was 3.8fold lower than
in the control (p<0.05, Fig. 7.60; J. L. Dugina et al., 2002; 2003a,b,c; S. G.
Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).
It should be emphasized that single administration of Epigam prevented
the stressinduced involution of the spleen and thymus and had a normalizing
effect on the weight of the adrenal glands (no differences from the intact
control).
Indomethacininduced ulcer. Mucosal damage in GIT is one of the most
common side effects of NAID (e.g., indomethacin). Ulcerogenic activity of
these drugs is associated with barrier dysfunction of the mucous membrane,
inhibition of glycosaminoglycan synthesis, blood flow disorders, impaired ability
of the mucosa for reepithelization, and increased secretion of acid and pepsin
(F. Bates, 1989). The gastroprotective effect of Epigam on the model of indo
methacininduced mucosal injury was studied with two species of animals. Indo
methacininduced damage to the gastric mucosa in mice was produced by
intragastric administration of indomethacin in a dose of 20 mg/kg twice daily
at a 4h interval. The number and severity of destructive changes were estimated
after 18 h (O. I. Epstein et al., 1001b). Indomethacininduced damage to the
gastric mucosa in rats was produced by intragastric administration of
indomethacin in 1 ml physiological saline (single dose 60 mg/kg; F. Bates et al.,
1989). The number of ulcers was evaluated 6 h after the last treatment with
indomethacin.
Average number of ulcers per mouse
10
control
Epigam
8
6
*
4
*
2
0
1
2
3
Fig. 7.60. Antiulcer activity of Epigam after prophylactic intragastric administration
to female mice. Neurogenic ulcer (0.3 ml × 1 day, 1); neurogenic ulcer (0.3 ml × 6
days, 2); and indomethacininduced damage (0.3 ml × 6 days, 3). *p<0.05 and
**p<0.01 compared to the control.
182
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
After preventive treatment with Epigam for 6 days, 25% mice did not have
gastric ulcers (p<0.01). The average number of ulcers in treated mice decreased
by 2.4 times (p<0.05; Fig. 7.61). Epigam also prevented the formation of large
ulcers and petechia. The average number of these lesions in mice of the Epigam
group decreased by 5.2 and 2.0 times, respectively (p<0.05). Moreover, prophy
lactic administration of Epigam completely abolished the formation of linear
ulcers. Under these conditions, the antiulcer activity of Epigam was 3.22 U.
Prophylactic administration of Epigam decreased the average number of
ulcers in rats with indomethacininduced damage to the gastric mucosa (by 1.33
times, p<0.01; Fig. 7.60). The length of petechia, linear lesions, and large ulcers
decreased by 1.4, 1.5, and 1.7 times (p<0.05), respectively, compared to the con
trol. Fig. 7.62 illustrates that treatment with Epigam was followed by a 1.5fold
decrease in the severity of gastric mucosal injury (p<0.05; J. L. Dugina et al., 2002,
2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).
Average number of ulcers per rat
25
control
Epigam
20
**
15
*
*
**
10
5
*
0
1
2
3
4
5
Fig. 7.61. Antiulcer activity of Epigam after prophylactic intragastric administration
to rats. Indomethacininduced damage (0.5 ml × 5 days, 1); acetylsalicylic acid
induced damage (0.5 ml × 7 days, 2); butadieneinduced damage (0.5 ml × 7 days,
3); ethanolinduced damage (0.5 ml × 8 days, 4); and Shay ulcer (0.5 ml × 7 days,
5). *p<0.05 and **p<0.01 compared to the control.
mm
75
control
treatment
60
45
*
30
15
**
0
1
2
3
Fig. 7.62. Severity of gastric mucosal injury in female rats after prophylactic
treatment with Epigam. Indomethacininduced damage (0.5 ml × 5 days, 1); buta
dieneinduced damage (0.5 ml × 7 days, 2); and ethanolinduced damage (0.5 ml
× 8 days, 3). *p<0.05 and **p<0.01 compared to the control.
183
Ultralow doses
Acetylsalicylic acidinduced ulcer. Gastric ulcer in rats was induced by two
fold intragastric administration of acetylsalicylic acid in a dose of 150 mg/kg at
a 4h interval. The animals were examined after 24 h. A direct effect of
acetylsalicylic acid on the gastric mucosa probably results in the loss of pro
tective and barrier properties, desquamation of the epithelium, and formation
of a large area of erosions and ulcers.
Petechia, linear ulcers, and large ulcers were revealed in all rats of the
control group 24 h after ulcerogenic treatment with acetylsalicylic acid. The
average number of ulcers was 15.31±1.89. Prophylactic treatment with Epigam
was followed by a decrease in the number of petechia (by 1.5 times, p<0.05) and
average number of gastric lesions per rat (by 1.7 times, p<0.05 compared to the
control; Fig. 7.61). The gastroprotective effect of Epigam was manifested in a
decrease in the incidence of linear ulcers (by 4.5 times, p<0.05) and number of
animals with this type of gastric lesions. Under these conditions, the antiulcer
activity of Epigam was 1.84 U (J. L. Dugina et al., 2002, 2003a,b,c; S. G.
Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).
Ethanolinduced ulcer. Ethanolinduced gastric mucosal injury in rats was
produced by single intragastric administration of 1 ml 96o ethanol per 200 g
body weight (C. F. BouAbbound et al., 1988). The antiulcer effect was eva
luated 1 h after ethanol administration.
Gastric lesions were found in 100% animals of the control group after
ethanol administration. The average number of ulcers was 14.00±0.95. Epigam
significantly decreased the area and severity of gastric mucosal injury. The
number of petechia, linear ulcers, and large ulcers in Epigamreceiving animals
decreased by 40.6, 72.1, and 39%, respectively, compared to control specimens.
The average number of ulcers in rats of the Epigam group was 1.9fold lower than
in control animals (p<0.01). The antiulcer activity of Epigam was 2.01 U (Fig.
7.61). The total length of mucosal lesions per rat decreased by 5.8 times (p<0.05).
The severity of damage decreased by 82.5% (p<0.05, Fig. 7.60; J. L. Dugina et al.,
2002, 2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).
Butadieneinduced ulcer. The rats received intramuscular injection of
butadiene in a single dose of 300 mg/kg (15% suspension in acetone). The
severity of ulceration was estimated after 24 h.
Butadieneinduced ulceration of the gastric mucosa was found in control
and treated animals. Epigam had an antiulcer effect on the model of butadiene
induced ulcer. The number of large ulcers and average number of ulcers in rats
of the Epigam group decreased by 1.8 and 1.4 times, respectively, compared to
the control (p<0.05). The severity of gastric mucosal injury in Epigamreceiving
animals decreased by 1.2 times (Fig. 7.62).
Shay ulcer. Laparotomy was performed along the white line of the anterior
abdominal wall under light ether anesthesia. The pylorus was ligated (H. Shay
184
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
et al., 1945). The tissues were sutured in layers. The animals were killed by ether
overdose after 16 h. The forceps were applied to the esophagus. The stomach
was removed. The gastric mucosa was examined under a microscope.
Administration of Epigam was followed by a significant decrease in the
number of animals with ulcers and average number of ulcers per rat. The
average number of large ulcers in 75% control rats was 2.1±0.64. After treatment
with Epigam only 57% animals had large ulcers. The average number of large
ulcers in Epigamreceiving rats decreased to 1.29±0.61 (p<0.05). The number
of rats with petechia decreased from 63 to 14% (p<0.05). Moreover, linear ulcers
were not detected after administration of Epigam. The average number of ulcers
in rats of the Epigam group decreased by 2.4 times (p<0.05; Fig. 7.61). Epigam
was potent in reducing the severity of gastric mucosal injury. The area of large
ulcers and petechia decreased by 2.3 and 7.3 times, respectively (p<0.05). The
average area of gastric lesions decreased by 2.5 times (p<0.05). Hence, the
severity of injury in treated animals decreased by 59.3% (p<0.05). The antiulcer
activity of Epigam was 3.31 U.
These data show that Epigam has a strong antiulcer effect on various mo
del of acute ulcerative lesion, which does not depend on the etiology of pa
thological process. This effect is manifested the prevention of ulceration and sig
nificant decrease in the severity of gastric mucosal injury (J. L. Dugina et al., 2002,
2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).
Antiulcer activity of Epigam on the model of chronic ulcer. Acid acetic
induced chronic ulcer was produced in rats (S. I. Budantseva, 1973; A. A.
Kalinichenko, 1973). Laparotomy was performed along the white line of the
anterior abdominal wall under light ether anesthesia. A solution of acetic acid
(0.05 ml, 5%) was administered into the subserous layer of the anterior wall of
the stomach. Epigam in a daily dose of 0.5 ml was administered intragastrically
for 21 days (immediately after the induction of ulceration). Control rats received
distilled water (solvent) according to the same regimen. The animals were killed
by ether overdose on days 7, 14, and 21. The antiulcer effect of Epigam was
estimated from the area of ulcerative lesions. For a morphological study, the
stomach was fixed in formalin and embedded into paraffin. Connective tissue
in deparaffinized sections (5 m in width) was stained with hematoxylin and
eosin by the Van Gieson technique. Acid glycosaminoglycans were stained by
Schiff reagent. RNA was stained by the method of Brachet with methyl green
pyronin (G. A. Merkulov, 1969). During examination of Brachetstained
samples, tissue basophils were counted in the ulcer margin (per 1 mm2 section).
By the number of granules, they were differentiated into strongly granulated,
partially degranulated, and strongly degranulated cells.
The effect of Epigam on rats with acetic acidinduced chronic gastric
ulcers was evaluated on days 7, 14, and 21. Mucosal ulcers of the stomach were
185
Ultralow doses
detected macroscopically in all animals of the control group on day 7 after
ulcerogenic treatment. The depth of ulcers was 12 mm. The area of the bottom
was 23.52±3.38 mm2. A swollen area around the ulcer was associated with
inflammatory infiltration and edema of the mucous membrane (Fig. 7.63). The
average size of ulcerandswelling in control animals was 98.04±15.04 mm2.
Ulcer size in control specimens remained practically unchanged on day 14. The
areas of ulcerandswelling and bottom decreased by 21.8 and 20.4%,
respectively. Healing of ulcerative lesions in control animals was observed only
on the 21st day. In this period the area of ulcerandswelling and ulcer bottom
decreased by 44.4 and 75.7%, respectively (compared to day 7). The size of
ulcerative lesions in Epigamreceiving rats was much lower than in control animals
(Fig. 7.63). On day 7 the area of ulcerandswelling and ulcer bottom decreased
by 14.6 and 19.9%, respectively, compared to the control. It should be emphasized
that ulcer healing in Epigamreceiving rats was revealed on day 14 after the induc
tion of ulceration. The area of ulcerandswelling and ulcer bottom decreased
by 61.2 (p<0.05) and 75.4% (p<0.01), respectively, compared to the control.
The degree of gastric ulcer healing in Epigamreceiving rats was much
higher than in the control (J. L. Dugina, 2002, 2003a,b,c; S. G. Krylova, 2002a;
O. I. Epstein, 2001b; J. L. Dugina, 2002). An ulcerative lesion was filled with
granulation tissue. The tissue had a greater degree of maturity, included a
smaller number of cells, and consisted of thick collagen fibers. The content of
RNA and glycosaminoglycans in the cytoplasm of epithelial cells was higher
compared to the control. Chief cells of the fundal glands had a normal
structure. RNA content in the cytoplasm of these cells was higher than in the
control. Hence, Epigam accelerates healing of experimental ulcers and
contributes to the formation of glycosaminoglycans in the stomach wall.
a
mm2
120
b
mm2
30
100
24
80
18
60
40
12
*
*
*
6
20
0
7
14
*
0
21
7
14
21
Time, days
control
Epigam
Fig. 7.63. Effect of Epigam on healing of acetic acidinduced chronic gastric ulcer
in male rats. Area of ulcerandswelling (a) and bottom of the ulcer (b). *p<0.05
and **p<0.01 compared to the control.
186
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Healing of chronic gastric ulcer was probably associated with an Epigam
induced increase in the number of mast cells in the zone of ulcerative lesion on
day 7 of study (by 1.4 times compared to the control, p<0.05). Similar results
were obtained on the 14th day. The count of mast cells in the gastric mucosa
increased by 1.5 times after administration of Epigam (p<0.05). These changes
should be considered as a positive event, which accelerates healing of the
mucous membrane. Previous studies showed that mast cells regulate trophic
processes in the gastric mucosa and play a role in ulcer healing (M. Barczyk et
al., 1995; S. Nakajima et al., 1996).
The model of chronic ulcers is most adequate to study the pathogenesis
of peptic ulcer disease. These experiments revealed a strong antiulcer effect of
Epigam.
Effect of Epigam on the functional state of GIT
Evaluation of secretory function of the stomach. The effect of Epigam on
gastric secretion in rats was studied on the model of H. Shay ulcer. Surgery was
performed under light ether anesthesia. The pylorus was ligated. The forceps
were applied to the esophagus. The stomach was removed after 16 h. The
stomach contents were placed in tubes and centrifuged at 1500 rpm for 10 min.
The following parameters were measured: volume and pH of gastric juice; total
acidity; and discharge of hydrochloric acid and pepsin (method of V. N.
Tugolukov, 1965).
Prophylactic administration of the test substance in a daily dose of 0.5 ml
for 5 days was followed by the decrease in gastric juice secretion over 1 h. These
changes reflect a decrease in the strain of gastric secretion. The increase in
gastric juice pH (p<0.05) was associated with a significant decrease in acidity.
Studying the proteolytic activity of gastric juice showed that Epigam significantly
decreases the discharge of free hydrochloric acid (by 9 times, p<0.01 compared
to the control). Basal H+ concentration was 3.7fold lower compared to the
control (p<0.05). These data show that preventive treatment with Epigam before
the exposure to an aggressive factor (pylorus ligation) is followed by a decrease in
acidpepsin aggression of gastric juice in rats (Table 7.11; J. L. Dugina et al., 2002,
2003a,b,c; S. G. Krylova, 2002a; O. I. Epstein, 2001b; J. L. Dugina, 2002).
Evaluation of excretory function of the intestine. Excretory function of the
intestine was studied by the method of G. V. Obolentseva (1984). The mice
received Epigam in 0.2 ml suspension of activated charcoal (10 mg/ml). The
appearance of blackcolored feces was considered as a positive result. The effect
in each animal was estimated after 3, 6, and 24 h and expressed in points.
Fourfold treatment with Epigam had a moderate laxative effect. The
laxative effect was most pronounced 3 and 6 h after the last administration of
187
Ultralow doses
Table 7.11.
Effect of prophylactic treatment with Epigam (daily dose 0.5 ml, 5 days) on
gastric secretion in rats with ligated pylorus (M±m)
Discharge
of free
hydrochloric acid,
mmol/liter/h
Gastric juice
secretion, ml/h
рН
H+ concentration,
mmol/liter
Control
0.87±0.14
2.03±0.20
17.21±6.80
3.902×104±
2.16×104
Epigam
0.80±0.17
2.97±0.33*
4.71±3.70*
4.35×103±
0.35×104**
Group
Note. *p<0.05 and **p<0.01 compared to the control.
Epigam (O. I. Epstein, 2001b; J. L. Dugina et al., 2002, 2003a,b,c; S. G.
Krylova, 2002a; J. L. Dugina, 2002).
Evaluation of motorevacuation function of GIT. Motorevacuation func
tion of the stomach and intestine was studied by the method of “labels” (G. P.
Coopman et al., 1977). Activated charcoal suspension of (0.5 ml, 10%) in 2%
potato starch served as a label and was administered into the digestive tract of
mice. The animals were killed 10 min after treatment. The effect of test
substance was evaluated.
Due to BaCl2induced spastic contraction of pyloric smooth muscles, a
suspension of activated charcoal remained in the stomach of 50% animals over
the first 10 min after injection of BaCl2. The degree of charcoal distribution in
the intestine of control animals was 31.43%. Administration of Epigam was
followed by an increase in evacuation function of the small intestine. Charcoal
transit in the intestine of Epigamreceiving mice was 52.3% (p<0.05) greater
than in control animals (O. I. Epstein, 2001b; J. L. Dugina et al., 2002,
2003a,b,c; S. G. Krylova, 2002a; J. L. Dugina, 2002).
Evaluation of spasmolytic activity. Spasmolytic activity was studied by the
method of J. Setnicar (1959). Experiments were performed on healthy mice and
animals with indomethacininduced ulcer. The animals received intraperitoneal
ly 0.2 ml 0.1% BaCl2. Activated charcoal suspension of (0.5 ml, 10%) in 2%
potato starch was administered intragastrically. The animals were killed after
10 min. Spasmolytic activity of the product was evaluated.
Epigam was potent in increasing the motor activity of GIT. In control
mice, the suspension of charcoal was shown to pass 73.1% intestinal length over
10 min. This parameter increased to 98.9% in Epigamreceiving animals
(p<0.01). A positive effect of Epigam on animals with indomethacininduced
ulcer was manifested in the increase in motor activity of GIT (by 25.8%, p<0.05
compared to the control; O. I. Epstein, 2001b; J. L. Dugina et al., 2002,
2003a,b,c; S. G. Krylova, 2002a; J. L. Dugina, 2002).
188
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Analgetic activity of Epigam
Analgetic activity of Epigam was studied on outbred mice with acetic
acidinduced writhing. The nociceptive response was induced by intraperitoneal
injection of acetic acid (0.2 ml, 3% solution; C. Cashik et al., 1977). The
reaction of animals to nociceptive stimulation was estimated from the number
of writhing episodes (20min period after acetic acid injection) and writhing LC.
Administration of Epigam in a dose of 0.3 ml was followed by a signifi
cant decrease in the number of writhing episodes (by 71.29%, p<0.05). Writhing
LC was shown to increase under these conditions. The analgetic effect of a
reference drug indomethacin was much smaller (Table 7.12; O. I. Epstein, 2001b;
J. L. Dugina et al., 2002, 2003a,b,c; S. G. Krylova, 2002b; J. L. Dugina, 2002).
Antiinflammatory activity of Epigam
Antiedematous effect of Epigam. The antiedematous effect of Epigam
was studied on the model of agarinduced edema, which results from the
induction of prostaglandin biosynthesis (S. K. Bhattacharya et al., 1989). The
acute inflammatory response (edema) was induced by subplantar injection of 1%
agar solution (50 ml) into the hindlimb pad. The animals were killed after 5 h.
The paws were cut off to the level of the knee joint and weighted on a torsion
balance. The antiinflammatory effect was evaluated from a change in the volume
of edema and expressed in percent of the control.
At the peak of agarinduced inflammation (5 h after agar injection), single
administration of Epigam caused a significant decrease in the degree of edema
(by 33.6%). We conclude that Epigam has a moderate antiinflammatory effect
on mice with agarinduced inflammation (Table 7.13; O. I. Epstein, 2001b;
J. L. Dugina et al., 2002, 2003a,b,c; S. G. Krylova, 2002b; J. L. Dugina, 2002).
Antiinflammatory (antiproliferative) activity of Epigam. The course of
chronic proliferative inflammation was studies in experimental rats. A sterile
Table 7.12.
Effect of Epiogam on pain sensitivity of female outbred mice on the model
of acetic acidinduced writhing (M±m)
Group
Control
Indomethacin
(10 mg/kg, 7 days)
Epigam (0.3 ml, 7 days)
Number of writhing
episodes over 20 min
Decrease in pain
sensitivity, %
24.00±3.67
Time to onset
of writhing, min
–
3.75±0.25
12.83±2.06*
46.54
4.00±0.68
6.89±1.71**
71.29
4.5±0.8
Note. *p<0.05 and **p<0.01 compared to the control.
189
Ultralow doses
Table 7.13.
Глава 7. Экспериментальная фармакология препаратов сверхмалых доз антител
Antiinflammatory (model of agarinduced edema) and antiproliferative
effects (model of cotton pellet granuloma) of Epigam in various doses (%)
Epigam dose
Parameter
0.3 ml, single treatment
0.5 ml, 8day course
33.6
—
Inhibition of proliferation
—
24.0*
Inhibition of exudation
—
30.0
Reduction of edema at the peak
of inflammation
Note. *p<0.05 compared to the control
cotton pellet (13 mg in weight) was implanted subcutaneously on the back of
animals (R. Meier et al., 1950). Epigam in a daily dose of 0.5 ml was
administered intragastrically for 8 days. The animals were killed on day 8.
Cotton pellets and surrounding granulation tissue were removed, weighted on
a torsion balance, and dried to a constant weight at 37oC. The proliferative
response was estimated from the difference in the weights of dried granuloma
and initial weight of a cotton pellet. The exudative response was evaluated from
the difference in wet granuloma weight and dry granuloma weight.
The cotton pellet granuloma test showed that Epigam decreases the initial
and dry weight of granulationandfibrous tissue. It should be emphasized that
Epigam had an inhibitory effect on proliferation in the early proliferative phase
of inflammation (p<0.05, Table 7.13). The degree of exudation tended to
decrease in Epigamreceiving rats. The weight of exudate in these animals
decreased by 1.5 times compared to the control. These data show that Epigam
produces a moderate antiproliferative effect (O. I. Epstein, 2001b; J. L. Dugina
et al., 2002, 2003a,b,c; S. G. Krylova, 2002b; J. L. Dugina, 2002).
The data suggest that Epigam affects the histaminedependent activation
of histamine H1, H2, and H3 receptors (Fig. 7.64). Epigam decreases acid
pepsin aggression of gastric juice, has a normalizing effect on motorevacuation
function of GIT, and possesses the antiinflammatory and analgetic properties.
Therefore, the antiulcer effect of Epigam is realized via central (H3 receptors)
and peripheral regulatory mechanisms (H1 and H2 receptors) of histamine
mediated functions.
7.6. Preclinical study of Afala
Antibodies to human prostatespecific antigen (PSA) in ULD constitute the
active substance of Afala. PSA is a promising molecular target in benign prostatic
hyperplasia (BPH; E. P. Diamandis, 2000; S. P. Balk et al., 2003). Expression of
190
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Histamine
H3
H1
4
3
1
H2
py
lo
ri
1
H.
2
4
2
2
H+
TS
NK
SMC
P
ECL
Vessel
BP
Fig. 7.64. Mechanisms for action of Epigam. Effect of Epigam on acidpepsin ag
gression of gastric juice (1); antiinflammatory effect (2); effect of Epigam on mo
torevacuation function of GIT (3); and analgetic effect of Epigam (4). His, histamine;
H 1, H 2, and H 3, histamine receptors; P, parietal cells; ECL, enterochromaffin cells;
BP, basophils; SM cells, smooth muscle cells; NK, natural killer cells; Ts, T sup
pressor cells.
this serine protease is regulated by androgens. PSA has antiangiogenic activity (A.
H. Fortier et al., 1999), plays a role in the regulation of stromal cell growth in the
prostate (D. M. Sutkowski et al., 1999), and modulates the expression of genes
for tumor growth in prostate tissue (B. Bindukumar et al., 2005).
Experimental preclinical studies on the models of acute and chronic
aseptic inflammation and hormoneinduced inflammation of the prostate
revealed that Afala exhibits the prostatotropic properties, reduces the severity of
edema and inflammation, has a normalizing effect on prostate function, and
prevents prostate sclerosis (Fig. 7.65). A toxicology study showed that Afala has
a good safety profile.
Antiinflammatory effect of Afala
Model of acute aseptic inflammation of the prostate. Experiments were
performed on 45 male outbred albino rats aging 3 months. The animals were
191
Ultralow doses
AFALA
Antiinflammatory
effect
Prostatotropic
effect
Young rats
Model of acute
prostatitis
Model of chronic
prostatitis
ГHormoneinduce
inflammation
Young
gonadectomized rats
Fig. 7.65. Studying the pharmacological activity of Afala.
divided into three groups. Group 1 rats (n=20) received Afala in a dose of 1.5
ml for 10 days. Distilled water in a dose of 1.5 ml was administered to 20
animals of group 2 (10day course). Group 3 consisted of 5 intact specimens.
On day 3 after administration of test substances, the prostate was sutured with
a silk thread to induce acute inflammation (B. A. Vertapetov, 1970). The animals
were killed 7 days after surgery. The internal organs were examined visually. A
histological study was performed with the prostate (A. P. Milovanov, 1986; G.
G. Avtandilov, 1990). Zn2+ concentration in prostate tissue was measured by
means of emission spectral analysis (G. V. Kashkan, 1988).
Administration of Afala prevented the formation of adhesions between the
prostate and surrounding tissues, had a normalizing effect on the density of the
prostate gland, and decreased the severity of edema and hyperemia (Fig. 7.66).
Zn2+ concentration in the ventral prostate of Afalareceiving rats was fourfold
higher than in control specimens (Fig. 7.67). Hence, Afala improves function
of the prostate in rats.
These data show that Afala has an antiinflammatory effect during acute
aseptic inflammation of the prostate.
Model of chronic prostatitis. Experiments were performed on 50 male
outbred rats weighing 250 g and aging 2.5 months. Chronic prostatitis was
induced by suturing of the prostate with a silk thread (B. A. Vertapetov, 1970).
The treatment group consisted of 20 animals. Afala therapy in these rats was
started 1 month after surgery. Afala in a daily dose of 1.5 ml was administered
intragastrically for 1 and 1.5 months. Control animals (n=20) were treated with
192
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
% of the control
100
!
80
!
!
60
40
20
0
Vessels
Edema
Fig. 7.66. Effect of Afala on the ratio of structural elements and severity of edema
in rats with acute aseptic inflammation of the prostate. *p<0.05 compared to the
control.
distilled water in a daily dose of 1.5 ml for 1 and 1.5 months after surgery. Ten
animals remained intact. The treated (n=10), control (n=10), and intact rats
(n=5) were killed 2 and 2.5 months after surgery (1 and 1.5 months after
administration of the test substance). The internal organs were examined
visually. The weight, volume, weight ratio, and density of the ventral prostate
were measured. The prostate was subjected to a histological study as described
by A. P. Milovanov (1986) and G. G. Avtandilov (1990). Zn2+ concentration in
prostate tissue was measured in 5 animals of each group (G. V. Kashkan, 1988).
Previous studies showed that chronic prostatitis is accompanied by a decrease
in sexual activity. In the present work, sexual activity of animals was estimated
by the method of Ya. Buresh et al. (1991).
Administration of Afala for 1 and 1.5 months contributed to a threefold
increase in Zn2+ concentration in the ventral prostate (p<0.05 compared to the
control, Table 7.14). Atrophy of prostate tissue due to chronic inflammation
(decrease in the relative area of the secretory epithelium) was less pronounced
in rats receiving Afala for 1.5 months (as compared to control specimens).
mg%
2.5
2.0
1.5
1.0
0.5
0
Control
Afala
2+
Fig. 7.67. Effect of Afala on Zn concentration in the ventral prostate of rats with
acute inflammation. *p<0.05 compared to the control.
193
Ultralow doses
Table 7.14.
Effect of Afala on Zn2+ concentration in the ventral prostate and area of
the epithelium in the distal region of the prostate in rats with chronic pro
statitis (M±m)
Parameter
Intact
Area of the epithelium in the
distal region, %
Zn2+ concentration, mg/liter
after 30 days
after 30 days
after 45 days
after 45 days
23.1±1.55
Control (distilled water)
19.2±2.80
16.60±0.96*
6.0±1.0
5.6±0.6
Treatment (Afala)
21.80±2.07
19.20±0.48*+
19.9±2.7+
20.9±2.3+
Note. p<0.05: *compared to intact animals; +compared to the control.
Sexual activity of animals with chronic prostatitis was much higher after
administration of Afala for 1.5 months (compared to the control group).
These data show that Afala has an antiinflammatory effect during chronic
prostatitis, reduces the degree of atrophy, and improves function of the prostate
(T. G. Borovskaya et al., 2001). Afala had a normalizing effect on sexual
behavior of animals, which was suppressed due to chronic prostatitis.
Prostatotropic effect of Afala
Model of gonadectomized infantile male rats with testosterone propionate
induced androgen deficiency. Experiments were performed on gonadectomized
infantile male rats aging 2325 days. Test substances were administered once
daily for 7 days. The animals were divided into the following groups:
1) intact specimens;
2) gonadectomized specimens, olive oil subcutaneously;
3) gonadectomized specimens, testosterone propionate (0.5 mg/kg sub
cutaneously) and distilled water (intragastrically); and
4) gonadectomized specimens, testosterone propionate (0.5 mg/kg sub
cutaneously) and Afala (intragastrically).
The animals were killed after the last treatment with test substances. The
internal organs were examined. The weight and weight ratio of target organs
were estimated (ventral prostate and seminal vesicles). The weight indexes were
calculated as the ratio of the weight of the organ to body weight.
The weight ratios of the prostate and seminal vesicles in gonadectomized
animals were lower (by 42.8% for the ventral prostate; and by 33.3% for the
seminal vesicles) than in intact specimens (Fig. 7.68). Testosterone propionate
contributed to an increase in the weight of androgensensitive organs in
gonadectomized male rats. The weight ratios of the prostate and seminal vesicles
in animals of the “distilled water + testosterone” group were 188.8 and 362.5%
194
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
%
300
а
b
+
200
х
+
100
!
!
0
1
2
3
1
2
3
Fig. 7.68. Effect of Afala on the weight ratio of the ventral prostate (a) and seminal
vesicles (b) in gonadectomized animals. 100%, intact animals. Treatment of rats
with olive oil (1), distilled water and testosterone (2), and Afala and testosterone
(3). p<0.05: *compared to intact animals; + compared to the olive oil group;
x
compared to animals receiving distilled water and testosterone propionate.
of those in rats of the “olive oil” group, respectively (p<0.05, Fig. 7.69). The
weight ratios of the ventral prostate and seminal vesicles in gonadectomized
animals of the “Afala + testosterone propionate” group were 135.2 and 103%
of those in rats of the “testosterone propionate” group, respectively (p=0.05).
These data show that Afala potentiates the androgenic effect of
testosterone on the prostate under conditions of androgen deficiency. However,
Afala does not modulate the androgenic effect of this drug on the seminal
vesicles (T. G. Borovskaya et al., 2001).
Prostatotropic effect of Afala in young animals. Experiments were
performed on 28 infantile male rats aging 23 days. The control and treatment
groups consisted of 14 animals each. Afala (treatment) or distilled water
(control) in a dose of 5 ml/kg was administered intragastrically for 10 days. The
animals were killed on day 11. The testes, ventral prostate, and seminal vesicles
were weighted. The weight ratios were calculated.
The weight ratios of the testes and seminal vesicles did not differ in animals
of the treatment and control groups. The weight ratio of the ventral prostate in
treated rats was 43.5% higher than in control animals (p<0.05, Fig. 7.69).
Therefore, Afala has a selective effect on the prostate in young animals
(prostatotropic action). The weight ratio of the prostate gland increases, while
the weight ratios of the testes and seminal vesicles remain unchanged under
these conditions (T. G. Borovskaya et al., 2001).
Efficacy of Afala in benign prostatic hyperplasia
BPH was induced by intraperitoneal injection of sulpiride (Eglonyl,
Sintelabo Grupp) in a dose of 40 mg/kg (F. Van Coppenolle, 2001) for 60 days.
This treatment resulted in the development of hyperprolactinemia and,
therefore, prostatic hyperplasia. Afala in a dose of 50 ml/kg was administered
195
Ultralow doses
mg/g
0.4
*
0.3
0.2
0.1
0
Control
Afala
Fig. 7.69. Effect of Afala on the weight ratio of the ventral prostate in young rats.
*p<0.05 compared to the control.
intragastrically for 60 days. Permixon (Serenoa Repens extract, Pierre Fabre
Medicament Production) in a dose of 50 mg/kg served as the positive control.
Distilled water (5 ml/kg intragastrically, 60 days) served as the negative control.
Sulpiride cause hyperprolactinemia. Plasma prolactin concentration in
creased on days 30 and 60 after sulpiride injection (by 2 and 1.6 times, re
spectively, compared to intact specimens; p<0.05). Hyperplasia of the lateral
prostate was observed on day 60 after sulpiride injection. The weight and weight
ratio of the lateral prostate in treated animals were twofold higher than in intact
specimens (p<0.05). A histological study revealed the signs of lateral prostate hyper
plasia on day 60 after sulpiride injection. They included a significant increase in
the relative area of the glandular epithelium, decrease in the lumen of distal
portions of the gland, and thickening of connective tissue layer between the acini.
Afala and Permixon had no effect on plasma prolactin concentration in
rats on days 30 and 60 of study. Both substances prevented an increase in the
weight ratio of the lateral prostate by the 60th day after sulpiride injection. The
efficacy of Afala was 1.7fold higher than that of Permixon. The weight ratios
of the lateral prostate in treated rats were 0.07±0.01 and 0.12±0.01 mg/g,
respectively (p<0.01; vs. 0.18±0.02 mg/g in the control group; Fig. 7.70). A
histological study of the prostate from animals of the Afala and Permixon groups
revealed a decrease in the degree of structural changes due to
hyperprolactinemia (compared to control animals). Administration of Afala and
Permixon was followed by a decrease in the relative area of epithelial structures
and increase in the relative area of the lumen in distal portions of the gland. The
thickness of connective tissue layer remained unchanged under these conditions.
These data indicate that Afala prevents the development of prolactin
dependent prostatic hyperplasia in rats of late reproductive age (810 months;
J. L. Dugina et al., 2006). The efficacy of Afala is similar to or higher than that
of Permixon (J. L. Dugina et al., 2006).
196
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
mg/g
0.4
1
2
3
4
0.3
!+ х
0.2
!
х
!
!
0.1
х
!
х
х
0
Anterior lobe
Median lobe
Posterior lobe
Fig. 7.70. Effect of Afala on the weight ratios of various lobes of the prostate in
rats after 60day treatment with sulpiride. Intact animals (1); distilled water +
sulpiride (2); Permixon + sulpiride (3); and Afala + sulpiride (4). p<0.05: *compared
to intact animals; +compared to the control (distilled water and sulpiride); xcompared
to Permixon and sulpiride.
Safety profile of Afala
A complete toxicology study was performed to evaluate the safety profile,
possible side effects, target organs, and safe dosage range of Afala. These
experiments were conducted in accordance with the recommendations given in
the Manual on Experimental (Preclinical) Study of New Pharmacological
Substances (2000). The purpose of studies with Afala was to determine the acute
toxicity (experiments on mice and rats), chronic toxicity (6month treatment of
rats and rabbits), reproductive and allergic toxicity (experiments on rats),
immunotoxicity, mutagenicity (chromosomal aberration assay in mouse bone
marrow cells), and genotoxicity (test system for somatic mosaicism in wing cells
of Drosophila melanogaster).
Our experiments demonstrated a good safety profile of Afala. An acute
toxicity study showed that Afala in the maximum permissible dose does not
cause death of animals. Drugrelated death of animals was not observed after
6month treatment with Afala in the highest dose. The product had no toxic
effect on organs and systems of experimental animals. A pathomorphological
study did not reveal damage to the internal organs or local irritation of the
gastric mucosa after drug administration. Afala did not cause reproductive
disorders in male and female rats. The embryotoxic effect of Afala was not
detected. Afala had no mutagenic, allergenic, and immunotoxic properties.
Some authors reported that chronic inflammation and hyperplasia are
related to an imbalance between growth factors in prostate tissue (C. L. Eaton,
2003). Moreover, BPH is accompanied by structural changes in PSA and de
velopment of the autoimmune response to PSA (A. Zisman et al., 1995, 1999).
Experimental studies revealed that the course of treatment with antibodies
to PSA in ULD modifies the effect of androgens on proliferative activity of prostate
197
Ultralow doses
tissue. Moreover, they have a normalizing effect on the morphofunctional state of
prostate tissue during chronic inflammation. Taking into account the pathogenesis
of BPH and possible regulatory role of PSA in proliferative activity of prostate
cells, it may be suggested that the pharmacological effect of Afala under
specified conditions is associated with functional modulation of endogenous
PSA. Our hypothesis is consistent with modern notions of the general
mechanism for action of products from ULD of antibodies to endogenous
regulators (i.e., modification of functional activity; O. I. Epstein et al., 2005).
7.7. Preclinical study of Kardos
ULD of antibodies to the Cterminal fragment of human angiotensin II
AT1 receptor constitute the active substance of Afala. Angiotensin II receptor
type 1 (AT1) mediates the key effects of angiotensin II, which has a role in the
pathogenesis of arterial hypertension, complications of this disorders, and
chronic heart failure (M. de Casparo et al., 2000; R. M. Touyz et al., 2000).
Previous studies showed that Kardos exhibits the hypotensive properties
under conditions of arterial hypertension (two strains of rats with inherited
arterial hypertension) and has a normalizing effect on cardiac function during
experimental chronic heart failure (Fig. 7.71).
Antihypertensive activity of Kardos
Hypotensive activity of Kados in ISIAH rats. Pharmacological activity of
Kardos was studied on adult (56 months) and young rats (28 days) with
inherited stressinduced arterial hypertension (ISIAH). The animals received
orally (through a pipette) 0.5 ml aqueous solution of Kardos, solvent (distilled
water, control), or reference drug losartan. Losartan was given in a dose of 10
mg/kg, which corresponds to the human daily dose (taking into account a
tenfold correction for interspecies metabolic differences).
The hypotensive effect of single administration and repeated treatment
with the test substance for 5 days or 4 weeks was studied in adult rats (mea
surement of systolic BP by the cuff method). ECG and behavioral parameters
of animals (openfield test and elevated plusmaze test) were recorded after a
4week course of treatment. The effect of Kardos (2week course) on the
hypertensive state was studied in young rats with inherited arterial hypertension.
The hypotensive effect of Kardos compared well with that of an
angiotensin II AT1 receptor antagonist losartan in a dose of 10 mg/kg (A. L.
Markel’ et al., 2002; O. I. Epstein et al., 2002c). However, the effect of losartan
developed more rapidly that that of Kardos (Fig. 7.72).
198
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
KARDOS
Efficacy on the model
of chronic heart failure
Antihypertensive activity
ISIAH rats
SHR rats
Izadrin model
Young rats
Direct measurement of blood pressure
Adult rats
Blood pressure measurement with a cuff
Fig. 7.71. Experimental study for pharmacological activity of Kardos.
Administration of Kardos for 2 weeks prevented the hypertensive state in
4weekold ISIAH rats (treatment from the 4th to the 6th week of life; Fig.
7.73).
A 4week course of treatment with Kardos was accompanied by the
following significant changes in ECG: shortening of the QT interval; decrease
in the QT/R ratio; and increase in RR. These results are consisted with
published data on the effect of antihypertensive drugs (ACE inhibitors) on ECG.
The observed changes reflect a positive effect of Kardos on the cardiac cycle.
Kardos had no negative (depriming) effect on locomotor, exploratory, and
other types of behavioral activity of hypertensive animals.
mm Hg
25
a
b
c
d
e
20
15
10
5
0
5
10
Fig. 7.72. Hypotensive effect of losartan (light bars) and Kardos (dark bars) on
adult male ISIAH rats. Single treatment (a); 5day course (b); 7 days after withdrawal
(c); repeated 5day course (d); and 4week course (e).
199
Ultralow doses
mm Hg
220
1
200
!
180
2
160
140
120
100
4
6
8
10
Age of animals, weeks
24
Fig. 7.73. Systolic BP in ISIAH rats receiving Kardos from the 4th to the 6th week
of life. Control (distilled water, 1); and Kardos (2). *p<0.05 compared to the control.
Hypotensive activity of Kardos in SHR rats. Experiments were performed
on the widely used model of inherited arterial hypertension (SHR rats). An
aqueous solution of Kardos in a dose of 2.5 ml/kg was administered
intragastrically. Control animals received the solvent (distilled water). Losartan
in a dose of 10 mg/kg was given to specimens of the reference group. Direct
measurements of BP and HR in the abdominal aorta were performed on day
28 of treatment. A catheter was introduced through the femoral artery under
sodium ethaminal anesthesia.
After 4week treatment of SHR rats, the hypotensive effect of Kardos was
similar to that of losartan. As compared to the placebo group, Kardos and
losartan decreased the mean BP (by 14.8 and 17.7%, respectively; Fig. 7.72),
systolic BP (by 16.5 and 20%, respectively), and diastolic BP (by 15.6 and
17.1%, respectively; N. A. Medvedeva et al., 2006a,b). As differentiated from
losartan, Kardos significantly decreased HR in SHR rats with hypertension and
tachycardia. HR decreased by 9.3 and 1.9% after administration of Kardos and
losartan, respectively (Fig. 7.74; N. A. Medvedeva et al., 2006a,b).
Efficacy of Kardos during experimental
chronic heart failure
The therapeutic efficacy of Kardos was studied in rats with CHF. CHF
was induced by twofold subcutaneous injection of isoproterenol (Izadrin) in a
dose of 80 mg/kg at a 24h interval (A. M. Chernukh et al., 1977; M. Ishizawa
et al., 2006; C. J. Trivedi et al., 2006). Izadrininduced hyperactivation of b
adrenoceptors is followed by necrotic damage to the myocardium, myocardial
dysfunction, and fibrosis and hypertrophy of the left ventricle (C. J. Friddle et
al., 2000; R. Bos et al., 2005). It is accompanied by the impairment of
excitation/inhibition in cardiomyocytes, actinmyosin interaction, and response
200
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
a
mm Hg
200
!
!
160
b
HR, bpm
300
290
280
120
270
80
260
!
250
40
240
230
0
1
2
3
1
2
3
Fig. 7.74. Systolic BP (direct measurement, a) and HR (b) in hypertensive SHR rats:
comparative efficacy of Kardos and losartan (28day course). Control (1); losartan
(2); and Kardos (3). *p<0.05 compared to the control.
to badrenergic stimulation (R. M. Saraiva, 2003). This model for the evaluation
of existing and potential cardiotonic drugs has several advantages. First, the
experiment involves standard laboratory animals (rats) with high rate of
reproduction (as differentiated from the model of epinephrine myocarditis).
Second, the results of this experiment are highly reproducible. And third, the
animals are not exposed to other factors and live under “standard”
environmental conditions during Izadrininduced intoxication and in the follow
up period. Experimental heart failure develops progressively, which is similar to
medical practice (Manual on Experimental (Preclinical) Study of New
Pharmacological Substances, 2005).
Experiments were performed on 80 Wistar rats (40 males and 40 females)
weighing 220250 g and aging 45 months.
Studying the tolerance of animals to physical exercise (swimming time
with a load of 15% body weight), recording of ECG (standard lead II), and
rheography (evaluation of the stroke volume, cardiac output, and other
hemodynamic parameters) were performed in the initial state, 7 days after the
second injection of Izadrin, and on the 14th and 28th days after administration
of test substances.
After the second test, all rats with signs of CHF (decrease in the swimming
time by not less than 20% of the initial level) were randomized into three groups.
Each group consisted of ten males and ten females. The animals were subjected
to daily intragastric treatment with Kardos (2.5 ml/kg), distilled water, or losartan
(10 mg/kg, 2.5 ml/kg aqueous solution; Cozaar, Merck Sharp & Dohme).
On day 28 of therapy, central hemodynamics and myocardial reserve in
50% rats were evaluated by the invasive method with an intraventricular catheter.
These experiments involved the volume loading test (intravenous infusion of
physiological saline, 0.3 ml per 100 g), intravenous injection of epinephrine, and
201
Ultralow doses
maximum isometric tension (30sec occlusion of the ascending aortic arch). The
maximum isometric tension in the left ventricular myocardium was calculated
as follows:
P×HR/M,
where P is left ventricular pressure; and M is left ventricular mass. This
parameter was expressed in mm Hg×g1×min1.
The tolerance to physical exercise in females and males decreased by 39
and 25.4%, respectively, on day 7 after the last injection of Izadrin. The signs
of CHF were more pronounced in females than in males.
Kardos was less potent than losartan in the ability to normalize physical
tolerance. The swimming time of CHF females increased by 30.2±7.7% after
4week administration of Kardos (placebo, by 9.9±6.8%; losartan, by 37.1±
5.7%). Fig. 7.75 shows that in males with CHF, the efficacy of test substances
was similar to that of placebo (Kardos, 7.9%; losartan, 11.9%; placebo, 5.1%).
Izadrin caused sinus tachycardia in females, which was manifested in a
16% increase in average HR. The degree of tachycardia decreased most
significantly after administration of Kardos (by 8%) as compared to the placebo
(by 7%) and losartan group (by 6%). Other parameters of ECG remained
practically unchanged under these conditions.
A rheographic study with males and females revealed a significant de
crease in the stoke volume, cardiac output (by more than 30%), and stroke
index and increase in total peripheral vascular resistance on day 7 after Izadrin
injection. A 4week course of treatment with Kardos and losartan was accom
panied by the improvement of these parameters.
Figs. 7.76 and 7.77 illustrate that statistically significant differences in
central hemodynamics (invasive functional tests) between the animals receiving
%
40
!
30
20
10
0
Females
distilled water
Males
losartan
Kardos
Fig. 7.75. Tolerance to physical exercise in CHF rats after a 4week course of
treatment with Kardos and losartan. Ordinate: Izadrine group. *p<0.05 compared
to the control.
202
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
а
mm Hg/sec
12 000
!
!
10 000
+
b
mm Hg/sec
12 000
10 000
8000
8000
6000
6000
4000
4000
2000
2000
0
!
+
!
0
1
2
3
1
2
3
Fig. 7.76. Rates of contraction (a) and relaxation (b) of the left ventricular myo
cardium in CHF females after a 4week course of treatment with Kardos and
losartan. Distilled water (1); losartan (2); and Kardos (3). *p<0.05 compared to
animals receiving distilled water.
mm Hg
116
*
112
108
104
100
96
Distilled water
Losartan
Kardos
Fig. 7.77. Left ventricular developed pressure in CHF rats after a 4week course
of treatment with Kardos and losartan. *p<0.05 compared to animals receiving
distilled water.
losartan or Kardos and specimens of the placebo group are most typical of
females with severe CHF.
The volume loading test showed that losartan and, particularly, Kardos
increase the rate of myocardial contraction and relaxation in rats with CHF (as
compared to the placebo group, distilled water). Volume loading was followed
by a greater increase in left ventricular pressure in animals of the Kardos and
losartan groups.
The epinephrine test revealed that animals of the Kardos and losartan
groups differ from placeboreceiving rats in a smaller increase in the rate of left
ventricular myocardial contraction. Therefore, administration of Kardos and
losartan is accompanied by a slight decrease in myocardial adrenoreactivity
during CHF.
Occlusion of the ascending aorta is used to evaluate the reserve capacity
of the left ventricular myocardium. Therapy of CHF rats with Kardos and lo
203
Ultralow doses
sartan was followed by a significant increase in left ventricular developed pres
sure and average maximum value of isometric tension in the left ventricular
myocardium (by 7 and 9.6%, respectively, compared to placebo). The observed
changes were particularly pronounced in females (19 and 20% for Kardos and
losartan, respectively).
These data show that Kardos significantly differs from placebo during a
4week course of intragastric administration to rats with Izadrininduced CHF.
Kardos was as good as losartan for the increase in physical tolerance, impro
vement of systemic hemodynamics, and elevation of myocardial reserve in the
left ventricle (S. Sergeeva et al., 2006; I. N. Tyurenkov et al., 2007).
Kardos safety
A complete toxicology study was performed to evaluate the safety profile,
possible side effects, target organs, and safe dosage range of Kardos. The pur
pose of studies with Kardos was to determine the acute toxicity (experiments on
mice and rats), chronic toxicity (6month treatment of rats and rabbits), repro
ductive and allergic toxicity (experiments on rats), immunotoxicity, mutagenicity
(chromosomal aberration assay in mouse bone marrow cells), and genotoxicity
(test system for somatic mosaicism in wing cells of Drosophila melanogaster).
The experiments demonstrated a good safety profile of Kardos. An acute
toxicity study showed that this substance in the maximum permissible dose does
not cause death of animals. Drugrelated death of animals was not observed
after 6month treatment with Kardos in the highest dose. The product had no
toxic effect on organs and systems of experimental animals. A pathomorpho
logical study did not reveal damage to the internal organs or local irritation of
the gastric mucosa after drug administration. HR in male and female rats
decreased by 1416% after 6month treatment with Kardos (p<0.01 compared
to the control). Kardos did not cause reproductive disorders in male and female
rats. The embryotoxic effect of Kardos was not observed. Kardos had no
mutagenic, allergenic, and immunotoxic properties.
7.8. Study for antidiabetic activity
of a new product from ultralow
doses of antibodies on the model
of streptozotocininduced diabetes in rats
According to the World Health Organization more than 180 million
people worldwide suffer from diabetes mellitus (DM). The number of these
204
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
patients is expected to increase by more than 2 times in 2030 (M. B. Antsiferov
et al., 2000). The main criteria of diabetes are an increase in fasting blood
glucose level (above 6.7 mmol/liter) and impaired glucose tolerance (Expert
Committee, 2003).
The common type of pharmacotherapy for type 1 DM is insulin replace
ment therapy. Blood sugarlowering drugs (sulfonylurea derivatives, biguanides,
meglitinides, etc.) are used in the therapy of type 2 DM. Despite the efficacy
of standard pharmacotherapy for DM, antidiabetic drugs cannot compensate
completely the associated disorders. Moreover, they cause some side effects
(M. B. Antsiferov et al., 2000; M. I. Balabolkin et al., 2005; R. K. Bidasee et
al., 2003). The search for new hypoglycemic drugs is an urgent problem (M. I.
Balabolkon, 1998).
Streptozotocininduced diabetes is an extensively used experimental
model of DM (D. A. Rees et al., 2005). Administration of streptozotocin is fol
lowed by progressive dysfunction of pancreatic βcells, impairment of glucose
tolerance, and development of associated disorders.
This study was designed to study the antidiabetic properties of a new
product, which belongs to a class of ULD antibodies (O. I. Epstein et al., 2004;
A. A. Spasov et al., 2007) and contains antibodies to the insulin receptor
βsubunit (ULD ABIRb) in ULD for oral administration.
Experiments were performed on 130 male outbred albino rats weighing 250
300 g. DM was induced by intravenous injection of streptozotocin in a single dose
of 50 mg/kg. Blood glucose level was measured after 72 h. Blood glucose level
in DM rats was not less than 15 mmol/liter. These animals were divided into
groups and treated in the followup period (50 days). The rats received distilled
water (2.5 ml/kg intragastrically once daily; control, n=60); insulin (Aktrapid,
daily dose 12 U/kg, subcutaneously twice daily; n=20); glybenclamide (Berlin
Chemie; daily dose 8 m/kg, intragastrically twice daily, n=20); or ULD ABIRβ
(“Materia Medica Holding” ResearchandProduction Company, 2.5 ml/kg
intragastrically once daily, n=20). The intact group consisted of ten rats.
Body weight, fasting blood glucose (glucose oxidase method with
Glyukoza FKD kits), and water consumption were estimated on days 3, 7, 14,
21, 28, 35, 42, and 50 of therapy. The glucose tolerance test (1 g/kg glucose
orally) was performed on days 14, 28, and 50 of therapy. The area under the
concentrationtime curve (AUC) was calculated by the method of trapezoids.
Streptozotocin caused hyperglycemia in rats. Blood glucose concentration
in control rats was 46.5 times higher than that in intact animals. By the end
of study, blood glucose level in control specimens reached 17.9±0.06 mmol/liter
(Fig. 7.78). The severity of DM was determined from the morality rate of
control rats. Only 15% animals of this group survived by the end of study (Table
7.15). On day 50 of observations, body weight loss in control specimens was
205
Ultralow doses
47%. By contrast, body weight of intact animals increased by 20%. Water
consumption in streptozotocinreceiving rats was 2.7fold higher than in control
specimens (Table 7.15).
Insulin injection was followed by a significant decrease in blood glucose
level (8.96±0.05 mmol/liter on day 50, p<0.001; Fig. 7.78), but had no effect
on the degree of polydipsia in rats with experimental diabetes. The survival rate
of these animals was 20% (Table 7.15). Body weight remained unchanged in rats
of the insulin group.
Glybenclamide also decreased the degree of hyperglycemia (10.01±0.03
mmol/liter on day 50, p<0.001; Fig. 7.78), which probably explains a slight
increase in the survival rate of animals (up to 20%; Table 7.15).
Blood glucose level in DM rats returned to normal on day 7 of treatment
with ULD ABIRβ (Fig. 7.78). In the followup period, blood glucose
concentration in animals of this group did not differ from that in intact
specimens. Moreover, blood glucose level in rats receiving ULD ABIRβ was
much lower than in animals of the insulin and glybenclamide groups.
Administration of ULD ABIRβ was also followed by a significant increase in
the survival rate of rats (up to 30%) compared to animals of other groups. The
external appearance, behavior, and body weight gain in rats of the ULD AB
IRβ group did not differ from those in intact animals. This product decreased
the volume of water consumption by 22.5% compared to the control (p<0.05).
The oral glucose loading test showed that streptozotocin injection is
followed by a 36fold decrease in glucose tolerance (Fig. 7.79). Glucose
tolerance increased by 2.53 and 22.5 times after intragastric administration of
reference drugs (insulin and glybenclamide, respectively; p<0.05 compared to
the control). Administration of ULD ABIRβ was also followed by the increase
mmol/liter
25
20
2
15
!
!
!
10
!
!
!
!
!
+
5
!
!
+
+
+
35
42
!
4
3
+5
1
0
3
7
14
21
28
50
Time, days
Fig. 7.78. Effect of ULD ABIRβ, insulin, and glybenclamide on blood glucose level
in rats with streptozotocininduced diabetes. Intact animals (1); control (2); dia
betes+insulin (3); diabetes+glybenclamide (4); and diabetes+ULD ABIRβ (5).
p<0.05: *compared to the control; +compared to the diabetes+insulin group and
diabetes+glybenclamide group.
206
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
Table 7.15.
Survival rate, body weight gain, and consumption of food and water in rats
with streptozotocininduced diabetes on day 50 of study
Total number
of animals
Survived
animals
Body weight
gain, %
Intact animals
10
10
+20
57.32±4.63
Control (diabetes)
60
9
47
151.78±10.5
Diabetes and insulin
20
4
0*
189.23±21.92
Diabetes and
glybenclamide
20
4
30.5
164.05±15.76
Diabetes and
ULD ABIRβ
20
6
+12.5*
117.57±8.38*
Group
Water consumption,
1 ml per rat
Note. *p<0.05 compared to the control.
in glucose tolerance in diabetic rats (by 2.43.7 times compared to the control,
p<0.05). It should be emphasized that the effect of ULD ABIRβ compared well
with that of reference drugs.
These data indicate that ULD ABIRβ have a strong antidiabetic effect
on the model of streptozotocininduced DM in rats. The product had a
normalizing effect on blood glucose level, glucose tolerance (oral glucose
loading), and body weight gain. The survival rate of rats significantly increased
after administration of ULD ABIRβ. The effect of ULD ABIRβ on glucose
tolerance was similar to that of standard drugs for the therapy of type 1 and 2
DM (insulin, 12 U/kg; and glybenclamide, 8 mg/kg). Moreover, ULD ABIRβ
had a greater hypoglycemic effect than reference drugs. We conclude that ULD
ABIRβ have high antidiabetic activity.
AUC, mmolхmin/liter
3000
2500
2000
1500
1000
500
!
!
! !
!
!
!
!
!
0
14
28
50
Duration of therapy, days
1
2
3
4
5
Fig. 7.79. Glucose tolerance (glucose AUC, oral glucose loading) in rats with
streptozotocininduced diabetes after administration of test substances. Intact
animals (1); control (diabetes, 2); diabetes+insulin (3); diabetes+glybenclamide
(4); and diabetes+ULD ABIRβ (5). *p<0.05 compared to the control.
207
Ultralow doses
* * *
Chapter 7 was devoted to the results of experimental studies with 11
products that contain ULD of antibodies to the following agents: S100 protein
(Proproten100, Tenoten, and Tenoten for children), NO synthase (Impaza),
IFNγ (Anaferon and Anaferon for children), TNFα (Artrofoon), histamine
(Epigam), PSA (Afala), Cterminal fragment of the angiotensin II AT1 receptor
(Kardos), and insulin receptor βsubunit.
These studies were designed to evaluate the range of pharmacological
activity of products from antibodies in ULD. ULD antiS100 have the
anxiolytic, antiasthenic, activating, antidepressant, antiaggressive, nootropic,
stressprotective, antihypoxic, antiischemic, and neuroprotective properties.
Antibodies to NO synthase in ULD improve endothelial function (e.g., erectile
function) and decrease the increased BP. ULD of antibodies to IFNγ have the
immunomodulatory and antiviral effects. Antibodies to TNFα in ULD possess
the antiinflammatory, analgetic, and antitumor properties on some models of
experimental tumors. ULD of antihistamine antibodies have the antiulcer effect
and improve motor activity of GIT. Antibodies to PSA in ULD exhibit the
prostatotropic activity (efficacy during experimental inflammation of the pro
state and BPH). ULD of antibodies to the Cterminal fragment of the angio
tensin II AT1 receptor produce the hypotensive and cardiotropic effects (reco
very of myocardial function and morphology on the model of CHF). ULD of
antibodies to the insulin receptor βsubunit have a strong hypoglycemic and
antidiabetic effect.
Each product was subjected to a complete toxicology study in accordance
with the recommendations given in the Manual on Experimental (Preclinical)
Study of New Pharmacological Substances (2000, 2005). The purpose of studies
with these products was to determine the acute toxicity (experiments on mice
and rats), chronic toxicity (6month treatment of rats and rabbits), reproductive
and allergic toxicity (experiments on rats), immunotoxicity, mutagenicity
(chromosomal aberration assay in mouse bone marrow cells), and genotoxicity
(test system for somatic mosaicism in wing cells of Drosophila melanogaster or
Ames test).
Antibodies in ULD demonstrated a good safety profile. An acute toxicity
study showed that intragastric and intraperitoneal administration of products in
the maximum permissible dose does not cause death of animals. Hence, ULD
of antibodies were classified to a group of lowhazard substances (GOST
12.1.00776). Drugrelated death of animals was not observed after 6month
treatment with ULD of antibodies in the highest dose. These products had no
toxic effect on organs and systems of experimental animals. A pathomorpholo
gical study did not reveal damage to the internal organs or local irritation of the
208
Chapter 7. Experimental pharmacology of products from ultralow doses of antibodies
gastric mucosa after drug treatment. Antibodies in ULD did not cause
reproductive disorders in male and female rats. The embryotoxic effect was not
observed. Products of antibodies in ULD had no mutagenic, allergenic, and
immunotoxic properties. A toxicology study revealed some specific effects of
products from ULD of antibodies.
Hence, preclinical studies demonstrated the efficacy of antibodies in
ULD. The activity of antibodies in ULD compares well with that of reference
drugs. Moreover, these products have a good safety profile. The results of
preclinical studies were confirmed by further clinical observations.
209
Ultralow doses
C h a p t e r
8
Clinical pharmacology
of products from ultralow
doses of antibodies
8.1. Use of medical products from antibodies
to S100 protein in the therapy for alcoholism
and anxiety disorders
P
harmacological activity of products from antibodies to S100 protein
(Proproten100, Tenoten, and Tenoten for children) is associated with
their ability to modify functional activity of the endogenous protein S100.
This property of antibodies in ULD was discovered in vitro on the model of
longterm posttetanic potentiation (Epstein et al., 2003a; O. I. Epstein et al.,
2003).
S100 protein has a wide range of biological functions, which formed a
theoretical basis for the development of medical products from antibodies to S
100 protein (new molecular target for drug treatment). This protein is involved
in neuronal plasticity, regulation of GABAergic neurotransmission, intracellular
calcium homeostasis, and neurotrophic processes (A. V. MartyushevPoklad et
al., 2004). Experimental studies showed that the products of antibodies to S100
protein in ULD have a wide range of pharmacological properties, which reflects
a variety of biological functions of this protein (O. I. Epstein et al., 2005). The
efficacy and safety of new medical products from antibodies to S100 protein
were confirmed in controlled clinical trials.
210
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Proproten100 in the therapy of alcoholism
Proproten100 was approved for the use in medical practice in 1999. This
drug was shown to be effective in the therapy of alcoholism (O. I. Epstein et
al., 2001c; A. G. Gofman et al., 2002; G. A. Livanov et al., 2003; N. A. Bokhan
et al., 2003; A. T. Davydov et al., 2004).
The efficacy and safety of Proproten100 in the therapy for alcohol
withdrawal syndrome (AWS) were studied in a doubleblind, randomized,
placebocontrolled trial with parallel groups (Proproten100 monotherapy and
placebo+detoxification; A. G. Gofman et al., 2003). An open comparative study
of Proproten100 vs. amitriptyline (75 mg daily + detoxification) and phena
zepam (2 mg daily + detoxification) was performed by E. N. Krylov (2003b).
Proproten100 monotherapy was much more effective than placebo treatment
and detoxification. The symptoms of mild and moderate AWS were reduced on
day 1 of treatment with Proproten100 (Fig. 8.1).
Anxiety, depression, alcohol craving, and somatovegetative symptoms of
AWS (tremor, hyperhidrosis, and tachycardia) were relieved in a greater number
of patients on days 13 of Proproten100 therapy. Dysphoria, asthenia, and
dyssomnia were not observed in a greater number of patients on days 23 of
treatment. Proproten100 had a strong anxiolytic, antidepressant, antiasthenic,
and vegetostabilizing effect, prevented the development of affective disorders,
and caused the reduction of alcohol craving in the acute period of alcohol
abstinence (Fig. 8.2; A. G. Gofman et al., 2003).
As differentiated from amitriptyline and phenazepam, Proproten100
significantly decreased the time to relief of AWS symptoms. It should be
emphasized that Proproten100 did not cause side effects. Amitriptyline was
more potent than Proproten100 in reducing alcohol craving. The severity of
anxiety and sleep disorders decreased most significantly after phenazepam
therapy (Fig. 8.3; E. N. Krylov, 2003b).
Points
25
20
15
10
1
5
2
0
1
2
3
Duration of therapy, days
4
Fig. 8.1. Effect of Proproten100 on the overall severity of AWS. Placebo and
detoxification (1); and Proproten100 (2).
211
Ultralow doses
a
%
100
ANXIETY
b
DEPRESSION
!
!
!
!
!
80
%
100
60
!
80
60
!
40
40
20
20
0
!
0
Placebo +
detoxification
Proproten
Placebo +
Proproten
detoxification
Placebo +
detoxification
Proproten
c
%
100
TREMOR
HYPERHIDROSIS
!
TACHYCARDIA
!
!
!
!
!
80
!
60
!
40
!
20
0
Placebo +
detoxification
Proproten
Placebo +
detoxification
Proproten
Placebo +
detoxification
Proproten
d
%
100
DYSSOMNIA
!
INSOMNIA
1st day
2nd day
3rd day
!
!
80
!
60
40
20
0
Placebo +
detoxification
Proproten
Placebo + Proproten
detoxification
Fig. 8.2. Effect of Proproten100 on the
symptoms of AWS. Anxiolytic and antide
pressant effect (a); antiasthenic effect (b);
effect on somatovegetative symptoms of
AWS (c); and effect on dyssomnia (d).
Ordinate: responding patients. *p<0.05
compared to the placebo group.
The efficacy and safety of Proproten100 in alcoholic patients with post
withdrawal disorders were studied in an open randomized controlled trial. After
the relief of AWS (N. A. Bokhan et al., 2003), hospital patients of the control
group received Proproten100 or individually prescribed antidepressants
(amitriptyline, up to 100 mg daily), drugs for behavioral disturbances (Neuleptil,
up to 30 mg daily; Sonapax, up to 50 mg daily), drugs for sleep disorders
(chlorprothixene, up to 100 mg daily), nootropic agents (piracetam, up to 800
mg daily), and vegetostabilizing drugs (Pyrroxan, up to 60 mg daily; Grandaxin,
up to 100 mg daily; N. A. Bokhan et al., 2003). The degree of affective
212
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Relief time, days
6
1
2
3
4
5
4
3
2
1
0
Alcohol craving
Anxiety
Depression
Sleep disorders
Somatovegetative disorders
Fig. 8.3. Comparative therapeutic efficacy of Proproten100 in AWS patients.
Placebo and detoxification (1); Proproten100 (2); amitriptyline (75 mg daily) and
detoxification (3); and phenazepam (2 mg daily) and detoxification (4).
disorders, dyssomnia, neurovegetative disturbances, and alcohol craving was
evaluated daily.
Proproten100 monotherapy was as good as combination therapy for the
reduction of AWS symptoms during the subacute period. The degree of anxiety
and depression (Hamilton’s scale) in patients receiving Proproten100 was re
duced more rapidly than in the control group. Proproten100 had a strong an
xiolytic and antidepressant effect during the subacute period of AWS. The effi
cacy of Proproten100 was higher compared to that of reference drugs (Fig. 8.4).
Studying the efficacy, safety profile, and activity showed that Proproten
100 monotherapy has some advantages over other psychopharmacological drugs
a
b
Degree of anxiety (Hamilton’s scale),
% of the baseline level
160
Degree of anxiety (Hamilton’s scale),
% of the baseline level
160
120
120
80
80
!
!
40
40
!
!
!
!
0
0
Baseline level 7 days 14 days 21 days
Baseline level 7 days 14 days 21 days
Time, days
reference group
Proproten100
Fig. 8.4. Anxiolytic (a) and antidepressant effects (b) of Proproten100 in the
therapy of alcoholic patients with postwithdrawal disorders. *p<0.05 compared
to the reference group.
213
Ultralow doses
during therapy of alcoholic patients with mild or moderate AWS and post
withdrawal disorders.
Previous experiments showed that the product of antibodies to S100
protein had an anxiolytic effect under standard conditions. Moreover, the
activity of Proproten100 compared well with that of diazepam. As differentiated
from diazepam, Proproten100 did not cause side effects (sedative, myorelaxant,
and amnesiainducing effects). Experimental studies revealed that Proproten100
has a wide range of pharmacological activity, including the antidepressant,
antiasthenic, stressprotective, antihypoxic, antiischemic, neuroprotective, and
nootropic (antiamnesic) properties. Therapeutic activity of the product from
antibodies to S100 protein is partly related to the GABAmimetic effect (O.
I. Epsyein et al., 2005). A favorable combination of the anxiolytic and activating
properties provides a basis for studying the clinical efficacy of Tenoten in anxiety
disorders.
Tenoten in the therapy of anxiety disorders
The therapy of anxiety disorders that constitute the most common type
of mental disturbances is an urgent problem of modern medicine. A deficiency
of GABAergic transmission in CNS is one of the major pathogenetic
mechanisms for anxiety disorders. The action of wellknown anxiolytic drugs
(e.g., benzodiazepines) and newly developed products is directed to compensate
for this deficiency. However, the most effective and rapidly acting drugs cause
a variety of side effects. This disadvantage limits the longterm use of standard
pharmaceuticals. Prolonged administration of some antidepressants with
anxiolytic activity may be accompanied by side effects, which limits their use
in clinical practice.
The efficacy and safety of Tenoten (product of antibodies to S100 protein,
ULD for oral administration) in the therapy of anxiety disorders were confirmed
in randomized controlled clinical trials at the Institute of Neurology (Russian
Academy of Medical Sciences), V. P. Serbskii State Research Center for
Forensic and Social Psychiatry (Moscow), V. M. Bekhterev Psychoneurological
Research Institute, Military Medical Academy (St. Petersburg) and other
institutions.
An openlabel randomized trial was designed to compare the efficacy and
tolerability of 4week monotherapy with Tenoten and diazepam (15 mg daily)
in patients with borderline disorders. The majority of patients had anxiety
disorders, including neurasthenia, adaptation disorder or mixed anxiety
depression, generalized anxiety disorder, and mixed anxietydepression disorder.
The anxiolytic effect of Tenoten was studied in patients of the treatment
group with borderline anxiety (primarily with generalized anxiety disorder).
214
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
The Hamilton anxiety scale (HAMA) is one of the most common and
valid methods to evaluate the severity of anxiety disorders. There are several
approaches to study the efficacy of anxiolytic drugs by HAMA (criteria for a
favorable response to treatment, remission criteria, etc.; A. Doyle et al., 2003).
This study also included a validated test with the Spielberger scale (StateTrait
Anxiety Inventory, STAI). As differentiated from HAMA, the patients were
asked to answer the STAI questions.
A multicenter, randomized, parallelgroup, comparative trial involved ambu
latory patients (men and women, 1865 years of age) who met the criteria of neura
sthenia (F48 by ICD10), adaptation disorders (mixed anxietydepression, F43.22),
generalized anxiety disorder (F41.1), and mixed anxietydepression disorder (F41.2)
and had moderate or severe anxiety (HAMA total score not less than 20).
The exclusion criteria were internal diseases, epilepsy, decompensated
personality disorders, comorbid neurological and somatic diseases (difficulties in
the evaluation of anxiety disorder), alcoholism, abuse of psychoactive drugs,
pregnancy and breastfeeding, and participation in another clinical trial within
the past 30 days.
The patients who met the inclusion criteria were selected during the first
visit. They were informed about the purpose of this trial. The informed consent
was obtained from each patient.
The patients were randomized into groups (1:1 ratio) at each medical center
(Institute of Neurology of the Russian Academy of Medical Sciences, V. P. Serbskii
State Research Center for Forensic and Social Psychiatry, V. M. Bekhterev
Psychoneurological Research Institute, Military Medical Academy, etc.). Therapy
was prescribed during the second visit (after entry into the trial). The time of entry
into the trial depended on the halflife of withdrawn drugs (usually 7 days).
The state of patients and adverse events were evaluated in each of the next
four visits at 7day intervals. A study of vital parameters (BP and HR), routine
blood test, and urine test were performed at the beginning (baseline visit) and
by the end of the trial (28 days). All data were recorded in the case report form.
The total duration of therapy was 28 days.
The study drug (Tenoten, 12 lozenges; 612 lozenges daily) or diazepam
(5 mg orally, three times daily) was given as monotherapy for 28 days.
Medication taking was not associated with food intake.
In patients of the Tenoten group, the frequency of drug treatment could
be increased to 1012 times daily (at low effectiveness) or decreased to 24 times
daily. The majority of patients required a higher dose of Tenoten. By the end
of study, the average daily dose of Tenoten was 10 tablets.
Psychotropic drugs, psychoactive compounds, and alcoholic beverages
could affect the results of therapy and, therefore, were strictly forbidden during
the trial period. The patients were allowed to take vitamins and to receive
215
Ultralow doses
physiotherapy or physiotherapy. Hypnotic drugs with a short halflife (Zolpidem
and Zopiclon) were prescribed in severe insomnia. The patient’s use of addi
tional drugs was recorded in the case report form during each visit.
The efficacy and safety of study drug were evaluated after 1, 2, and 4
weeks of therapy. A decrease in the severity of anxiety (HAMA total score) and
symptoms of anxiety (STAI scale) was considered as the primary efficacy
endpoint. The secondary endpoints were a favorable response to treatment
(decrease in the HAMA total score by at least 50%) and achievement of partial
remission (decrease in the HAMA total score to 10 points or less).
The safety profile was determined after 1, 2, and 4 weeks of therapy using
a structured scale for adverse events (AE). Vital functions (BP and HR) and key
laboratory parameters were measured.
This study was designed to test the hypothesis that Tenoten and diazepam
are equally potent in reducing the overall degree of anxiety (HAMA and STAI)
and providing a 50% decrease in the anxiety score (HAMA).
The data from all patients who met the inclusion criteria and received
drug therapy were subjected to statistical analysis (intenttotreat analysis). The
mean values were compared with the baseline and diazepam group (Student’s
t test). The data of patients from both groups who demonstrated a favorable
response to treatment and signs of remission were compared by x2 test for
homogeneity of proportions. All statistical tests were twosided. The hypotheses
were tested at a significance level of 5%. The number of patients in each group
was selected to achieve a statistical power of 80%.
Among 300 patients enrolled in the trial, 272 patients were randomized
into the groups of diazepam (130 subjects) and Tenoten (142 subjects). The final
stage was performed with 247 patients. Six patients of the diazepam group and
fifteen patients of the Tenoten group were excluded from the trial due to
protocol noncompliance. Drug safety was evaluated in 272 patients.
Demographic data and key clinical parameters for all patients enrolled in
the trial and further analysis are shown in Table 8.1.
The efficacy of Tenoten and diazepam according to HAMA (primary
endpoints) is illustrated in Fig. 8.5.
Small differences in the HAMA total score were found between patients
of various groups in the basal state. Significant betweengroup differences were
revealed in the final total score (HAMA) and average decrease in the HAMA
total score. However, these differences were clinically irrelevant. In clinical
practice, a statistically significant difference may be characterized by variations
in effect size. Two drugs are considered to be clinically equivalent when the
deviation of effect size does not exceed 0.5. Effect size is calculated as the ratio
of the difference between mean values in groups to the standard deviation (SD)
of the combined sample. A difference in the efficacy (decrease in the HAMA
216
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Table 8.1.
Baseline characteristics of patients with anxiety disorders enrolled in the
trial (M±m)
Group
Parameter
diazepam
Tenoten
Number of patients in group
120
127
Number of women in group
73 (60.8%)
81 (63.8%)
Average age, years
39.0±1,0
38.6±1.0
Mean duration of disease, months
34.0±3.8
27.7±3.5
Mean total score by HAMA
28.6±0.5
27.4±0.5*
Number of patients with the diagnosis
GAD (F41.1)
40 (33.3%)
40 (31.5%)
MADD (F41.2)
26 (21.7%)
31 (24.4%)
adaptation disorder (F43.22)
16 (13.3%)
17 (13.4%)
neurasthenia (F48.0)
38 (31.7%)
39 (30.7%)
Note. *p<0.05 compared to another group.
total score) of diazepam and Tenoten is 2.3 points, which corresponds to the
effect of 0.33. SD of the combined sample is 6.96. Hence, a statistically
significant advantage of diazepam over Tenoten is clinically irrelevant. These
data indicate that diazepam and Tenoten have similar efficacy.
Small betweengroup differences were found in the baseline level of
situational and trait anxiety (STAI). Diazepam and Tenoten caused a similar
decrease in situational and trait anxiety. The patients reported that these drugs
are equally potent in decreasing the degree of anxiety.
Points
35
a
b
Points
25
!
30
20
!
25
15
20
!
15
10
10
5
5
0
0
Baseline
After 4 weeks
STAIS
STAIТ
Fig. 8.5. Efficacy of 4week treatment with diazepam (light bars) and Tenoten (dark
bars) in patients with anxiety disorders. (a) Decrease in the total score by the
Hamilton’s scale; and (b) reduction of situational (STAIS) and trait anxiety (STAIT)
by the Spielberger scale. *p<0.05 compared to the diazepam group.
217
Ultralow doses
Fig. 8.6 illustrates the secondary efficacy endpoints. The number of
patients who achieved a good response to treatment was insignificantly higher
in the diazepam group. The incidence of partial remission was similar in patients
receiving diazepam and Tenoten.
Diazepam demonstrated a slight advantage over Tenoten in inducing the
rapid response to treatment (50% decrease in the total score by the Hamilton
anxiety scale and achievement of partial remission; Fig. 8.7). However, no
betweengroup differences were found in the number of patients with remission
and favorable response to treatment after 4 weeks of therapy. The “delayed
response” of Tenotenreceiving patients could be related to selecting the optimal
dose of this drug at the beginning of treatment. At the start of our study, the
optimal dose of Tenoten was at least 10 tablets daily. In patients receiving less
than 10 tablets daily over the 1st week of therapy (n=52), partial remission or
favorable response to treatment was not observed after 2 weeks. The patients
receiving 1012 tablets daily from the start of the study (n=75) demonstrated a
favorable response (22.7% subjects) and partial remission (9.3% subjects) after
2 weeks. By the 4th week of therapy, small differences were found between
subgroups of patients receiving various doses of Tenoten. The exception was the
achievement of remission. Among 82 patients receiving 12 tablets daily,
remission was achieved in 17.1% subjects. After treatment with a lower dose of
Tenoten (36 patients), remission was observed only in 2.8% subjects. These data
indicate that the optimal dose of Tenoten is 1012 tablets daily. By the 14th day
of therapy, the HAMA total score in these subjects decreased more significantly
than in patients receiving less than 10 tablets daily (10.1 and 7.1 points,
respectively).
Fig. 8.8 illustrates the baseline, final level, and average decrease in the
degree of psychic (psychic anxiety factor; items 16 and 14) and somatic anxiety
%
100
a
b
!
80
60
40
20
0
Fig. 8.6. Efficacy of 4week treatment with diazepam (light bars) and Tenoten (dark
bars) in patients with anxiety disorders: percentage of patients with a favorable
response to treatment (a) and partial remission (b). *p<0.05 compared to the
diazepam group.
218
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
1 week
100
2 weeks
4 weeks
80
60
40
20
0
PR
RPT
PR
RPT
PR
RPT
Fig. 8.7. Partial response to diazepam (light bars) and Tenoten (dark bars) during
a 4week course of therapy for anxiety disorders. Patients with a favorable response
to treatment (decrease in the HAMA score by at least 50%) and partial remission
(decrease below 11 points). RPT, response to treatment; PR, partial remission.
(somatic anxiety factor; items 713; Hamilton’s scale) in patients receiving
diazepam and Tenoten. Before the start of therapy, psychic anxiety in the
diazepam group was more pronounced than in the Tenoten group. After a 4
week course of therapy, diazepam was more potent that Tenoten in reducing the
psychic and somatic symptoms of anxiety. Similarly to the HAMA total score,
diazepam had a slight advantage over Tenoten in effect size.
The primary efficacy endpoints are shown in Fig. 8.9.
The efficacy of diazepam and Tenoten practically did not depend on
patient’s sex. Among patients of the Tenoten group, a favorable response to
treatment was most typical of women. Similar results were obtained for patients
of the diazepam group (statistically insignificant).
In subgroups of patients below 45 years of age, baseline anxiety, decrease
in the HAMA total score, and ratio of responding subjects were slightly higher
after therapy with diazepam. The advantage was clinically irrelevant. However,
18
16
14
12
10
8
6
4
2
0
a
b
16
14
12
10
8
6
4
2
0
!
Baseline
After 4 weeks
!
Baseline
After 4 weeks
Fig. 8.8. Efficacy of Tenoten (4week course of treatment) in patients with anxiety
disorders. HAMA psychic anxiety (a) and somatic anxiety (b). Light bars, diazepam;
dark bars, Tenoten. *p<0.05 compared to the diazepam group.
219
Ultralow doses
a
%
100
!
80
60
40
30
0
All diagnoses
GAD
MADD
%
100
Adaption disorder
Neurasthenia
b
!
80
60
40
30
0
1
2
3
4
5
6
7
Fig. 8.9. Therapeutic efficacy of Tenoten in patients with anxiety disorders. Analysis
of subgroups. (a) Diagnosis. (b) Baseline characteristics of patients: (1) all
parameters; (23) sex (women and men, respectively); (45) age (not older than
45 years of age, older than 45 years of age); and (67) baseline anxiety score (less
than 30 points by HAMA, not more than 30 points by HAMA). Ordinate: percentage
of patients with a favorable response. Light bars, diazepam; dark bars, Tenoten.
GAD, generalized anxiety disorder; MADD, mixed anxietydepression disorder.
*p<0.05 compared to the diazepam group.
these drugs did not differ in the posttreatment level of anxiety and ratio of
patients with partial remission.
The efficacy of study drugs was much lower in patients older than 45
years of age (compared to younger subjects). It is probably related to the greater
inertia of pathological processes that require a longer time for regression. In
patients of this group, Tenoten was as good as diazepam for the reduction of
anxiety. Moreover, the percentage of patients with a favorable response to
treatment and achievement of remission did not differ after therapy with
Tenoten and diazepam.
Onethird of patients in both groups had severe anxiety disorder (HAMA
total score > 30). The effects of diazepam and Tenoten (decrease in the HAMA
total score; and percentage of patients with a goof response to treatment) were
particularly pronounced in subgroups of patients with severe anxiety. Diazepam
220
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
was more effective than Tenoten in patients with moderate anxiety. However, the
efficacy of Tenoten compared well with that of diazepam in subgroups of
patients with high level of anxiety.
The size of subgroups with various diagnoses (GAD, MADD, adaptation
disorder, and neurasthenia) was similar in the diazepam and Tenoten groups. A
statistically significant advantage of diazepam over Tenoten was revealed in
patients with MADD and neurasthenia (decrease in the total score). This score
decreased most significantly in subgroups of MADD patients, which was
probably related to high level of baseline anxiety. Other criteria for the efficacy
of Tenoten and diazepam were similar in all nosological groups. A statistically
significant advantage of Tenoten over diazepam (percentage of patients with
partial remission) was revealed in the group of patients with adaptation disorder.
A statistically significant advantage of diazepam over Tenoten in
decreasing the HAMA total score was observed not only in main groups, but
also in subgroups (except for patients with severe anxiety, GAD, and adaptation
disorder). However, the percentage of patients with a favorable response to
treatment and partial remission did not differ in subgroups of patients receiving
Tenoten and diazepam.
The safety evaluation was performed with all patients who received study
drugs (n=272).
Significant betweengroup differences were found in the incidence of AE.
The percentage of patients reporting AE was sevenfold lower in the Tenoten
group than in the diazepam group. The total number of AE (per 100 patients)
was 15fold lower in Tenotenreceiving patients (Fig. 8.10).
The majority of AE in diazepamreceiving patients were the typical side
effects of this drug. The relationship between AE and drug therapy was probable
or possible. The most common AE were observed in more than 10% patients
and included daytime sleepiness, muscle weakness, orthostatic disorders, vertigo,
and dry mouth.
a
b
300
300
200
200
100
100
!
!
0
0
Fig. 8.10. Reporting of adverse events during a 4week course of therapy with
diazepam and Tenoten. (a) Percentage of patients; and (b) number of AE per 100
patients. *p<0.05 compared to diazepam.
221
Ultralow doses
Mild AE were revealed in the majority of Tenotenreceiving patients. A
causeandeffect relationship between AE and drug treatment was ambiguous or
absent. Daytime sleepiness and tympanites were observed most frequently (1.4%
patients). One patient of the Tenoten group and six patients of the diazepam
group were excluded from the trial due to side effects.
The results of laboratory tests and vital parameters remained practically
unchanged during this trial.
A randomized controlled clinical trial showed that 4week monotherapy
with Tenoten (average daily dose 10 tablets) is less potent than diazepam (15 mg
daily) in reducing anxiety in patients with anxiety disorders. The severity of
anxiety was evaluated by a physician (HAMA scale) and patient (STAI scale,
effect size 0.33). The efficacy of Tenoten compared well with that of diazepam
in patients with severe anxiety (more than 30 points by the HAMA scale).
The effect of Tenoten developed more slowly than that of diazepam. By
the 4th week of therapy, Tenoten and diazepam were equally potent in
producing a favorable response (at least twofold decrease in anxiety) and partial
remission (HAMA total score < 11). The effect of drug is determined by an
initial daily dose. The optimal dose of Tenoten is not less than 10 tablets daily.
Tenoten (average daily dose 10 tablets) had a better safety profile than the
reference drug diazepam (15 mg daily) during 4week monotherapy of patients
with anxiety disorders (1865 years of age). Serious AE were not observed in
patients of both groups. In the majority of Tenotenreceiving patients, the
relationship between AE and drug treatment was ambiguous or absent. The
design of this trial suggested an open comparative study of Tenoten and
diazepam. However, the possibility of system errors due to the placebo effect
cannot be excluded. Moreover, the duration of this trial could not exceed the
maximum length of diazepam treatment (4 weeks).
The trial allowed us to make several conclusions (A. V. Martyushev
Poklad et al., 2005a). During shortterm treatment of patients with anxiety
disorders (4 weeks), the product of antibodies to S100 protein (Tenoten) shows
a better efficacysafety ratio (benefiteffect) than diazepam in a daily dose of 15
mg. The efficacy of Tenoten in patients with GAD and severe anxiety was
similar to that of diazepam. Due to the absence of sedative and myorelaxant
properties, Tenoten may be considered as a firstline daytime tranquilizer.
A clinical trial at the Institute of Neurology (Russian Academy of Medical
Sciences, Moscow) included the patients who had not only anxiety disorders, but
also chronic cerebrovascular diseases and Parkinson’s disease. Tenoten was well
tolerated and exhibited the high antianxiety activity (above the placebo effect).
Therefore, Tenoten can be used for the therapy of patients with serious diseases.
Controlled clinical trials showed that the original products of antibodies
to S100 protein demonstrate high efficacy and good safety profile in the
222
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
therapy for alcoholism (Proproten100) and anxiety disorders (Tenoten). By the
range of pharmacological properties and benefit/risk ratio, both products have
advantage over standard pharmaceuticals. Hence, Proproten100 and Tenoten
hold much promise for the use in clinical practice. These products should be
subjected to largescale clinical trials.
8.2. Use of Impaza in monotherapy and
combined treatment for erectile dysfunction
Although the development of erectile dysfunction (ED) is associated with
a variety of factors, the major pathogenetic types of this disorder have a com
mon mechanism. It suggests functional insufficiency of the peripheral me
chanism for erection (signal transduction cascade of NO synthase — NO gua
nylate cyclase — cGMP) and, primarily, inadequate production of NO (T. F.
Lue, 2000; K. E. Andersson, 2001). Type 5 phosphodiesterase (PDE5) inhi
bitors are most effective in the therapy for ED. They provide temporal control
over a deficiency in the cavernous tissue, which is related to a modulatory effect
on the final stage of this cascade. However, the therapy for ED of various etio
logies should be directed to an increase or recovery of normal NO production.
Impaza consists of antibodies to endothelial NO synthase (ULD for oral
administration). This medical product was approved for the therapy of ED in
2001. Preclinical studies showed that Impaza improves copulative function of
male rats (T. G. Borovskaya et al., 2001, 2002; I. V. Smolenov et al., 2002) and
does not exhibit the general or reproductive toxicity. The peripheral effects of
Impaza in ED are related to its influence on the signal pathway of NO synthase
— NO guanylate cyclase — cyclic GMP in the cavernous tissue (Fig. 8.11). The
course of treatment with Impaza increased the activity of NO synthase,
production of NO, and concentration of cGMP in the cavernous tissue of male
rats.
The efficacy and safety of Impaza in monotherapy and combination
therapy for ED were studied in controlled clinical trials (A. MartyushevPoklad
et al., 2005c).
A randomized placebocontrolled trial of the efficacy and safety of
Impaza during ED was performed in 20012003 (common protocol). This trial
involved the following five clinical centers in Russia: Department of Clinical
Pharmacology, Volgograd State Medical University; Institute of Pharmacology,
Volgograd State Medical University; Department of Urology and Surgical
Nephrology, Russian State Medical University (Moscow); R. M. Fronshtein
Urology Clinic, I. M. Sechenov Moscow Medical Academy; and S. P. Fedorov
St. Petersburg Urology Society.
223
Ultralow doses
IMPAZA
+++
eNOS
eNOS
Vascular endothelium
NO
NO
GTO
Phosphate
Smooth
muscle cell
cGMP
Cell relaxation
Guanylate cyclase activation
Fig. 8.11. Effect of Impaza on the regulatory cascade of NO synthase NO cyclic
AMP (experimental data). NO, nitric oxide; eNO, endothelial NO synthase; GTP,
guanosine triphosphate; cGMP, cyclic guanosine triphosphate.
After the primary clinical and laboratory examination, the trial enrolled
ambulatory patients (1870 years of age) in heterosexual relationships that com
plained of decreased erection. The diagnosis of ED was made by the Inter
national Index of Erectile Function (IIEF). The integral index of “erectile func
tion” varies from 7 to 25 points. The patients were asked to sign informed con
sent to participate in a clinical trial.
Before enrollment in the trial, all patients were examined for case history,
history of sexual activity, and IIEF questionnaire. Laboratory examination
included the routine blood test, urine test, measurement of plasma glucose and
creatinine, and study of hormonal status and lipid profile.
The exclusion criteria were alcoholism, drug abuse, anatomical de
formation of the penis, endocrine causes of ED, uncontrolled or decompensated
somatic disease, therapy with other pharmaceuticals for ED, treatment with
medical products that may cause ED, and participation in other clinical trials.
Impaza and placebo lozenges were prescribed to take in the evening time
(1 tablet) every other day. The patents could also receive 1 tablet of Impaza
13 h before the proposed sexual activity.
The efficacy was evaluated after therapy for 4 and 12 weeks. Patients were
invited to complete the IIEF questionnaire and to evaluate the overall efficacy
224
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
of therapy. The primary efficacy endpoints were a 3point increase in the
integral index of IIEF for “erectile function” (percentage of patients with a fa
vorable response), achievement of a normal criterion for “erectile function”
(more than 25 points), and patient’s evaluation of the overall efficacy (per
centage of patients who evaluated drug efficacy as “excellent” and “good”).
Three months after the main trial, some patients receiving Impaza (n=18,
Volgograd Medical University) were enrolled in the prospective evaluation of
drug efficacy. The degree of ED (according to IIEF) and patient’s subjective
evaluation of ED over 3 months after completion of the main trial were
analyzed in the followup visit.
The safety profile was evaluated by monitoring of drugrelated adverse
events (side effects) and drug interactions. Clinical examination of patients and
study of vital indexes were performed by the 4th and 12th weeks.
The patients who refused to participate in a clinical trial or demonstrated
serious drugrelated adverse events were excluded from the research.
During the first examination in the Department of Urology and Surgical
Nephrology (Russian State Medical University, Moscow), all patients were
subjected to intracavernous pharmacological testing, Doppler ultrasonography of
penile vessels before and after artificial erection, and penile electromyography.
In the Department of Urology and Surgical Nephrology (Russian State
Medical University) and Urology Clinic (I. M. Sechenov Moscow Medical
Academy), the hormonal status of patients (morning blood test for thyrotropic
hormone, triiodothyronine, thyroxin, total testosterone, and serum prolactin)
was assayed before enrollment in the trial and after the 4th and 12th weeks of
therapy.
One hundred and sixtynine patients met the inclusion criteria. They were
randomized to the placebo group (30 patients) and Impaza group (139 patients).
The trial involved 1960 patients in each center. After 12 weeks, 23 patients of
the placebo group (ineffectiveness of therapy) and 1 patient of the Impaza group
(irrationality of further treatment due to the recovery of erectile function)
discontinued participation in the trial. Six of seven patients from the placebo
group had ED of psychogenic origin.
Study groups were comparable by the major indexes (except for ED
etiology; Table 8.2). The placebo group mainly consisted of patients with
psychogenic ED. These patients could be expected to exhibit the maximum
favorable response to treatment. Therefore, a possible system error of the trial
might be related to overestimation of placebo efficacy.
Impaza was much more potent than placebo in the ability to improve
erectile function (Table 8.3, Fig. 8.12). As differentiated from placebo, Impaza
efficacy significantly increased with an increase in the duration of therapy from
4 to 12 weeks.
225
Ultralow doses
Table 8.2. Data on patients enrolled in the trial (M±m)
Group
Parameter
Average age, years
Impaza (n=139)
placebo (n=30)
47.8±0.98 (1969)
47.5±1.8 (3367)
Mean duration of ED, years
3.6±0.32
4.1±0.64
Patients with ED of primarily
psychogenic origin, %
51.1
73.3
17.70±0.35
16.3±0.7
Average score of “erectile function”
according to IIEF (30 points maximum)
ED patients, %
severe (EF < 11 points)
7.2
10
moderate (EF = 1116 points)
29.5
33.3
mild (EF = 1725 points)
63.3
56.7
Note. EF, erectile function.
Impaza had a stronger effect on various components of erectile function
compared to placebo (IIEF items 15 and 15; Table 8.4).
Table 8.4 shows the intentiontotreat analysis of groups without regard
to 77% placebo patients who discontinued the trial. The perprotocol analysis
revealed a greater difference between patients of the Impaza and placebo groups
on the 12th week of study.
It may be concluded that the efficacy of Impaza is much higher than the
placebo effect (Tables 8.28.4). Impaza therapy was followed by significant
improvement of the integral index for “erectile function”. Impaza had a positive
effect not only on erectile function, but also on other aspects of sexual activity
in patients with ED (Table 8.5).
Percentage of patients, %
80
!
!
!
!
60
!
40
30
0
Favorable response
to treatment
Recovery
of EF
Impaza, 4 weeks
Impaza, 12 weeks
Patient’s evaluation
as “excellent”/”good”
Placebo, 4 weeks
Placebo, 12 weeks
Fig. 8.12. Impaza efficacy in a placebocontrolled trial: patients who achieved the
key efficacy endpoints. *p<0.05 compared to the control.
226
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Table 8.3. Therapy efficacy in the Impaza and placebo groups (key endpoints, %)
Time of observation, weeks
4
Parameter
12
Impaza
(n=138)
placebo
(n=30)
Impaza
(n=138)
placebo
(n=7)
Patients with improved
erectile function
(EF, increase by 13 points)
68.3*
30
76.9**
3.3
Patients who achieved a normal
level of EF (> 25 points)
19.4*
6.6
33.8**
0
Patients who evaluated
the efficacy of therapy
as “excellent” or “good”
62.6*
23.3
69.6**
3.3
Note. *p<0.05 and **p<0.01 compared to the placebo group.
In the Impaza group, three patients suffered from headache (2%) and one
patient had nausea (0.7%) during the 1st week of treatment. These events were
believed to be associated with drug therapy. Headache was reported by one
patient of the placebo group (3.3%). Serious drugrelated adverse events were
not revealed in the Impaza and placebo groups. Adverse drug interactions with
Impaza were not found in patients who received medical products for the
underlying disease (e.g., nitrates in CHD).
These data illustrate the efficacy and safety of Impaza in patients with
ED. The effect was particularly pronounced after longterm treatment with
Impaza (12 weeks or more).
Table 8.4.
Effect of Impaza and placebo on various components of erectile function
(completion of the IIEF questionnaire, M±m)
Impaza
Parameter
Baseline
Placebo
4 weeks
12 weeks
4 weeks
(n=30)
12 weeks
(n=7)
3.2±0.2
3.0±0.3
Frequency of erection
3.1±0.1
3.8±0.1*+
4.2±0.1*+
Success in insertion
of the penis
3.0±0.1
3.7±0.1*+
4.0±0.1*+
2.9±0.2
2.7±0.3
Ability to achieve erection
3.1±0.1
3.8±0.1*
+
4.1±0.1*+
2.9±0.15
3.2±0.3
Ability to maintain erection
2.9±0.1
3.7±0.1*+
3.9±0.1*
3.0±0.15
3.4±0.3
Ability to complete
sexual intercourse
2.8±0.1
3.5±0.1*+
3.8±0.1*+
3.1±0.15*
2.8±0.2
Erection confidence
2.5±0.1
3.5±0.1*
+
3.8±0.1*+
2.7±0.2
Erectile function
17.4±0.4
22.1±0.3*+ 24.0±0.3*+ 17.8±0.8*
2.6±0.3
17.9±0.7
Note. p<0.05: *compared to the baseline; +compared to the placebo group.
227
Ultralow doses
Table 8.5.
Effect of Impaza and placebo on the integral criteria of IIEF (average
increase; M±m)
Impaza
Parameter
Placebo
Baseline
4 weeks
12 weeks
4 weeks
(n=30)
12 weeks
(n=7)
Satisfaction with
sexual intercourse
8.3±0.2
2.0±0.2**+ 3.0±0.2**+
0.6±0.2*
0.7±0.9
Orgasm
6.9±0.2
1.0±0.1**+ 1.2±0.2**+
0.3±0.1*
0.14±0.4
Libido
6.1±0.2
1.0±0.1**+ 1.5±0.2**+ 0.4±0.1**
0.3±0.2
Overall satisfaction
5.1±0.2
+
1.6±0.2** 2.3±0.2**+
0.4±0.2*
0.1±0.4
Note. *p<0.05 and **p<0.01 compared to the baseline; +p<0.01 compared to placebo.
A detailed study revealed the specific drug effects in patients of various
age groups (Fig. 8.13). A favorable response to treatment was observed in
patients of the following age groups: younger than 40 years (n=31), 90%
patients; 4049 years of age (n=39), 77% patients; 5059 years of age (n=48),
73% patients; and 6069 years of age (n=24), 70.8% patients. After Impaza
therapy, normal erectile function was achieved in 71, 28, 25, and 8.3% patients,
respectively. A favorable response was observed only in 3.3% patients of the
placebo group.
Therefore, Impaza was effective in the majority of ED patients from all
age groups. Impaza had a normalizing effect on erectile function in the majority
of patients younger than 40 years (primarily psychogenic ED). Impaza was
effective in more than twothirds of ED patients from the older age group
Percentage of patients, %
120
100
80
60
40
20
0
Impaza, total
(n=139)
Placebo
(n=30)
Younger than 4049 years
40 years (n=31)
(n=30)
response to treatment
recovery of EF
5059 years
(n=45)
6069 years
(n=24)
patient’s positive evaluation
Fig. 8.13. Efficacy of Impaza in patients of various age groups: achievement of
the key efficacy endpoints.
228
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
(primarily organic ED). A complete recovery of erectile function did not occur
in the majority of these patients, which was probably related to progressive
organic changes during ED.
The efficacy of oral pharmacotherapy differed in patients with various
pathogenetic variants of ED. Hence, a detailed analysis of Impaza effect was
performed by the results of a placebocontrolled trial. Table 8.6 shows the
characteristics of patients from various groups.
Impaza was most effective in patients with primarily psychogenic ED
(Table 8.7; Figs. 8.14 and 8.15). The effect of Impaza in these patients
developed more rapidly compared to subjects of other groups. Moreover, this
effect remained practically unchanged with an increase in the duration of
therapy from 4 to 12 weeks. The efficacy of Impaza in patients with psychogenic
ED is probably related to the influence on central and peripheral components
of erectile function.
Study drug had a progressive effect on patients with organic ED. The
percentage of responding patients was shown to increase by 1015% with an
increase in the duration of therapy to 12 weeks. The efficacy of Impaza was
highest in patients with the prevalence of arterial factors for ED, but lowest in
Table 8.6.
Subgroups of patients with various pathogenetic factors for ED in a placebo
controlled trial of Imapza
Group
n
Age
Patients with venous factors
11
51.5±2.4
Patients with neurogenic factors (except for venous factors)
15
53.6±0.8
Patients with arterial factors (except for venous and neurogenic factors)
38
54.4±1.0
Patients with all organic factors
68
53.6±0.8
Patients with primarily psychogenic factors of ED
71
42.3±1.5
Note. n, number of patients.
Points
10
8
6
4
2
0
Venous
Neurogenic
Arterial
All organic
factors
Psychogenic
ED
Fig. 8.14. Effect of pathogenetic factors for ED on the efficacy of 12week therapy
with Impaza: average increase in the index of “erectile function”.
229
Ultralow doses
Table 8.7.
Effect of pathogenetic factors for ED on the efficacy of Impaza therapy
(percentage of patients who achieved the efficacy endpoints)
Favorable
response to
treatment, %
Pathogenetic factor
4
weeks
12
weeks
27.3
45.5
Venous (group 1)
Patient’s eva
Average
luation as “good”
increase in EF
or “excellent”, %
(12 weeks,
points)
4
12
weeks
weeks
45.5
45.5
2.9±0.9
Neurogenic (group 2)
46.7
60
60
66.7
3.4±0.7
Arterial (group 3)
63.2
76.3
47.4
55.3
4.7±0.5
53
67.6
50
57.4
4.2±0.4
83.1
85.9
74.6
80.3
8.6±0.6
All organic factors
Prevalence of psychogenic factors
vascular venous ED. These results are consistent with published data on the
peripheral mechanism of drug effect (recovery of endothelial function).
Particular attention was paid to the efficacy of Impaza in patients with
cardiovascular diseases (arterial hypertension and atherosclerosis). A favorable
response to therapy for ED in 56 and 69% patients of this group (n=48) was
observed by the 4th and 12th weeks of treatment, respectively. An average
increase in the index of “erectile function” was 3.1±0.4 (4 weeks) and
4.3±0.5 (12 weeks). Normal erectile function was achieved in 16.7 and 18.8%
patients by the 4th and 12th weeks of Impaza therapy, respectively. The
patients with cardiovascular diseases did not report side effects or adverse drug
interactions between Impaza and medical products for the underlying disease
(e.g., nitrates).
A clinical trial of Impaza efficacy in ED patients with chronic CHD
(Institute of Cardiology, Russian Ministry of Health, Saratov) showed that the
course of drug treatment not only improves erectile function, but also decreases
the incidence of anginal attacks (K. S. Umetskii et al., 2005, 2006).
Patients, %
100
4 weeks
12 weeks
80
60
40
20
0
Venous
Neurogenic
Arterial
All organic Psychogenic
factors
ED
Fig. 8.15. Effect of pathogenetic factors for ED on the efficacy of 12week therapy
with Impaza: favorable response to treatment (percentage of responding patients).
230
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
An initiative controlled clinical study of Impaza efficacy in patients with
CHD was performed at the Altai State Medical University (A. I. Neimark et al.,
2006a,b). Impaza not only improves erectile function, but also has a positive
effect on clinical manifestations of angina pectoris, microcirculation, and
endothelial function in these patients. The product and standard drugs for CHD
therapy are a good combination. These data indicate that Impaza holds much
promise for the therapy of CHD.
All patients were asked to complete the IIEF questionnaire 3 months after
completion of the trial.
The course of drug treatment was followed by the relief of ED and
withdrawal of medical products to improve erectile function in 44.4% patients.
The subjective evaluation was confirmed by the results of IIEF testing. This
group consisted of patients with primarily psychogenic factors of ED.
Drug withdrawal was accompanied by the decrease in erectile function in
33.3% patients. Therapy should be continued in these patients. The effect of
Impaza was preserved after reinstitution of therapy. This group mainly consisted
of patients with arterial factors of ED (arterial hypertension). The remaining
patients (22.3%) were not satisfied with Impaza therapy and refused to use this
drug in the followup period.
Drugrelated adverse events were not reported over 3 months after
completion of the main trial.
Serum testosterone (TS) concentration was measured in 60 patients.
Impaza therapy for 12 weeks was followed by improvement of erectile function
in 67% patients. Table 8.8 shows that 10% variations in total serum TS were
typical of 72% patients (increase in 52% patients, decrease in 20% patients).
TS concentration significantly increased in 50% responding patients
(group 1). The mean values did not differ between patients of this subgroup and
entire group (except for an average increase in erectile function).
TS concentration increased by more than 10% in group 2 patients not
responding to Impaza. The average increase in TS level and improvement of
libido in these subjects were twofold lower than in group 1 patients. These
groups differed in the etiological structure. The percentage of ED patients with
venous and/or neurogenic factors in group 2 was twofold higher than in group
1 (55 and 22.5%, respectively). By contrast, the percentage of patients with
primarily psychogenic ED in group 2 was three times lower than in group 1 (5
and 15%, respectively).
The reduced baseline level of TS (group 3) was associated with low index
of erectile function. These patients exhibited an intermediate frequency of
response to treatment and improvement of erectile function. By contrast, the
increase in TS concentration was most pronounced in group 3 patients (by more
than 25%). Orgasmic function and libido improved in these subjects.
231
Ultralow doses
Table 8.8. Effect of 12week treatment with Impaza on serum total TS in ED patients
Responding to treatment, %
Subgroup
Number
of patients,
% of the
entire group
Average age,
years
Improved erectile function
67
53.5±0.9
No improvement
of erectile function
33
Baseline TS < 14 nM
42
Increase in TS after 12
weeks, by more than 10%
Decrease in TS after 12
weeks, by more than 10%
TS
baseline, nM
increase,
% of the
baseline
value
100
14.9±0.8
14.1±4.5*
53.7±0.9
0
15.1±1.3
7.2±4.1
55.6±1.2
64
9.9±0.4
27.1±5.5*
52
55.2±1.1
68
12.7±0.7
30.0±3.8*
20
51.8±1.7
75
18.3±1.5
20.2±2.6*
No changes in TS level
28
52.3±0.6
59
16.3±1.3
2.4±1.4
Total
100
53.7±0.7
(4569)
67
14.8±0.7
12.1±3.3*
Note. *p<0.05 compared to the baseline value.
Impaza therapy was followed by the decrease in total TS concentration
in group 5 patients, which demonstrated high baseline level of TS and relatively
low index of erectile function. The percentage of group 5 patients responding
to treatment was higher than the average. These patients were characterized by
an intermediate increase in erectile function. The increase in orgasmic function,
libido, and overall satisfaction was most pronounced in group 5 patients.
Impaza had no effect on TS level in group 6 patients with the higher in
tegral index of orgasmic function, libido, and overall satisfaction. TS concen
tration in these patients was above the average. The incidence of psychogenic/
psychological factors for ED in group 6 patients was twofold lower than the
average. The response to treatment was rarely observed in these patients.
TS concentration increased by more than 10% in six of seven patients
with primarily psychogenic ED (85% vs. 52% on average). These data suggest
that Impaza has a modulatory effect on the central mechanisms of erectile
function and central regulation of androgen status.
Hence, longterm treatment with Impaza is accompanied by the increase
in total serum TS concentration in 50% patients. This effect is particularly
pronounced in patients with low baseline level of TS.
Among patients with baseline total serum TS > 14 nM, this parameter
remained unchanged in 50% subjects, increased in 20% subjects, and decreased
in 30% subjects. The decrease in TS level was associated with a greater in
cidence of response to treatment and significant increase in orgasmic function,
232
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
libido, and overall satisfaction (above the average). A causeandeffect rela
tionship between Impaza treatment and decrease in TS concentration should be
evaluated in further studies.
The effect of drug therapy for ED depends on the relative contribution
of central and peripheral components in the impairment of erectile function in
each patient. A change in androgen status seems to be secondary to the correc
tion of central components of erectile function. It may be suggested that the
effect of Impaza on androgen status is mediated by the central regulation of
androgen biosynthesis.
The second clinical trial extended the notion of a possible role of Impaza
in clinical practice (E. B. Mazo et al., 2004a,b). This trial was performed at the
Department of Urology (Russian State Medical University, Moscow) in 20032004.
A prospective, openlabel, randomized, parallelgroup study was designed to
evaluate the efficacy and safety of various schemes of pharmacotherapy for ED.
After clinical and laboratory examination, ambulatory patients (2075
years of age) with complaints of reduced potency were enrolled in the trial.
All patients were examined as follows: case history; history of sexual ac
tivity; IIEF questionnaire; physical examination; Viagra test; intracavernous phar
macological testing with prostaglandin E1; Doppler pharmacoultrasonography of
penile vessels with audiovisual sexual stimulation before and after artificial
erection; penile electromyography; blood hormone test; and standard laboratory
tests (routine blood test, urine test, blood glucose, creatinine, and lipid profile).
All patients were divided into three groups of comparable size, age,
possible etiology, pathogenesis, and severity of ED.
The duration of therapy was 6 months. Group 1 patients (n=81) received
Viagra (sildenafil, Pfizer) in an individual dose. The initial dose of Viagra was
100 mg. Viagra dose could be reduced, which depended on the effect,
tolerability, and degree of undesired reactions. Viagra was given twothree times
a week. Group 2 patients (n=64) received Sialis (tadalafil, Eli Lilly) in a dose
of 20 mg twothree times a week. Group 3 patients (n=73) received Impaza (1
sublingual lozenge) every other day.
The effects of PDE5 inhibitors (Viagra and Sialis) and Impaza are
mediated by various mechanisms. These drugs were combined in patients that
did not respond to monotherapy and had serious side effects.
An additional group consisted of patients who did not respond to Viagra
or Sialis in the previous time, received intracavernous injections of vasoactive
drugs (n=22), or were untreated (n=32). The patients received Levitra
(vardenafil, Bayer) at an initial dose of 20 mg. This drug was given 2040 min
before sexual activity. The dose of Levitra was then selected individually, which
depended on drug effect and tolerability (reducton to 10 ml and 5 mg; or no
changes, 20 mg). The course of Levitra therapy was 3 months.
233
Ultralow doses
The efficacy of therapy in all groups was evaluated from an increase in
the index of “erectile function” by at least 3 points (IIEF questionnaire,
favorable response to treatment) or up to 26 points (recovery of EF).
The percentage of subjects who responded to treatment, achieved normal
EF, and rated the efficacy as “good” or “excellent” was evaluated in each group
of patients enrolled in the trial and receiving therapy (ITT). All statistical tests
were twosided. The hypotheses were tested at a significance level of 5%. The
number of patients in each group was selected to achieve a statistical power of
80%. The analysis of proportions was performed by x2 test for homogeneity of
proportions. Parametric variables were analyzed by Student’s t test for dependent
(difference from the baseline) and independent variables (difference from
placebo or reference drugs).
The main part of this trial (groups 13) involved 218 patients with ED
(2173 years of age, average age 58.1±13.2 years): younger than 35 years, 58
patients; 3555 years of age, 69 patients; and older than 55 years of age, 91
patients. The possible etiologic factors for ED in 174 patients were essential
arterial hypertension (n=81), diabetes mellitus (n=27), CHD (n=15),
lumbosacral osteochondrosis (n=23), chronic pelvic pain syndrome (n=21), and
postoperative period after radical surgery (organs of the pelvis minor, n=7).
Eight patients had organic disorders of unknown etiology. Psychogenic ED was
diagnosed in 36 patients. Mild (1825 points), moderate (1117 points), and
severe ED (not more than 10 points) was found in 74 patients (33.9%), 91
patients (41.7%), and 53 patients (24.4%), respectively. According to the results
of a complex andrological examination, these patients had ED of primarily
psychogenic (n=36, 16.5%), arteriogenic (n=87, 39.9%), venoocclusive (n=54,
24.8%), and neurogenic origin (n=41, 18.8%).
The groups were comparable by size, age of patients, possible etiology,
pathogenesis, and severity of ED (Table 8.9).
The overall efficacy of therapy with Viagra, Sialis, and Impaza was 77.8,
81.3, and 56.2%, respectively. The dependence of drug efficacy on the age of
patients, pathogenesis of ED, and severity of ED is shown in Table 8.10.
The efficacy of Viagra, Sialis, and Impaza was similar in patients younger
than 35 years. Sialis was much more potent than other drugs in patients of the
older age group (55 years of age or older). The advantage of Sialis over Viagra
is probably associated not only with physical capacities of these patients, but
also with the role of prelude to sexual activity and main attributes of sex (e.g.,
romantic situation).
Both inhibitors of PDE5 were pathogenetically effective in all groups of
patients. The efficacy of Sialis was higher only in patients with venoocclusive
ED. These differences are probably related to the pharmacodynamic and
pharmacokinetic properties of Sialis (e.g., prolonged circulation in blood
234
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Table 8.9.
Characteristics of ED patients enrolled in a comparative efficacy study of
monotherapy with PDE5 inhibitors and Impaza
Viagra
Sialis
Impaza
Parameter
abs.
%
abs.
%
abs.
%
Age
younger than 35 years (n=58)
22
27.2
17
26.5
19
26.0
3555 years (n=69)
26
32.1
20
31.3
23
31.5
older than 55 years (n=91)
33
40.7
27
42.2
31
42.5
16.4
Etiology
psychogenic ED (n=36)
13
16.1
11
17.2
12
essential hypertension (n=81)
30
37.0
24
37.5
27
37.0
diabetes mellitus (n=27)
10
12.3
8
12.5
9
12.3
11.0
osteochondrosis (n=23)
8
9.9
7
10.9
8
chronic pelvic pain syndrome (n=21)
8
9.9
6
9.4
7
9.7
CHD (n=15)
6
7.4
3
4.7
6
8.2
after radical surgeries (n=7)
2
2.5
3
4.7
2
2.7
unknown etiology (n=8)
4
4.9
2
3.1
2
2.7
16.4
Pathogenesis
psychogenic (n=36)
13
16.1
11
17.2
12
arteriogenic (n=87)
33
40.7
25
39.0
29
39.7
venoocclusive (n=54)
20
24.7
16
25.0
18
24.7
neurogenic (n=41)
15
18.5
12
18.8
14
19.2
Degree
mild (n=74)
27
33.3
21
32.8
26
35.6
moderate (n=91)
34
42.0
27
42.2
30
41.1
severe (n=53)
20
24.7
16
25.0
17
23.3
81
100
64
100
73
100
Total (n=218)
plasma). Impaza was most effective in patients with compensated and
subcompensated arteriogenic ED.
The efficacy of study drugs decreased with an increase in the severity of ED.
The efficacy of Impaza progressively increased from 33.2% (1 month of
therapy) to 56.2% (6 months of therapy). Significant changes were observed 19
patients (26.2%) by the 4th month of therapy (Fig. 8.16). These patients initially
reported the improvement and recovery of spontaneous erection, penile swelling,
and increase in the size of the penis (state of continuous partial tumescence).
The recovery of erections was observed after 34 months. These changes
probably serve as the early signs for therapeutic action of Impaza. They reflect
the cumulative effect and physiological activity of Impaza. Hence, Impaza
should be taken for at least 34 months to achieve a stable therapeutic effect.
235
Ultralow doses
Table 8.10. Efficacy of PDE5 inhibitors and Impaza as monotherapy for ED
Number of patients
with a favorable response
Parameter
Viagra
(n=81)
Sialis
(n=64)
Impaza
(n=73)
abs.
%
abs.
%
abs.
%
younger than 35 years (n=58)
21
95.5
15
88.2
16
84.2
3555 years (n=69)
22
84.6
17
85.0
12
52.2
older than 55 years (n=91)
20
60.6
20
74.1
13
41.9
Age
Pathogenesis
psychogenic (n=36)
11
84.6
10
90.9
9
75.0
arteriogenic (n=87)
25
75.6
19
76.0
18
62.1
venoocclusive (n=54)
14
70.0
13
81.3
6
33.3
neurogenic (n=41)
13
86.7
10
83.3
8
57.1
Degree
mild (n=74)
25
92.6
19
90.5
20
76.9
moderate (n=91)
26
76.5
22
81.5
15
50.0
severe (n=53)
12
60.0
11
68.8
6
35.3
63
77.8
52
81.3
41
56.2
Total (n=218)
Examination of an additional group showed that Levitra is effective in 44
patients (81.5%), including 28 of 32 primary patients (87.5%) and 16 of 22
patients not responding to Viagra or Sialis (72.7%). A favorable response to
Levitra (10 mg) was observed in 19 primary patients (59.4%). A positive effect
of Levitra (20 mg) was also revealed in the majority of patients not responding
to Viagra or Sialis.
Side effects of Viagra therapy (100 mg) were mainly found in fasting
patients. They included headache (11 patients, 13.6%), reddening of the skin
and neck (7 patients, 8.6%), dyspepsia (5 patients, 6.2%), and change in color
perception (3 patients, 3.7%). These symptoms persisted for up to 4 h.
AE of Sialis therapy included headache (eight patients, 12.5%), dyspepsia
(six patients, 9.4%), reddening of the skin and neck (four patients, 6.3%), and
back pain (two patients, 3.1%). The duration of side effects varied from several
hours to several days. In some patients, side effects persisted for the period of
drug action (36 h or more; up to 3 days).
Levitra was well tolerated. The efficacy and side effects of Levitra did not
depend on food intake and alcohol consumption. Side effects were most
pronounced 5090 min after the use of Levitra in a dose of 20 mg, which
corresponded to the maximum concentration of this product in the blood. They
236
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
%
60
50
40
30
20
10
0
1
2
3
4
5
Duration of therapy, months
Fig. 8.16. Efficacy of Impaza therapy: percentage of patients with a favorable
response.
included headache (seven patients, 12.9%), hot flashes to the face or neck (six
patients, 11.1%), stuffiness of the nose (six patients, 11.1%), and dyspepsia (two
patients, 3.7%). The events were of mild severity, persisted for up to 1.5 h, and
did not require treatment with additional pharmaceuticals or drug withdrawal.
AE were not revealed during Impaza therapy. Impaza had no adverse
effect on the course of underlying diseases. Therapy correction was not required.
The design of study and results of combination therapy are shown in
Fig. 8.17.
Viagra monotherapy was ineffective in 18 patients. The therapy was
supplemented by Impaza (1 tablet every other day). Combination therapy had
a positive effect in eight patients. Ten patients not responding to combined
treatment were prescribed to take Sialis. Sialis was effective in two of these
patients. The remaining eight patients were prescribed to receive intracavernous
injections. Combination therapy with Impaza and Viagra was given to 12
patients with serious side effects (severe headache), which resulted from the use
of Viagra in a dose of 100 mg. The dose of Viagra was reduced to 50 mg in eight
of these patients (previously ineffective dose). This therapy was effective and did
not cause side effects. The dose of Viagra could not be reduced in four patients.
These patients were successfully treated with Sialis, which did not cause side effects.
Combined treatment with Impaza was followed by a positive effect in 6 of
12 patients from the Sialis group. The remaining six patients demonstrated a good
response to Viagra in a dose of 100 mg. Five patients received therapy with
intracavernous injections and entered the Levitra group in the followup period.
Patients not responding to Impaza (n=32) were divided into two equal
groups (group 1, Viagra; group 2, Sialis). Viagra and Sialis had a positive effect
in 11 patients (68.7%) and 12 patients (75%). The remaining nine patients
received intracavernous injections.
These data show that combination therapy with two inhibitors of PDE
5 and Impaza increases the overall efficacy of oral therapy for ED up to 90%.
237
Ultralow doses
218 patients
81 patients
64 patients
73 patients
Viagra
Sialis
Impaza
+
63
18
77,8%
100 mg
Side effects
12
52
12
56,2%
+Impaza
+
+Impaza
+
8
6
+Impaza
+
+
81,3%
10
Sialis
+
+
41
32
6
16
16
Viagra
+
Sialis
Viagra
+
12
4
75%
8
50 mg
4
Sialis
2
90,1%
8
1
5
92,2%
+
11
5
68,7%
87,7%
Other forms of therapy
Fig. 8.17. Efficacy of PDE5 inhibitors and Impaza in combination therapy for ED.
“+”, presence of effect; “”, no effect.
Four primary patients did not respond to Levitra monotherapy (Fig. 8.18).
None of the PDE5 inhibitors had a positive effect in six patients. After three
unsuccessful attempts of sexual intercourse during Levitra treatment, ten
patients were prescribed to receive combination therapy with Impaza (1
sublingual lozenge every other day). This scheme of treatment had a positive
effect in seven patients (3 months of therapy). Therefore, combination therapy
with Impaza and Levitra was followed by an increase in the efficacy of therapy
from 81.5 to 94.4%.
The efficacy and safety of Impaza for ED of various etiologies were
confirmed in accordance with the principles of evidencebased medicine.
The course of prolonged treatment with Impaza was most effective.
Impaza differs from other products for pharmacotherapy of ED in the
mechanism of action, profile of therapeutic activity, safety profile, and influence
on androgen status.
Preclinical and clinical studies showed that Impaza has a pathogenetic
effect during ED, including the recovery (increase) of nitric oxide production
in the endothelium (key process in erectile function).
238
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
The pathogenetic mechanism for action of Impaza explains the fact that
a clinical effect of this product develops more slowly compared to other oral
drugs for ED.
An individual approach is required to develop the scheme of drug
treatment for each patient. It is necessary to take into account all factors that
modulate the clinical effect of study drug.
High efficacy of Impaza in patients with primarily psychogenic ED and
vascular arterial ED, as well as a good combination of Impaza and nitrates in
CHD patients indicate that Impaza holds promise as a firstline drug for ED
in patients of these groups.
Taking into account the mechanisms for action of Impaza and oral
inhibitors of PDE5, combination therapy is required to increase the clinical
efficacy of these drugs.
The therapeutic algorithm for ED patients was developed from the results
of combined treatment (Fig. 8.19).
If a young patient has psychogenic, isolated neurogenic (osteochondrosis),
compensated and subcompensated arteriogenic ED of mild or moderate severity,
and CHD (nitrate therapy), treatment can be started from Impaza. PDE5
54 patients
22 patients:
ineffectiveness
of Viagra
and Sialis
32 primary patients
with ED
Levitra
+
72,7%
16
Levitra
+
28
4
87,5%
6
10
+Impaza
+
7
3
94,4%
Fig. 8.18. Efficacy of combination therapy with Impaza and Levitra.
239
Ultralow doses
inhibitors are prescribed for patients with Impaza inefficacy, as well as for
patients with severe or moderate venooclusive ED.
Pharmacotherapy with PDE5 inhibitors should not be withdrawn when
one of these drugs appears to be ineffective. Another inhibitor of PDE5 can
be prescribed under these conditions. At low efficacy of PDE5 inhibitors, they
should be combined with Impaza. This scheme of treatment will allow us to
reduce side effects of monotherapy due to a decrease in the dose of PDE5
inhibitors. The efficacy of therapy will remain unchanged.
When therapy is chosen, a physician should perform dynamic monitoring
and evaluation of the patient’s state.
From the first days of treatment, Impaza should be given in combination
with PDE5 inhibitors. Besides the above mentioned advantages, this
combination allows us to increase the interval between drug intakes. These
patients maintain the ability to complete successful sexual intercourse.
Prolonged therapy with Impaza and PDE5 inhibitors is accompanied by the
recovery of adequate and spontaneous erections. This conclusion is derived from
the patient’s report, increase in cavernous blood flow (Doppler ultrasonography
of the penis with audiovisual sexual stimulation), and elevation of cavernous
Complex andrological examination
Mild
Impaza
+
Moderate
Arteriogenic
and neurogenic ED
Another pathogenesis
of ED
“yes” •sexual activity more than twice a week
Sialis
•sexual intercourse in the morning
•necessity of spontaneous intercourse
+
Severe
“not” for everything
“not” for •combination of sex with food
everything intake or alcohol consumption
Viagra
•necessity of the rapid effect
“yes” •severe ED
+
Combination of Impaza
and PDE5 inhibitors
Levitra
Six months
of therapy
+
Other forms
of therapy
no effect
Fig. 8.19. Algorithm of differential combined pharmacotherapy with Impaza for
erectile dysfunction (E. B. Mazo et al., 2004).
240
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
electrical activity (penile electromyography). It is possible to reduce the mini
mum effective dose and to withdraw PDE5 inhibitors. The patient is transferred
to Impaza monotherapy. This drug may be withdrawn in the followup period.
The proposed scheme of combined pharmacotherapy with Impaza has a
positive effect in more than 90% ED patients, increases the safety of treatment,
and significantly decreases the cost of therapy.
8.3. Clinical effectiveness and mechanisms
for action of Anaferon
Among a variety of products for therapy of acute respiratory viral
infections (ARVI), much attention is given to pathogenetic drugs that affect the
natural mechanisms of antiviral resistance (primarily the IFN system). Despite
much progress in vaccine prevention of influenza, the antigenic variability of this
virus and high incidence of mixed infections in clinical practice require the
development of universal remedies for ARVI prophylaxis. Modulation of the
endogenous IFN system as a natural mechanism of antiviral resistance holds
much promise in this respect.
The adequate induction of endogenous IFN provides a mild or abortive
course of viral infections. Therefore, IFNinducing agents have a particular
place in the therapy for ARVI. Moreover, viral infections often cause or
accompany secondary immunodeficiency. The strategy of immunorehabilitation
with immunomodulatory drugs should be used for the therapy and prevention
of ARVI, particularly in children. IFN inductors are effective in the therapy of
viral infections. A variety of side effects limit the prolonged use of these drugs
for prevention of viral infections, particularly in children.
Experimental and clinical studies showed that Anaferon, a new
immunomodulator with antiviral activity (Russia), holds promise for therapy and
prevention of ARVI. Anaferon contains antibodies to human IFNγ. The drug
formulation for children was designated as “Anaferon for children” (V. F.
Uchaikin et al., 2003). Anaferon induces the production of endogenous IFNγ
and IFNα/β and affects the expression of functionally related cytokines,
including IL2, IL4, and IL10 (A. V. MartyushevPoklad, 2003).
Previous experiments showed that endogenous IFNγsensitive immune
cells serve as a target for Anaferon (Fig. 8.20; A. V. MartyushevPoklad, 2003).
The most probable mechanisms for therapeutic activity of Anaferon in
ARVI are shown in Figs. 8.21 and 8.22 (according to the results of experimental
and clinical studies).
The early and frequent use of Anaferon from the onset of viral infection
provides the induction of IFNα, IFNβ, and IFNγ and activation of NK cells.
241
Ultralow doses
IFN suppresses viral replication and prevents infection of other cells. NK cells
lyse infected cells. These processes determine the rapid antiviral effect of Anaferon.
The induction of endogenous IFNγ as a key immunoregulatory cytokine
triggers the cascade of other events in the immune system. They include the
induction of regulatory components for the immune response (type 1 and 2 T
helper cells) and activation of macrophages. Activation of effector components
of the cellular and humoral response is realized via cytotoxic lymphocytes and
antibodies and contributes to lysis of infected cells, binding of viral particles, and
elimination of the virus.
Sufficient activation of macrophages and antibody production prevent the
development of bacterial complications after viral infection.
Randomized controlled clinical studies of the efficacy and safety of Anaferon
for children in children with influenza and other ARVI were performed at the In
stitute of Influenza (Russian Academy of Medical Sciences, St. Petersburg), Rus
sian State Medical University (Moscow), and Volgograd State Medical University.
The trial involved 390 children (from 1 month to 14 years of age). The children
received inpatient or outpatient therapy for influenza (verified by an immunofluor
ANAFERON
IL12
TNFα
IFNγ
IFNγ
AG
IL12
NK
Cellular immune response
Monocyte
IL12
IFNγ
Th 1
IL2
IFNγ
TNFβ
MP
Tc
NK
Th 1
cytokines
Th 0
Th 2
cytokines
Humoral immune response
IL4
Basophil
PL
Th 2
IL2
IL10
IL13
B
AB
IL10
IL6
AG
Monocyte
ANAFERON
Fig. 20. Natural regulation of the immune response and targets for Anaferon. Th, T
helper cells; AG, antagonists; AB, antibodies; MP, macrophages; NK, natural killer cells.
242
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
ANAFERON
IFNγ
IFNα/β
Activation
of NK cells
Suppression
of viral
replication
Lysis
of infected
cells
Induced
resistance
of uninfected
cells
ANTIVIRAL EFFECT
Fig. 8.21. “Rapid” mechanisms for the antiviral effect of Anaferon.
ANAFERON
IFNγ
Activation
of NK
Activation
of macrophages
Induction of
regulatory
components
for the immune
response
Type 2
T helper cells
B lymphocytes
(proliferation
and differentiation)
Type 1 T helper cells
Antibody
production
Cytotoxic
T lymphocytes
(proliferation,
differentiation,
and activation)
Lysis
of infected
cells
IgM, IgG,
IgA
Binding
of viral
particles
ELIMINATION
OF VIRUSES
Fig. 8.22. Mechanisms for the immunomodulatory effect of Anaferon.
243
Ultralow doses
escence study) and other ARVI, including those complicated by laryngotracheo
bronchitis with stenosis or bacterial infection. Anaferon lozenges were given three
seven times a day, which depended on the stage of disease. The product was dis
solved in water for treatment of infants. Patients of the reference groups received
placebo, Immunal, or Arbidol in combination with symptomatic and/or antibac
terial therapy (when prescribed). The main symptoms of disease (fever, intoxica
tion, and catarrhal symptoms) and possible side effects were evaluated. The in
terferon system and immune status (subpopulations of peripheral blood lympho
cytes, serum IgE, and sIgA in nasal lavage fluid) were studied in some children.
Anaferon therapy was followed by a significant decrease in the severity and
duration of main clinical manifestations of ARVI and complications in children
(p<0.05 compared to the placebo group). The duration of fever, intoxication,
rhinitis, and cough decreased by 3540, 4050, 20, and 30%, respectively.
Fig. 8.23 illustrates the main results of a placebocontrolled trial of the
efficacy of Anaferon for children during influenza (Institute of Influenza,
Russian Academy of Medical Sciences). The diagnosis of influenza was verified
by laboratory tests.
The trial involved 105 patients (110 years of age, average age 5.40±0.02
years). Besides the presence of influenza A and/or B virus, 59% patients were
shown to have the adenovirus, respiratory syncytial virus, parainfluenza virus,
coronavirus, or mycoplasma as etiologic factors of the disease.
In addition to the reduction of disease duration, patients of the Anaferon
for children group were characterized by a significant decrease in the incidence
of purulent rhinitis as a major complication of influenza (p<0.05; Fig. 8.24).
The use of Anaferon was followed by a decrease in the period of inpatient
treatment and volume of antibacterial and symptomatic therapy. Drugrelated
adverse events were not observed in patients of the Anaferon group.
Anaferon for children improved the IFN status of patients. This remedy
not only promoted the induction of endogenous IFN on days 23 of therapy,
Days
7
6
5
4
3
2
1
0
placebo
!
Anaferon
!
!
!
Fever
(>37°С)
Intoxication
Rhinitis
Cough
Fig. 8.23. Effect of Anaferon for children on the duration of main clinical mani
festations of influenza. *p<0.05 compared to placebo.
244
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
but also prevented a decrease in IFN production by lymphocytes at the stage
of reconvalescence (Fig. 8.25).
The ability of Anaferon to induce not only endogenous IFNγ (as shown
in a preclinical study), but also IFNα in patients with viral infection is related
to the existence of a close functional relationship between IFNγ and IFNα.
Published data show that IFNγ potentiates the induction of IFNα during viral
infection (A. P. CostaPereira et al., 2002). These data are of particular import
ance since one of the major “strategies” of influenza virus in overcoming the
mechanisms of natural resistance includes the inhibition of early interferon in
duction (X. Wang et al., 2000).
The content of various subpopulations of peripheral blood lymphocytes
(CD3+, CD4+, CD8+, CD20+, and CD16+) returned to normal after drug ther
apy (Fig. 8.26). These changes were accompanied by a decrease in IgE level and
increase in sIgA level (Fig. 8.27).
Percentage of patients, %
30
25
20
15
!
10
5
0
Placebo
Anaferon
Fig. 8.24. Effect of Anaferon for children on the incidence of purulent rhinitis after
influenza. *p<0.05 compared to placebo.
a
pg/ml
400
b
pg/ml
60
!
!
50
300
!
40
200
30
!
20
100
10
0
0
1
2
3
1
placebo
2
3
Anaferon
Fig. 8.25. Effect of Anaferon for children on the IFN status of influenza patients:
stimulated production of IFNγ (a) and IFNα by peripheral blood leukocytes (b).
Days 12 of disease (1); days 23 of therapy (2); and convalescence (3). *p<0.05
compared to placebo.
245
Ultralow doses
Children with normal content of lymphocytes, %
!
100
90
!
!
!
!
80
!
70
60
50
40
30
20
10
0
CD3+
CD4+
CD8+
CD20+
Lymphocyte subpopulation
before therapy (control)
before therapy (Anaferon)
CD16 +
CD3+/CD8+
after therapy (control)
after therapy (Anaferon)
Fig. 8.26. Effect of Anaferon for children on subpopulations of peripheral blood
lymphocytes in influenza patients. *p<0.05 compared to placebo.
Hence, the therapeutic use of Anaferon has an immunomodulatory effect
on influenza patients. These data are consistent with the results of preclinical
studies and confirm the fact that Anaferon modulates the mechanisms of
antiviral resistance (innate and adaptive cellular and humoral responses; L. V.
Osidak et al., 2003).
a
% of the baseline
160
b
% of the baseline
!
160
120
!
120
!
80
!
80
40
40
0
0
Control
Anaferon
Control
before therapy
Anaferon
after therapy
Fig. 8.27. Parameters of humoral immunity during therapy of influenza patients
with Anaferon for children: secretory IgA in nasal lavage fluid (a); and IgE in blood
serum (b). *p<0.05 compared to the baseline value.
246
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Among a variety of ARVI, respiratory syncytial virus (RSV) infections have
a particular place. These infections most often cause complications (disorders of
the lower respiratory tract) and require hospitalization. A randomized placebo
controlled study of the efficacy of Anaferon for children in patients with RSV
infection was performed at the Institute of Influenza (Russian Academy of
Medical Sciences). The patients with confirmed RSV infection (110 years of
age) received Anaferon (n=40) or placebo (n=36) in combination with
symptomatic drugs for 710 days (from the 1st or 2nd day of disease). Anaferon
therapy was followed by a decrease in the mean duration of hyperthermia (by
1.2 days), period of intoxication (by 1 day) and catarrhal syndrome (by 2.2
days), and total length of disease (by 2.3 days, p<0.05). Similarly to influenza
patients, Anaferon induced the production of endogenous IFNα and IFNγ by
peripheral blood leukocytes (days 23 of therapy) and prevented a change in the
IFN status (period of reconvalescence; E. G. Golovacheva et al., 2003).
A randomized placebocontrolled study of the efficacy of Anaferon for
children in ARVI prevention was performed at the Institute of Influenza (Rus
sian Academy of Medical Sciences, St. Petersburg) and Russian State Medical
University (Moscow). The trial involved 400 children (from 1 month to 4 years
of age, 70% patients with poor health). The drug or placebo (lozenges) was
given daily for 3 months. The product was dissolved in water for treatment of
infants. ARVI patients were prescribed to take Anafaron as a therapeutic drug
in combination with symptomatic therapy. The morbidity rate, severity of ARVI,
incidence of complications, and drug tolerability were evaluated during Anaferon
therapy. The IFN status in some patients was studied before and after the start
of preventive treatment. Latent infection with various viruses was determined by
an immunofluorescent study of nasal lavage fluid.
Both trials in various clinical institutions yielded the same results (Fig.
8.28). The average number of ARVI in one patient decreased by 1.51.8 times.
This drug had a stronger effect on the incidence of severe (febrile) ARVI, which
was reduced by 2.12.4 times.
The prophylactic and therapeuticandprophylactic effects of this drug were
studied in details at the Institute of Influenza. During this trial, the patients were
examined daily. The preventive and therapeutic use of Anaferon for children was
followed by a decrease in the incidence and severity of ARVI and complications.
The duration of all symptoms (fever, intoxication, and/or catarrhal symptoms)
decreased by 2.2 times. The duration of fever and purulent rhinitis decreased by
3.8 and 3 times, respectively (Fig. 8.29). The incidence of ARVI complications
due to purulent rhinitis and otitis decreased by 2.4 and 2.7 times, respectively.
The decrease in ARVI morbidity was most pronounced in children with
poor health. The percentage of children not suffering from ARVI over a
3month trial increased from 3 to 24.7%.
247
Ultralow doses
а
b
ARVI morbidity
per one patient
ARVI morbidity
per 100 patients per month
3
60
50
!
2
40
!
!
30
1
!
20
10
0
0
I
II
I
placebo
II
Anaferon
Fig. 8.28. Effect of prophylactic treatment with Anaferon for children (3month
course) on the overall incidence of ARVI (a) and incidence of febrile ARVI (t>37.5 oC,
b). Results of two placebocontrolled trials: Institute of Influenza (I) and Russian
State Medical University (II). *p<0.05 compared to placebo.
Anaferon did not cause AE. An immunofluorescent study of nasal lavage
fluid showed that latent infection of children (occurrence of typical agents for
ARVI) decreased from 48.1 to 22.9%. Latent infection with herpes simplex virus
decreased from 13.5 to 4.4%. After the course of treatment with Anaferon for
children, the ability of peripheral blood lymphocytes to produce IFNα/β upon
stimulation increased by 2.2 times.
The efficacy and safety of Anaferon for children as a drug for the therapy
and prevention of influenza and other ARVI in children were confirmed in
randomized clinical trials (in accordance with the principles of evidencebased
medicine). This remedy not only decreases the duration and severity of clinical
manifestations of ARVI and reduces the morbidity rate from viral infections, but
Incidence per 100
patients per month
a
Number of days
35
30
25
20
15
10
5
0
b
60
50
40
!
Any symptom
of ARVI/
complication
30
!
20
!
!
!
10
0
Subfebrile/
febrile state
Purulent
rhinitis
Purulent
rhinitis
placebo
Otitis
Anaferon
Fig. 8.29. Effect of therapeutic and prophylactic treatment with Anaferon for
children (3month course) on the total duration of ARVI symptoms (a) and incidence
of ARVI complications in children (b). *p<0.05 compared to placebo.
248
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
also contributes to immunorehabilitation of patients with clinical and laboratory
signs of secondary immunodeficiency (L. V. Osidak et al., 2003; A. V. Martyu
shevPoklad et al., 2004a,b; S. A. Sergeeva et al., 2004).
A deficiency of immune resistance and inadequacy of the immune
response play an important role in the pathogenesis of viral infections (including
herpes virus infections, enterovirus infections, and viral hepatitides). A combina
tion of immunomodulatory and antiviral properties of Anaferon is related to the
induction of endogenous IFN and provides a significant clinical effect of the
drug during viral infections.
Studying the prophylactic efficacy of Anaferon showed that this remedy
promotes the elimination of herpes simplex virus under conditions of latent virus
infection (Institute of Influenza, Russian Academy of Medical Sciences). These
results were confirmed by further observations. The family of herpes viruses also
includes the agents that cause infectious mononucleosis (EpsteinBarr virus) and
chickenpox (Herpes zoster).
The therapeutic efficacy and safety of Anferon for children in patients
with infectious mononucleosis (EpsteinBarr virus) were evaluated in a double
blind placebocontrolled trial at the Siberian State Medical University (Tomsk).
The efficacy of Anaferon in various therapeutic schemes for infectious mono
nucleosis was also studied at the Ural State Medical Academy (Ekaterinburg).
The trials involved 140 children (314 years of age) with moderatetosevere and
severe infectious mononucleosis. The efficacy of Anaferon for children,
acyclovir, cycloferon, combination of Anaferon and acyclovir, and placebo was
evaluated. The patients also received antibacterial and symptomatic therapy
(when prescribed). The therapeutic efficacy was estimated from main clinical
manifestations, including the duration of fever and acute tonsillitis. The trial in
Tomsk showed that Anaferon significantly decreases the duration of clinical
symptoms (as compared to placebo). The duration of acute tonsillitis, hepato
megaly, and lymphadenopathy decreased by more than 1, 3, and 4 days, respec
tively. Body temperature rapidly returned to normal after Anaferon therapy.
Studying the efficacy of Anaferon in various therapeutic schemes for infectious
mononucleosis was performed in Ekaterinburg. A combination of Anaferon and
acyclovir was most effective under these conditions. The mean duration of fever
and acute tonsillitis in patients of various groups appeared as follows: 2.4 and 4.3
days, respectively, in the Anaferon group; 2.2 and 2.8 days, respectively, in the
Anaferon+acyclovir group; 5.1 and 4.1 days, respectively, in the acyclovir group;
5.4 and 4.8 days, respectively, in the cycloferon group; and 8.7 and 6.5 days,
respectively, in the placebo group. Therefore, Anaferon and other inductors of in
terferon hold much promise for the therapy of infectious mononucleosis. IFNin
ducing agents should be used in combination with antiviral drugs (L. A. Zhu
ravleva et al., 2003a,b; K. I. Chuikova et al., 2004; V. V. Fomin et al., 2004).
249
Ultralow doses
A doubleblind, randomized, placebocontrolled trial of the efficacy and
safety of Anaferon for children in chickenpox was performed at the Vol’sk
Children’s Hospital (Saratov oblast). The trial involved 236 children (115 years
of age). The duration of disease did not exceed 48 h. Anaferon for children
(n=136) or placebo (n=100) was given therapeutically in combination with
symptomatic drugs (treatment of rash with 2% solution of brilliant green).
The time to normalization of body temperature, new crops of skin rash,
relief of itching, and incidence and severity of complications were evaluated.
Anaferon significantly decreased the duration and severity of disease (Fig.
8.30). Body temperature in Anaferonreceiving children returned to normal by
the 3rd day of therapy (vs. 6th day in the reference group). A correlation was
found between the occurrence of skin rash and body temperature, which is
consistent with published data. Anaferon prevented the appearance of new spots
in an earlier period (by 3 days). The duration of itching decreased by 4 days in
the Anaferon group (as compared to placebo). The percentage of children with
bacterial infectionassociated pustules was sevenfold higher in the reference
group compared to the Anaferon group. These data illustrate the efficacy and
safety of Anaferon for children in the therapy of chickenpox (M. V. Kudin,
2005a,b; A. V. MartyushevPoklad et al., 2005b).
The development of acute intestinal infections is one of the urgent
problems in treatment of children’s infections. According to the international
statistics, 7080% of intestinal infections are caused by viruses. Rotaviruses,
caliciviruses, and coronaviruses are the major etiologic agents for these diseases.
The efficacy and safety of Anaferon for children in acute intestinal
infections of viral etiology were confirmed by several clinical studies.
A doubleblind randomized clinical trial was performed at the Institute of
Influenza (St. Petersburg). The trial involved 79 patients (from 6 months to 10
a
Duration, days
8
7
6
5
4
3
2
1
0
!
Fever
b
!
New spots
!
Itching
°С
38,0
37,9
37,7
37,5
37,3
37,1
36,9
36,7
36,5
1
2
1
1e
2
2e
3
3e
4
4e
5
Period of therapy, daysлечения, сут
placebo
Anaferon
Fig. 8.30. Efficacy of Anaferon for children in chickenpox: effect on the duration
of main clinical symptoms (a) and temperature curve (b). Placebo (1) and Anaferon
(2). (b) Evening temperature, e. *p<0.05 compared to placebo.
250
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
years of age). The diagnosis of coronavirus infection was confirmed by
laboratory tests. Patients of the main (n=51) and control groups (n=28) received
Anaferon and placebo, respectively, in combination with standard therapy. The
clinical state at admission was moderatetosevere. The main symptoms of
coronavirus infection were rapidly relieved in Anaferonreceiving patients. These
patients were characterized by a significant decrease (p<0.05 compared to the
placebo group) in the duration of gastrointestinal symptoms (from 4.4±0.5 to
2.5±0.2 days), fever (from 3.4±0.3 to 2.0±0.1 days), intoxication (from 5.1±0.4
to 2.9±0.1 days), and catarrhal symptoms in the nasopharynx (from 7.0±0.4 to
4.3±0.2 days; E. V. Obraztsova et al., 2006).
Twoday treatment of children with coronavirus infection of the main and
control groups was followed by a significant decrease in the number of patients
with fever (by 75 and 44%, respectively).
A doubleblind, placebocontrolled clinical trial of the efficacy and safety
of Anaferon for children in calicivirus infection was performed at the Institute
of Children’s Infections (St. Petersburg). The trial involved 60 children (from
6 months to 15 years of age) that were hospitalized with symptoms of acute
intestinal infection. The diagnosis of calicivirus infection was verified by means
of scanning electron microscopy. The patients were divided into two groups of
Anaferon for children (n=30) and placebo (n=30). The groups were represen
tative of age, sex, and severity of disease. The use of Anaferon for 57 days was
followed by a significant decrease in the duration of symptoms of calicivirus
infection. The duration of fever, vomiting, and diarrhea in patients of the main
group was 1.3±0.2, 1.5±0.2, and 1.2±0.1 days, respectively. Fever, vomiting, and
watery diarrhea in patients of the control group persisted for 2.9±0.3, 2.4±0.2,
and 2.0±0.2 days, respectively (p<0.05). Anaferon had a strong effect on the
duration of virus elimination. By the end of treatment, caliciviruses were
detected in the feces from 1 patient of the main group (3%) and 16 patients of
the control group (53%; I. V. Razdyakonova et al., 2005).
A pilot study of the efficacy of Anaferon for children in rotavirus
gastroenteritides was performed at the Rostov State Medical University (Rostov
onDon). The trial involved 27 children (1.55.5 years of age). The diagnosis of
rotavirus gastroenteritis was confirmed by laboratory tests. Thirteen patients
received Anaferon (therapeutic treatment) and standard therapy. Fourteen
patients received only standard drugs. The introduction of study drug into
combination therapy was followed by a significant decrease in the duration of
main symptoms of this disease (p<0.001). The duration of fever and diarrhea
decreased by 1.5 and 2.3 days, respectively (E. N. Simovan’yan et al., 2004).
Clinical studies demonstrated the efficacy and safety of Anaferon for
children in treatment of acute intestinal infection of viral etiology. Anaferon
significantly decreased the time to virus elimination from an organism, which
251
Ultralow doses
reflects the antiviral effect of this remedy. The product was well tolerated and
did not cause AE. These data indicate that Anaferon for children should be used
in combination therapy of children with acute viral infections. Anaferon had a
positive effect in patients with tickborne encephalitis (A. V. Skripchenko et al.,
2005), hemorrhagic fever with renal syndrome (E. B. Egorov et al., 2004),
pseudotuberculosis (V. N. Timchenko et al., 2004), tubulointerstitial nephritis (I.
F. Vladimirtseva et al., 2005), and enteroviral and meningococcal meningitis
(Yu. B. Khamanova et al., 2005). Anaferon was also used in emergency pro
phylactic therapy of ARVI in children with bronchial asthma (E. I. Kondrat’eva
et al., 2004, 2005, 2006), as well as in the treatment of ARVI in children with
heart diseases (L. V. Yakovleva et al., 2005).
Controlled clinical trials in leading medical institutions of the Russian
Federation showed that Anaferon holds much promise for the prevention and
therapy of influenza, viral infections of the respiratory tract, and herpes
infection (including chickenpox and infectious mononucleosis). Anaferon can
be used in combination therapy for acute intestinal infections of viral etiology,
prevention and treatment of complications of viral infections, therapy of
secondary immunodeficiency of various etiologies, and combination therapy for
bacterial infections.
8.4. Artrofoon as a promising drug
for pathogenetic therapy
of chronic arthropathies
The introduction of biological antirheumatic drugs into clinical practice
in various countries has opened new perspectives in the therapy of RA, osteo
arthritis (OA), and other autoimmune inflammatory diseases. Unfortunately,
these drugs are of limited use in Russia due to the high cost. Artrofoon (ULD
of antibodies to TNFα, Russia) is a new drug for the therapy of RA. Six
month clinical studies were designed to evaluate the efficacy and safety of Artro
foon in RA and OA. The drug was also used in inflammatory and degenerative
diseases of the joints.
The clinical efficacy of Artrofoon during RA was evaluated in an open
randomized study at the Volgograd State Medical University (V. I. Petrov et al.,
2003, 2005; A. V. MartyushevPoklad et al., 2003; V. I. Petrov et al., 2003; J.
L. Dugina et al., 2005a,b). Diclofenac was used as a reference drug. The trial
involved 81 patients with RA (according to the criteria of the American
Rheumatology Association, ARA). Thirtyone patients (average age 54.0±1.1
years, mean duration of disease 11.6±1.2 years) received diclofenac in a daily
dose of 100 mg. Fifty patients (average age 51.2±1.5 years, mean duration of
252
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
disease 8.9±1.0 years) were treated with Artofoon (4 tablets twice daily) for 6
months. The average number of damaged joints in patients of the diclofenac and
Artrofoon groups was 23.8±1.3 and 22.7±1.3, respectively. Seronegative RA was
diagnosed in 48.4 and 42% patients of the diclofenac and Artrofoon groups,
respectively. Among patients enrolled in the trial, 12.9% patients of the
diclofenac group and 25% patients of the Artrofoon group previously received
standard therapy (methotrexate, 7.5 mg weekly). This treatment was ineffective
in 45 and 26% patients of the diclofenac and Artrofoon groups, respectively.
During the trial, standard therapy was continued in 16.1% patients of the
diclofenac group and in 18% patients of the Artrofoon group.
A 6month course of treatment with diclofenac was followed by a
decrease in the number of painful joints (from 19.2±1.3 to 17.4±1.2, by 1.9±0.5
or 9.4%) and swollen joint (from 8.3±1.0 to 5.7±0.7, by 2.6±0.6 or 27.5%) and
reduction of the duration of morning stiffness (from 131±13 to 102±9 min, by
29±7 min or 17.3%). The improvement of clinical symptoms was more
pronounced in patients of the Artrofoon group. Artrofoon therapy was followed
by a decrease in the average number of painful joints (from 17.4±1.1 to
13.6±0.9, by 3.8±0.6 or 20.5%) and swollen joint (from 6.2±0.7 to 3.8±0.4, by
2.4±0.5 or 33.6%) and reduction of the duration of morning stiffness (from
136±10 to 87±6 min, by 49±5 min or 33.2%).
After 6 months of therapy with diclofenac, 25.8% patients reached the
ACR20 criterion (20% improvement of clinical symptoms, CI 95% = 13.7
43.3%). The number of Artrofoonreceiving patients who reached the ACR20
criterion was twofold higher than in the diclofenac group (58% patients, CI
95% = 44.270.6; Fig. 8.31).
The symptoms of RA in patients were reduced after 1 month of therapy
with diclofenac. In the followup period, the effect of diclofenac increased less
significantly than that of Artrofoon. Artrofoon had a positive effect on local and
general symptoms of inflammation in RA patients. A significant improvement
%
60
50
40
30
20
10
0
Artrofoon
Diclofenac
(100 mg daily)
Fig. 8.31. Patients with rheumatoid arthritis who reached the ACR 20 improvement
criterion after 6 months of therapy.
253
Ultralow doses
of the patient’s state was observed 3 months after the start of therapy. The effect
of Artrofoon developed progressively and reached maximum by the 6th month
of therapy (Fig. 8.32).
The severity of pain in patients of the diclofenac and Artorfoon groups
decreased by 21.0 and 34.7%, respectively. Roentgenologically, progression of the
disease was not found in patients of both groups. The effect of study drug was
rated as “good” by 54% patients of the Artrofoon group and 32.3% patients of
the diclofenac group.
All patients who were enrolled in the trial completed a 6month course
of therapy. Artrofoon had a better safety profile than diclofenac. Serious adverse
events were not reported in the Artrofoon group. AE in 22.6% patients of the
diclofenac group were associated with drug effect on GIT (pain of discomfort
in the epigastric region, eructation, and nausea). These events required the
prescription of antacid drugs and/or antiemetic agents.
Eleven patients of the Artrofoon group continued to take the drug after
6 months of therapy (five patients for 12 months; one patient for 11 months;
one patient for 10 months; one patient for 9 months; and three patients for 8
months). By the end of treatment, all patients reached the ACR20 criterion.
The relief of clinical symptoms for joint disease in Artrofoonreceiving
patients was accompanied by a significant improvement in laboratory signs of
inflammation, including the concentration of proinflammatory cytokines. In
some patients the concentration of proinflammatory cytokines significantly
decreased (>25%) after 6 months of therapy with Artrofoon. They demonstrated
a decrease in the concentrations of plasma TNFα (50% patients), IL1 (70%
a
% of the baseline
60
50
!
40
!
30
20
!
!
10
0
Number
of painful
joints
Severity
of pain
b
% of the baseline
Duration
of morning
stiffness
CRP level
Artrofoon
90
80
70
60
50
40
30
20
10
0
Number
of painful
joints
Severity
of pain
Duration
of morning
stiffness
CRP level
diclofenac, 100 mg daily
Fig. 8.32. Effect of 6month therapy with Artrofoon (light bars) and diclofenac (dark
bars) on the severity of rheumatoid arthritis. Mean improvement of the RA activity
(a); and percentage of patients with an improvement by at least 20% (b). CRP, C
reactive protein. *p<0.05 compared to the diclofenac group.
254
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
patients), and IL6 (50% patients). These changes were not found in patients
of the control group.
These data suggest that antiinflammatory activity of Artrofoon is related
to the regulation of systemic production of proinflammatory cytokines.
An open comparative clinical trial of the efficacy and safety of Artrofoon
vs. diclofenac in OA patients was performed at the Volgograd State University
(I. V. Kostryukova et al., 2005; I. V. Kostryukova et al., 2006). The trial involved
120 patients with the diagnosis of OA (112 women, 93.33%; and 8 men, 6.66%;
average age 63.84±0.88 years). The majority of patients were older than 60 years
of age (66.6% disabled persons). They had polyarticular (101 patients, 84.16%),
oligoarticular (11 patients, 9.16%), monoarticular OA (8 patients, 6.66%). The
majority of OA patients (n=72, 60%) suffered from severe synovitis, which was
manifested in pain at rest, swelling, hyperthermia, and functional disorders.
Subclinical synovitis in 48 patients (40%) was manifested in spontaneous nighttime
pain, morning stiffness, and local tenderness in palpation. Pain sensation in all
patients was revealed during joint movement and palpation of the joints and
periarticular tissue. Damage was most often observed in the knee joints (90.83%),
ankle joints (50.83%), hip joints (20.83%), and small joints of the hand (31.66%).
All patients with OA were randomized into the following three groups:
group 1 (60 patients), Artrofoon (4 tablets twice daily); group 2 (30 patients),
reference drug diclofenac (100 mg daily); and group 3 (30 patients), com
bination therapy with Artrofoon (8 tablets daily) and diclofenac (100 mg daily).
Drug efficacy was evaluated 1, 3, and 6 months after the start of therapy.
The following clinical parameters were evaluated: WOMAC index (Western
Ontario and McMaster Universities Osteoarthritis Index); overall pain; articular
index; Ritchie index; swelling index; and Leken’s indexes for gonarthrosis and co
xarthrosis. Artrofoon safety was determined from the incidence of AE (Table 8.11).
The WOMAC total score in Artrofoonreceiving patients was 49.92±1.75
(vs. 52.00±0.72 and 51.5±1.97 in the diclofenac and diclofenac+Artrofoon
groups, respectively). The WOMAC total score in patients of these groups
decreased by 10.94, 10.23, and 22.03 points, respectively, 1 month after the start
of therapy. The WOMAC total score in these patients decreased by 24.22, 20.6,
and 32 points, respectively, 3 months after the start of therapy. Six months after
the start of therapy, the WOMAC total score in Artrofoonreceiving patients was
18.72±1.81 points (decrease by 31.2 points, p<0.001 compared to the diclofenac
group). This index also decreased in patients receiving diclofenac (by 25 points,
27.53±2.52 points) and combination therapy (by 40.4 points, 11.10±1.12 points;
p<0.001 and p<0.01 compared to the diclofenac and Artrofoon groups, respec
tively; Fig. 8.33).
Similar changes were found in clinical manifestations of OA (WOMAC
subscales of pain, stiffness, and function). Six months after the start of therapy,
255
Ultralow doses
256
Table 8.11. Main clinical and laboratory parameters of osteoarthritis during therapy with Artrofoon and diclofenac
Artrofoon (2 tablets, 4 times daily)
Parameter
baseline
after
baseline
3 months
Severity of pain
2.17±0.10
Duration
of morning stiffness
Ritchie index
Articular index
Lee index
Diclofenac (100 mg daily)
6 months
after
3 months
Artrofoon + diclofenac
baseline
after
3 months
6 months
6 months
1.12±0.10** 0.58±0.10**
2.27±0.14
1.53±0.12** 1.57±0.12**
2.43±0.18
19.37±1.77
8.72±1.13*
3.72±0.81*
18.83±2.71
12.17±1.57*
13.43±1.46
17.83±1.06
2.83±0.92*
6.45±0,40
3.73±0.31*
2.43±0.18**
7.23±1.01
5.47±0.87
4.87±0.87
5.70±0.38
2.77±0.31* 1.90±0.22**
5.03±0.31
3.23±0.26*
2.30±0.21**
6.33±1.05
4.60±0.64
3.97±0.52*
4.67±0.25
2.27±0.25* 1.90±0.20**
8.53±0.41
5.75±0.41*
4.00±0.30**
9.87±0.79
8.07±0.67
7.83±0.59*
9.03±0.33
5.20±0.27*
4.33±0.25*
Leken’s index
14.95±0.49
10.97±0.58*
8.28±0.54*
16.07±0.59
13.27±0.51* 13.00±0.53* 14.70±0.42
9.93±0.44*
8.00±0.32*
WOMAC index
49.92±1.75
25.70±1.89* 18.72±1.81*
52.00±1.72
31.40±2.49* 27.53±2.52*
51.5±1.97
19.50±1.23* 11.10±1.12*
ESR
17.90±1.16
12.92±0.73*
11.3±0.69*
20.77±1.96
14.83±0.78* 14.13±0.83* 18.13±1.20
11.87±0.88* 10.50±0.62*
Creactive protein
9.30±0.58
7.20±0.37** 6.80±0.27**
10.20±0.87
7.80±0.77*
7.40±0.55*
Note. *p<0.05 and **p<0.01 compared to the baseline value.
7.60±0.64*
10.20±0.96
1.40±0.24** 0.57±0.10**
1.00±0.56*
7.00±0.42*
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
a decrease in the severity of pain, stiffness, and dysfunction was most significant
in the combined treatment group and least pronounced in the diclofenac group.
After 6 months of monotherapy for OA, Artrofoon (4 tablets twice daily)
was more effective than diclofenac (100 mg daily) for the relief of pain and
improvement of functional state and quality of life. The effect of Artrofoon
developed more slowly than that of diclofenac. One month after the start of
therapy, the clinical efficacy of diclofenac was higher than that of Artrofoon.
However, Artrofoon and diclofenac were equally potent by the 3rd month of
therapy. After 6 months of therapy, Artrofoon had a stronger effect than
diclofenac. The improvement of clinical symptoms in patients of the diclofenac
group was not observed from the 3rd to the 6th month of treatment. The clinical
effect was most pronounced in patients receiving combination therapy with
Artrofoon and diclofenac. A significant clinical effect in these patients was
detected after 1 month of therapy. By the end of therapy, the efficacy of combined
treatment did not differ from that of Artrofoon monotherapy. Diclofenac therapy
was followed by a variety of gastrointestinal complications in 80% patients. By
contrast, Artrofoon had an excellent safety profile and did not cause AE.
As differentiated from diclofenac, Artrofoon had a longterm antiinflamma
tory effect and demonstrated an excellent safety profile. Therefore, Artrofoon holds
much promise for the therapy of OA. These properties are related to the action of
Artrofoon on pathogenetic mechanisms of degenerative and atrophic processes in
the osteoarthritic joint. During the 1st month of therapy, Artrofoon should be
used in combination with diclofenac to produce a rapid effect. Artrofoon
monotherapy can be given from the 2nd month to reduce the incidence of AE.
Similar results were obtained by Prof. B. A. Alikhanov at the Moscow
State Medical Stomatological University (B. A. Alikhanov, 2004, 2006). The
clinical efficacy of Artrofoon in various doses was studied in OA patients after
longterm therapy with this drug (from 6 months to 2 years). The trial involved
WOMAC total score
60
Artrofoon
50
Artrofoon+diclofenac
diclofenac, 100 mg
40
!
!
!
30
!
20
!
+
!
+
10
0
Baseline
After 3 months
After 6 months
Fig. 8.33. WOMAC total score in osteoarthritic patients after 6month treatment
with Artrofoon and diclofenac. p<0.05: *compared to the baseline value; +compared
to the diclofenac group.
257
Ultralow doses
90 OA patients (stage IIIII gonarthrosis) receiving various doses of Artrofoon
as monotherapy or in combination with NAID for 624 months. The efficacy
of therapy was evaluated from the severity of joint syndrome, laboratory and
clinical parameters, and ultrasound examination of the joints. Artrofoon had a
significant clinical effect, which was observed on days 2030 of treatment and
reached maximum after 3 months of therapy. After 3 months, the overall effect
of therapy was most pronounced in patients receiving Artrofoon as monotherapy
(8 tablets daily) or combination with NAID. This effect persisted for up to 6
months during therapy with Artrofoon at the specified dose or half dose. The
positive effect was maintained for 2 years of treatment with Artrofoon in a dose
of 24 tablets daily. NAID monotherapy had a smaller effect. Longterm
therapy with Artrofoon (for up to 2 years) did not cause AE. Combination
therapy with Artrofoon and NAID was followed by the reduction of NAID
produced adverse reactions. The efficacy and safety of Artrofoon in OA
patients were confirmed by Prof. N. A. Shostak at the Russian State Medical
University (N. A. Shostak et al., 2005).
The effect of Artrofoon on production of cytokines and growth factors in
RA patients was studied by Prof. V. I. Mazurov in St. Petersburg (V. I. Mazurov
et al., 2007). Besides the high clinical efficacy, Artrofoon decreased the
production of proinflammatory cytokines IL1β and TNFα, stimulated the
secretion of antiinflammatory cytokines IL4 and IL10, and had a normalizing
effect on the concentrations of epidermal growth factor (EGF) and vascular
endothelial growth factor (VEGF).
The efficacy and safety of Artrofoon in patients with urogenic RA (UA)
were evaluated in a pilot study at the Volgograd State Medical University (V. I.
Petrov et al., 2005).
An open randomized comparative study of Artrofoon efficacy was per
formed with UA patients (diagnostic criteria of E. R. Agababova). The diagnoses
of ureaplasma infection and chlamydial infection were made in 66.7 and 33.3%
patients, respectively.
A clinicalandanatomical type of UA was presented by OA with primary
damage to the joints of the lower extremities. The patients received Artrofoon
(8 tablets daily) or diclofenac (100 mg daily) for 3 months in combination with
antibacterial therapy (depending on the infectious agent). Clinical and labora
tory parameters were monitored for 1 month at 2week intervals. The patients
were examined monthly in the followup period. Artrofoon therapy was followed
by a decrease in the severity of pain (83.3% patients), Ritchie index, and
swelling (66.6% patients). The severity of pain, Ritchie index, and swelling were
reduced in 66.6% patients of the diclofenac group. Drugrelated adverse events
were not observed in patients of the Artrofoon group. The efficacy of Artrofoon
in UA patients should be confirmed in largescale clinical trials.
258
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Artrofoon was effective in the therapy of patients with other diseases of
the joints and musculoskeletal system, including psoriatic and gouty arthritis,
ankylosing spondyloarthritis, osteochondrosis, and periarthritis of the shoulder
joint (V. V. Badokin et al., 2005; O. V. Inamova et al., 2005; I. V. Kudryavtseva
et al., 2005; O. I. Epstein et al., 2005; N. A. Khitrov, 2006).
The therapeutic efficacy of Artrofoon in patients with nonspecific ulcerative
colitis was demonstrated at the Samara Military Medical Institute. TNFα has an
important role in the pathogenesis of this disease (M. A. Osadchuk et al., 2005).
The results of randomized controlled clinical studies of Artrofoon allow
us to make the following conclusions. Pharmacodynamically, Artrofoon holds
promise as a diseasemodifying drug with the longterm clinical effect in RA
patients. Artrofoon may be used in combined treatment and monotherapy of
these patients. Artrofoon has a complex effect on the cytokine cascade during
systemic autoimmune inflammation, which differs from the action of TNFα
antagonists (e.g., Infliximab). The antiinflammatory effect of Artrofoon on RA
patients is probably associated with a decrease in the systemic production of
TNFα. The clinical efficacy of Artrofoon should be evaluated in patients with
TNFαrelated diseases.
8.5. Epigam in the therapy for gastric ulcer
and duodenal ulcer
An open, randomized, comparative pilot study of the clinical efficacy and
safety of Epigam (ULD to histamine for oral administration, 28day triple
therapy) during gastric ulcer and duodenal ulcer was performed at the Volgograd
State Medical University in 20022003 (P. A. Bakumov et al., 2003; J. L.
Dugina, 2003a,b; O. I. Epstein et al., 2003b; J. L. Dugina et al., 2002).
The trial involved stationary patients 1850 years of age. Ulcerative lesions
of the gastric or duodenal mucosa (20 mm in diameter) were revealed during
endoscopic examination. The patients were confirmed to have Helicobacter pylori
infection.
The patients were divided into two groups of ten subjects each. Group 1
patients received Epigam (1 tablet, six times daily for 28 days), amoxicillin (500
mg, three times daily for 14 days), and metronidazole (500 mg, twice daily for
14 days). Ranitidine (150 mg, twice daily for 28 days), amoxicillin (500 mg,
three times daily for 14 days), and metronidazole (500 mg, twice daily for 14
days) were given to group 2 patients. During the study, all patients could receive
symptomatic antacid therapy. The main symptoms of peptic ulcer were evaluated
in the basal state and after 1, 2, 3, and 4 weeks of therapy. The overall severity
of symptoms was determined during a pretrial period of 1 week. Endoscopic
259
Ultralow doses
examination with an OlympusGIFE gastrofiberscope was performed before
and after 4week therapy. The number and area of ulcerative lesions, endoscopic
and histological signs of gastroduodenal mucosal inflammation, and secretory
status (Congo red test) were evaluated. The benign nature of mediogastric ulcers
was confirmed histologically. Helicobacter pylori was detected by invasive
(histological, molecular genetic, and rapid urease test) and noninvasive methods
for infection diagnostics (indirect solidphase enzyme immunoassay). The rapid
urease test (KhELPIL test, St. Petersburg) was used for primary rapid diag
nostics of H. pylori. This test is based on the measurement of urease activity in
biopsy specimens of the gastric mucosa after endoscopic examination.
The average age of patients in both groups (n=20) was 32.2 years. Twelve
men (60%) and eight women (40%) were enrolled in the trial. The majority of
patients (n=19, 95%) had a history of peptic ulcer. In one patient (5%), the
diagnosis was made for the first time. The mean duration of disease was 3.2 years.
The time to relief of pain syndrome and dyspepsia did not differ in
patients receiving Epigam and ranitidine. Epigastric pain in group 1 patients was
reduced after therapy for 3 (two patients, 20%), 10 (eight patients, 80%), or 14
days (all patients). These changes were accompanied by the disappearance of
dyspeptic disorders. Epigastric pain in group 2 patients was reduced after therapy
for 2 (five patients, 50%), 10 (eight patients, 80%), or 14 days (all patients). The
mean time to pain relief was 7.50±0.81 days (Fig. 8.34).
The severity of epigastric pain syndrome (including nighttime pain),
heartburn, and nausea and volume of symptomatic therapy in patients of the
Epigam group decreased significantly after 4week treatment (p<0.01, Table
8.12). The mean time to pain relief was 10.6±1.2 days (67 days in the majority
of patients). Pain syndrome and dyspeptic disorders were reduced in the early
period after combination therapy with ranitidine, amoxicillin, and metronidazole
(p<0.05). Epigastric and pyloroduodenal pain and dyspeptic disorders were
relieved over 311 and 214 days, respectively. Pain syndrome in 53.8% patients
Duration of symptom, days
15
Epigam
ranitidine
12
9
6
3
0
Pain
Heartburn
Eructation
Nausea
Fig. 8.34. Mean time to relief of peptic ulcer symptoms in a controlled clinical
study of Epigam.
260
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Table 8.12.
Reduction of peptic ulcer symptoms in patients receiving Epigam in
combination with amoxicillin and metronidazole (points, M±m)
Symptom
Epigam+
amoxicillin+metronidazole
baseline
after 4 weeks
Ranitidine+
amoxicillin+metronidazole
baseline
after 4 weeks
Epigastric pain
2.8+0.4
0.8+0.04*
2.4±0.5
0.70±0.03*
Nighttime pain
1.8+0.2
0.1+0.01*
1.6±0.1
0.20±0.01*
Heartburn
1.3+0.2
0.5+0.01*
1.6±0.1
0.30±0.01*
Eructation
1.1+0.1
0.70+0.03
0.90±0.04
0.80±0.01
Nausea
0.9+0.1
0.60+0.02
1.20±0.06
0.40±0.02*
Palpatory tenderness
2.9+0.2
0.70+0.03*
2.7±0.2
0.50±0.06*
Note. *p<0.05 compared to the baseline value.
was not observed after drug therapy for 5 days. The mean time to relief of
epigastric pain and dyspepsia (mainly of nausea) was 7.5±0.8 and 4.9±0.8 days,
respectively.
The duration and severity of symptoms did not depend on basal gastric
acid secretion and length of the disease.
The area of gastroduodenal ulcers decreased significantly after 4week
triple therapy with Epigam (p<0.001). Ulcer cicatrization in nine of ten patients
(90%) was observed by the 4th week treatment. The area of ulcerative lesion in
one patient decreased by 7580%. Epigam significantly decreased the severity of
erosive changes. In six patients (60%) ulcer cicatrization was accompanied by
epithelization of gastric and/or duodenal erosions after 4week therapy (p<0.05,
Table 8.13).
Fourweek combination therapy with ranitidine, amoxicillin, and metro
nidazole was also followed by a decrease in the area of mucosal erosions and
ulcers and reduction of macroscopic (endoscopic) signs for gastritis. Erosive
gastroduodenitis was not found in treated patients. Ulcer healing was observed
in 100% patients (Table 8.14).
Table 8.13.
Clinical efficacy of 4week combination therapy with Epigam in patients with
peptic ulcer of the stomach and duodenum (n=10)
Parameter
Epigam+
amoxicillin+metronidazole
Ranitidine+
amoxicillin+metronidazole
abs.
%
abs.
%
Incidence of ulcer cicatrization
9
90
10
100
Incidence of epithelization
of erosions
6
60
8
80
261
Ultralow doses
Table 8.14.
Endoscopic examination for reparative and inflammatory processes in the
gastric and duodenal mucosa of peptic ulcer patients receiving combination
therapy with Epigam (points, M±m)
Parameter
Epigam+
amoxicillin+metronidazole
baseline
Ranitidine+
amoxicillin+metronidazole
after 4 weeks
baseline
after 4 weeks
Area of ulcers
1.21±0.07
0*
0.96±0.02
0*
Number of ulcers
1.06±0.04
0*
1.45±0.04
0.07±0.01*
Erosions
0.90±0.03
0.46±0.02*
0.73±0.01
0.24±0.01*
Gastritis
1.29±0.06
1.05±0.01
1.17±0.06
1.03±0.03
Duodenitis
1.35±0.04
1.30±0.05
1.08±0.02
1.01±0.02
Duodenogastric reflux
0.18±0.01
1.17±0.04
0.35±0.01
0.27±0.01
Note. *p<0.05 compared to the baseline value.
The clinical improvement was accompanied by a decrease in the volume
of symptomatic therapy in patients receiving Epigam (from 3.21±0.16 to
0.53±0.09 tablets/spoons daily, p<0.001) and ranitidine (from 2.96±0.11 to
0.22±0.04 tablets/spoons daily, p<0.001).
A pilot clinical study confirmed the efficacy of combination therapy with
Epigam (6 tablets daily for 28 days) and standard drugs (amoxicillin and me
tronidazole) in patients with H. pyloriassociated peptic ulcer of the stomach
and duodenum. The efficacy of Epigam compared well with that of the refer
ence drug ranitidine (300 mg daily).
8.6. Afala in the therapy for benign
prostatic hyperplasia
The drugs for BPH have two main molecular targets, 5αreductase and
αadrenoceptors in the lower urinary tract. 5αReductase inhibitors are
effective in severe hyperplasia of the prostate gland, but do not affect clinical
symptoms of BPH during the first 34 months of therapy. However, these drugs
improve the longterm prognosis in BPH patients. αAdrenoceptor antagonists
do not influence prostate tissue, but have a strong effect on the dynamic
component of obstruction and rapidly relive the symptoms of BPH. Clinical
symptoms (total IPSS, International Prostate Symptom Score) and urodynamics
improve by 2050 and 2030%, respectively (S. Madersbacher et al., 2004).
Among a variety of physiotherapeutic products, much attention was paid
to Serenoa repens extract. The efficacy of Serenoa repens extract compares well
with that of finasteride (T. Wilt et al., 2004).
262
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
The target for S. repens was not identified. This extract has the pleiotropic
properties (modulation of cholesterol metabolism; antiestrogenic, antiandro
genic, and antiinflammatory effects; etc.).
PSA is one of the molecular targets during BPH (E. P. Diamandis, 2000;
S. P. Balk et al., 2003). The expression of this serine protease is regulated by
androgens. PSA has antiangiogenic activity (A. H. Fortier et al., 1999) and plays
a role in the regulation of stromal cell growth in the prostate gland (D. M.
Sutkowski et al., 1999).
BPH is accompanied by a strong immune response to PSA, which serves
as one of the pathogenetic factors and reflects the progression of other
pathological processes (A. Zisman et al., 1995, 1999).
The pathogenetic role of PSA in BPH requires further investigations.
Afala was developed at the “Materia Medica Holding” Researchand
Production Company. The active components of this product are rabbit poly
clonal antibodies to PSA (ULD for oral administration). Preclinical studies
showed that Afala decreases the severity of acute and chronic aseptic inflam
mation of the prostate gland (T. G. Borovskaya, 2002). Moreover, this product
reduces the degree of prostatic hyperplasia under conditions of sulpiride
induced hyperprolactinemia (K. V. Savel’eva et al., 2007). The effect of Afala
is probably related to functional modification of endogenous PSA, which is
impaired during BPH.
Here we describe the results of two randomized, controlled, parallelgroup
studies to evaluate the efficacy and safety of Afala in BPH (V. N. Pavlov et al.,
2005a,b; A. A. MartyushevPoklad et al., 2005; Z. A. Yurmazov et al., 2005;
V. I. Petrov et al., 2006; A. MartyushevPoklad et al., 2005d).
The trials of similar design were performed at the Volgograd State Medical
University, Bashkirian State Medical University (Ufa), Institute of Pharmaco
logy (Tomsk Research Center of the Siberian Division of the Russian Academy
of Medical Sciences), and Central Military Medical Hospital No. 32 (Moscow).
The patients were randomized into the Afala group, placebo group, and open
control group. After 4 weeks of a placebocontrolled phase, the Afala group was
opened. An open randomized comparative study was performed with the active
reference drug. The total duration of therapy was 16 weeks.
The study involved patients (4070 years of age, average age 63.50±0.49
years) with moderate symptoms of chronic prostate disease. The diagnosis of
stage III BPH (total IPSS 925) was confirmed by transrectal ultrasound
(TRUS) examination. The volume of the prostate gland was more than 25 cm3.
The maximum urine flow rate (Qmax) was 515 ml/sec. The informed consent
was obtained from each patient. The exclusion criteria were a history of surgical
treatment for diseases of the prostate or urinary bladder, residual urine volume
> 150 ml, suspected prostate cancer, serum PSA > 4 ng/ml, etc.
263
Ultralow doses
The patients received Afala (16 weeks) or placebo (4 weeks) in a daily
dose of two lozenges. The parallel open control group was treated with an active
product of Serenoa repens extract (Prostamol Uno, 320 mg daily).
The trial involved 241 patients (stage III BPH) receiving Afala (n=132),
Prostamol Uno (n=54), or placebo (n=55, control group).
The severity of disease symptoms (total IPSS), quality of life (integral
criterion, IPSS), urodynamics (uroflowmetry), state of the prostate gland
(TRUS), and serum PSA level were evaluated during followup visits. AE were
recorded during each visit. Routine blood test, urine test, and measurement of
serum glucose or creatinine were performed before and after the study.
The efficacy endpoints were the relief of symptoms, improvement of
urodynamic parameters, and increase in patients’ quality of life.
After 4 weeks of therapy, the efficacy of Afala significantly differed from that
of placebo (according to clinical manifestations and urodynamics). IPSS decreased
by 0.9±0.4 points, while Qmax remained unchanged in the placebo group. IPSS
was 1.9±0.3 points, and Qmax increased by 13.3±2.4% in the Afala group.
Fig. 8.35 illustrates the results of a 16week open comparative study. Drug
treatment was followed by a significant decrease in the total IPSS (by more than
40% after 16 weeks). In 50% patients, IPSS decreased below 8 points.
Pharmacotherapy was not required under these conditions. The improvement of
urodynamics (Qmax) in patients of the Afala and Prostamol groups was 45 and
36%, respectively. The quality of life (qualityoflife score in IPSS) improved
more significantly in the Afala group.
TRUS examination revealed a significant decrease in residual urine
volume (from 31.0±3.2 to 12.9±1.7 ml, p<0.001) and size of the prostate gland
(from 44.6±1.3 to 41.9±1.3 cm3, p<0.01) after Afala therapy.
Afala (8 tablets daily for 16 weeks) had a good safety profile in patients
with stage III BPH. Drugrelated AE were not reported. The results of routine
blood test, urine test, and biochemical blood analysis did not differ from normal
before and after therapy.
The concentrations of total and free PSA in blood serum from Afala
receiving patients not only remained within normal limits (less than 4 ng/ml),
but even decreased (by 17.6 and 21.7%, respectively). The PSAfree/PSAtotal ratio
did not change after Afala therapy (within normal limits). Serum PSA level
remained unchanged in the Prostamol group.
These data indicate that Afala is an effective and safety drug for the
therapy of stage III BPH. The product improves clinical symptoms of this
disease (particularly those associated with urination disorders), parameters of
urodynamics, size of the prostate, and residual urine volume. A modulatory
effect of Afala on serum PSA level indirectly reflects the pathogenetic action of
this product.
264
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
a
Qmax, ml/sec
16
Points
16
12
12
8
8
4
4
0
c
0
Baseline
4
8
12
16
Baseline
4
b
Qaver, ml/sec
9
8
12
16
12
16
d
Points
4
6
3
2
3
1
0
0
Baseline
4
8
12
16
Baseline
4
8
Time, weeks
Afala
Prostamol (320 mg daily)
Fig. 8.35. Course of BPH during therapy with Afala (light bars) and Prostamol (dark
bars). Q max (a); average flow rate of urine (b); reduction of disease symptoms (total
IPSS, c); and quality of life (QoL index, d). *p<0.05 compared to the other group.
8.7. Clinical pharmacology of Kardos
Chronic heart failure is an urgent problem of public health. The incidence
of CHF in the European population reaches 0.42.0%. The disease is most
common in elderly people. The average age of CHF patients is 74 years. The
percentage of patients with systolic dysfunction not accompanied by CHF is
similar to that of subjects with CHF symptoms.
Nearly half of patients with the diagnosis of CHF die over 4 years. The
survival time of 50% patients with severe CHF does not exceed 1 year. CHF
patients have a poor longterm prognosis.
CHF is most often associated with CHD. The left ventricular ejection
fraction in many patients decreases below 4550%. However, there is no direct
relationship between symptoms and degree of cardiac dysfunction (as shown by
instrumental methods). Hence, the severity of CHF is mainly evaluated from
functional parameters.
265
Ultralow doses
There are a variety of clinical, electrophysiological, and other signs of
poor prognosis of CHF. The major signs are a persistent decrease in BP, NYHA
functional class III or IV CHF (clinical parameter), reduced level of maximum
oxygen consumption (functional parameter), and low left ventricular ejection
fraction (LVEF, central hemodynamic parameter).
Therapy for CHF is directed to the prevention of disease progression,
maintenance or improvement of the quality of life, decrease in the incidence of
hospitalization for CHF, and increase in the lifespan of patients.
The drugs affecting the reninangiotensinaldosterone system are widely
used to decrease the mortality rate of patients and incidence of hospitalization.
They include angiotensinconverting enzyme (ACE) inhibitors, angiotensin II
receptor antagonists (AT II; mainly in combination therapy), aldosterone
antagonists, and βadrenoreceptor antagonists (Guidelines for the Diagnosis and
Treatment of Chronic Heart Failure, 2005).
The angiotensin II receptor type 1 (AT1) mediates the key effects of AT
II, which plays a role in the pathogenesis of arterial hypertension, complications
of this disease, and CHF.
Various fragments of the receptor have different functions. The extracellu
lar fragment is responsible for binding of peptide agonists. The transmembrane
fragment has a role in binding of peptide agonists, signal transduction to G
proteins (rapid effects of AT), and binding of nonpeptide antagonists (including
those used in clinical practice). The Cterminal intracellular fragment is in
volved in signal transduction (phosphorylation and, therefore, longterm effect
of angiotensin) and receptor internalization.
The longterm effects of AT II include longterm regulation of vascular
tone and remodeling of the vascular wall. Internalization is a key event of the
receptor cycle, which serves as one of the regulatory mechanisms for AT II
signal transduction. Arterial hypertension is accompanied by abnormalities in
receptor internalization (impairment of signal transduction from the receptor)
and overphosphorylation (M. de Gasparo et al., 2000; R. M. Touyz et al., 2000).
The Cterminal fragment of the AT II AT1 receptor serves as a molecular
target for Kardos. Antibodies to this fragment are the active component of
Kardos (ULD for oral administration; Fig. 8.36).
Previous experiments showed that Kardos has a hypotensive effect on
animals with inherited arterial hypertension and exhibits the cardioprotective
properties on the model of CHF. Toxicology studies revealed that this product
has a good safety profile.
Clinical trials at the Volgograd State Medical University were performed to
evaluate Kardos efficacy in patients with arterial hypertension and CHF (P. A. Ba
kumov et al., 2005; V. I. Petrov et al., 2005, 2006; V. V. Ivanenko et al., 2005;
S. A. Sergeeva et al., 2006).
266
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
NH2
Agonists
С
С
K 102
108 V
111 N
74
N
R 167
163
D
A P
198
T K 199
D R
Y
Y 292
N 295
N P
L 300
301 F
Y
Antagonists
252
W
L
H P
W S 294 N
256
P
Y
Kardos
COOH
Fig. 8.36. Functional fragments of the angiotensin II AT 1 receptor. Targets for
peptide agonists and nonpeptide antagonists.
Clinical studies of Kardos efficacy in patients with arterial hypertension. An
open clinical study at the Volgograd State Medical University was designed to
evaluate the safety profile and effect of Kardos in various doses on BP in
normotensive subjects (healthy volunteers).
During the first stage of the trial, Kardos pharmacodynamics was studied
in 48 healthy volunteers (2148 years of age). These volunteers were divided into
the following four groups (according to the dosage regimen of Kardos): group
1, 1 tablet three times daily; group 2, 2 tablets three times daily; group 3, 2
tablets five times daily; and group 4, 2 tablets six times daily. Kardos was given
2 h after the start of BP monitoring. The 24h profile of BP and incidence of
AE were evaluated.
According to the results of 24h BP monitoring, Kardos did not cause the
decrease of BP in all four groups (Fig. 8.37). AE were not reported.
These data indicate that Kardos has a good safety profile and does not cause
firstdose hypotension in normotensive patients (E. A. Zernyukova et al., 2005).
Kardos safety was demonstrated in CHD patients (no firstdose hypoten
sion; N. A. Davydova et al., 2005).
Pharmacodynamics and efficacy of Kardos during therapy of patients with
arterial hypertension. An open randomized clinical study at the Volgograd State
Medical University was designed to evaluate the antihypertensive properties,
optimal dosage regimen, and safety profile of Kardos during therapy of patients
with arterial hypertension.
267
Ultralow doses
mm Hg
140
120
100
80
60
40
20
0
0 1
1
2
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time, h
Fig. 8.37. 24h BP monitoring in healthy volunteers. Acute pharmacological test
with Kardos, 2 tablets six times daily (maximum dose). Systolic BP (1) and diastolic
BP (2).
The trial involved 40 patients (1870 years of age) with mild arterial
hypertension. The patients did not receive hypotensive drugs in the pretrial
period (2 weeks before the start of study). BP was 140/160 mmHg (systolic)
and/or 90/100 mmHg (diastolic).
The patients were divided into four groups. Kardos was given orally
(lozenges) in the following four regimens: 1 tablet three times daily (group 1);
2 tablets three times daily (group 2); 2 tablets six times daily (group 3); and 1
tablet six times daily (group 4).
The duration of therapy was 3 months (12 weeks). The state of patients
was estimated at 4week intervals. The BP profile was estimated from the results
of 24h BP monitoring. BP was measured manually.
In group 1, the hypotensive effect of Kardos became more pronounced
with an increase in the duration of therapy (significant decrease in diastolic BP
during the nighttime).
The hypotensive effect of Kardos in group 2 patients was observed after
1 month of therapy and remained unchanged over the next 2 months. Systolic
BP decreased by 10.7% compared to the baseline value (manual measurement;
5.6% according to the results of 24h BP monitoring). A decrease in systolic and
diastolic BP was particularly pronounced in the nighttime (by 18.7 and 27.2%,
respectively).
The hypotensive effect of Kardos on systolic and diastolic BP in group 3
patients was significant after 2week therapy (decrease by 7.3 and 7.7%,
respectively; p<0.05; manual measurement). After 3 months of therapy, systolic
BP decreased by 8.4 (manual measurement) or 11.4% (24h monitoring). These
changes were accompanied by a 20.3% decrease in nighttime systolic BP (24
h monitoring). Nighttime diastolic BP decreased by 26.6%.
The hypotensive effect of Kardos in group 4 patients was similar to that
in group 3 patients.
268
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Drugrelated AE were not reported by patients of all groups.
In various regimens of 3 monthtreatment, Kardos had a moderate
hypotensive effect on patients with “mild” arterial hypertension. A stable
hypotensive effect of Kardos was observed after 2 months of therapy. Increasing
the dose of Kardos was accompanied by a more rapid development of the effect.
This product was most potent in modulating the nighttime level of BP.
Therefore, Kardos had a normalizing effect on the 24h profile of BP. The
product was well tolerated in various dosage regimens.
A randomized, controlled, parallelgroup study of the efficacy of 4week
therapy with various pharmaceutical formulations of Kardos (monotherapy and
combination therapy for stage III arterial hypertension) was performed at the
Volgograd State Medical University. A placebocontrolled study was performed
with the drop formulation of Kardos. Patients of two open control groups
received an ACE inhibitor and AT II receptor antagonist. All patients enrolled
in the trial were divided into six groups of 15 subjects each: group 1, placebo
(612 drops, three times daily); group 2, Kardos (612 drops, three times daily);
group 3, Kardos (12 tablets, three times daily); group 4, Kardos (12 tablets,
three times daily) and enalapril (5 mg twice daily); group 5, lisinopril (1020 mg
once daily); and group 6, losartan (5010 mg three times daily). The duration
of therapy was 4 weeks. The results of this study are shown in Table 8.15 and
Fig. 8.38.
Biochemical parameters remained practically unchanged during the
course of monotherapy or combination therapy with Kardos.
% of the baseline
14
SBP 24h
DBP 24h
BPm
12
10
8
6
4
2
0
2
1
2
3
4
5
6
Group
Fig. 8.38. Decrease in BP in patients with stage III arterial hypertension receiving
antihypertensive therapy for 4 weeks (24h monitoring). SBP 24h, 24h systolic BP;
DBP 24h, 24h diastolic BP; BP m, mean BP. *p<0.01 compared to group 1.
269
Ultralow doses
270
Table 8.15.
Results of 24h BP monitoring in patients with stage III arterial hypertension during 4week hypotensive therapy (mmHg,
M±m)
Group
Parameter
1
2
3
4
5
6
24h systolic BP
baseline
149.5±2.7
149.7±2.1
149.2±2.8
151.2±5.5
150.8±5.4
151.0±2.8
after therapy
150.6±2.9
139.9±2.4*
139.3±2.1
136.0±4.8
134.2±4.6
135.9±2.2
baseline
90.3±1.9
90.5±1.1
91.4±1.6
91.6±3.2
91.7±3.1
94.4±1.8
after therapy
91.2±2.3
84.9±0.9*
85.3±1.7
80,7±3.4
80.7±3.2
86.3±1.5
24h diastolic BP
Mean BP
baseline
118.9±3.8
113.1±1.9
113.3±1.0
115.4±2.9
114.7±2.8
115.2±3.6
after therapy
120.4±2.7
105.8±1.2*
105.7±0.9
102.8±1.7
101.5±1.9
104.3±2.8
Note. *p<0.05 compared to group 1.
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
Side effects were not observed during Kardos therapy (including dry cough
and firstdose hypotension).
We conclude that 4week treatment with the drop formulation of Kardos
has a strong hypotensive effect (as differentiated from placebo). Various
pharmaceutical formulations of the product (drops and tablets, 4week therapy)
produce the same hypotensive effect. Combination therapy with Kardos and
ACE inhibitor potentiates the hypotensive effect of treatment. The hypotensive
effect of lisinopril and losartan was greater than that of Kardos.
Therapeutic efficacy of Kardos in patients with chronic heart failure. A
blind, randomized, placebocontrolled study was performed at the Department
of Cardiology and Functional Diagnostics (Faculty of Postgraduate Medical
Education, Volgograd State Medical University). The trial involved ambulatory
patients (2080 years of age) with a stable course of functional class IIIV
CHF. LVEF did not exceed 35%. The patients received standard therapy for
CHF (ACE inhibitors, βadrenoceptor antagonists, and diuretics). The infor
med consent was obtained from each patient. The patients were randomized
into groups to receive 6month therapy with Kardos (1 tablet three times
daily; 30 patients, including 27 male subjects) or placebo (30 patients, inclu
ding 24 male subjects).
The efficacy and safety of therapy were evaluated during outpatient visits
after 4, 12, and 24 weeks of treatment.
The percentage of patients with improved functional class of CHF was
considered as a primary efficacy endpoint. The secondary efficacy endpoints
were an increase in physical tolerance (6min walking distance), variations in
LVEF (echocardiography), and maximum oxygen consumption (treadmill test).
The safety profile was determined by recording of AE.
The statistical analysis was performed with patients who met the inclusion
criteria and received therapy. The mean values were compared by Student’s t test
for independent variables. The percentage of patients was compared by χ2 test
for homogeneity of proportions.
Kardos significantly surpassed placebo in the primary efficacy endpoint or
one of three secondary efficacy endpoints. No betweengroup differences were
observed in the safety profile.
The average age of patients enrolled in the study and receiving placebo
and Kardos was 53.9±1.0 and 57.3±1.6 years, respectively. CHF was associated
with CHD in 28 patients of the placebo group and in 30 patients of the Kardos
group. Arterial hypertension served as an etiologic factor of CHF in two patients
of the placebo group and in three patients of the Kardos group. The baseline
severity of disease did not differ in patients of both groups. Patients of the
placebo and Kardos groups had FC III (15 and 19 patients, respectively) or FC
IV (3 and 1 patients, respectively; Fig. 8.39).
271
Ultralow doses
Placebo
Kardos
10%
3%
40%
50%
34%
63%
FC II
FC III
FC IV
Fig. 8.39. Severity of CHF in patients enrolled in the study of Kardos efficacy for CHF.
After 6 months of therapy, FC of CHF was reduced in 8 patients of the
placebo group (26.7%; CI 95% = 14.244.5) and in 16 patients of the Kardos
group (53.3%; CI 95% = 36.169.8). Among patients with baseline FC IIIIV,
the improvement was observed in 8 subjects of the placebo group (44.4%; CI
95% = 24.666.3) and in 14 subjects of the Kardos group (70%; CI 95% = 48.1
85.5; Fig. 8.40).
Sixmonth therapy was followed by an increase in LVEF in patients of the
placebo group (from 26.4±1.1 to 28.0±1.4%) and Kardos group (from 27.1±0.9
to 33.6±1.5%; p<0.01 compared to the baseline value and placebo group; Fig.
8.41). Effect size was 1.01. The standard deviation of baseline LVEF for the
combined sample was 5.52.
The 6min walking distance increased in patients receiving placebo (from
390.5±11.9 to 409.1±11.5 m; p=0.12 compared to the baseline value; average
a
b
Percentage of patients, %
80
LVEF, %
36
60
32
40
28
20
24
!
20
0
Placebo
Kardos
Baseline
24th week
placebo
Kardos
Fig. 8.40. Kardos efficacy during 6month treatment of CHF patients. Percentage
of patients with improved FC of CHF (CI 95%, a); and systolic function of the left
ventricle during therapy (b). *p<0.05 compared to placebo.
272
Chapter 8. Clinical pharmacology of products from ultralow doses of antibodies
a
m
440
b
VO2max, ml/kg/min
22
!
420
!
20
400
18
380
16
360
14
340
12
320
10
Placebo
Kardos
Placebo
baseline
Kardos
24th week
Fig. 8.41. Efficacy of 6month therapy with Kardos in CHF patients. Influence on
physical tolerance (6min walking distance, a) and maximum oxygen consumption
(treadmill test, b). *p<0.05 compared to the baseline value.
increase 6.7±3.7%) and Kardos (from 378.7±12.4 to 419.6±13.7 m; p<0.05
compared to the baseline value; average increase 11.9±2.9%).
After 6 months of therapy, maximum oxygen consumption in the
treadmill test increased in patients of the placebo group (from 16.2±1.4 to
17.8±1.3 ml/kg/min, statistically insignificant) and Kardos group (from
17.4±1.2 to 19.5±1.3 ml/kg/min; p<0.05).
After 6 months of therapy, the total score of the Minnesota questionnaire
for assessing quality of life was reduced in patients of the placebo group (from
48.3±3.7 to 42.4±3.4 points) and Kardos group (from 47.5±2.8 to 39.1±3.0
points; p<0.05).
Patients of the Kardos group were characterized by a significant
improvement of myocardial remodeling (size and volume of the left ventricle),
heart rate variability, and endothelial function (pulse wave propagation and
endotheliumdependent vasodilation; V. V. Ivanenko et al., 2004a,b,c). Fatal
outcome was not observed during 6 months of therapy. None of the patients
withdrew from the trial. One AE was reported in the placebo group. Cardiac
fibrillation was followed by decompensation of CHF, which required
hospitalization of the patient.
Combined treatment with Kardos (3 tablets daily for 6 months) and
standard drugs was much more effective than standard therapy for CHF.
Kardos significantly improved the clinical state, morphofunctional parameters
of the heart, and tolerance to physical exercise. We conclude that Kardos is
an effective drug, which has a good safety profile and may be used in
combination with standard therapy for CHF. Kardos improves the prognosis
of this disease.
273
Ultralow doses
***
Chapter 8 reviews the results of clinical studies with ULD of antibodies.
All clinical trials were performed in accordance with the Manual on Clinical
Studies of New Pharmaceutical Substances in Russia (2005) and International
Principles of Good Clinical Practice (GCP).
Placebocontrolled clinical trials confirmed the results of preclinical
studies. It was concluded that products of antibodies in ULD demonstrate the
efficacy and good safety profile. Proproten100 was effective and had a good
safety profile in the therapy of alcoholism and reduction of alcohol withdrawal
syndrome. Tenoten was successfully used in the therapy of anxiety disorders.
Anaferon and Anaferon for children were effective in the therapy of ARVI
(influenza, adenovirus infection, respiratory syncytial virus infection, etc.),
herpes virus infections (chickenpox, infectious mononucleosis, and genital
herpes), and acute intestinal infections (rotavirus infection, coronavirus
infection, and calicivirus infection). The efficacy and safety of Impaza were
revealed in treatment for ED of various etiologies. Impaza was used as
monotherapy or in combination with other drugs to improve potency (Viagra
and Sialis). The efficacy and safety of Artrofoon were demonstrated in patients
with RA, OA, and other autoimmune diseases. Some trials were performed with
Epigam (therapy for gastric ulcer and duodenal ulcer), Afala (treatment of
BPH), and Kardos (CHF and arterial hypertension).
It should be emphasized that the possibility of using antibodies in ULD
is not limited to the described nosological forms. ULD of antiTNFα (Art
rofoon), antiNOS (Impaza), and antiIFNγ (Anaferon and Anaferon for
children) hold much promise for therapy of autoimmune disease (e.g.., non
specific ulcerative colitis), endothelial dysfunction, and tickborne encephalitis,
respectively.
274
CONCLUSION
T
he method for potentiation (activation) of ultradiluted solutions was
empirically discovered by S. Hahnemann at the end of the 18th century.
This approach was used to prepare medical products that have a good safety
profile and do not cause drugrelated adverse events. Treatment with activated
(potentiated) substances in ultralow doses was a part of the therapeutic method
of S. Hahnemann (homeopathy). Hence, these products received the name
“homeopathic substances”.
A famous botanist K. Negeli (19th century) and other researches (20th
century) showed that ultradiluted solutions of various substances exhibit the
biological activity. There was no direct relationship between these properties and
homeopathic doctrine. Experimental studies of ultralow doses by J. Benveniste
and E. B. Burlakova were of high methodological quality and gained general
acceptance in the scientific community.
Homeopathy is based on a particular property of ultralow doses. They
cause a hyperergic reaction, which increases the effect of potentiated products.
Homeopathy is an effective, but casuistic approach. The method requires
individualization of therapy, which limits the use of homeopathic remedies.
Potentiated products have low biological activity. Therefore, ultralow
concentrations cannot substitute for standard (therapeutic) doses of medical
products.
Our studies revealed that the activated form of a substance, which is
prepared by the method of Hahnemann, can modify the effect of this substance
(phenomenon of bipathy). The phenomenon of bipathy was demonstrated on
biological and simple physicochemical models. Hence, ultralow doses are not
the prerogative of biology. By contrast, they are the subject matter of various
sciences. Ultralow doses may be used not only in medicine, but also in other
fields of science and technics.
The modifying (bipathic) activity of ultralow doses holds much promise
for modern pharmacology. This property may be used to potentiate the action
of pharmaceutical products, to reduce the adverse effects, and to prevent the
development of drug resistance.
275
Ultralow doses
We showed for the first time that activated antibodies do not inhibit, but
modify the activity of the corresponding antigen. The modifying effect is a
variant of the phenomenon of bipathy, which contributes to the development of
a new class of medical products with ultralow doses of antibodies.
The results of experimental and clinical observations are considered as a
step to pharmacology of ultralow doses.
The resolution power of modern scientific methods does not allow us to
evaluate a physical basis for the activity of ultradiluted potentiated solutions.
At the present stage of science, the mechanisms for action of ultralow doses are
hypothetically explained. Our hypothesis of dual spatiotemporal organization of
vital activity provides an explanation for the pleiotropic effects of activated
substances.
The reproducibility is a distinctive feature of the phenomenon of bipathy.
New medical products developed on the basis of modifying activity of antibodies
in ultralow doses meet the requirements of evidencebased medicine. In recent
years, the number of approved pharmaceuticals decreased sharply throughout
the world. The efficacy of newly synthesized compounds does not surpass that
of less selective analogues. There is reason to hope that further studies of
activated products will extend the possibilities of modern pharmacology, which
requires the development of new approaches.
276
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