Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMBINATION PHARMACEUTICAL COMPOSITION AND
METHODS OF TREATING AND PREVENTING THE INFECTIOUS DISEASES
FIELD
The present invention relates to a pharmaceutical composition and method of
treating and preventing the infectious diseases, including bacterial
infections caused
by different infectious agents such as pseudotuberculosis, whooping cough,
yersiniosis, pneumonitis of different etiology, and acute and chronic viral
infections such as acute respiratory tract infections, influenza of different
types, acute
viral hepatitis A, B, C and other types of hepatitis, the diseases and
conditions
caused by HIV or associated with HIV, including AIDS.
The invention relates to the area of medicine and may be used for the
BACKGROUND
treatment and preventing the infectious diseases, including bacterial
infections
caused by different infectious agents such as pseudotuberculosis, whooping
cough, yersiniosis, pneumonitis of different etiology, and acute and chronic
viral
infections such as acute respiratory tract infections, influenza of different
types, acute
viral hepatitis A, B, C and other types of hepatitis, the diseases and
conditions
caused by HIV or associated with HIV, including AIDS.
Treatment of viral diseases based on ultra-low doses of antibodies to
interferon is known in the art (RU 2192888 Cl, A61K39/395, 11/20/2002).
However,
the given medical product can be not effective enough for treatment of the
diseases
associated with HIV.
The therapeutic effect of an extremely diluted form (or ultra-low form) of
antibodies potentized by homeopathic technology (activated-potentiated form)
has
been discovered by Dr. Oleg I. Epshtein. For example, U.S. Patent No.
7,582,294
discloses a medicament for treating Benign Prostatic Hyperplasia or
prostatitis by
administration of a homeopathically activated form of antibodies to prostate
specific
antigen (PSA). Ultra-low doses of antibodies to gamma interferon have been
shown
to be useful in the treatment and prophylaxis of diseases of viral etiology.
See U.S.
Patent No. 7,572,441, which is incorporated herein by reference in its
entirety.
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The present invention is directed to a pharmaceutical composition and
methods of its use in treatment and preventing of the infectious diseases ,
including
bacterial infections caused by different infectious agents such as
pseudotuberculosis, whooping cough, yersiniosis, pneumonitis of different
etiology, and acute and chronic viral infections such as acute respiratory
tract
infections, influenza of different types, acute viral hepatitis A, B, C and
other types of
hepatitis, the diseases and conditions caused by HIV or associated with HIV,
including AIDS.
The solution to the existing problem is presented in form of a combination
pharmaceutical composition for treatment and prophylaxis (prevetion) of
infectious
diseases, which comprises a) an activated-potentiated form of antibodies to
cytokine
and b) an activated-potentiated form of antibodies to receptor.
SUMMARY
In one aspect, the invention provides a combination pharmaceutical
composition comprising a) an activated-potentiated form of an antibody to at
least
one cytokine and b) an activated-potentiated form of an antibody to at least
one
receptor. In an embodiment, the pharmaceutical composition further comprises a
solid carrier, wherein said activated-potentiated forms of antibodies are
impregnated
onto said solid carrier. In a variant, the pharmaceutical composition is in
the form of a
tablet.
Preferably, the pharmaceutical composition including said activated-
potentiated forms of antibodies is in the form of a mixture of C12, C30, and
C200
homeopathic dilutions. It is specifically contemplated that said mixture of
C12, C30,
and C200 homeopathic dilutions is impregnated onto a solid carrier.
The activated-potentiated forms of said antibodies may be activated-
potentiated forms of a monoclonal, Polyclonal or natural antibody. It is
specifically
contemplated that the activated-potentiated form of said antibodies is
activated-
potentiated form of a polyclonal antibody. The invention provides activated-
potentiated forms of antibodies to antigen(s) having sequences described in
the
specification and claimed in the appended claims.
In a variant, the pharmaceutical composition includes activated-potentiated
forms of antibodies prepared by successive centesimal dilutions coupled with
shaking
of every dilution. Vertical shaking is specifically contemplated.
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In another aspect, the invention provides a method of treating and preventing
the infectious diseases, said method comprising administering to a patient in
need
thereof a) an activated-potentiated form of an antibody to at least one
cytokine and b)
an activated-potentiated form of an antibody to at least one receptor.
Preferably, the
activated-potentiated forms of antibodies are administered in the form of
pharmaceutical composition.
In an embodiment, the pharmaceutical composition is administered in the form
of a solid oral dosage form which comprises a pharmaceutically acceptable
carrier
and an activated-potentiated form of an antibody to at least one cytokine and
activated-potentiated form of an antibody to at least one receptor, said
activated-
potentiated forms impregnated onto said carrier. In a variant, said solid oral
dosage
form is a tablet. Variants and embodiments are provided.
In accordance with the method aspect of the invention, the pharmaceutical
composition may be administered in one to three unit dosage forms, each of the
dosage form being administered from once daily to six times daily. In a
variant, the
pharmaceutical composition is administered twice daily, each administration
consisting of two oral dosage forms. In a variant, the pharmaceutical
composition is
administered in one to two unit dosage forms, each of the dosage forms being
administered twice daily. In a variant, the pharmaceutical composition is
administered in one to two unit dosage forms, each of the dosage forms being
administered four times daily. All variants and embodiments described with
respect
to the composition aspect of the invention may be used with the method aspect
of the
invention.
DETAILED DESCRIPTION
The invention is defined with reference to the appended claims. With respect
to the claims, the glossary that follows provides the relevant definitions.
The term "antibody" as used herein shall mean an immunoglobulin that
specifically binds to, and is thereby defined as complementary with, a
particular
spatial and polar organization of another molecule. Antibodies as recited in
the claims
may include a complete immunoglobulin or fragment thereof, may be natural,
polyclonal or monoclonal, and may include various classes and isotypes, such
as
IgA, IgD, IgE, IgG '1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may
include
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Fab, Fv and F(abl)2, Fab', and the like. The singular "antibody" includes
plural
"antibodies."
The term "activated-potentiated form" or "potentiated form" respectively, with
respect to antibodies recited herein is used to denote a product of
homeopathic
potentization of any initial solution of antibodies. "Homeopathic
potentization" denotes
the use of methods of homeopathy to impart homeopathic potency to an initial
solution of relevant substance. Although not so limited, 'homeopathic
potentization"
may involve, for example, repeated consecutive dilutions combined with
external
treatment, particularly vertical (mechanical) shaking. In other words, an
initial
solution of antibody is subjected to consecutive repeated dilution and
multiple vertical
shaking of each obtained solution in accordance with homeopathic technology.
The
preferred concentration of the initial solution of antibody in the solvent,
preferably
water or a water-ethyl alcohol mixture, ranges from about 0.5 to about 5.0
mg/ml. The
preferred procedure for preparing each component, i.e. antibody solution, is
the use
of the mixture of three aqueous or aqueous-alcohol dilutions of the primary
matrix
solution (mother tincture) of antibodies diluted 10012, 1003 and 100200
times,
respectively, which is equivalent to centesimal homeopathic dilutions (C12,
C30, and
0200) or the use of the mixture of three aqueous or aqueous-alcohol dilutions
of the
primary matrix solution of antibodies diluted 10012, 1003 and 1005 times,
respectively, which is equivalent to centesimal homeopathic dilutions (C12,
C30 and
C50). Examples of homeopathic potentization are described in U.S. Patent. Nos.
7,572,441 and 7,582,294, which are incorporated herein by reference in their
entirety
and for the purpose stated. While the term "activated-potentiated form" is
used in the
claims, the term "ultra-low doses" is used in the examples. The term "ultra-
low
doses" became a term of art in the field of art created by study and use of
homeopathically diluted and potentized form of substance. The term "ultra-low
dose"
or "ultra-low doses" is meant as fully supportive and primarily synonymous
with the
term 'activated-potentiated" form used in the claims.
In other words, an antibody is in the "activated-potentiated" or "potentiated"
form when three factors are present. First, the "activated-potentiated" form
of the
antibody is a product of a preparation process well accepted in the
homeopathic art.
Second, the "activated-potentiated" form of antibody must have biological
activity
determined by methods well accepted in modern pharmacology. And third, the
biological activity exhibited by the "activated potentiated" form of the
antibody cannot
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be explained by the presence of the molecular form of the antibody in the
final
product of the homeopathic process.
For example, the activated potentiated form of antibodies may be prepared by
subjecting an initial, isolated antibody in a molecular form to consecutive
multiple
dilutions coupled with an external impact, such as mechanical shaking. The
external
treatment in the course of concentration reduction may also be accomplished,
for
example, by exposure to ultrasonic, electromagnetic, or other physical
factors. V.
Schwabe "Homeopathic medicines", M., 1967, U.S. Patents Nos. 7,229,648 and
4,311,897, which are incorporated by reference in their entirety and for the
purpose
stated, describe such processes that are well-accepted methods of homeopathic
potentiation in the homeopathic art. This procedure gives rise to a uniform
decrease
in molecular concentration of the initial molecular form of the antibody. This
procedure is repeated until the desired homeopathic potency is obtained. For
the
individual antibody, the required homeopathic potency can be determined by
subjecting the intermediate dilutions to biological testing in the desired
pharmacological model. Although not so limited, 'homeopathic potentization"
may
involve, for example, repeated consecutive dilutions combined with external
treatment, particularly vertical (mechanical) shaking. In other words, an
initial solution
of antibody is subjected to consecutive repeated dilution and multiple
vertical shaking
of each obtained solution in accordance with homeopathic technology. The
preferred
concentration of the initial solution of antibody in the solvent, preferably,
water or a
water-ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml. The
preferred
procedure for preparing each component, i.e. antibody solution, is the use of
the
mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix
solution
(mother tincture) of antibodies diluted 10012, 1003 and 100200 times,
respectively,
which is equivalent to centesimal homeopathic dilutions C12, C30 and C200 or
the
mixture of three aqueous or aqueous-alcohol dilutions of the primary matrix
solution
(mother tincture) of antibodies diluted 10012, 1003 and 1005 times,
respectively,
which is equivalent to centesimal homeopathic dilutions C12, C30 and C50.
Examples of how to obtain the desired potency are also provided, for example,
in
U.S. Patent Nos. 7,229,648 and 4,311,897, which are incorporated by reference
for
the purpose stated. The procedure applicable to the "activated-potentiated"
form of
the antibodies described herein is described in more detail below.
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There has been a considerable amount of controversy regarding homeopathic
treatment of human subjects. While the present invention relies on accepted
homeopathic processes to obtain the "activated-potentiated" form of
antibodies, it
does not rely solely on homeopathy in human subjects for evidence of activity.
It has
been surprisingly discovered by the inventor of the present application and
amply
demonstrated in the accepted pharmacological models that the solvent
ultimately
obtained from consecutive multiple dilution of a starting molecular form of an
antibody
has definitive activity unrelated to the presence of the traces of the
molecular form of
the antibody in the target dilution. The "activated-potentiated" form of the
antibody
provided herein are tested for biological activity in well accepted
pharmacological
models of activity, either in appropriate in vitro experiments, or in vivo in
suitable
animal models. The experiments provided further below provide evidence of
biological activity in such models. Human clinical studies also provide
evidence that
the activity observed in the animal model is well translated to human therapy.
Human
studies have also provided evidence of availability of the "activated
potentiated"
forms described herein to treat specified human diseases or disorders well
accepted
as pathological conditions in the medical science.
Also, the claimed "activated-potentiated" form of antibody encompasses only
solutions or solid preparations the biological activity of which cannot be
explained by
the presence of the molecular form of the antibody remaining from the initial,
starting
solution. In other words, while it is contemplated that the "activated-
potentiated" form
of the antibody may contain traces of the initial molecular form of the
antibody, one
skilled in the art could not attribute the observed biological activity in the
accepted
pharmacological models to the remaining molecular form of the antibody with
any
degree of plausibility due to the extremely low concentrations of the
molecular form of
the antibody remaining after the consecutive dilutions. While the invention is
not
limited by any specific theory, the biological activity of the "activated-
potentiated' form
of the antibodies of the present invention is not attributable to the initial
molecular
form of the antibody. Preferred is the "activated-potentiated" form of
antibody in liquid
or solid form in which the concentration of the molecular form of the antibody
is below
the limit of detection of the accepted analytical techniques, such as
capillary
electrophoresis and High Performance Liquid Chromatography. Particularly
preferred
is the "activated-potentiated" form of antibody in liquid or solid form in
which the
concentration of the molecular form of the antibody is below the Avogadro
number. In
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the pharmacology of molecular forms of therapeutic substances, it is common
practice to create a dose-response curve in which the level of pharmacological
response is plotted against the concentration of the active drug administered
to the
subject or tested in vitro. The minimal level of the drug which produces any
detectable response is known as a threshold dose. It is specifically
contemplated and
preferred that the "activated-potentiated" form of the antibodies contains
molecular
antibody, if any, at a concentration below the threshold dose for the
molecular form of
the antibody in the given biological model.
The present invention provides a combination pharmaceutical composition that
includes activated-potentiated form of antibodies to cytokine and activated-
potentiated form of antibodies to receptor, prepared according to the
homeopathic
technology of potentiation by repeated, consistent dilution and intermediate
external
action of shaking as described in more detail herein below. The pharmaceutical
composition of the invention is particularly useful in the treatment and
prophylaxis of
the infectious diseases, including bacterial infections caused by different
infectious
agents such as pseudotuberculosis, whooping cough, yersiniosis, pneumonitis of
different etiology, and acute and chronic viral infections such as acute
respiratory
tract infections, flu of different types, acute viral hepatitis A, B, C and
other types of
hepatitis, the diseases and conditions caused by HIV or associated with HIV,
including AIDS. As shown in the Examples, the pharmaceutical composition of
the
invention possesses unexpected synergetic therapeutic effect, which manifest
itself in
particular therapeutic effectiveness in treating and preventing the infectious
diseases,
including bacterial infections caused by different infectious agents such as
pseudotuberculosis, whooping cough, yersiniosis, pneumonitis of different
etiology, and acute and chronic viral infections such as acute respiratory
tract
infections, influenza of different types, acute viral hepatitis A, B, C and
other types of
hepatitis, the diseases and conditions caused by HIV or associated with HIV,
including AIDS.
The pharmaceutical composition of the invention expands the arsenal of
preparations available for the treatment prophylaxis of the infectious
diseases,
including bacterial infections and acute and chronic viral infections.
The combination pharmaceutical composition in accordance with this aspect of
the invention may be in the liquid form or in solid form. Activated-
potentiated form of
the antibodies included in the pharmaceutical composition is prepared from an
initial
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molecular form of the antibody via a process accepted in homeopathic art. The
starting antibodies may be monoclonal, or polyclonal antibodies prepared in
accordance with known processes, for example, as described in
lmmunotechniques,
G. Frimel, M., "Meditsyna", 1987, p. 9-33; "Hum. Antibodies. Monoclonal and
recombinant antibodies, 30 years after" by Laffly E., Sodoyer R. ¨2005 ¨ Vol.
14. ¨ N
1-2. P.33-55, both incorporated herein by reference.
Monoclonal antibodies may be obtained, e.g., by means of hybridoma
technology. The initial stage of the process includes immunization based on
the
principles already developed in the course of polyclonal antisera preparation.
Further
stages of work involve the production of hybrid cells generating clones of
antibodies
with identical specificity. Their separate isolation is performed using the
same
methods as in the case of polyclonal antisera preparation.
Polyclonal antibodies may be obtained via active immunization of animals. For
this purpose, for example, suitable animals (e.g. rabbits) receive a series of
injections
of the appropriate antigen (cytokine and receptor). The animals' immune system
generates corresponding antibodies, which are collected from the animals in a
known
manner. This procedure enables preparation of a monospecific antibody-rich
serum.
If desired, the serum containing antibodies may be purified, for example by
using affine chromatography, fractionation by salt precipitation, or ion-
exchange
chromatography. The resulting purified, antibody-enriched serum may be used as
a
starting material for the preparation of the activated-potentiated form of the
antibodies. The preferred concentration of the resulting initial solution of
antibody in
the solvent, preferably water or a water-ethyl alcohol mixture, ranges from
about 0.5
to about 5.0 mg/ml.The preferred procedure for preparing each component of the
combination
drug according to the present invention is the use of the mixture of three
aqueous-
alcohol dilutions of the primary matrix solution of antibodies diluted 10012,
1003 and
1005 times, respectively, which is equivalent to centesimal homeopathic
dilutions
C12, C30, and C50 or diluted 10012, 1003 and 100200 times, respectively,
which is
equivalent to centesimal homeopathic dilutions C12, C30 and C200. To prepare a
solid dosage form, a solid carrier is treated with the desired dilution
obtained via the
.homeopathic process. To obtain a solid unit dosage form of the combination of
the
invention, the carrier mass is impregnated with each of the dilutions. Both
orders of
impregnation are suitable to prepare the desired combination dosage form.
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In a preferred embodiment, the starting material for the preparation of the
activated potentiated form that comprise the combination pharmaceutical
composition
of the invention is polyclonal, animal-raised antibody to the corresponding
antigen. To
obtain the activated-potentiated form of polyclonal antibodies to cytokine or
receptor,
the desired antigen may be injected as immunogen into a laboratory animal,
preferably, rabbits.
Polyclonal antibodies to CD4 receptor may be obtained using the whole
molecule of human CD4 receptor of the following sequence:
SEQ. ID. NO. 1
Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gin
1 5 10 15
Leu Ala Leu Leu Pro Ala Ala Thr Gin Gly Lys Lys Val Val Leu
16 20 25 30
Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gin
31 35 40 45
Lys Lys Ser Ile Gin Phe His Trp Lys Asn Ser Asn Gin Ile Lys
46 50 55 60
Ile Leu Gly Asn Gin Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys
61 65 70 75
Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gin Gly
76 80 85 90
Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp
91 95 100 105
Thr Tyr Ile Cys Glu Val Glu Asp Gin Lys Glu Glu Val Gin Leu
106 110 115 120
Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gin
121 125 130 135
Gly Gin Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser
136 140 145 150
Pro Ser Val Gin Cys Arg Ser Pro Arg Gly Lys Asn Ile Gin Gly
151 155 160 165
Gly Lys Thr Leu Ser Val Ser Gin Leu Glu Leu Gin Asp Ser Gly
166 170 175 180
Thr Trp Thr Cys Thr Val Leu Gin Asn Gin Lys Lys Val Glu Phe
181 185 190 195
Lys Ile Asp Ile Val Val Leu Ala Phe Gin Lys Ala Ser Ser Ile
196 200 205 210
Val Tyr Lys Lys Glu Gly Glu Gin Val Glu Phe Ser Phe Pro Leu
211 215 220 225
Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp
226 230 235 240
Gin Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp
241 245 250 255
Leu Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gin Asp Pro
256 260 265 270
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Lys Leu Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro
271 275 280 285
Gln Ala Leu Pro Gln Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala
286 290 295 300
Leu Glu Ala Lys Thr Gly Lys Leu His Gln Glu Val Asn Leu Val
301 305 310 315
Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu Val
316 320 325 330
Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu
331 335 340 345
Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val
346 350 355 360
Leu Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp Ser
361 365 370 375
Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp
376 380 385 390
Ser Thr Pro Val Gln Pro Met Ala Leu Ile Val Leu Gly Gly Val
391 395 400 405
Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val
406 410 415 420
Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln Ile
421 425 430 435
Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His Arg
436 440 445 450
Phe Gln Lys Thr Cys Ser Pro Ile
451 445 458
The polyclonal antibodies to CD4 receptor can be obtained using a polypeptide
fragment of C04 receptor chosen, for example, from the following amino-acid
sequences:
SEQ. ID. NO. 2
Gly Lys Lys Val Val Leu
26 30
Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gln
31 35 40 45
Lys Lys Ser Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys
46 50 55 60
Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys
61 65 70 75
Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly
76 80 85 90
Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp
91 95 100 105
Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu
106 110 115 120
Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gln
121 125 130 135
Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser
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136 140 145 150
Pro Ser Val Gin Cys Arg Ser Pro Arg Gly Lys Asn Ile Gin Gly
151 155 160 165
Gly Lys Thr Leu Ser Val Ser Gin Leu Glu Leu Gin Asp Ser Gly
166 170 175 180
Thr Trp Thr Cys Thr Val Leu Gin Asn Gin Lys Lys Val Glu Phe
181 185 190 195
Lys Ile Asp Ile Val Val Leu Ala Phe Gin Lys Ala Ser Ser Ile
196 200 205 210
Val Tyr Lys Lys Glu Gly Glu Gin Val Glu Phe Ser Phe Pro Leu
211 215 220 225
Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp
226 230 235 240
Gin Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp
241 245 250 255
Leu Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gin Asp Pro
256 260 265 270
Lys Leu Gin Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro
271 275 280 285
Gin Ala Leu Pro Gin Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala
286 290 295 300
Leu Glu Ala Lys Thr Gly Lys Leu His Gin Glu Val Asn Leu Val
301 - 305 310 315
Val Met Arg Ala Thr Gin Leu Gin Lys Asn Leu Thr Cys Glu Val
316 320 325 330
Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu
331 335 340 345
Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val
346 350 355 360
Leu Asn Pro Glu Ala Gly Met Trp Gin Cys Leu Leu Ser Asp Ser
361 365 370 375
Gly Gin Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp
376 380 385 390
Ser Thr Pro Val Gin Pro Met Ala Leu Ile Val Leu Gly Gly Val
391 395 400 405
Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val
406 410 415 420
Arg Cys Arg His Arg Arg Arg Gin Ala Glu Arg Met Ser Gin Ile
421 425 430 435
Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gin Cys Pro His Arg
436 440 445 450
Phe Gin Lys Thr Cys Ser Pro Ile
451 445 458
SEQ. ID. NO. 3
Ile Gly Leu Gly Ile Phe Phe Cys Val
412 415 420
Arg Cys Arg His Arg Arg Arg Gin Ala Glu Arg Met Ser Gin Ile
421 425 430 435
Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gin Cys Pro His Arg
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436 440 445 450
Phe Gin Lys Thr Cys Ser Pro Ile
451 445 458
SEQ. ID. NO. 4
Gly Lys Lys Val Val Leu
26 30
Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gin
31 35 40 45
Lys Lys Ser Ile Gin Phe His Trp Lys Asn Ser Asn Gin Ile Lys
46 50 55 60
SEQ. ID. NO. 5
Asp
91 95 100 105
Thr Tyr Ile Cys Glu Val Glu Asp Gin Lys Glu Glu Val Gin
106 110 115 119
SEQ. ID. NO. 6
Lys Glu Glu Val Gin Leu
115 120
Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gin
121 125 130 135
Gly Gin Ser Leu
136 139
The exemplary procedure for preparation of the starting polyclonal antibodies
to CD4 receptor may be described as follows. In 7-9 days before blood
sampling, 1-3
intravenous injections of the desired antigen are made to the rabbits to
increase the
level of polyclonal antibodies in the rabbit blood stream. Upon immunization,
blood
samples are taken to test the antibody level. Typically, the maximum level of
immune
reaction of the soluble antigen is achieved within 40 to 60 days after the
first injection
of the antigen. Upon completion of the first immunization cycle, rabbits have
a 30-day
rehabilitation period, after which re-immunization is performed with another 1-
3
intravenous injections.
To obtain antiserum containing the desired antibodies, the immunized rabbits'
blood is collected from rabbits and placed in a 50m1 centrifuge tube. Product
clots
formed on the tube sides are removed with a wooden spatula, and a rod is
placed
into the clot in the tube center. The blood is then placed in a refrigerator
for one night
at the temperature of about 40 C. On the following day, the clot on the
spatula is
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removed, and the remaining liquid is centrifuged for 10 min at 13,000
rotations per
minute. Supernatant fluid is the target antiserum. The obtained antiserum is
typically
yellow. 20% of NaN3 (weight concentration) is added in the antiserum to a
final
concentration of 0.02% and stored before use in frozen state at the
temperature of -
20 C or without NaN3 at the temperature of -70 C. To separate the target
antibodies
to gamma interferon from the antiserum, the following solid phase absorption
sequence is suitable:
ml of the antiserum of rabbits is diluted twofold with 0.15 M NaCl, after
which 6.26g Na2SO4 is added, mixed and incubated for 12-16 hours at 4 C. The
10 sediment is removed by centrifugation, diluted in 10m1 of phosphate buffer
and
dialyzed against the same buffer during one night at ambient temperature.
After the
sediment is removed, the solution is applied to a DEAE-cellulose column
balanced by
phosphate buffer. The antibody fraction is determined by measuring the optical
density of the eluate at 280 nm.
The isolated crude antibodies are purified using affine chromatography
method by attaching the obtained antibodies to CD4 antigen located on the
insoluble
matrix of the chromatography media, with subsequent elution by concentrated
aqueous salt solutions.
The resulting buffer solution is used as the initial solution for the
homeopathic
dilution process used to prepare the activated potentiated form of the
antibodies. The
preferred concentration of the initial matrix solution of the antigen-purified
polyclonal
rabbit antibodies to CD4 receptor is 0.5 to 5.0 mg/ml, preferably, 2.0 to 3.0
mg/ml.
The polyclonal antibodies to gamma interferon may also be obtained by a
similar methodology to the methodology described for C04 receptor antibodies
using an adjuvant. Polyclonal antibodies to gamma interferon may be obtained
using the whole molecule of gamma interferon of the following sequence:
SEQ ID NO: 7
Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gin Leu Cys Ile Val
1 5 10 15
Leu Gly Ser Leu Gly Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu
16 20 25 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
46 50 55 60
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Glu Glu Ser Asp Arg Lys Ile Met Gin Ser Gin Ile Val Ser Phe
61 65 70 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser Ile Gin
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
136 140 145 150
Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser
151 155 160 165
Gin
166
Polyclonal antibodies to gamma interferon may be obtained using the whole
molecule of gamma interferon of the following sequence:
SEQ ID NO: 8
Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gin Leu Cys Ile Val
1 5 10 15
Leu Gly Ser Leu Gly Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu
16 20 25 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
46 50 55 60
Glu Glu Ser Asp Arg Lys Ile Met Gin Ser Gin Ile Val Ser Phe
61 65 70 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser Ile Gin
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
136 140 145 150
Lys Arg Lys Arg Ser Gln Met Leu Phe Gin Gly Arg Arg Ala Ser
151 155 160 165
Gin
166
The use of gamma interferon fragments as antigen is also contemplated.
The suitable sequence for such antigen is as follow:
SEQ ID NO: 9
Ile Leu Ala Phe Gin Leu Cys Ile Val
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7 10 15
Leu Gly Ser Leu Gly Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu
16 20 25 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile
46 50 55
SEQ ID NO: 10
Gin Asp Pro Tyr Val Lys Glu
24 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
46 50 55 60
Glu Glu Ser Asp Arg Lys Ile Met Gin Ser Gln Ile Val Ser Phe
61 65 70 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser Ile Gin
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
136 140 145 150
Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser
151 155 160 165
Gin
166
SEQ ID NO: 11
Gin Asp Pro Tyr Val Lys Glu
24 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
46 50 55 60
Glu Glu Ser Asp Arg Lys Ile Met Gin Ser Gin Ile Val Ser Phe
61 65 70 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser Ile Gin
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
136 140 145 150
Lys Arg Lys Arg Ser Gin Met Leu Phe Gin Gly Arg Arg Ala Ser
151 155 160 165
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Gin
166
SEQ ID NO: 12
Gin Ser Gin Ile Val Ser Phe
69 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser Ile Gin
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val
121 123
SEQ ID NO: 13
Met Asn Val Lys Phe Phe
100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gin Val Met Ala Glu Leu Ser Pro
136 140 145
SEQ ID NO: 14
Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
92 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gin Arg
121 125 130
SEQ ID NO: 15 ,
Val Thr Asp Leu Asn Val Gin Arg Lys Ala Ile His Glu
123 125 130 135
Leu Ile Gin Val Met Ala Glu Leu Ser Pro Ala Ala
136 140 145 147
SEQ ID NO: 16
Ser Tyr Ile Leu Ala Phe Gin Leu Cys Ile Val
5 10 15
Leu Gly Ser Leu Gly Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu
16 20 25 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
SEQ ID NO: 17
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Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
94 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp
106 110 114
Polyclonal antibodies to gamma interferon may be obtained using the
molecule of recombinant gamma interferon of one of the following sequences:
SEQ ID NO: 18
Met Gln Asp Pro Tyr Val Lys Glu
24 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
46 50 55 60
Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe
61 65 70 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
136 140 145 150
Lys Arg Lys Arg Ser Gln Met Leu Phe Gln Gly Arg Arg Ala Ser
151 155 160 165
Gln
166
SEQ ID NO: 19
Met Gln Asp Pro Tyr Val Lys Glu
24 30
Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val
31 35 40 45
Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
46 50 55 60
Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe
61 65 70 75
Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln
76 80 85 90
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
91 95 100 105
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
106 110 115 120
Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
121 125 130 135
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
136 140 145 150
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Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser
151 155 160 165
Gin
166
The polyclonal antibodies to alpha interferon may also be obtained by a
similar methodology to the methodology described for C04 receptor antibodies
using an adjuvant. Polyclonal antibodies to alpha interferon may be obtained
using the whole molecule of human alpha interferon type 8 of the following
sequence:
SEQ ID NO: 20
Met Ala Leu Thr Phe Tyr Leu Leu Val Ala Leu Val Val Leu Ser
1 5 10 15
Tyr Lys Ser Phe Ser Ser Leu Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gin Met Arg
31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Glu
46 50 55 60
Phe Pro Gin Glu Glu Phe Asp Asp Lys Gin Phe Gin Lys Ala Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gin Gin Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp Glu Thr Leu Leu
91 95 100 105
Asp Glu Phe Tyr Ile Glu Leu Asp Gin Gin Leu Asn Asp Leu Glu
106 110 115 120
Ser Cys Val Met Gin Glu Val Gly Val Ile Glu Ser Pro Leu Met
121 125 130 135
Tyr Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Ser Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Ile Asn
166 170 175 180
Leu Gin Lys Arg Leu Lys Ser Lys Glu
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 2 of the following sequence:
SEQ ID NO: 21
Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser
1 5 10 15
Cys Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Arg
31 35 40 45
Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
46 50 55 60
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Phe Pro Gin Glu Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr
61 65 70 75
Ile Pro Val Leu His Glu Met Ile Gin Gin Ile Phe Asn Leu Phe
76 80 85 90
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp
91 95 100 105
Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala
106 110 115 120
Cys Val Ile Gin Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
121 125 130 135
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile Thr
136 140 145 150
Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
151 155 160 165
Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu
166 170 175 180
Gin Glu Ser Leu Arg Ser Lys Glu
181 185 188
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 17 of the following sequence:
SEQ ID NO: 22
Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser
1 5 10 15
Tyr Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gin Met Gly
31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
46 50 55 60
Leu Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Thr Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gin Gin Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gin Ser Leu Leu
91 95 100 105
Glu Lys Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asn Leu Glu
106 110 115 120
Ala Cys Val Ile Gin Glu Val Gly Met Glu Glu Thr Pro Leu Met
121 125 130 135
Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn
166 170 175 180
Leu Gin Lys Ile Leu Arg Arg Lys Asp
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 4 of the following sequence:
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SEQ ID NO: 23
Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser
1 5 10 15
Tyr Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gin Met Gly
31 35 40 45
Arg Ile Ser His Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
46 50 55 60
Phe Pro Glu Glu Glu Phe Asp Gly His Gin Phe Gin Lys Ala Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gin Gin Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gin Ser Leu Leu
91 95 100 105
Glu Lys Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu
106 110 115 120
Ala Cys Val Ile Gin Glu Val Gly Val Glu Glu Thr Pro Leu Met
121 125 130 135
Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn
166 170 175 180
Leu Gin Lys Arg Leu Arg Arg Lys Asp
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 21 of the following sequence:
SEQ ID NO: 24
Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser
1 5 10 15
Tyr Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gin Met Gly
31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
46 50 55 60
Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gin Gln Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Glu Gin Ser Leu Leu
91 95 100 105
Glu Lys Phe Ser Thr Glu Leu Asn Gin Gin Leu Asn Asp Leu Glu
106 110 115 120
Ala Cys Val Ile Gin Glu Val Gly Val Glu Glu Thr Pro Leu Met
121 125 130 135
Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
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151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Lys Ile
166 170. 175 180
Phe Gln Glu Arg Leu Arg Arg Lys Glu
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type1/13 of the following sequence:
SEQ ID NO: 25
Met Ala Ser Pro Phe Ala Leu Leu Met Val Leu Val Val Leu Ser
1 5 10 15
Cys Lys Ser Ser Cys Ser Leu Gly Cys Asp Leu Pro Glu Thr His
16 20 25 30
Ser Leu Asp Asn Arg Arg Thr Leu Met Leu Leu Ala Gln Met Ser
31 35 40 45
Arg Ile Ser Pro Ser Ser Cys Leu Met Asp Arg His Asp Phe Gly
46 50 55 60
Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Pro
61 65 70 75
Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile Phe Asn Leu
76 80 85 90
Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp Leu Leu
91 95 100 105
Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
106 110 115 120
Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met
121 125 130 135
Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile
136 140 145 150
Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn
166 170 175 180
Leu Gln Glu Arg Leu Arg Arg Lys Glu
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 10 of the following sequence:
SEQ ID NO: 26
Met Ala Leu Ser Phe Ser Leu Leu Met Ala Val Leu Val Leu Ser
1 5 10 15
Tyr Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gln Thr His
16 20 25 30
Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Gly Gln Met Gly
31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Arg
46 50 55 60
Ile Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Gln
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gln Ser Leu Leu
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91 95 100 105
Glu Lys Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu
106 110 115 120
Ala Cys Val Ile Gin Glu Val Gly Val Glu Glu Thr Pro Leu Met
121 125 130 135
Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn
166 170 175 180
Leu Gin Lys Arg Leu Arg Arg Lys Asp
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 5 of the following sequence:
SEQ ID NO: 27
Met Ala Leu Pro Phe Val Leu Leu Met Ala Leu Val Val Leu Asn
1 5 10 15
Cys Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Ser Asn Arg Arg Thr Leu Met Ile Met Ala Gin Met Gly
31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
46 50 55 60
Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gin Gin Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Asp Glu Thr Leu Leu
91 95 100 105
Asp Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu
106 110 115 120
Ala Cys Met Met Gin Glu Val Gly Val Glu Asp Thr Pro Leu Met
121 125 ' 130 135
Asn Val Asp Ser Ile Leu Thr Val Arg Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Ala Asn
166 170 175 180
Leu Gin Glu Arg Leu Arg Arg Lys Glu
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 7 of the following sequence:
SEQ ID NO: 28
Met Ala Arg Ser Phe Ser Leu Leu Met Val Val Leu Val Leu Ser
1 5 10 15
Tyr Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pro Gin Thr His
16 20 25 30
Ser Leu Arg Asn Arg Arg Ala Leu Ile Leu Leu Ala Gin Met Gly
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31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Glu Phe Arg
46 50 55 60
Phe Pro Glu Glu Glu Phe Asp Gly His Gin Phe Gin Lys Thr Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Ile Gin Gin Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gin Ser Leu Leu
91 95 100 105
Glu Lys Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu
106 110 115 120
Ala Cys Val Ile Gin Glu Val Gly Val Glu Glu Thr Pro Leu Met
121 125 130 135
Asn Glu Asp Phe Ile Leu Ala Val Arg Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Phe Ser Thr Asn
166 170 175 180
Leu Lys Lys Gly Leu Arg Arg Lys Asp
181 185 189
Polyclonal antibodies to alpha interferon may be obtained using the
whole molecule of human alpha interferon type 14 of the following sequence:
SEQ ID NO: 29
Met Ala Leu Pro Phe Ala Leu Met Met Ala Leu Val Val Leu Ser
1 5 10 15
Cys Lys Ser Ser Cys Ser Leu Gly Cys Asn Leu Ser Gin Thr His
16 20 25 30
Ser Leu Asn Asn Arg Arg Thr Leu Met Leu Met Ala Gin Met Arg
31 35 40 45
Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Glu
46 50 55 60
Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin
61 65 70 75
Ala Ile Ser Val Leu His Glu Met Met Gin Gin Thr Phe Asn Leu
76 80 85 90
Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu
91 95 100 105
Glu Lys Phe Tyr Ile Glu Leu Phe Gin Gin Met Asn Asp Leu Glu
106 110 115 120
Ala Cys Val Ile Gin Glu Val Gly Val Glu Glu Thr Pro Leu Met
121 125 130 135
Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gin Arg Ile
136 140 145 150
Thr Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
151 155 160 165
Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn
166 170 175 180
Leu Gin Lys Arg Leu Arg Arg Lys Asp
181 185 189
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The polyclonal antibodies to CD8 receptor may also be obtained by a
similar methodology to the methodology described for CD4 receptor antibodies
using an adjuvant. Polyclonal antibodies to CD8 receptor may be obtained
using the whole molecule of CD8 receptor of the following sequence: =
SEQ ID NO: 30
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
1 5 10 15
Leu His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp
16 20 25 30
Arg Thr Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gin Val
31 35 40 45
Leu Leu Ser Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gin Pro
46 50 55 60
Arg Gly Ala Ala Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gin
61 65 70 75
Asn Lys Pro Lys Ala Ala Glu Gly Leu Asp Thr Gin Arg Phe Ser
76 80 85 90
Gly Lys Arg Leu Gly Asp Thr Phe Val Leu Thr Leu Ser Asp Phe
91 95 100 105
Arg Arg Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala Leu Ser Asn
106 110 115 120
Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala
121 125 130 135
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
136 140 145 150
Thr Ile Ala Ser Gin Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
151 155 160 165
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
166 170 175 180
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
181 185 190 195
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn
196 200 205 210
Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser Gly
211 215 220 225
Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val
226 230 235
The use of CD8 receptor fragments as antigen is also contemplated. The
suitable sequences for such antigen are as follow:
SEQ ID NO: 31
Pro Leu Ala Leu Leu
11 15
Leu His Ala Ala Arg Pro Ser Gin Phe Arg Val Ser Pro Leu Asp
16 20 25 30
SEQ ID NO: 32
Ala Glu Gly Leu Asp Thr Gin Arg Phe Ser
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81 85 90
Gly Lys Arg Leu Gly Asp Thr Phe Val Leu
91 95 100
SEQ ID NO: 33
Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala
121 125 130 135
Lys Pro Thr Thr Thr
136 140
SEQ ID NO: 34
Val Ile Thr Leu Tyr Cys Asn His Arg Asn
201 205 210
SEQ ID NO: 35
Val Val Lys Ser Gly
221 225
Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val
226 230 235
Polyclonal antibodies to tumor necrosis factor alpha (TNF-a) may be
obtained by the above-mentioned method of obtaining antibodies to CD4
receptor using a whole molecule of tumor necrosis factor alpha of the
following
sequence:
SEQ ID NO: 36
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu
1 5 10 15
Ala Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys
16 20 25 30
Leu Phe Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr
31 35 40 45
Thr Leu Phe Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg
46 50 55 60
Glu Glu Phe Pro Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln
61 65 70 75
Ala Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala
76 80 85 90
His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu
91 95 100 105
Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg
106 110 115 120
Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr
121 125 130 135
Ser Gln Val Leu Phe Lys Gly. Gln Gly Cys Pro Ser Thr His Val
136 140 145 150
Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr
151 155 160 165
Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu
166 170 175 180
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Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr
181 185 190 195
Leu Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala
196 200 205 210
Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gin
211 215 220 225
Val Tyr Phe Gly Ile Ile Ala Leu
226 230 233
To obtain polyclonal antibodies to tumor necrosis factor alpha (TNF-a), it
is also possible to use a polypeptide fragment of the tumor necrosis factor,
selected, for example, from the following sequences:
SEQ ID NO: 37
Pro Ser Asp Lys Pro
84 88
SEQ ID NO: 38
Val Ala Asn Pro Gin
93 97
SEQ ID NO: 39
Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gin
65 70 75
Ala Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala
76 80 85 90
His Val Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu
91 95 100 105
Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg
106 110 115 120
Asp Asn Gin Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr
121 125 130 135
Ser Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Ser Thr His Val
136 140 145 150
Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gin Thr
151 155 160 165
Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gin Arg Glu
166 170 175 180
Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr
181 185 190 195
Leu Gly Gly Val
196 199
SEQ ID NO: 40
Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala
77 80 85 90
His Val Val
91 93
SEQ ID NO: 41
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Phe Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr
32 35 40 45
Thr Leu Phe Cys Leu Leu His Phe Gly
46 50 54
SEQ ID NO: 42
Ile Gly Pro Gin Arg
56 60
Glu Glu Phe Pro Arg Asp Leu Ser Leu Ile Ser Pro Leu
61 65 70 73
SEQ ID NO: 43
Gin Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr
123 125 130 135
Ser Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Ser Thr His Val
136 140 145 150
Leu Leu Thr His Thr Ile Ser Arg Ile Ala
151 155 160
SEQ ID NO: 44
Pro Cys Gin Arg Glu
176 180
Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp
181 185 190
SEQ ID NO: 45
Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu
5 10 15
Ala Leu Pro Lys Lys Thr Gly Gly Pro Gin Gly Ser Arg Arg Cys
16 20 25 30
Leu Phe Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr
31 35 40 45
SEQ ID NO: 46
Val
150
Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gin Thr
151 155 160 165
Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gin Arg Glu
166 170 175 180
Thr Pro Glu Gly
181 184
SEQ ID NO 47
Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala
77 80 85 90
His Val Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu
91 95 100 105
Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg
106 110 115 120
Asp Asn Gin Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr
121 125 130 135
Ser Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Ser Thr His Val
136 140 145 150
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Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr Gin Thr
151 155 160 165
Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gin Arg Glu
166 170 175 180
Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr
181 185 190 195
Leu Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala
196 200 205 210
Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gin
211 215 220 225
Val Tyr Phe Gly Ile Ile Ala Leu
226 230 233
Polyclonal antibodies to histamine, which is a biogenic amine (4(2-
aminoethyl)-imidazole or beta-imidazolylethylamine with the chemical formula
C5H9N3), may be obtained by the above-mentioned method of obtaining
antibodies to CD4 using adjuvant and industrially produced histamine
dihydrochloride as immunogen (antigen) for immunization of rabbits.
The activated-potentiated form of an antibody to cytokine or receptor
may be prepared from an initial solution by homeopathic potentization,
preferably using the method of proportional concentration decrease by serial
dilution of 1 part of each preceding solution (beginning with the initial
solution)
in 9 parts (for decimal dilution), or in 99 parts (for centesimal dilution),
or in 999
parts (for millesimal dilution) of a neutral solvent, starting with a
concentration
of the initial solution of antibody in the solvent, preferably, water or a
water-
ethyl alcohol mixture, in the range from about 0.5 to about 5.0 mg/ml, coupled
with external impact. Preferably, the external impact involves multiple
vertical
shaking (dynamization) of each dilution. Preferably, separate containers are
used for each subsequent dilution up to the required potency level, or the
dilution factor. This method is well-accepted in the homeopathic art. See,
e.g.
V. Schwabe "Homeopathic medicines", M., 1967, p. 14-29, incorporated herein
by reference for the purpose stated.
For example, to prepare a 12-centesimal dilution (denoted C12), one part of
the initial matrix solution of antibodies to CD4 receptor with the
concentration of 3.0
mg/ml is diluted in 99 parts of neutral aqueous or aqueous-alcohol solvent
(preferably, 15%-ethyl alcohol) and then vertically shaked many times (10 and
more)
to create the 1st centesimal dilution (denoted as Cl). The 2nd centesimal
dilution
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(C2) is prepared from the 1st centesimal dilution Cl. This procedure is
repeated 11
times to prepare the 12th centesimal dilution C12. Thus, the 12th centesimal
dilution
C12 represents a solution obtained by 12 serial dilutions of one part of the
initial
matrix solution of antibodies to gamma interferon with the concentration of
3.0 mg/ml
in 99 parts of a neutral solvent in different containers, which is equivalent
to the
centesimal homeopathic dilution C12. Similar procedures with the relevant
dilution
factor are performed to obtain dilutions C30, C50 and C 200.The intermediate
dilutions may be tested in a desired biological model to check activity. The
preferred
activated-potentiated form for the composition of the invention are a mixture
of C12,
C30, and C50 dilutions or C12, C30 and C200 dilutions. When using the mixture
of
various homeopathic dilutions (primarily centesimal) of the active substance
as
biologically active liquid component, each component of the composition (e.g.,
C12,
C30, C50, C200) is prepared separately according to the above-described
procedure
until the next-to-last dilution is obtained (e.g., until C11, C29, and C199
respectively),
and then one part of each component is added in one container according to the
mixture composition and mixed with the required quantity of the solvent (e.g.
with 97
parts for centesimal dilution).
It is possible to use the active substance as mixture of various homeopathic
dilutions, e.g. decimal and/or centesimal (D20, C30, C100 or C12, C30, C50 or
C12,
C30, C200, etc.), the efficiency of which is determined experimentally by
testing the
dilution in a suitable biological model, for example, in models described in
the
examples herein.
In the course of potentiation and concentration decrease, the vertical shaking
may be substituted for external exposure to ultrasound, electromagnetic field
or any
similar external impact procedure accepted in the homeopathic art.
Preferably, the pharmaceutical composition of the invention may be in the form
of a liquid or in the solid unit dosage form. The preferred liquid carrier is
water or
water-ethyl alcohol mixture.
The solid unit dosage form of the pharmaceutical composition of the invention
may be prepared by impregnating a solid, pharmaceutically acceptable carrier
with
the mixture of the activated potentiated form aqueous or aqueous-alcohol
solutions of
active component. Alternatively, the carrier may be impregnated consecutively
with
each requisite dilution. Both orders of impregnation are acceptable.
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Preferably, the pharmaceutical composition in the solid unit dosage form is
prepared from granules of the pharmaceutically acceptable carrier which was
previously saturated with the aqueous or aqueous-alcoholic dilutions of the
activated
potentiated form of antibodies to at least one cytokine and activated
potentiated form
of antibodies to at least one receptor. The solid dosage form may be in any
form
known in the pharmaceutical art, including a tablet, a capsule, a lozenge, and
others.
As an inactive pharmaceutical ingredients one can use glucose, sucrose,
maltose,
amylum, isomaltose, isomalt and other mono- olygo- and polysaccharides used in
manufacturing of pharmaceuticals as well as technological mixtures of the
above
mentioned inactive pharmaceutical ingredients with other pharmaceutically
acceptable excipients, for example isomalt, crospovidone, sodium cyclamate,
sodium
saccharine, anhydrous citric acid etc), including lubricants, disintegrants,
binders and
coloring agents. The preferred carriers are lactose and isomalt. The
pharmaceutical
dosage form may further include standard pharmaceutical excipients, for
example,
microcrystalline cellulose, magnesium stearate and citric acid.
To prepare the solid oral form, 100-300 pm granules of lactose are
impregnated with aqueous or aqueous-alcoholic solutions of the activated-
potentiated
forms of antibodies in the ratio of 1 kg of antibody solution to 5 or 10 kg of
lactose
(1:5 to 1:10). To effect impregnation, the lactose granules are exposed to
saturation
irrigation in the fluidized boiling bed in a boiling bed plant (e.g. "Mifflin
Pilotlab" by
Main GmbH) with subsequent drying via heated air flow at a temperature below
40 C. The estimated quantity of the dried granules (10 to 34 weight parts)
saturated
with the activated potentiated form of antibodies is placed in the mixer, and
mixed
with 25 to 45 weight parts of "non-saturated" pure lactose (used for the
purposes of
cost reduction and simplification and acceleration of the technological
process
without decreasing the treatment efficiency), together with 0.1 to 1 weight
parts of
magnesium stearate, and 3 to 10 weight parts of microcrystalline cellulose.
The
obtained tablet mass is uniformly mixed, and tableted by direct dry pressing
(e.g., in a
Korsch ¨ XL 400 tablet press) to form 150 to 500 mg round pills, preferably,
300 mg.
After tableting, 300mg pills are obtained that are saturated with aqueous-
alcohol
solution (3.0-6.0 mg/pill) of the activated-potentiated form of antibodies in
the form of
a mixture of centesimal homeopathic dilutions C12, C30, and C50 or a mixture
of
centesimal homeopathic dilutions C12, C30 and C200.
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While the invention is not limited to any specific theory, it is believed that
the
activated potentiated form of the antibodies described herein do not contain
the
molecular form of the antibody in an amount sufficient to have biological
activity
attributed to such molecular form. The biological activity of the combination
drug
(pharmaceutical composition) of the invention is amply demonstrated in the
appended examples.
Preferably, for the purpose of treatment, the combination of the invention is
administered from once daily to six times daily, preferably twice daily or
four times
daily, each administration including one or three combination unit dosage
forms.
The invention is further illustrated with reference to the appended non-
limiting
examples.
EXAMPLES
Example 1.
Study of the effect of a complex preparation containing ultralow doses of
activated¨potentiated forms of polyclonal affinity purified rabbit antibodies
to
CD4 receptor (anti-CD4) and gamma interferon (anti-IFN-y), obtained by super-
dilution of initial matrix solution (concentration: 2.5 mg/ml) (10012, 1003 ,
1005
times), equivalent to a mixture of centesimal homeopathic dilutions C12, C30,
C50 (ratio: 1:1) ( anti-CD4+anti-IFN-y>>), as well as its components:
activated¨
potentiated form of polyclonal affinity purified rabbit antibodies to CD4
receptor,
purified on antigen, obtained by super-dilution of initial matrix solution
(10012,
1003 , 10 05 times, equivalent to a mixture of centesimal homeopathic
dilution
C12, C30, C50 ("anti-CD4"), and activated¨potentiated form of polyclonal
rabbit
antibodies to gamma interferon, obtained by super-dilution of initial matrix
solution (10012, 10030, 10050 times), equivalent to a mixture of centesimal
homeopathic dilution C12, C30, C50 ("anti-IFN-y") on in vitro on binding of
standard ligand [31-I]pentazocine to human recombinant cr1 receptor was
evaluated using radioligand method. Potentiated distilled water (mixture of
homeopathic dilutions C12+C30+C50) was used as test preparations control.
The sigma-1 (c:r1) receptor - an intracellular one which is localized in the
cells of central nervous system, the cells of the most of peripheral tissues
and
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PCT/1B2011/002470
immune competent cells. This receptor via control of homeostasis of
intracellular calcium regulates intracellular signaling events leading to
activation of the corresponding transcription factors and transcription of a
whole gene family coding in particular the factors of resistance to infectious
agents and cytokines. In this regard, the ability of drugs to influence to the
efficiency of interaction of ligands with sigma-1 receptor indicates the
presence
of antiviral and immunomodulating components in the spectrum of its
pharmacological activity that allows to consider these preparations as
effective
ones for the treatment and prophylaxis of various infectious diseases.
During the test (to measure total binding) 20 pl of the complex preparation
anti-CD4+anti-IFN-y or 10 pl of anti-CD4 or 10 pl of anti-IFN-y were added to
the incubation medium. Thus, the quantity of ULD of anti-CD4+anti-IFN-y
transferred into the test basin when testing the complex preparation was
identical to that of anti-CD4 and ULD of anti-IFN-y tested as
monopreparations,
which allows for a comparison of the efficiency of the preparation to its
separate components. 20 pl and 10 pl of potentiated water were transferred
into the incubation medium.
Further, 160 pl (about 200pg of protein) of Jurkat cell line membranes
homogenate (human leukemic T-Iymphocyte line), and finally, 20 pl of tritium-
labeled radioligand [3H]pentazocine (15 nm) were transferred.
In order to measure non-specific binding, 20 pl of non-labeled ligand-
haloperidol (10 pM) were transferred in the incubation medium instead of the
preparations or potentiated water.
Radioactivity was measured using a scintillometer (Topcount, Packard)
and scintillation blend (Microscint 0, Packard) following the incubation
within
120 minutes at 22 C in 50 mM Tris-HCI buffer (pH = 7.4) and filtration using
fiberglass filters (GF/B, Packard). Specific binding (during the test or
control)
was calculated as a difference between total (during the test or control) and
non-specific binding.Results are represented as percentage of specific binding
inhibition in
control (distilled water was used as control) (Table 1).
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Table 1
% of radioligand specific of
Quantity binding in control radioligan
Test group per test Pt test 2" test Average d binding
basin inhibition
in control
anti-CD4+anti-
IFN-y 20 pl 50.8 49.1 49.9 50.1
anti-CD4 10 pl 74.0 76.2 75.1 24.9
anti-IFN-y 10 pl 158.9 149.8 154.3 -54.3
Potentiated
water 20 pl 98.1 75.8 86.9 13.1
Potentiated
water 10 pl 140.1 106.2 123.2 -23.2
Effect of the preparations and potentiated water on binding of standard
ligand [3H]pentazocine to human recombinant a 1 receptor
Note: ')/0 of specific binding in control = (specific binding during the test/
specific binding in control)* 100%;
A of specific binding inhibition in control = 100% - (specific binding
during the test/specific binding in control) * 100%).
The results reflecting inhibition above 50% represents significant effects
of the tested compounds; inhibition from 25% to 50% confirms mild to moderate
effects; inhibition less than 25% is considered to be insignificant effect of
the
tested compound and is within background level.
Therefore, this test model showed that the complex preparation of anti-
CD4+anti-IFN-y is more efficient than its separate components (anti-CD4 and
anti-IFN-y) in inhibiting the binding of standard radioligand [3H]pentazocine
to
human recombinant al receptor; anti-CD4, transferred into the test basin,
namely 10 pl, inhibit the binding of standard radioligand [3H]pentazocine to
human recombinant al receptor, but the effect intensity is inferior to that of
the
complex preparation of anti-CD4+anti-IFN-y; anti-IFN-y, transferred into the
test
well, namely 10 pl, had no effect on the binding of standard radioligand
[3H]pentazocine to human recombinant al receptor; potentiated water,
transferred into the test basin, namely 10 pl or 20 pl, had no effect on the
binding of standard radioligand [3H]pentazocine to human recombinant al
receptor.
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Example 2 (mononuclear cells; reverse transcriptase; mode "treatment")
List of acronyms:
= TCID50 stands for 50% Tissue Culture Infective Dose
Evaluation of antiretroviral activity of complex medication that contains
ultra-low doses of rabbit polyclonal antibodies to CD4 (mixture of homeopathic
dilutions C12+C30+C50) and ultra-low doses of rabbit polyclonal antibodies to
interferon gamma (mixture of homeopathic dilutions C12+C30+C50) in 1:1
ratio(hereinafter referred to as Complex drug) and components that form part
of
it (ultra-low doses of rabbit polyclonal antibodies to CD4 (mixture of
homeopathic dilutions C12+C30+C50) (hereinafter referred to as ULD AB to
CD4) and ultra-low doses of rabbit polyclonal antibodies to interferon gamma
(mixture of homeopathic dilutions C12+C30+C50 (hereinafter referred to as
ULD AB to IFN gamma)) was performed with use of mononuclear cells of
peripheral blood of a human being infected with in vitro strain HIV-1-LAI. As
comparative drug azidothymidine was used (Sigma ¨ AZ169-100mg, lot 107
K1578).
Mononuclear cells of peripheral blood of a human being were separated
from blood of healthy seronegative donor through centrifugation in density
gradient ficcol-gipaque. The cells were activated during 3 days with use of 1
mkg/ml phytohemagglutinin P and 5 ME/ml of recombinant interleukine-2 of a
human being in medium RPMI1640 (DIFCO) with 10% fetal calf serum
(complement was removed through heating during 45 minutes within
temperature of 56 C), 1% solution of antibiotics (PSN Gibco containing 50
pg/ml penicillin, 50 pg/ml streptomycin and 100 p/ml neomycin).
For evaluation of antiretroviral activity combination medications were
introduced to well 15-30 minutes after contamination of cells with strain HIV-
1-
LAI with dose of 100 TCID50 (50 mkl inoculums of strain HIV-1-LAI). On the 7th
day after infection of cells supernatant used for evaluation of influence of
medications to inhibition of HIV replication was selected.
Before introduction to well containing 150 pl of cell culture medications
were diluted with medium RPMI1640 (DIFCO) till final volume of 50 pl was
reached. ULD AB to CD4 and ULD AB to IFN gamma were diluted in medium
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RPMI1640 (DIFCO) in 8 times (degree of dilution 1/4). So quantity of ULD AB
to CD4 and ULD AB to IFN gamma being introduced to experimental well
during testing of complex drug similar to the quantity of ULD AB to CD4 and
ULD AB to IFN gamma tested as mono-component that allows to make
comparison of efficiency of complex drug with its separate components.
Azidothymidine was diluted with medium RPMI1640 (DIFCO) up to
concentration of 8 nM was achieved.
Efficiency medications was defined on inhibition of HIV replication that
was evaluated on enzymatic activity of HIV-reverse transcriptase in
supernatants of macrophages of peripheral blood of a human being with use of
HIV RI RetroSys production set INNOVAGEN (lot 10-059C). For calculation of
% of inhibition of HIV replication as control supernatant of cells was used to
which tested medications were not introduced (see Table 2).
Table 2.
Antiretroviral activity of medications with use of mononuclear cells of
peripheral blood of a human being infected in vitro with strain HIV-1-LAI
Degree of dilution Inhibition of enzymatic activity of
in media HIV-reverse transcriptase
Medication RPMI1640 ( /0 from control)
(DIFCO) 7th day
ULD AB to IFN gamma 1/8 0 5
ULD AB to CD4 1/8 67 22
Complex drug (ULD AB to
IFN gamma and ULD AB to 1/4 85 1
CD4 in 1: 1 ratio)
Azidothymidine (8 nM) 58 7
Thus, in conditions of this experimental model it is shown that
antiretroviral activity of complex drug exceeds antiretroviral activity of its
separate components (ULD AB to IFN gamma and ULD AB to CD4).
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Example 3 (macrophages; reverse transcriptase; mode "prophylaxis")
List of acronyms:
= TCID50 ¨ dose infecting 50% cells of tissue culture.
Evaluation of antiretroviral activity of complex medication that contains
ultra-low doses of rabbit polyclonal antibodies to CD4 (mixture of homeopathic
dilutions C12+C30+C50) and ultra-low doses of rabbit polyclonal antibodies to
interferon gamma (mixture of homeopathic dilutions C12+C30+C50) in 1:1
ratio(hereinafter referred to as Complex drug) and components that form part
of
it (ultra-low doses of rabbit polyclonal antibodies to CD4 (mixture of
homeopathic dilutions C12+C30+C50) (hereinafter referred to as ULD AB to
CD4) and ultra-low doses of rabbit polyclonal antibodies to interferon gamma
(mixture of homeopathic dilutions C12+C30+C50 (hereinafter referred to as
ULD AB to IFN gamma)) was performed with use of macrophages that were
received from mononuclear cells of peripheral blood of a human being infected
with in vitro strain HIV-1-LAI. As comparative drug azidothymidine was used
(Sigma ¨ AZ169-100mg, lot 107 K1578).
Macrophages of donor peripheral blood received from mononuclear cells
of human peripheral blood were isolated from blood of two healthy seronegative
donors through centrifugation in density gradient ficcol-gipaque. Mononuclear
cells of human peripheral blood were grown for 3 days in medium RPMI1640
(DIFCO) that was added with 10% fetal calf serum (complement was removed
through heating during 45 minutes within temperature of 56 C), 1% solution of
antibiotics (PSN Gibco containing 50 pg /ml penicillin, 50 pg/ml streptomycin
and 100 pg /ml neomycin), 15 ng/ml GM-CSF (granulocyte macrophagal
colony-stimulating factor). Then the cells were placed to culture plates
(150000
cells/well in 48-well plate), grown during 7 days together with 1 ng/ml GM-CSF
(granulocyte macrophagal colony-stimulating factor) and 10 ng/ml M-CSF
(macrophagal colony-stimulating factor) so that the cells could be completely
differentiated to macrophages.
For evaluation of antiretroviral activity combination medications were
introduced to well 24 hours before contamination of cells with strain HIV-1-
LAI
with dose of 1000 TCID50 (100 mkl inoculums of strain HIV-1-Ba-L) and on the
3rd, 7th, 1 oth, 1 4, 1 = I-.thday after contamination. On the 3rd, 7th, 10th,
14th, 17th
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day after infection of cells supernatant used for evaluation of influence of
medications to inhibition of HIV replication was selected.
Before introduction to well containing 750 pl of cell culture medications
were diluted with medium RPMI1640 (DIFCO) up to final volume of 250 pl was
reached. ULD AB to CD4 and ULD AB to IFN gamma were diluted in medium
RPMI1640 (DIFCO) in 8 times (degree of dilution 1/4). So quantity of ULD AB
to C04 and ULD AB to IFN gamma being introduced to experimental well
during testing of complex drug similar to the quantity of ULD AB to CD4 and
ULD AB to IFN gamma tested as mono-component that allows to make
comparison of efficiency of complex drug with its separate components.
Azidothymidine was diluted with medium RPMI1640 (DIFCO) till concentration
of 8 nM was achieved.
Efficiency medications was defined by inhibition of HIV replication that
was evaluated on enzymatic activity of HIV-reverse transcriptase in
supernatants of supernatant macrophages of peripheral blood of a human
being with use of HIV RI RetroSys production set INNOVAGEN (lot 10-059C).
For calculation of % of inhibition of HIV replication as control supernatant
of
cells was used to which tested medications or azidothymidine were not
introduced (see table 3 and 4).
Table 3.
Antiretroviral activity of medications with use of macrophages of human
peripheral blood (donor No. 1) infected in vitro with strain HIV-1-Ba-L
Degree of Inhibition of enzymatic activity of HIV-
dilution in reverse transcriptase ( /0 from control)
Medication media
RPMI1640 14th day 17th day 21st day
(DI FCO)
ULD AB to IFN 1/8 24 4 24 4 0 0
gamma
ULD AB to CD4 1/8 353 1 37 7 0 0
Complex drug (ULD
AB to IFN gamma and 1/4 69 1 74 9 37 3
ULD AB to CD4 in 1: 1
ratio)
Azidothymidine (8 nM) 97 1 97 0 98 2
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Table 4.
Antiretroviral activity of medications with use of macrophages of human
peripheral blood (donor No. 2) infected in vitro with strain HIV-1-Ba-L
Degree of Inhibition of enzymatic activity of HIV-
dilution in media reverse transcriptase (% from control)
Medication
RPMI1640
14th day 17th day 21st day
(DIFCO)
ULD AB to IFN 1/8 39 20 0 0 0 0
gamma
ULD AB to CD4 1/8 0 0 0 0 0 0
Complex drug
(ULD AB to IFN
gamma and ULD 1/4 50 5 42 4 30 6
AB to CD4 in 1:1
ratio)
Azidothymidine (8 82 2 54 1 41 1
nM)
So in conditions of this experimental model it is shown that:
1. Antiretroviral activity of complex medication exceeds antiretroviral
activity of its separate components (ULD AB to IFN gamma and ULD
AB to CD4).
2. Antiretroviral activity of complex medication is lasted during the whole
experiment period in contrast to antiretroviral activity of its separate
components (ULD AB to IFN gamma and ULD AB to CD4).
3. Only complex medication showed antiretroviral activity in in vitro
model of infected macrophages of human peripheral blood received
from different seronegative donors, which is the evidence of more
pronounced antiretroviral effect of complex medication in comparison
with its components (ULD AB to IFN gamma and ULD AB to CD4),
antiretroviral activity of which was registered in in vitro model of
infected macrophages of human peripheral blood received only from
one seronegative donor.
Example 4 (mononuclear cells; reverse transcriptase; therapy regimen)
List of abbreviations:
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= TCID50 stands for 50% Tissue Culture Infective Dose.
The assessment of antiretroviral activity of a complex product consisting
of ultra low-dose rabbit polyclonal antibodies to interferon alpha, ultra low-
dose
rabbit polyclonal antibodies to interferon gamma, ultra low-dose rabbit
polyclonal antibodies to CD4 and ultra low-dose rabbit polyclonal antibodies
to
CD8 as 1:1:1:1 ratio (a mixture of homoeopathic dilutions C12+C30+C50)
(hereinafter referred to as the Complex product), was carried out using human
peripheral blood mononuclear cells infected with the strain HIV-1LAI in vitro.
Azidothymidine (Sigma ¨ AZ169-100 mg, Lot 107 K1578) was used as a
comparator product.
Human peripheral blood mononuclear cells were isolated from blood of a
seronegative healthy donor by centrifugation on a Ficoll-Hypaque density
gradient. The cells were stimulated for 3 days with 1 pg/mL of
phytohemagglutinin P and 5 IU/mL of recombinant human interleukin-2 in
RPMI1640 (DIFCO) medium supplemented with 10% fetal calf serum (the
complement was removed by heating for 45 minutes at 56 C), 1% antibiotic
solution (PSN Gibco containing 50 pg/mL of penicillin, 50 pg/mL of
streptomycin and 100 pg/mL of neomycin).
In order to assess antiretroviral activity the products were placed in a
well 15-30 minutes after cells infection with the strain HIV-1- LAI at the
dose of
100 TCID50 (50 pL inoculum of the strain HIV-1-LAI). Supernatant fluids used
to assess the effect of products on the inhibition of HIV replication were
also
collected on day 7 after infection of cells.
Before placing in a well, which contained 150 pL of cell culture, the
complex product was diluted with RPMI1640 (DIFCO) medium at a 4-fold
dilution (at a 1/4 dilution) to a final volume of 50 pL. Azidothymidine was
diluted
with RPMI1640 (DIFCO) medium to yield a 8 nM concentration.
The products' efficiency was established by the inhibition of HIV
replication which was assessed by HIV-reverse transcriptase activity in the
supernatant fluid from human peripheral blood mononuclear cells using the HIV
RT RetroSys kit made by INNOVAGEN (Lot 10-059C). The supernatant fluid of
cells, to which test products or azidothymidine were not inoculated, was used
as control to calculate the percentage of inhibition of HIV replication (see
Table
5).
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Table 5.
Antiretroviral activity of the complex product using human peripheral
blood mononuclear cells infected with the strain HIV-1-LAI in vitro
Inhibition of HIV-reverse
Medium Dilution
transcriptase activity (`)/0 of
Product Ratio RPMI1640
control)
(DIEGO)
Day 7
Complex product (Ultra low-
dose antibodies to IFN-
alpha, Ultra low-dose
antibodies to IFN-gamma,
1/4 81 11
Ultra low-dose antibodies to
CD4 and Ultra low-dose
antibodies to CD8 as 1:1:1:1
ratio)
Azidothymidine (8 nM) 58 7
Thus, this experimental model demonstrated the antiretroviral activity of
the complex product comprising ultra low-dose rabbit polyclonal antibodies to
interferon alpha, ultra low-dose rabbit polyclonal antibodies to interferon
gamma, ultra low-dose rabbit polyclonal antibodies to CD4 and ultra low-dose
rabbit polyclonal antibodies to CD8 as 1:1:1:1 ratio (a mixture of
homoeopathic
dilutions C12+C30+C50).
Example 5 (mononuclear cells; nucleocapsid protein p24;
prevention and therapy regimen)
The assessment of antiretroviral activity of ultra low-dose of rabbit
polyclonal antibodies to interferon-alpha (a mixture of homoeopathic dilutions
C12+C30+C50), ultra low-dose of rabbit polyclonal antibodies to interferon-
gamma (a mixture of homoeopathic dilutions C12+C30+C50) (ULD IFN-y)),
ultra low-dose of rabbit polyclonal antibodies to C04 receptor (a mixture of
homoeopathic dilutions C12+C30+C50) and ultra low-dose of rabbit polyclonal
antibodies to CD8 receptor (a mixture of homoeopathic dilutions
C12+C30+C50) (ULD Ab IFN-a+ IFN-y+CD4+CD8)was carried out using human
peripheral blood mononuclear cells infected with the strain HIV-1LAI in vitro.
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Human peripheral blood mononuclear cells were isolated from blood of a
seronegative healthy donor by centrifugation on a Ficoll-Hypaque density
gradient. The cells were stimulated for 3 days with 1 pg/mL of
phytohemagglutinin P and 5 IU/mL of recombinant human interleukin-2.
In order to assess antiretroviral activity the products were placed in a
well containing 100 pL of activated mononuclears 24 hours before or 15 min
after cell infection with the strain HIV-1- LAI at the dose of 100 TCID50 (50
pL
inoculum of the strain HIV-1-LAI). Before adding to a well, ULD Ab IFN-a+ IFN-
y+CD4+CD8 (12.5 pL) or reference azidotimidine (1000 nM) were mixed with
RPMI1640 medium (DIFCO) to achive a final probe volume of 50 pL
The supernatant fluids were collected on day 7 after infection of cells.
The products' activity was measured by the inhibition of HIV replication which
was assessed by the level of core nucleocapsid protein p24 in the supernatant
fluid from human peripheral blood mononuclear cells using Retrotek Elisa kit.
It was shown that ULD Ab IFN-a+ IFN-y+CD4+CD8 inhibited HIV
replication by 94 6% when added to a well 24 hours before the infection, a
and by 46 13 % when added to a well 15 min after the infection of cells with
the strain HIV-1LAI. Azidotimidine at a dose of 1000 nM inhibited HIV
replication by 99 0 and 99 1 % added to a well 24 hours before and 15 min
after the infection of cells with the strain HIV-1LAI, respectively.
Thus, this experimental model demonstrated the antiretroviral activity of
ultra low-doses of rabbit polyclonal antibodies to ULD Ab IFN-a+ IFN-
y+CD4+CD8 (a mixture of homoeopathic dilutions C12+C30+C50.
Example 6
Investigation of efficiency of combined use of ultra-low doses of
antibodies to interferon alpha (mixture of homeopathic dilutions C12+C30+C50)
(hereinafter referred to as ULD AB to IFNalpha) and ultra-low doses of
antibodies to CD4 (mixture of homeopathic dilutions C12+C30+C50)
(hereinafter referred to as ULD AB to CD4) and ULD AB to IFNalpha and ULD
AB to CD4 separately in the context of influenza infection at mice-female of
the
line Balb/c was performed on the basis of FSBI "SRI of influenza" Ministry of
health of social development of Russia (Saint Petersburg) in two stages. At
the
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first stage efficiency of ULD AB to IFNalpha and ULD AB to CD4 was
investigated, at the second stage efficiency of combined use of ULD AB to
IFNalpha and ULD AB to CD4 (in 1:1 ratio) (hereinafter referred to as
combination medication) was investigated. Both during testing of combination
medication and during testing of ULD AB to IFNalpha and ULD AB to CD4
oseltamivir was used as comparative drug.
Infectious process was simulated through intranasal introduction of
influenza virus A/California/07/2009swl (H1N1) with a dose 1OLD50.
ULD AB to IFNalpha, ULD AB to CD4 and combination medication was
intragastrically introduced to mice (n=20 in each group) at 0.2 ml/mouse twice
a
day (daily dose 0.4 ml/mouse) during 5 days before infection and during 10
days after infection. Additionally ULD AB to IFNalpha, ULD AB to CD4 and
combination medication were added to drinking bowls of animals of
corresponding experimental groups (free access was allowed).
Reference drug oseltamivir was intragastrically introduced to mice (n=20)
twice a day with a dose of 10 mg/kg (daily dose 20 mg/kg) starting 1 hour
before infection. Oseltamivir was introduced during 5 days after infection.
During 4 days before infection and starting 6 days after infection distilled
water
at a dose of 0,2 ml/mouse twice a day (daily dose 0,4 ml/mouse) was
intragastrically introduced instead of oseltamivir to mice of this
experimental
group. Distilled water was intragastrically introduced to mice of control
group
(n=20) twice a day at a dose of 0,2 ml/mouse (daily dose 0,4 ml/mouse). During
the whole experiment period drinking bowls of animals of these two
experimental groups contained distilled water (free access was allowed).
Efficacy of medications was evaluated by survival rate of animals.
Results of study of antiviral activity of ULD AB to IFNalpha and ULD AB to CD4
(stage 1) see in Table 6, results of study of antiviral activity of
combination
medication (stage 2) see in Table 7. Statistical significance of differences
between experimental groups and control (distilled water) was calculated with
use of non-parametric chi-square criterion.
Table 6.
Antiviral activity of ULD AB to IFNalpha and ULD AB to CD4 in the model
of influenza infection at female Balb/c mice infected through intranasal
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introduction of influenza virus A/California/07/2009swl (H1N1) with a dose of
1OLD50 (10th day after infection).
Survival, % Difference between `)/0 of survival
in the group that received
No. Experimental
medication and % of survival in
group 1OLD50
the group that received distilled
water
1. ULD AB to
+5%
25
IFNalpha
2. ULD AB to CD4 30
+10%
3. Oseltamivir 80*
+60%
4. Distilled water 20
* - p < 0.05 vs control
Table 7.
Antiviral activity combination medication containing ULD AB to IFNalpha
and ULD AB to CD4 in the model of influenza infection at female Balb/c mice
infected through intranasal introduction
of influenza virus
A/California/07/2009swl (H1N1) with a dose of 1OLD50 (10th day after
infection).
Survival, % Difference between A of survival
in the group that received
Experimental
No.
medication and A of survival in
group 1OLD50
the group that received distilled
water
Combination
medication (ULD
1 AB to IFNalpha 30*
+25%
+ ULD AB to
CD4 in 1:1 ratio)
2 Oseltamivir 70*
+65%
3 Distilled water 5
* - p < 0.05 vs control.
It is shown that survival of mice infected with influenza
A/California/07/2009swl (H1N1) with a dose of 1OLD50 was higher at the stage
1 than at the stage 2: survival in the group that received distilled water was
20% and 5% respectively; survival in the group of comparative drug oseltamivir
was 80% and 70% respectively. It is the evidence of more expressed lethal
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effect induced through intranasal introduction of influenza virus
A/California/07/2009swl (H1N1) with a dose of 1OLD50, at the stage 2 of the
study.
However, combination medication increased in 25% survival of
experimental animals infected with influenza virus A/California/07/2009sw1
(H1N1) with a dose of 1OLD50 as compared with control. Whereas survival in
the group that received ULD AB to IFNalpha was only 5% higher than the
survival in control group, and survival rate in the group received ULD AB to
CD4 was only 10% higher than the survival in control group.
So as the result of performed study it was shown that combined use of
ULD AB to IFNalpha and ULD AB to CD4 (combination medication) provides
more pronounced antiviral effect than separate components, in spite of the
fact
that the dose of ULD AB to IFNalpha and ULD AB to CD4 as part of
combination medication is twice lower than the dose of ULD AB to IFNalpha
and ULD AB to CD4 tested as separate medications.
Example 7.
Investigation of efficiency of combined use of ultra-low doses of
antibodies to tumor necrosis factor alpha (mixture of homeopathic dilutions
C12+C30+C50) (hereinafter referred to as ULD Ab to TNFalpha) and ultra-low
doses of antibodies to CD4 (mixture of homeopathic dilutions C12+C30+C50)
(hereinafter referred to as ULD Ab to CD4) and ULD AB to TNFalpha and ULD
AB to CD4 separately in the context of influenza infection at mice-female of
the
line Balb/c was performed on the basis of FSBI "SRI of influenza" Ministry of
health of social development of Russia (Saint Petersburg) in two stages. At
the
first stage efficiency of ULD Ab to TNFalpha and ULD Ab to CD4 was
investigated, at the second stage efficiency of combined use of ULD AB to
TNFalpha and ULD Ab to CD4 (in 1:1 ratio) (hereinafter referred to as
combination medication) was investigated. Both during testing of combination
medication and during testing of ULD AB to TNFalpha and ULD AB to CD4
oseltamivir was used as comparative drug.
Infectious process was simulated through intranasal introduction of
influenza virus A/California/07/2009sw1 (H1N1) with a dose 1OLD50.
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45
ULD Ab to TNFalpha, ULD Ab to CD4 and combination medication was
intragastrically introduced to mice (n=20 in each group) at 0.2 ml/mouse twice
a
day (daily dose 0.4 ml/mouse) during 5 days before infection and during 10
days after infection. Additionally ULD AB to TNFalpha, ULD AB to CD4 and
combination medication were added to drinking bowls of animals of
corresponding experimental groups (free access was allowed).
Reference drug oseltamivir was intragastrically introduced to mice (n=20)
twice a day with a dose of 10 mg/kg (daily dose 20 mg/kg) starting 1 hour
before infection. Oseltamivir was introduced during 5 days after infection.
During 4 days before infection and starting 6 days after infection distilled
water
at a dose of 0,2 ml/mouse twice a day (daily dose 0,4 ml/mouse) was
intragastrically introduced instead of oseltamivir to mice of this
experimental
group. Distilled water was intragastrically introduced to mice of control
group
(n=20) twice a day at a dose of 0,2 ml/mouse (daily dose 0,4 ml/mouse). During
the whole experiment period drinking bowls of animals of these two
experimental groups contained distilled water (free access was allowed).
Efficacy of medications was evaluated by survival rate of animals.
Results of study of antiviral activity of ULD Ab to TNFalpha and ULD Ab to CD4
(stage 1) see in table 8, results of study of antiviral activity of
combination
medication (stage 2) see in table 9. Statistical significance of differences
between experimental groups and control (distilled water) was calculated with
use of non-parametric chi-square criterion.
Table 8.
Antiviral activity of ULD Ab to TNFalpha and ULD Ab to CD4 in the model
of influenza infection at female Balb/c mice infected through intranasal
introduction of influenza virus A/California/07/2009swl (H1N1) with a dose of
1OLD50 (10th day after infection).
Survival, % Difference between % of survival in
Experimental the group that received
No. medication and % of survival in
group 1OLD50
the group that received distilled
water
1. ULD Ab to 25 +5%
TNFalpha
2. ULD Ab to CD4 30 +10%
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3. Oseltamivir 80* +60%
4. Distilled water 20
* - p < 0.05 vs control
Table 9.
Antiviral activity combination medication containing ULD Ab to TNFalpha
and ULD Ab to CD4 in the model of influenza infection at female Balb/c mice
infected through intranasal introduction of influenza
virus
A/California/07/2009sw1 (H1N1) with a dose of 1OLD50 (10th day after
infection).
Survival, % Difference between % of survival
in the group that received
No Experimental medication and % of
survival in
group 1OLD50 the group that received distilled
water
Combination
medication (ULD
1. Ab to TNFalpha + 30* +25%
ULD Ab to CD4 in
1:1 ratio)
2. Oseltamivir 70* +65%
3. Distilled water 5
* - p < 0.05 vs control.
It is shown that survival of mice infected with influenza
A/California/07/2009sw1 (H1N1) with a dose of 1OLD50 was higher at the stage
1 than at the stage 2: survival in the group that received distilled water was
20% and 5% respectively; survival in the group of comparative drug oseltamivir
was 80% and 70% respectively. It is the evidence of more expressed lethal
effect induced through intranasal introduction of influenza virus
A/Califoinia/07/2009sw1 (H1N1) with a dose of 1OLD50, at the stage 2 of the
study.
However, combination medication increased in 25% survival of
experimental animals infected with influenza virus A/California/07/2009swl
(H1N1) with a dose of 1OLD50 as compared with control. Whereas survival in
the group that received ULD Ab to TNFalpha was only 5% higher than the
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survival in control group, and survival rate in the group received ULD Ab to
CD4 was only 10% higher than the survival in control group.
So as the result of performed study it was shown that combined use of
ULD Ab to TNFalpha and ULD Ab to CD4 (combination medication) provides
more pronounced antiviral effect than separate components, in spite of the
fact
that the dose of ULD Ab to TNFalpha and ULD AB to CD4 as part of
combination medication is twice lower than the dose of ULD Ab to TNFalpha
and ULD AB to CD4 tested as separate medications.
Example 8.
Pharmaceutical composition (tablets) containing activated potentiated form
of ultra-low doses (ULD) antibodies to interferon gamma (Ab IFNgamma),
antibodies to CD4 (Ab CD4), antibodies to histamine (Ab His), impregnated
onto lactose in the form of aqueous alcoholic solution of mixture of
homeopathic dilutions C12, C30, C200 of each (Ab IFNgamma + Ab CD4 +Ab
to His) was used in the study.
In the double blind placebo-controlled study being conducted at present
both men and women aged 18-60 years with viral URI's accompanied with
intoxication, catarrh signs are enrolled. Patients with body temperature 37.8
C
and higher (provided that the temperature is registered at the onset of the
disease), with the duration of the disease not exceeding 48 hours by the time
of
the therapy onset, not having severe complications were included in the study.
Express test to detect influenza virus antigens was conducted. Patients with
positive test results were not included in the study. Prior to the beginning
of all
the procedures the patients sign Informed consent to participate in the study.
The patients were given diaries, in which body temperature twice daily,
concomitant therapy, etc were registered. The patients receive Ab IFNgamma +
Ab CD4 +Ab His or placebo at a dose of 8 tablets daily on Day 1 and at a dose
of 3 tablets daily on Days 2-5. If required the patients were allowed to take
antipyretics. The intake of antiviral, immunomodulating, antihistamines and
antibiotics is not allowed. Prior to start of therapy and at the last visit
blood and
urine samples are collected for assessment of laboratory parameters aimed at
= monitoring the safety of the conducted therapy. The overall therapy
duration is
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days, the duration of follow-up observation period is 2 days. Thus the
duration of each patient's participation in the study is 7 days.
Time to reducing body temperature down to 37.0 C and lower was
considered as the therapy efficacy criterion; besides the number of
antipyretics
5 intakes was compared.
By the time when the analysis was conducted 78 patients finished the
therapy (40 patients received Ab IFNgamma + Ab CD4 +Ab His, 38 patients
received placebo). The proportion of patients with the body temperature
reduced down to 37.0 C and lower are represented on Figure 1. The Figure
shows that Ab IFNgamma + Ab CD4 +Ab His administration by the end of Day
2 from onset of the therapy resulted in 17.4% reduction in the patients' body
temperature as compared to placebo group (p<0.05). At that the number of
antipyretics intakes in the groups was significantly lower than in anti- Ab
IFNgamma + Ab CD4 +Ab His group (3.5 0.25 intake of antipyretics by the end
of Day 2 of the treatment vs 3.9 0.32 in placebo group, p<0.05). Ab IFNgamma
+ Ab CD4 +Ab His superiority over placebo group was seen as early as in the
morning of Day 2 of the treatment and maintained all over therapy period.
Data of all 78 patients involved in the study and having finished the
treatment in due terms were included in safety analysis; no discharge of
patients were registered. Good drug tolerability was seen during the whole
observation period. No adverse events related to Ab IFNgamma + Ab CD4 +Ab
His administration was registered. Blood tests conducted at the onset of the
treatment and at the end of it did not show any pathologic deviations from
norm. Urine analysis made on Day 1 and the last day of the study also did not
reveal pathological changes in all patients.
When comparing the data with the results obtained during double blind
placebo controlled randomized study of clinical efficacy and safety of Ab
IFNgamma administration in influenza and other viral URI's conducted in 2005
(Influenza RI, RAMS, Saint- Petersburg, 2005) it was revealed that Ab
IFNgamma + Ab CD4 +Ab His reduces body temperature more effectively than
Ab IFNgamma (Figure1, Table 10 and Table 11).
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Table 10. Proportion of patients with body temperature reduced down to
37,0 C and lower on the background Ab IFNgamma + Ab CD4 +Ab His
/placebo administration
Day Day Day Day Day Day Day
1, 1 2, 2 3, 3 4,
morn even morn even morn even morn
ing ing ing ing ing ing ing
Total number of
+ 40 40 40 40 40 40 40
E patients
c.) The number of
2 z patients with
LL
.0 - normal
19 20 24 28 27 30 31
< temperature
+<
Proportion of
(.9
patients with
12 < 7r normal
< + c temperature , %
47.5 50.0 60.0 70.0 67.5 75.0 77.5
Total number of
38 38 38 38 38 38 38
patients
The number of
co patients with
ii normal
18 17 19 20 23 24 27
temperature
0
.0 Proportion of
patients with
normal
47.4 44.7 50.0 52.6 60.5 63.2 71.1
temperature, %
o Total number of
t=-) 30 30 30 30 30 30 30
ii patients
The number of
patients with
normal
0 3 14 12 18 19 25
temperature
cn
Proportion of
u_ patients with
.o normal
0 10.0 46.7 40.0 60.0 63.3 83.3
temperature , %
Total number of
30 30 30 30 30 30 30
patients
The number of
patients with
II
normal
0 0 10 7 16 15 28
temperature
.o Proportion of
4:u patients with
ca normal
6". 0 0 33.3 23.3 53.3 50.0 93.3
temperature , %
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50
Table 10 ¨ Continue
Day Day Day Day Day Day 7
4 5, 5 6, 6 ,
eveni morni eveni morni eveni morni
ng ng ng ng ng ng
Total number of
co 40 40 40
40 40 40
+ E patients
E ci The number of
O g E
patients with
oz.
u. < normal
33 36 38 40 40 40
temperature
< -12
+ < ".CI Proportion of
3 = 0 patients with
normal
temperature , % 82.5 90.0 95.0 100.0
100.0 100.0
<< 4.
Total number of
38 38 38 38 38 38
patients
The number of
patients with
03
0.) normal
II 24 29 30
33 37 38
temperature
C
o Proportion of
.c)
0) patients with
c.) normal
cci 63.2 76.3 78.9 86.8 97.4 100.0
E. temperature, %
Total number of
4,- 30 30 30
30 30 30
03 patients
E
The number of
E
03 patients with
Izi
z normal
29 29 30 29 30 29
u_
¨ temperature
Proportion of
c, patients with
ce,
normal
96.7 96.7 100 96.7 100 96.7
< c temperature, ')/c,
Total number of
30 30 30 30 30 30
_ patients
The number of
c,
cr) patients with
II normal
c 28 28 30
30 30 30
- temperature
O Proportion of
.0
w patients with
0
co normal
ii93.3 93.3 100 100 100 100
temperature, %
* According to the results of double blind placebo controlled randomized
study of clinical efficacy and safety of Ab IFNgamma administration in
influenza
and other viral URI's conducted in 2005 (Influenza RI, RAMS, Saint-
Petersburg, 2005)
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JA
Table 11. Mean values of body temperature in patients depending on
treatment groups, C, M SD
Day 1 , Day 1 Day 2 , Day 2 Day 3 , Day 3 Day 4 ,
mornin evenin mornin evenin mornin evenin mornin
9 9 9 9 g 9
+
.0 co < o
< E
E
co 37.5 0 37.7 37.2 37.1 36.8 0 36.8 36.7
co u)
z .54
0.56 0.67 0.53 .43 0.49 0.31
u_ <
-0
a) co 37.6 0 37.6 37.2 37.1 36.9 0 36.9 36.8
y_ .71 0.63 0.48 0.49 .41 0.36 0.49
.0 2
< E
Q 38.1 0 38.0 37.4 37.3 37.1 0 37.0 36.8
cv)
z
c .62 0.58 0.80 0.61 .50 0.47 0.35
.0 0
(1) cv) 38.0 0 38.0 37.4 37.4 37.0 0 37.0 36.8
co c
cT_ .48 0.50 0.60 0.47 .37 0.42 0.23
Table 11 Continue
Day 4 Day 5 , Day 5 Day 6 , Day 6 Day 7 ,
evening morning evening morning evening morning
+
.0 ( < o
< E
E c
36.6 0. 36.6 0. 36.6 0. 36.6 36.6 36.6
= (...)
z 33
u_ < 25 23 0.22 0.15 0.18
a) 36.7 0. 36.6 0. 36.6 0. 36.6 36.5 36.5
c.)
co ce)
32 21 0.28 0.18 0.18
cT_ 37
co
= E
< E
36.6 0. 36.6 0. 36.5 0. 36.6 36.6 36.6
a) co
z
u- c 32 21 26 0.21 0.26 0.26
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52
0
36.6 0. 36.6 0. 36.6 5. 36.6 36.5 36.6
co cn 34 28 42 0.21 0.24 0.18
* According to the results of double blind placebo controlled randomized
study of clinical efficacy and safety of Ab IFNgamma administration in
influenza and other viral URI's (Influenza RI, RAMS, Saint- Petersburg,
2005)
Example 9.
Pharmaceutical composition (tablets) containing activated potentiated
forms of ultra-low doses (ULD) antibodies to interferon ¨ gamma (Ab
IFNgamma), antibodies to CD4 (Ab to CD4), antibodies to histamine (Ab to
His), impregnated onto lactose in the form of aqueous alcoholic mixture of
homeopathic dilutions C12, C30, C200 of each (Ab IFNgamma + Ab CD4 +Ab
His) was used in the study.
In the open- label comparative controlled clinical study of Ab IFNgamma +
Ab CD4 +Ab His and Tamiflu0 (F.Hoffmann-La Roche Ltd ¨ Switzerland,
Oseltamivir) study being conducted at present both men and women aged 18-
60 years with influenza accompanied by intoxication, catarrh signs are
enrolled.
Patients with body temperature 37.8 C and higher (provided that the
temperature is registered at the onset of the disease), with the duration of
the
disease not exceeding 48 hours by the time of the therapy onset, not having
severe complications were included in the study. Express test to detect
influenza virus antigens was conducted. Patients with positive test results
were
included in the study. Prior to the beginning of all the procedures the
patients
sign Informed consent to participate in the study. The patients were given
diaries, in which body temperature twice daily, concomitant therapy, etc were
registered. The patients receive Ab IFNgamma + Ab CD4 +Ab His at a dose of
8 tablets daily on Day 1 and at a dose of 3 tablets daily on Days 2-5 or or
Tamiflu at a dose of 75 mg 2 TID according to patient's information leaflet.
If
required the patients were allowed to take antipyretics. The intake of
antiviral,
immunomodulating, antihistamines and antibiotics is not allowed. Prior to
start
of therapy and at the last visit blood and urine samples are collected for
assessment of laboratory parameters aimed at monitoring the safety of the
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conducted therapy. The overall therapy duration is 5 days, the duration of
follow-up observation period is 2 days. Thus the duration of each patient's
participation in the study is 7 days.
Time to reducing body temperature down to 37.0 C and lower was
considered as the therapy efficacy criterion; besides the number of
antipyretics
intakes was compared.
By the time when the analysis was conducted 17 patients have finished
the therapy (6 patients in Ab IFNgamma + Ab CD4 +Ab His group and 11
patient in Oseltamivir group).
Proportions of patients with the body temperature reduced down to 37.0 C
and lower in the groups did not significantly differ in the course of therapy.
As
early as by Day 4 of the treatment patients of both groups practically
recovered
(see Figure 2). As early as by Day 2 of the treatment in 1/3 of patients of
both
groups normalization of body temperature was registered. The difference in
mean number of antipyretic intakes also was not significant and by the morning
of Day 4 of the therapy was 7.6 0.8 in the group receiving Ab IFNgamma + Ab
CD4 +Ab His and 7.4 0.90 in Oseltamivir group respectively.
Data of all 17 patients involved in the study and having terminated the
treatment in due terms were included in safety analysis; no discharge of
patients were registered. Good drug tolerability was seen during the whole
observation period. No adverse events related to Ab IFNgamma + Ab CD4 +Ab
His administration was registered. Blood tests conducted at the onset of the
treatment and at the end of it did not show any pathologic deviations from
norm. Urine analysis made on Day 1 and the last day of the study also did not
reveal pathology in all patients.
When comparing the data with the results obtained during double blind
placebo controlled randomized study of clinical efficacy and safety of Ab
IFNgamma administration in influenza and other viral URI's conducted in 2005
(Influenza RI, RAMS, Saint- Petersburg, 2005) it was revealed that Ab
IFNgamma + Ab CD4 +Ab His reduces body temperature more effectively than
Ab IFNgamma (Figure 2, Table 12 and Table 13).
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Table 12. Proportion of patients with body temperature reduced to 37.0
C and lower values on the background of Ab IFNgamma + Ab CD4 +Ab His
/Oseltamivir administration
Morni Eveni Morni Eveni Morni Eveni Morni Eveni
ng ng, ng ng,
ng ng, ng
ng, =
Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4
,o= Total number of
o patients
6 6 6 6 6 6 6 6
The number of
< patients
with
+ normal
co temperature
Ec
0 0
2 3 4 4
6 5
E
co c Proportion
of
= -
z 2 patients
with
normal temperature
^ < %
100.
= +
0 0 33.3 50.0 66.7 66.7 0
83.3
Total number of
patients
11 11 11 11
11 11 11
10
The number of
patients with
normal
temperature
0 1 4 5 5 6 10 8
'5 Proportion
of
E patients
with
g. normal temperature
Cl ) to
0 9.1 36.4 45.5 45.5 54.5 90.9 80.0
= Total number of
patients
30 30 30 30 30 30 30 30
The number of
patients with
co normal
2' temperature
0 3 14_ 12 18 19 25 29
u_ Proportion
of
co patients with
.0 I, normal temperature
< c , %
0 10.0 46.7_ 40.0 60.0 63.3 83.3 96.7
Total number of
patients
30 30 30 30 30 30 30 30
The number of
0 patients
with
ii normal
C
temperature
0 0 10 7 16 15 28 28
lo Proportion
of
a) patients
with
as normal temperature
0 ,
0 0 33.3 23.3 53.3 50.0
93.3 93.3
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Table 12. Continue
Mornin Evenin Mornin Evenin Mornin Evenin Mornin
g Day g, Day g Day g, Day g Day g, Day g Day
5 5 6 6 7 7 8
.1 Total number of
0 patients 6 6, 5 5 4 4 N/A
.0 The number of
patients with
normal
E (0., temperature 6 6 5 5 4 4 N/A
g Proportion of
cn cr; patients with
z
= ¨ normal temperature
¨JD , %
< + 100.0 100.0 100.0 100.0 100.0 100.0 N/A
Total number of
patients
10 10 9 7 5 4 3
The number of
patients with
normal
temperature
9 9 9 7 5 4 3
- Proportion of
3 patients with
E normal temperature
8 90.0 90.0 100.0 100.0 100.0 100.0 100.0
Total number of
Q patients
30 30 30 30 30 30 30
The number of
(ti patients with
E normal
E temperature 29 30 29 30 29 30 30
co
Proportion of
L.L.. patients with
.0 normal temperature
96.7 100 96.7, 100 96.7 100 100
Total number of
patients 30 30 30 30 30 30 30
The number of
c, patients with
cf)
II normal
. temperature 28 30 30 30 30 30 30
so Proportion of
.0
.a> patients with
õc0 normal temperature
o.. 0/0 93.3 100 100 100 100 100 100
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56
* According to the results of double blind placebo controlled randomized
study of clinical efficacy and safety of Ab IFNgamma administration in
influenza
and other viral URI's (Influenza RI, RAMS, Saint- Petersburg, 2005)
Table 13.Mean values of body temperature in patients depending on
treatment groups, C, M SD
Morni Eveni Morni Eveni Morni Eveni Morni Eveni
ng ng, ng ng, ng ng, ng ng,
Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Day 4
cis
E _
E
crs cy) c 38.5 38.1 37.2 37.2 36.5 36.8 36.5 36.6
cnz a
z u_ v; 0.49. 0.62 1.01 0.67 0.61 0.47 0.37 0.46
<
<
< +
5
=E
38.1 37.3 37.3 36.9 36.9 36.7 36.8 36.5
0.82 0.71 0.72 0.53 0.47 0.46 0.37 0.38
c
38.1 38.0 37.4 37.3 37.1 37.0 36.8 36.6
0.62 0.58 0.80 0.61 0.50 0.47 0.35 0.32
z
õ
< = c
00
cf, 38.0 38.0 37.4 37.4 37.0 37.0 36.8 36.6
ii
co c 0.48 0.50 0.60 0.47 0.37 0.42 0.23 0.34
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57
Table 13.Continue
Mornin Evenin Mornin Evenin Mornin Evenin Mornin
g Day g, Day g Day g, Day g Day g, Day g Day
5 5 6 6 7 7 8
`J
E <
E
2.8 36.5 0. 36.6 0. 36.4 0. 36.5 0. 36.4 0. 36.5 0.
z u_ (..) 0 26 28 35 20 26
22 ND
.0 <
<
< +
:=5
E 36.7 0. 36.5 0. 36.6 0. 36.3 0. 36.5 0. 36.6 0. 36.5 0.
03 7
25 21 21 19 10 12 14
TD. c
36.6 0. 36.5 0. 36.6 0. 36.6 0. 36.6 0. 36.5 0. 36.5 0.
21 26 21 26 26 27 23
0)0
n
< ¨ c
cp 36.6 0. 36.6 5. 36.6 0. 36.5 0. 36.6 0. 36.5 0. 36.4 0,
c 28 42 21 24 18 19
21
* According to the results of double blind placebo controlled randomized
study of clinical efficacy and safety of Ab IFNgamma administration in
influenza
and other viral URI's (Influenza RI, RAMS, Saint- Petersburg, 2005)
Example 10.
Pharmaceutical composition (tablets) containing activated potentiated
forms of ultra-low doses (ULD) antibodies to interferon ¨ gamma (Ab
IFNgamma), antibodies to CD4 (Ab to CD4), antibodies to histamine (Ab to
His), impregnated onto lactose in the form of aqueous alcoholic mixture of
homeopathic dilutions C12, C30, C200 of each (Ab IFNgamma + Ab CD4 +Ab
His) was used in the study.
In the present double blind placebo controlled study of efficacy and
safety of Ab IFNgamma + Ab CD4 +Ab His in viral URI's (Example 8) and in the
present open-label comparative study of Ab IFNgamma + Ab CD4 +Ab His
efficacy and safety in influenza (Example 9) the number of complications
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58
including bacterial ones (bacterial pneumonia, tracheitis, otitis,
glomerulonephritis, etc) developed on the background of acute infectious
process were assessed in addition.
If the body defense system works properly infectious process can be
arrested or localized, thus does not lead to the development of evident
clinical
symptoms, i.e. adequate defense reaction causes quick infectious agent
inactivation, restoration of the body impaired functions and the recovery.
Different situation can be seen in the subjects highly sensitive to infectious
agent and lacking the proper mechanism of specific and non- specific defense
(immunocompromized , patients). In such cases increasingly replicated
infectious agents and products of their interaction with epithelial and immune
cells as well as damaged cells penetrate into blood causing the development of
severe disease course, development of complications and potential poor
outcome.
The use of Ab IFNgamma + Ab CD4 +Ab His both in influenza and viral
URI's caused considerable reduction in the frequency of bacterial
complications
as compared to placebo (Table 14) and therefore to reduction in antibacterial
therapy. It seems that the drug inhibits the development of secondary immune
deficit at the stage of recovery exerting immunomodulating effect and
enhancing the body natural defense. The ability of Ab IFNgamma + Ab CD4
+Ab His to reduce the frequency of bacterial complications development
exceeded that of Ab IFNgamma.
Table 14. The frequency of bacterial complications
Bacterial complications
Drug Number of Otitis Tracheiti Pneumon Total
patients n/ % s ia n/ %
n / (1/0 n / %
Ab IFNgamma +
Ab CD4 +Ab His 40 0 / 0 1 / 2.5 0 / 0 1 / 2.5
(viral URI's)
Placebo (viral 38 3 / 7.9 7 / 18.4 0 / 0 10 / 26.3
URI's)
Ab IFNgamma +
Ab CD4 +Ab His 6 0 / 0 0 / 0 0 / 0 0 / 0
(influenza)
OseItamivir 11 0 / 0 2 / 18.2 1 / 9.1 3 / 27.2
(influenza )
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59
Ab IFNgamma
(influenza and viral 30 1 / 3.3 2 / 6.7 0 / 0 3 / 10.0
URI's )*
Placebo (influenza 30 4 / 13.3 5 / 16.7 0 / 0 9 / 30
and viral URI's)*
* According to the results of double blind placebo controlled randomized
study of clinical efficacy and safety of Ab IFNgamma administration in
influenza
and other viral URI's (Influenza RI, RAMS, Saint- Petersburg, 2005)
Example 11.
To study the activity of pharmaceutical compositions for the treatment of
patients of the group No.1 tablets 300 mg impregnated onto pharmaceutical
composition containing aqueous-alcoholic solutions (6 mg/tablet) of activated-
potentiated forms of rabbit polyclonal affinity purified antibodies to human
interferon gamma (anti-IFN-y) and CD4 (anti-CD4) in ultra-low doses (ULD)
obtained by means of ultra dilution of initial matrix solution in 10012, 1003
,
1005 times equal to mixture of centesimal homeopathic dilutions C12, C30,
C50 were used; for treatment of patients of group No. 2 300 mg impregnated
onto pharmaceutical composition containing aqueous-alcoholic solutions (6
mg/tablet) of activated-potentiated forms of rabbit polyclonal affinity
purified
antibodies to human interferon gamma (anti-IFN-y) and CD4 (anti-CD4) and
histamine (anti-His) in ultra-low doses (ULD) obtained by means of ultra
dilution
of initial matrix solution in 10012, 1003 , 1005 times equal to mixture of
centesimal homeopathic dilutions C12, C30, C50 were used; for treatment of
patients of group No. 3 tablets 300 mg impregnated onto pharmaceutical
composition containing aqueous-alcoholic solutions (3 mg/tablet) of activated-
potentiated forms of rabbit polyclonal affinity purified antibodies to human
interferon gamma (anti-IFN-y) in ultra-low doses (ULD) obtained by means of
ultra dilution of initial matrix solution in 10012, 1003 , 1005 times equal
to
mixture of centesimal homeopathic dilutions C12, C30, C50 were used.
Antiretroviral activity of pharmaceutical compositions ULD anti-IFN- y +
anti-CD4 and ULD anti-IFN- y + anti-CD4 + anti-His has been evaluated in the
course of the open-label comparative clinical trial with participation of the
human immunodeficiency virus (HIV) infected patients at Local Centre for
Prevention and Fight Against AIDS and Infectious Diseases. The study
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included 97 patients (65 men and 32 women) aged 18 - 48 years old, with viral
load of HIV-1 RNA 150 copies/ml in blood plasma and CD-4 lymphocyte
counts 250 cells/pi (or n,25x109/1). Thirty four out of 97 study participants
were treatment naïve patients. Sixty three out of 97 patients have been
receiving antiretroviral therapy (ART) for one or two years. Patients with
liver
cirrhosis, viral hepatitis C, severe concomitant diseases in exacerbation
period,
pregnant women, as well as ones taking narcotic substances intravenously
were not included in the study. The trial was carried out during autumn winter
period when seasonal rise in influenza and acute respiratory viral infection
is
common.
Seventy five study participants were randomized into three groups
prescribed either the study pharmaceutical compositions (groups No 1 and No
2) or reference pharmaceutical composition (group No 3) in a regimen
corresponding to ARVI prophylaxis ¨ 1 tablet once a day for 6 weeks:
patients of group No 1 (n=25) were prescribed with ULD anti-IFN-
y + anti-CD4 (subgroup 1A: treatment naïve patients, n=12) or prescribed with
ULD anti-IN- y + anti-CD4 + ART (subgroup 1B, n=13);
= patients of group No 2 (n=23) were prescribed with ULD anti-IFN-
y + anti-CD4 + anti-His (subgroup 2A: treatment naïve patients, n=11) or
prescribed with ULD anti-IFN- y + anti-CD4 + anti-His + ART (subgroup 2B,
n=12);
= patients of group No 3 (n=27) were prescribed with ULD anti-IFN-
y (subgroup 3A: treatment naïve patients, n=11) or prescribed with ULD anti-
IFN- y + ART (subgroup 3B, n=16).
The control group (group No 4, n=22) included patients who continued
receiving ART alone in accordance with the earlier prescription (ART group).
At a baseline and after 6-week therapy viral load, CD4 ii CD8
lymphocytes counts, CD4/CD8 immunoregulatory index were assayed in all the
patients. To detect HIV-1 RNA copies in blood plasma the COBAS AMPLICOR
HIV-1 MONITOR Kit (version 1,5 for automatic PCR-analyzer COBAS
AMPLICOR, Roche, Switzerland) were used. Phenotyping of peripheral blood
circulating lymphocytes was carried out on flow cytofluorometer FACSCount
(Becton Dickinson, USA) using FACSCount Reagent Kit, which contain FITC
PE fluorochrome-labeled antibodies to CD3, CD4, CD8.
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Date on viral load (the number of copies of HCV RNA) presented in the
table 15 as median (Me) and the range between first and third quartiles [Q1-
Q3]. The study results indicate that 6-week treatment with ULD anti-IFN- y +
anti-CD4 decreased the number of RNA HIV-1 copies in 58% treatment naïve
patients (in 7 out of 12 people of 1A subgroup), the average viral load
decrease
was 16.9%. Combination of ULD anti-IFN- y + anti-CD4 and ART showed
comparable efficacy, the number of HIV-1 RNA copies decreased in 62% of
patients (in 8 out of 13 people in 1B subgroup), and the average viral load
decrease from the baseline was 18.2%. Similar results were obtained in
patients received ULD anti-IFN- y + anti-CD4 + anti-His: antiviral activity
was
registered in 55% HIV-infected treatment naïve patients (in 6 out of 11 people
in 2A subgroup) and in 67% of patients receiving combination of ULD anti-IFN-
y + anti-CD4 + anti-His and ART (in 8 out of 12 people in 2B subgroup); the
average viral load decrease was 17.3% and 18.9% respectively. Antiretroviral
activity observed in the first two groups was somewhat higher compared with
the treatment outcome in control group. ULD anti-IFN- y monotherapy for 6
weeks decreased the number of HIV-1 RNA copies in 36% treatment naïve
patients (in 4 out of 11 people in 3A subgroup), the average viral load
decrease
was 9.5%. The combination of ULD anti-IFN- y and ART improved the efficacy
of therapy: the viral load decrease was registered in 50% of patients (in 8
out of
16 people in 3B subgroup), the average viral load decrease was 14.2%. In
patients taking only ART (group No 4) the decrease in viral load were detected
in 32% of patients (in 7 out of 22 patients) and an average viral load
decrease
13.3%.An assessment of circulating lymphocytes subpopulations during the
study (Table 16) revealed more pronounced as compared to the control group
increase in number of CD4 lymphocytes after 6-week therapy in ULD anti-IFN-
y + anti-CD4, ULD anti-IFN- y + anti-CD4 + anti-His and ULD anti-IFN-y as a
monotherapy in treatment naïve patients (groups 1A, 2A and 3A) or in
combination with ART (subgroups 1B, 2B in 3B). The number of CD8
lymphocytes after 6-week therapy (without or in combination with ART)
remained unchanged in all study groups. The positive dynamics in CD4-
lymphocytes count in the course of the treatment resulted in increase in
CD4/CD8 immunoregulatory index, which was most significant in the subgroups
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of patients taking ULD anti-IFN- y + anti-CD4 and ULD anti-IFN- y + anti-CD4 +
anti-His (without or in combination with ART, i.e. groups 1 and 2) and ULD
anti-
IFN- y +ART (subgroup 3B).
No drugs-related adverse events were registered during the study, which
evidences of their good tolerance. Absence of pathological variations in blood
and urine analysis including markers of renal and hepatic insufficiency
confirmed safety of the treatment.
Thus, the present study demonstrated antiretroviral activity of ULD anti-
IFN- y + anti-CD4 and ULD anti-IFN- y + anti-CD4 + anti-His pharmaceutical
compositions, possibly mediated by the change in functional activity of CD4
receptors, which blocks HIV penetration into the cells, and also suppresses
HIV
replication inside the cell due to activation of transcription of mRNA of
antiviral
proteins. It was shown that the viral load decrease at the end 6-week course
of
ULD anti-IFN- y + anti-CD4 and ULD anti-IFN- y + anti-CD4 + anti-His in the
dose of 1 tablet a day was more pronounced compared to that of 6-week
treatment with ULD anti-IFN- y in the same dose or in .patients continued
receiving ART alone in accordance with the earlier prescription. The
combination of ULD anti-IFN- y + anti-CD4, ULD anti-IFN- y + anti-CD4 + anti-
His or ULD anti-IFN- y medication with ART somewhat increases the antiviral
activity of the latter, which was revealed as the decrease of average viral
load
after 6 weeks in a larger proportion of patients.
The influence of ULD anti-IFN- y + anti-CD4 and ULD anti-IFN- y + anti-
CD4 + anti-His on CD4/CD8 lymphocytes ratio in HIV-infected patients (due to
a decrease in the number of CD4 cells) was shown, which was most evident
when combined with ART,. Taking into consideration a simultaneous viral load
decrease in patients taking ULD anti-IFN- y + anti-CD4 and ULD anti-IFN- y +
anti-CD4 + anti-His, one can assume that the increase in the number of CD4
cells is associated with population recruitment at the expense of healthy (non-
infected) cells. Combination of ART with ULD anti-IFN- y + anti-CD4, ULD anti-
IFN- y + anti-CD4 + anti-His or ULD anti-IFN- y more effectively recovers
CD4/CD8 immunoregulatory index than ART alone does.
The observed antiretroviral activity of pharmaceutical compositions
containing ULD anti-IFN-y + anti-CD4 and ULD anti-IFN- y + anti-CD4 + anti-
His makes it possible to use them for the treatment and prophylaxis of HIV
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63
infection both in treatment naïve HIV-infected patients and in patients taking
ART.
Table 15
Viral Load Dynamics Depending on Therapy
Average Decrease of
Viral Load. copies/ml
Viral Load. %
ULD anti-IFN- y + anti-CD4 (Me [Q1-Q3])
Baseline 5769 [368-62584]
16.9
After 6 weeks of treatment 4575 [337-58526]
ART and ULD anti-IFN- y + anti-CD4 (Me [Q1-Q3])
Baseline 5238 [385-59695]
18.2
After 6 weeks of treatment 4408 [320-50197]
ULD anti-IN- y + anti-CD4 + anti-H (Me [Q1-Q3])
Baseline 5638 [385-61742]
17.3
After 6 weeks of treatment 4754 [278-57426]
ART and ULD anti-IN- y + anti-CD4 + anti-H (Me [Q1-Q3])
Baseline 5189 [350-59798]
18.9
After 6 weeks of treatment 46108 [269-47987]
ULD anti-IN- y (Me [Q1-Q3])
Baseline 5813 [150-33356]
9.5
After 6 weeks of treatment 5786 [150-38359]
ART and ULD anti-IN- y (Me [Q1-Q3])
Baseline 4680 [274-9838]
14.2
After 6 weeks of treatment 4652 [272-8874]
ART (Me [Q1-Q3])
Baseline 5547 [385-58996] I
13.3
After 6 weeks of treatment 5308 [338-57709]
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Table 16
Circulating Lymphocytes Subpopulation level in Patients of Study Groups
Observation Period CD4. cl/mcl (M SE) CD4 /
CD8 (M SE)
ULD anti-IFN- y + anti-CD4 (n=12)
Baselint 516 33 0.46
0.09
After 6 weeks of treatment 712 24
0.58 0.07*
ART and ULD anti-IFN- y + anti-CD4 (n=13)
Baseline 499 41 0.50
0.08
After 6 weeks of treatment 728 29
0.60 0.06*
ULD anti-IFN- y + anti-CD4 + anti-H (n=11)
Baseline 509 45 0.49
0.06
After 6 weeks of treatment 706 27
0.58 0.08*
ART and ULD anti-IFN- y + anti-CD4 + anti-H (n=12)
Baseline 521 37 0.48
0.09
After 6 weeks of treatment 734 22
0.62 0.10*
ULD anti-IFN- y (n=11)
Baseline 513 98 0.38
0.19
After 6 weeks of treatment 563 26
0.44 0.12
ART and ULD anti-IFN- y (n=16)
Initially 491 49 0.55
0.06
After 6 weeks of treatment 623 45
0.67 0.05*
ART (n=22)
Initially 510 29 0.44
0.06
After 6 weeks of treatment 595 35
0.50 0.12
* difference is significant vs baseline at p<0,05
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Example 12.
To study the activity of pharmaceutical compositions for the treatment of
patients of the group No.1 tablets 300 mg impregnated onto pharmaceutical
composition containing aqueous-alcoholic solutions (6 mg/tablet) of activated-
potentiated forms of rabbit polyclonal affinity purified antibodies to human
interferon gamma (anti-IFN-y) and CD4 (anti-CD4) in ultra-low doses (ULD)
obtained by means of ultra dilution of initial matrix solution in 10012, 1003
,
1005 times equal to mixture of centesimal homeopathic dilutions C12, C30,
C50 were used; for treatment of patients of group No. 2 300 mg impregnated
onto pharmaceutical composition containing aqueous-alcoholic solutions (6
mg/tablet) of activated-potentiated forms of rabbit polyclonal affinity
purified
antibodies to human interferon gamma (anti-IFN-y) and CD4 (anti-CD4) and
histamine (anti-His) in ultra-low doses (ULD) obtained by means of ultra
dilution
of initial matrix solution in 10012, 1003 , 1005 times equal to mixture of
centesimal homeopathic dilutions C12, C30, C50 were used; for treatment of
patients of group No. 3 tablets 300 mg impregnated onto pharmaceutical
composition containing aqueous-alcoholic solutions (3 mg/tablet) of activated-
potentiated forms of rabbit polyclonal affinity purified antibodies to human
interferon gamma (anti-IFN-y) in ultra-low doses (ULD) obtained by means of
ultra dilution of initial matrix solution in 10012, 1003 , 1005 times equal
to
mixture of centesimal homeopathic dilutions C12, C30, C50 were used.
Evaluation of efficacy of three pharmaceutical compositions containing
ULD anti- IFN-y+anti-CD4, ULD anti- IFN-y+anti-CD4+anti-His and ULD anti-
IFN-y in the treatment of chronic viral hepatitis C was performed in the
course
of comparative parallel group study. Eighteen patients (14 men and 4 women)
at the age of 27-52 were enrolled. Diagnosis of hepatitis C was confirmed by
serum markers (anti-HVC and HCV RNA). All patients included to the study had
2nd or 3rd genotype HCV, mild slowly progressive course of chronic hepatitis C
with low disease activity (serum aminotransferases < 3-fold normal values or
<100 U/I); none of the patients receive specific antiviral therapy before. The
patients with positive result of serologic analysis for HIV, RW, anti-HCA,
HBsAg
or HBcorAg Ab, with cirrhosis, severe concomitant diseases at the stage of
exacerbation, thalassemia or other hemoglobinopathy, alcoholic and\or
medication/drug dependence, patients after transplantation of organs who
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66
constantly took immunosuppressive medications as well as pregnant women
and lactating women were not included in the study. The patients of three
study
groups were given the pharmaceutical compositions according to the following
regimen: 1 tablet three times a day for 24 weeks: patients of the 1st group
(n=5)
¨ULD anti- IFN-y+anti-CD4; patients of the 2nd group (n=4) ¨ULD anti- IFN-
y+anti-CD4+anti-His; patients of the 3rd group (n=4) - ULD anti- IFN-y.
Control
group consisted of 5 patients with persistent viremia and stable normal levels
of
aminotransferases (<20 UM) received no specific therapy. During the study
course regular examinations, control of viral load and laboratory rates were
carried out, concomitant therapy was registered as well as undesirable adverse
events. Therapy efficacy was assessed on week 24 by viral load with HCV RNA
and activity of alanine-aminotransferase (ALT).
Date on viral load (the number of copies of HCV RNA) presented in the
table as median (Me) and the range between first and third quartiles [Q1-Q3],
evidenced positive effect of therapy in patients of groups 1-3 by the end of
24-
week treatment. Intake of pharmaceutical composition of ULD anti- IFN-y+anti-
CD4 caused a reduction in the number of copies of HCV RNA in 2 out of 5
persons of the 1st group and an average reduction of viral load was 75%.
Similar results were obtained in patients administered with pharmaceutical
composition of ULD anti- IFN-y+anti-CD4+anti-His: its antiviral activity was
registered in all patients (4 out of 4 subjects of the group 2), average
reduction
of viral load was 70%. Moreover, complete virus clearance was registered in 2
patients (one of group 1 and one of group 2) by the end of therapy. Antiviral
activity of monocomponent ULD anti- IFN-y was somewhat lower and a
reduction in the number of copies of HCV RNA was recorded in 3 out of 4
patients of 31d group, an average reduction of viral load was 55%. In control
group, no positive changes in viral load were revealed.
Antiviral activity of the studies pharmaceutical compositions was
accompanied with positive changes in ALT level registered in patients of
groups 1-3 by the end of 24-week therapy. Normalization of ALT level was
found in 2 patients of ULD anti- IFN-y+anti-CD4 group, in 1 patient of ULD
anti-
IFN-y+anti-CD4+anti-His group and in 1 patient ULD anti- IFN-y groupin 1
patient of control group ALT level exceeded upper border of norm (>20 UM) due
to an increase of viral load at the end of 24-week study period.
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No drugs-related adverse events were registered during the study, which
evidences of their good tolerance. Absence of pathological variations in blood
and urine analysis including markers of renal and hepatic insufficiency
confirmed safety of the treatment.
Thus, the study of efficacy and safety of pharmaceutical compositions
containing ULD anti- IFN-y+anti-CD4, ULD anti- IFN-y+anti-CD4+anti-His and
ULD anti- IFN-y in patients with chronic hepatitis C were carried out. The
strongest antiviral effect was registered for ULD anti- IFN-y+anti-CD4, ULD
anti- IFN-y+anti-CD4+anti-His, which was confirmed by positive dynamics of
viral load and viral clearance by the end of 24-week therapy in 2 patients.
Antiviral efficacy of ULD anti- IFN-y+anti-CD4, ULD anti- IFN-y+anti-CD4+anti-
His and ULD anti- IFN-y was accompanied with a reduction of activity of
chronic hepatitis C, which was confirmed by the reduction and even
normaluzation of ALT level in some patients at the end of 24-week course of
treatment.
Table 17
Dynamics of viral load in the study groups
Average reduction
HCV RNA, copies/ml
of viral load, %
ULD anti- IFN-y+anti-CD4 (Me [Q1-Q3])
Baseline 66200 [450-181400]
75
24-week treatment 12500 [50-30560]
ULD anti- IFN-y+anti-CD4+anti-His (Me [Q1-Q3])
Baseline 58900 [600-124500]
70
24-week treatment 15600 [50-45700]
ULD anti- IFN-y (Me [Q1-Q3])
Baseline 84700 [350-172800]
55
24-week treatment 22400 [150-58500]
Control group (Me [Q1-Q3])
Baseline 79500 [300-155600]
24-week treatment 87900 [450-164300]
