RU2519862C2 - Complex medication and method of prevention and treatment of infectious viral diseases - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine, namely to therapy, and deals with prevention and treatment of infectious viral diseases. For this purpose a complex medication, containing a mixture of several components in a form of activated-potentiated forms of antibodies to a dissolved antigen and an antigen, associated with an external membrane of immune system cells, is introduced.

EFFECT: medication ensures efficient treatment of viral diseases of different etiology due to synergic antiviral action of active medication components.

29 cl, 17 tbl, 2 dwg, 11 ex

Description

The invention relates to medicine and can be used for the effective prevention and treatment of infectious viral diseases, including, for example, acute and chronic viral infections, acute viral hepatitis A, B, C and other viral hepatitis, herpes viruses, influenza of various types, including including influenza A and B.

The prior art knows a medicine for the treatment of infectious diseases, including viral etiology, based on the activated form of ultra-low doses of antibodies to interferon (RU 2192888 C1, A61K 39/395, 11/20/2002). However, this drug may not be effective enough to treat a wide range of acute and chronic infectious viral diseases, including, for example, acute viral hepatitis A, B, C and other viral hepatitis, herpes virus infections.

The invention is directed to the creation of a comprehensive drug without pronounced side effects, which provides both effective primary prevention of infectious viral diseases and the treatment of a wide range of infectious viral diseases.

The solution to this problem is provided by the fact that the complex drug for the prevention and treatment of infectious viral diseases, according to the invention, contains a mixture of several components, each of which is presented in the form of an activated-potentiated form of antibodies to an antigen-protein or a peptide of the immune system involved in the pathogenesis of infectious viral disease.

At the same time, at least one of the components of the claimed drug may be an activated-potentiated form of antibodies to an antigen associated with the outer membrane of cells of the immune system.

In addition, at least one of the components of the claimed drug can be an activated-potentiated form of antibodies to a dissolved antigen (or a soluble antigen, i.e., an antigen that is not associated with the outer membrane of cells of the immune system).

At the same time, cytokines can be used as a dissolved antigen.

As an antigen associated with the outer membrane of cells of the immune system, receptors of immunocompetent cells can be used.

Moreover, differentiation clusters can be used as receptors for immunocompetent cells.

In addition, the activated-potentiated form of antibodies to dissolved antigens and the activated-potentiated form of antibodies to antigen associated with the outer membrane of the cells of the immune system are used in the form of an activated-potentiated aqueous or aqueous-alcoholic solution, the activity of which is due to the process of sequential multiple dilution of the matrix - initial - a solution of antibodies in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - vertical shaking each dilution, with the final dilutions containing ultra-low doses (SMD) of antibodies.

The complex drug can be made in the form of a pharmaceutical composition in solid dosage form and contain the technologically necessary (effective) amount of a neutral carrier saturated with a mixture of aqueous or aqueous-alcoholic solutions of an activated-potentiated form of antibodies to dissolved antigens and an activated-potentiated form of antibodies to antigen, associated with the outer membrane of the cells of the immune system, and pharmaceutically acceptable additives, which include, for example, lactose, cellulose microcrystalline and magnesium stearate.

Preferably, aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to dissolved antigens and to an antigen associated with the outer membrane of cells of the immune system can be obtained by multiple serial dilutions of a matrix - initial - solution of antibodies to the antigen bound to the outer membrane of cells of the immune system , and / or antibodies to the dissolved antigen in combination with an external mechanical action - vertical shaking of each dilution, while the concentration of the matrices th solution is 0.5 ÷ 5.0 mg / ml.

Each of the components based on ultra-low doses of antibodies can be used in the form of a mixture of various, mainly hundred, dilutions prepared according to homeopathic technology.

In addition, the solution of the problem is ensured by the fact that in the method for the prevention and treatment of infectious viral diseases, according to the invention, a mixture (complex) of several components is used in the form of activated-potentiated forms of antibodies to various proteins or peptides of the immune system involved in the pathogenesis of an infectious viral disease .

Moreover, the claimed method simultaneously and combined uses the activated-potentiated form of antibodies to the dissolved antigen and the activated-potentiated form of antibodies to the antigen associated with the outer membrane of the cells of the immune system.

As an antigen associated with the outer membrane of cells of the immune system, receptors of immunocompetent cells can be used.

As receptors for immunocompetent cells, differentiation clusters can be used.

As dissolved antigens, cytokines can be used.

In this case, a mixture of activated-potentiated forms of antibodies is used in the form of an activated-potentiated aqueous or aqueous-alcoholic solution of each component, the activity of which is due to the process of multiple dilution of the matrix - initial - solution of antibodies in an aqueous or aqueous-alcoholic solvent in combination with an external mechanical action - vertical shaking each dilution.

It is preferable to use a mixture of various dilutions of antibodies to a dissolved antigen in combination with a mixture of various dilutions of antibodies to an antigen associated with the outer membrane of cells of the immune system prepared by homeopathic technology, prepared as a single drug - a single dosage form.

Aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to dissolved antigens and to antigens associated with the outer membrane of cells of the immune system can be obtained by repeated dilution of a matrix - initial - solution of antibodies to antigens bound to the outer membrane of cells of the immune system, and / or antibodies to dissolved antigens in combination with an external mechanical action - vertical shaking of each dilution, while the concentration of the matrix solution is 0.5 ÷ 5.0 mg / ml.

According to the invention, the activated-potentiated form is an antigen antibody form prepared according to the homeopathic potentiation technique by repeatedly diluting the matrix - initial - antibody solution in combination with an external action - vertical shaking of each dilution, which is active in pharmacological models and / or clinical methods HIV infection prevention; prevention and effective treatment of infectious viral diseases.

The proposed combination of activated-potentiated forms of antibodies to dissolved antigens (e.g., human gamma-interferon) and antigen associated with the outer membrane of cells of the immune system (e.g., CD4 receptor) in the claimed drug provides an unexpected synergistic therapeutic effect, experimentally confirmed on an adequate model, which consists in the high effectiveness of the drug both in the prevention and treatment of a wide range of infectious diseases, due to the high immunotropic action components, e.g., increased interferon-dependent activation of CD4 lymphocytes and an increase in the number of receptors on the surface of CD4 cell receptor gamma interferon, enhancement of antiviral activity.

The drug is prepared mainly as follows.

To prepare the activated-potentiated form of the active components, monoclonal or, mainly, polyclonal antibodies are used, which can be obtained according to well-known technology-methods described, for example, in the book: Immunological methods, ed. G. Frimel, M., "Medicine", 1987, S. 9-33; or, for example, in an article by Laffly E., Sodoyer R. Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

Monoclonal antibodies are obtained, for example, using hybridoma technology. Moreover, the initial stage of the process includes immunization, based on the principles already developed in the preparation of polyclonal antisera. Further stages of the work include obtaining hybridoma cells producing clones of antibodies of the same specificity. Their isolation is carried out by the same methods as in the case of polyclonal antisera.

Polyclonal antibodies can be obtained by active immunization of animals. For this purpose, according to a specially developed scheme, the animals are given a series of injections of the substance required in accordance with the invention — an antigen or a conjugated antigen (for example, human gamma-interferon or CD4 receptor). As a result of this procedure, a monospecific antiserum with a high antibody content is obtained, which is used to obtain the activated-potentiated form. If necessary, the antibodies present in the antiserum are purified, for example, by affinity chromatography, using fractionation by salt precipitation or ion exchange chromatography.

Preferred for the preparation of the claimed complex drug is the use of polyclonal antibodies, for example, to human gamma-interferon, and, for example, to the CD4 receptor, which are used as a matrix (initial) solution with a concentration of 0.5 ÷ 5.0 mg / ml for subsequent preparation of the activated-potentiated form.

Preferred for the preparation of each component is the use of a mixture of three water-alcohol dilutions of the initial matrix solution of antibodies diluted, respectively, 100 ¹² , 100 ³⁰ , and 100 ⁵⁰ times, which is equivalent to hundreds of dilutions C12, C30 and C50 prepared according to homeopathic technology. When performing the claimed drug in solid dosage form, a mixture of these components is applied to lactose.

Preferred for the preparation of the claimed drug is the use of polyclonal antibodies, for example to human gamma interferon, and, for example, to the CD4 receptor, which can be obtained by immunization of rabbits as follows.

To obtain polyclonal antibodies to human gamma-interferon, an adjuvant and, for example, a whole human gamma-interferon molecule of the following sequence are used as immunogen (antigen) for immunization of rabbits:

1 MKYTSYILAF QLCIVLGSLG CYCQDPYVKE AENLKKYFNA GHSDVADNGT LFLGILKNWK

61 EESDRKIMQS QIVSFYFKLF KNFKDDQSIQ KSVETIKEDM NVKFFNSNKK KRDDFEKLTN

121 YSVTDLNVQR KAIHELIQVM AELSPAAKTG KRKRSQMLFR GRRASQ

It is possible to obtain a polyclonal antibody to human gamma interferon as an immunogen (antigen) for immunization of rabbits using an adjuvant and, for example, one polypeptide fragment of human gamma interferon selected from the following sequences:

7-55:

ILAF QLCIVLGSLG CYCQDPYVKE AENLKKYFNA GHSDV

24-166:

QDPYVKEAE NLKKYFNAGH SDVADNGTLF LGILKNWKEE SDRKIMQSQI VSFYFKLFKN FKDDQSIQKS VETIKEDMNV KFFNSNKKKR DDFEKLTNYS VTDLNVQRKA IHELIQVMAGRSRA

24-166:

QDPYVKEAE NLKKYFNAGH SDVADNGTLF LGILKNWKEE SDRKIMQSQI VSFYFKLFKN FKDDQSIQKS VETIKEDMNV KFFNSNKKKR DDFEKLTNYS VTDLNVQRKA IHELIQVMAGRSPA

69-123:

QS QIVSFYFKLF KNFKDDQSIQ KSVETIKEDM NVKFFNSNKK KRDDFEKLTN YSV

100-145:

M NVKFFNSNKK KRDDFEKLTN YSVTDLNVQR KAIHELIQVM AELSP

92-130:

SVETIKEDM NVKFFNSNKK KRDDFEKLTN YSVTDLNVQR

123-147:

VTDLNVQR KAIHELIQVM AELSPAA

24-166:

MQDPYVKEAE NLKKYFNAGH SDVADNGTLF LGILKNWKEE SDRKIMQSQI VSFYFKLFKN FKDDQSIQKS VETIKEDMNV KFFNSNKKKR DDFEKLTNYS VTDLNVQRKA IHELIQQMGRR RAS

5-45:

SYILAF QLCIVLGSLG CYCQDPYVKE AENLK

94-114:

ETIKEDM NVKFFNSNKK KRDD,

24-166:

MQDPYVKEAE NLKKYFNAGH SDVADNGTLF LGILKNWKEE SDRKIMQSQI VSFYFKLFKN FKDDQSIQKS VETIKEDMNV KFFNSNKKK DDFEKLTNYS VTDLNVQRKA IHELIQQMRRSR

Before blood sampling for 1-3 days, 1-3 intravenous injections are performed to increase the level of antibodies. During immunization, small blood samples are taken from rabbits to evaluate the amount of antibody. The maximum level of the immune response to the administration of most antigens is achieved 40-60 days after the first injection. After the end of the first cycle of immunization of rabbits for 30 days, they restore health and re-immunization, including 1-3 intravenous injections. To obtain antisera from immunized rabbits, blood is collected in a 50 ml centrifuge tube. Using a wooden spatula, the formed clots are removed from the walls of the tube and the wand is placed in the clot formed in the center of the tube. Blood is placed in a refrigerator (temperature 4 ° C) at night. The next day, the clot attached to the spatula is removed and the remaining liquid is centrifuged at 13000 g for 10 minutes. The supernatant (supernatant) is an antiserum. The resulting antiserum should be yellow. You can add 20% NaN ₃ to the antiserum to a final concentration of 0.02% and store until use in a frozen state at a temperature of -20 ° C (or without adding NaN ₃ at a temperature of -70 °). The selection of anti-gamma interferon antibodies from the antiserum is possible as follows:

1. 10 ml of rabbit antiserum is diluted 2 times with 0.15 M NaCl, 6.26 g of Na ₂ SO ₄ are added, stirred and incubated for 12-16 hours at 4 ° C;

2. The precipitate formed is removed by centrifugation, dissolved in 10 ml of phosphate buffer and then dialyzed against the same buffer overnight at room temperature;

3. after removal of the precipitate by centrifugation, the solution is applied to a column with DEAE-cellulose, balanced with phosphate buffer;

4. The antibody fraction is determined by measuring the optical density of the eluate at 280 nm.

Antibodies are purified by affinity chromatography on a column with antigen by binding of antibodies to gamma interferon with an antigen (gamma interferon) attached to an insoluble matrix of the column, followed by elution of the antibodies with concentrated salt solutions.

Thus obtained buffer solution of polyclonal rabbit antibodies to human gamma-interferon purified on an antigen with a concentration of 0.5 ÷ 5.0 mg / ml, preferably 2.0 ÷ 3.0 mg / ml, is used as a matrix ( initial) solution for the subsequent preparation of the activated-potentiated form.

Polyclonal antibodies to the CD4 receptor are obtained according to the above method, using an immunogen (antigen) for immunization of rabbits with an adjuvant and a whole molecule or one polypeptide fragment of the CD4 receptor, selected, for example, from the following amino acid sequences:

412-458:

IGLGIFFCV RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPI

26-60:

MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGD

105-119:

A DSRRSLWDQG NFPL

115-139:

G NFPLIIKNLK IEDSDTYICE VEDQ

26-458:

MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWW QAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQ YAGSGNLTLA LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLML SLKLENKEAK VSKREKAVWV LNPEAGMWQC LLSDSGQVLL ESNIKVLPTW STPVQPMALI VLGGVAGLLL FIGLGIFFCV RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPI

Thus obtained buffer solutions of each component of polyclonal antibodies to human gamma interferon and to the CD4 receptor with a concentration of 0.5 ÷ 5.0 mg / ml, preferably 2.0 ÷ 3.0 mg / ml, are used as matrix (initial) solutions for the subsequent preparation of activated-potentized forms of the drug.

The activated-potentiated form of each component is prepared by uniformly decreasing the concentration as a result of successive dilution of 1 part of each solution to be diluted, starting from the above matrix solution, in 9 parts (for decimal dilution D) or in 99 parts (for hundredth dilution C) or in 999 parts (for a thousandth dilution M) of a neutral solvent in combination with multiple vertical shaking (potentiation, or "dynamization") of each dilution obtained and using tdelnyh containers for each subsequent dilution to the required potency-the multiplicity of homeopathic dilution method (see., eg, B. Schwabe "Homeopathic medicines", M., 1967, s.14-29).

External processing in the process of reducing the concentration can also be performed by ultrasound, electromagnetic or other physical effects.

For example, to prepare the 12th hundredth C12 dilution, one part of a matrix (initial) antibody solution, for example, human gamma interferon (or, for example, antibodies to the CD4 receptor) with a concentration of 2.5 mg / ml, is diluted in 99 parts of neutral aqueous or aqueous -alcohol solvent and repeatedly (10 or more times) vertically shake - potentiate, getting the 1st hundredth C1 dilution. From the 1st hundredth C1 dilution prepare the 2nd hundredth dilution C2. This operation is repeated 11 times, obtaining the 12th hundredth dilution of C12. Thus, the 12th hundredth dilution of C12 is a solution obtained by diluting successively 12 times in different containers of the 1st part of the initial matrix solution of antibodies to human gamma-interferon with a concentration of 2.5 mg / ml in 99 parts of a neutral solvent, those. the solution obtained by diluting the matrix solution in 100 ¹² times. Similar operations with an appropriate dilution ratio are carried out to obtain a dilution of C 30 and C 50.

When using, for example, an activated-potentiated form of antibodies to human gamma-interferon (or, for example, antibodies to the CD4 receptor) in the form of a mixture of various, mainly hundred, dilutions, each dilution (for example, C12, C30, C50) is prepared separately according to the technology described above before dilution, 3 dilutions are less than the final one (respectively, until C9, C27, C47 are obtained), and then, in accordance with the composition of the mixture, one part of each component is added to one container and mixed with the required amount of solvent (corresponding to but with 97 parts for centesimal dilution). Next, the resulting mixture was successively diluted twice in a ratio of 1 to 100, potentiating the resulting solution after each dilution. In this case, an activated-potentiated form of antibodies is obtained, for example, to human gamma-interferon (or, for example, antibodies to the CD4 receptor) in an ultra-low dose obtained by diluting the matrix solution 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundred dilutions of C12, C30, C50.

It is possible to use each component in the form of a mixture of other different, dilutions, for example, decimal and or hundredths (D20, C30, C100 or C12, C30, C50, etc.) prepared according to homeopathic technology, the effectiveness of which is determined experimentally.

To obtain a medicinal product, aqueous or aqueous-alcoholic solutions of the active substances of the components are mixed, mainly in a volume ratio of 1: 1 and used in liquid dosage form.

The claimed drug can also be used in solid dosage form in the form of a pharmaceutical composition, which contains the technologically necessary (effective) amount of a neutral carrier - for example, lactose - saturated, by impregnation to saturation with a mixture prepared by the above technology, of aqueous or aqueous-alcoholic solutions activated -potentiated forms of antibodies, for example, to human gamma interferon, and, for example, to the CD4 receptor and pharmaceutically acceptable additives, including, pre uschestvenno, lactose, microcrystalline cellulose and magnesium stearate.

To obtain a solid oral form of the claimed drug, a fluidized bed is installed (for example, type “Hüttlin Pilotlab” manufactured by Hüttlin GmbH) irrigation until saturation of the granules of a neutral substance - lactose (milk sugar) with a particle size of 50 ÷ 500 is introduced into the fluidized - boiling layer microns, pre-prepared aqueous or aqueous-alcoholic solution of the activated-potentiated form of antibodies to the CD4 receptor, mainly in the ratio of 1 kg of antibody solution to 5 or 10 kg of lactose (1: 5-1: 10) with simultaneous drying in the flow of heated air supplied under the grate at a temperature not exceeding 40 ° C. The estimated amount of lactose (10 ÷ 91% of the tablet mass) saturated with the activated-potentiated form of antibodies according to the above procedure is loaded into the mixer and mixed with lactose, moistened with the activated-potentiated form of antibodies, in an amount of 3 ÷ 10% of the tablet mass and with pure lactose in an amount of not more than 84% of the tablet mass (to reduce the cost and some simplification and acceleration of the process without reducing the effectiveness of the therapeutic effect). Then, microcrystalline cellulose is added to this mixture in an amount of 5-10% of the tablet mass and magnesium stearate in an amount of 1% of the tablet mass. The resulting tablet mass is uniformly mixed and tabletted by direct dry pressing (for example, in a Korsch-XL 400 tablet press). After tabletting, 300 mg tablets are obtained, impregnated with an aqueous-alcoholic solution with an activated-potentiated form of antibodies to human gamma-interferon and to the CD4 receptor in an ultra-low dose, each component prepared from a matrix solution diluted in 100 ¹² , in 100 ³⁰ in 100 ⁵⁰ , which is equivalent to a mixture of hundreds of dilutions of C12, C30 and C50, prepared according to homeopathic technology.

Preferably, the claimed drug is recommended to take 1-2 tablets 2-4 times a day.

For experimental studies, antibodies were used, prepared by order of a specialized biotechnological company.

Example 1

The antiviral effect of the claimed drug was studied on an HIV model, inhibiting HIV replication in a culture of human peripheral blood mononuclear cells infected with an in vitro strain of HIV-1-LAI. The effectiveness of inhibiting HIV replication was evaluated by the content of the main nucleocapid protein p24 of HIV in supernatants.

For conducting experimental studies, affinity-purified rabbit polyclonal antibodies to tumor necrosis factor alpha and affinity-purified rabbit polyclonal antibodies to gamma-interferon, prepared by order of a specialized biotechnological company, were used, on the basis of which activated-potentiated forms of antibodies to gamma-interferon and antibodies were prepared to the tumor necrosis factor alpha in an ultra-low dose in the form of a mixture of dilution C12 + C30 + C200 obtained by homeopathic technology (far complex preparation).

Evaluation of the antiviral activity of the complex preparation was carried out using mononuclear cells of human peripheral blood infected with an in vitro strain of HIV-1-LAL

Human peripheral blood mononuclear cells were isolated from the blood of healthy seronegative donors by centrifugation in a density gradient of ficoll-hypak. Cells were activated for 3 days using 1 μg / ml phytohemmagglutin R and 5 IU / ml recombinant human interleukin-2.

To evaluate antiretroviral activity, the preparations were added to a well containing 100 μl of activated human peripheral blood mononuclear cells 24 hours or 15 minutes after infection of cells with HIV-1-LAI strain at a dose of 100 TCID50 (50 μl of HIV-1-LAI strain inoculum). Before introducing into the well, a complex preparation (12.5 μl) or azidothymidine (active ingredient - zidovudine) at a dose of 1000 nM (reference preparation) was mixed with RPMI1640 medium (DIFCO) until a final volume of 50 μl was reached.

Cell culture supernatants were harvested 7 days after infection. The effectiveness of the drugs was determined by inhibiting HIV replication, which was assessed by the content of the main nucleosapid HIV p24 protein in cell supernatants by ELISA (Retrotek Elisa kit).

The complex preparation was shown to inhibit HIV replication by 91 ± 3% when added to the well 24 hours before, and by 34 ± 30% when added to the well 15 minutes after infection of cells with the HIV-1-LAI strain, respectively. Azidothymidine at a dose of 1000 nM inhibited HIV replication by 99 ± 0 and 99 ± 1% when added to the well 24 hours before and 15 minutes after infection of cells with HIV-1-LAI strain, respectively.

Thus, an in vitro study showed high antiviral activity of a combination of ultra-low doses of rabbit polyclonal antibodies to tumor necrosis factor-alpha and rabbit polyclonal antibodies to interferon-gamma (a mixture of homeopathic dilution C12 + C30 + C200).

Note:

- TCID50 dose infecting 50% of tissue culture cells.

Example 2

The antiviral effect of the claimed drug was studied on an HIV model, inhibiting HIV replication in a culture of human peripheral blood mononuclear cells infected with an in vitro strain of HIV-1-LAI. The effectiveness of inhibiting HIV replication was evaluated by the content of the main nucleocapid protein p24 of HIV in supernatants.

For experimental studies, affinity purified rabbit polyclonal antibodies to interferon-alpha, affinity purified rabbit polyclonal antibodies to gamma-interferon, affinity purified rabbit polyclonal antibodies to T4 lymphocyte CD4 receptor and affinity purified rabbit polyclonal CD8 receptor antibodies were used. prepared by order of a specialized biotechnological company, on the basis of which activated-potentiated forms of antibodies to gamma-inte were prepared Rferon, antibodies to interferon-alpha, antibodies to the CD4 receptor for T-lymphocytes and antibodies to the CD8 receptor for T-lymphocytes in an ultra-low dose in the form of a mixture of dilution C12 + C30 + C50 obtained by homeopathic technology (hereinafter referred to as the complex preparation).

Evaluation of the antiviral activity of the complex preparation was carried out using human peripheral blood mononuclear cells infected with an in vitro strain of HIV-1-LAI.

Human peripheral blood mononuclear cells were isolated from the blood of healthy seronegative donors by centrifugation in a density gradient of ficoll-hypak. Cells were activated for 3 days using 1 μg / ml phytohemmagglutin R and 5 IU / ml recombinant human interleukin-2.

To evaluate antiretroviral activity, the preparations were added to a well containing 100 μl of activated human peripheral blood mononuclear cells 24 hours or 15 minutes after infection of cells with HIV-1-LAI strain at a dose of 100 TCID50 (50 μl of HIV-1-LAI strain inoculum). Before introducing into the well, a complex preparation (12.5 μl) or azidothymidine (active ingredient - zidovudine) at a dose of 1000 nM (reference preparation) was mixed with RPMI1640 medium (DIFCO) until a final volume of 50 μl was reached.

Cell culture supernatants were harvested 7 days after infection. The effectiveness of the drugs was determined by inhibiting HIV replication, which was assessed by the content of the main nucleosapid HIV p24 protein in cell supernatants by ELISA (Retrotek Elisa kit).

It was shown that the complex preparation inhibits HIV replication by 94 ± 6% when introduced into the well 24 hours before, and by 46 ± 13% when introduced into the well 15 minutes after infection of cells with the HIV-1-LAI strain, respectively. Azidothymidine at a dose of 1000 nM inhibited HIV replication by 99 ± 0 and 99 ± 1% when added to the well 24 hours before and 15 minutes after infection of cells with HIV-1-LAI strain, respectively.

Thus, an in vitro study showed high antiviral activity of a combination of ultra-low doses of rabbit polyclonal antibodies to interferon-alpha, rabbit polyclonal antibodies to interferon-gamma, rabbit polyclonal antibodies to CD4, rabbit polyclonal antibodies to CD8 (a mixture of homeopathic dilution C12 + C30 + C50 + )

Note:

- TCID50 dose infecting 50% of tissue culture cells.

Example 1

A study of the effectiveness of the use of the claimed complex drug containing activated-potentized forms of aqueous dilution of polyclonal affinity purified rabbit antibodies to human alpha interferon in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50, and activated-potentiated form of polyclonal affinity-purified rabbit antibodies to CD4 receptors y in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) 100 ^12, 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 (SMD AT to IFN-α + SMD AT to CD4) in a 1: 1 ratio of components, as well as a drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to interferon alpha in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , 100 ^{3 0} , 100, ⁵⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions of C12, C30, C50 (SMD AT to IFN-α) and a medicinal product containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to CD4 receptor in ultra-low doses (DMD), obtained by over-dilution of the initial matrix solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵ times, equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C50 (SMD AT to CD4) under conditions of influenza infection in female mice Balb / c was carried out on the basis of FSBI “NI flu "Health Ministry of Russia in two stages. In the first stage, the effectiveness of the preparation of SMD AT to IFN-α and the preparation of SMD AT to CD4 was investigated, in the second stage, the effectiveness of the preparation of SMD AT to IFN-α + SMD AT to CD4 was studied. Both when testing the preparation of SMD AT for IFN-α + SMD AT for CD4, and when testing the preparation of SMD AT for IFN-α and the preparation SMD AT for CD4, the drug oseltamivir was used as a comparison drug, as a control for mice (n = 20 ) was introduced distilled water (control group).

The infection process was modeled by intranasal administration of the influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 (dose infecting 50% of tissue culture cells).

The preparation of SMD AT to IFN-α, the preparation of SMD AT to CD4 and the preparation of SMD AT to IFN-α + SMD AT to CD4 was administered to mice (n = 20 in each group) intragastrically at 0.2 ml / mouse twice a day (daily) dose of 0.4 ml / mouse) within 5 days before infection and within 10 days after infection with influenza virus A / California / 07 / 2009swl (H1N1). Additionally, the preparation of SMD AT to IFN-α, the preparation of SMD AT to CD4 and the preparation of SMD AT to IFN-α + SMD AT to CD4 were added to drinkers for animals of the corresponding experimental groups. Drinking bowls were in the public domain.

Comparison drug oseltamivir was administered to mice (n = 20) intragastrally twice a day at a dose of 10 mg / kg (daily dose of 20 mg / kg), starting 1 hour before infection with influenza virus A / California / 07 / 2009swl (H1N1). The drug oseltamivir was administered within 5 days after infection. Within 4 days before infection and starting from 6 days after infection, mice of this experimental group were injected intragastrally with distilled water of 0.2 ml / mouse twice a day (daily dose of 0.4 ml / mouse) instead of oseltamivir.

In the control group, mice (n = 20) were injected intragastrically with distilled water 2 times a day at 0.2 ml / mouse (daily dose of 0.4 ml / mouse). Throughout the study, distilled water was used in animal drinkers of these two experimental groups. Drinking bowls were in the public domain.

The effectiveness of the drugs was evaluated by animal survival. The results of the study of the antiviral activity of the preparation of SMD AT to IFN-α and the preparation of SMD AT to CD4 (stage 1) are shown in table 1, the results of the study of the antiviral activity of the preparation of DMD AT to IFN-α + SMD AT and CD4 (stage 2) are shown in table 2 The statistical significance of the differences between the experimental groups and the control (distilled water) was calculated using the non-parametric chi-square test.

Table 1. The results of a study of the antiviral activity of the preparation of SMD AT to IFN-α and the preparation of SMD AT to CD4 in a model of influenza infection in female Balb / c mice infected with intranasal administration of influenza virus A / California / 07 / 2009swl CH1N1) at a dose of 10 LD50 (10 days after infection). No. p / p Experimental group Survival% 10LD50 The difference between% survival in the group receiving the drug and% survival in the group receiving distilled water one. SMD AT to IFN-α 25 + 5% 2. SMD AT to CD4 thirty + 10% 3. Oseltamivir (comparison drug) 80 * + 60% four. Distilled water (control) twenty - * - p <0.05 (significance of differences from control).

Table 2. The results of a study of the antiviral activity of the preparation of SMD AT to IFN-α + SMD AT to CD4 in a model of influenza infection in female Balb / c mice infected with intranasal administration of influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 (10 days after infection). No. p / p Experimental group Survival rate,%
10LD50 The difference between% survival in the group receiving the drug and% survival in the group receiving distilled water one. The drug SMD AT to IFN-α + SMD AT to CD4, the ratio of 1: 1 thirty* + 25% 2. Oseltamivir (comparison drug) 70 * + 65% 3. Distilled water (control) 5 - * - p <0.05 (significance of differences from control).

It was shown that the survival rate of mice infected with influenza A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 was higher in stage 1 of the study than in stage 2: the survival rate in the group treated with distilled water was 20% and 5%, respectively ; survival in the oseltamivir comparison drug group was 80% and 70%, respectively. This indicates a more pronounced lethal effect induced by the intranasal administration of influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50, in stage 2 of the study.

At the same time, the preparation of SMD AT for IFN-α + SMD AT for CD4 increased the survival of experimental animals infected with influenza A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 by 25% compared with the control, while survival in the group receiving the drug SMD AT to IFN-α was only 5% higher than the survival in the control group, and the survival rate in the group receiving the drug SMD AT to CD4 was only 10% higher than the survival in the control group.

Thus, as a result of the study, it was shown that the use of the preparation of SMD AT to IFN-α + SMD AT to CD4 has a more pronounced antiviral effect than the use of the preparation of SMD AT to IFN-α or the preparation of SMD AT to CD4, despite the fact that the dose of each component in the composition of SMD AT to IFN alpha + SMD AT to CD4 is two times lower than the dose of components in the composition of SMD AT to IFN alpha and the dose of components in the composition of SMD AT to CD4 tested as separate drugs.

Example 2

A study of the effectiveness of the use of the claimed complex drug containing activated-potentiated forms of aqueous dilution of polyclonal affinity-purified rabbit antibodies to tumor necrosis factor alpha in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , ^3, 100, 100, equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 and activated-potentiated form of polyclonal affinity-purified rabbit antibodies to CD4 receptors y in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) 100 ^12, 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 (SMD AT TNF-α + SMD AT to CD4) in a 1: 1 ratio, as well as a drug containing activated-potentized forms of aqueous dilution of polyclonal affinity purified rabbit antibodies to tumor necrosis factor alpha in ultra-low doses (SMD) obtained by over-dilution of the initial matrix solution (concentration 2, 5 mg / ml) in 100 ¹ , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions of C12, C30, C50 (SMD AT to TNF-α) and a drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to CD4 receptor in ultra-low doses (DMD) obtained super-dilution of the initial matrix solution (concentration of 2.5 mg / ml) 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C50 (SMD AT to CD4) under conditions of influenza infection in female mice of the Balb strain / c was carried out on the basis of FSBI “Research Institute influenza "Ministry of Health and Social Development of Russia in two stages. In the first stage, the effectiveness of the preparation of SMD AT for name and surname-α and the preparation of SMD AT to CD4 was investigated, in the second stage, the effectiveness of the use of SMD AT for full name-α + SMD AT for CD4 was investigated. Oseltamivir was used as a comparison drug, distilled water was given to the control group (control).

The infection process was modeled by intranasal administration of the influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 (dose infecting 50% of tissue culture cells).

The preparation of SMD AT to TNF-α, the preparation of SMD AT to CD4, as well as the preparation of SMD AT to TNF-α + SMD AT to CD4 was administered to mice (n = 20 in each group) intragastrically at 0.2 ml / mouse twice a day (daily dose of 0.4 ml / mouse) for 5 days before infection and for 10 days after infection. Additionally, the preparation of SMD AT to TNF-α, the preparation of SMD AT to CD4, and the preparation of SMD AT to TNF-α + SMD AT to CD4 were added to drinkers for animals of the corresponding experimental groups. Drinking bowls were in the public domain.

Comparison drug oseltamivir was administered to mice (n = 20) intragastrally twice a day at a dose of 10 mg / kg (daily dose of 20 mg / kg), starting 1 hour before infection. The drug was administered within 5 days after infection with the influenza virus A / California / 07 / 2009swl (H1N1). Within 4 days before infection and starting from 6 days after infection, mice of this experimental group were injected intragastrally with distilled water of 0.2 ml / mouse twice a day (daily dose of 0.4 ml / mouse) instead of oseltamivir.

In the control group, mice (n = 20) were injected intragastrically with distilled water 2 times a day at 0.2 ml / mouse (daily dose of 0.4 ml / mouse). Throughout the study, distilled water was used in animal drinkers of these two experimental groups. Drinking bowls were in the public domain.

The effectiveness of the drugs was evaluated by animal survival. The results of the study of the antiviral activity of the preparation of SMD AT to name-α and the preparation of SMD AT to CD4 (stage 1) are shown in table 3, the results of the study of the antiviral activity of the preparation of DMD AT to TNF-α + SMD AT to CD4 (stage 2) are shown in table 4 The statistical significance of the differences between the experimental groups and the control (distilled water) was calculated using the non-parametric chi-square test.

Table 3. The results of a study of the antiviral activity of the preparation of SMD AT to TNF-α and the preparation of SMD AT to CD4 in a model of influenza infection in female Balb / c mice infected with intranasal administration of influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 (10 day after infection). No. p / p Experimental group Survival% 10LD50 The difference between% survival in the group receiving the drug and% survival in the group receiving distilled water one. SMD AT to TNF-α 25 + 5% 2. SMD AT to CD4 thirty + 10% 3. Oseltamivir (comparison drug) 80 * + 60% four. Distilled water (control) twenty - * - p <0.05 (significance of differences from control).

Table 4. The results of a study of the antiviral activity of the preparation of SMD AT to TNF-α + SMD AT to CD4 in a model of influenza infection in female Balb / c mice infected with intranasal administration of influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 (10 days after infection). No. p / p Experimental group Survival% 10LD50 The difference between% survival in the group receiving the drug and% survival in the group receiving distilled water one. SMD AT to TNF-α + SMD AT to CD4, in the ratio 1: 1 thirty* + 25% 2. Oseltamivir (comparison drug) 70 * + 65% 3. Distilled water (control) 5 - * - p <0.05 (significance of differences from control).

It was shown that the survival rate of mice infected with influenza A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 was higher in stage 1 of the study than in stage 2: the survival rate in the group treated with distilled water was 20% and 5%, respectively ; survival in the oseltamivir comparison drug group was 80% and 70%, respectively. This indicates a more pronounced lethal effect induced by the intranasal administration of influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50, in stage 2 of the study.

At the same time, the preparation of SMD AT for the name, α + SMD AT for CD4 increased the survival of experimental animals infected with the influenza virus A / California / 07 / 2009swl (H1N1) at a dose of 10 LD50 by 25% compared with the control, while the survival rate in the group receiving the drug DMD AT to TNF-α was only 5% higher than the survival in the control group, and the survival rate in the group receiving the drug DMD AT to CD4 was only 10% higher than the survival in the control group.

Thus, as a result of the study, it was shown that the use of the drug SMD AT for TNF-α + SMD AT for CD4 has a more pronounced antiviral effect than the use of the drug SMD AT for TNF-α or the drug SMD AT for CD4, despite the fact that the dose of each component in the composition of SMD AT to TNF-α + SMD AT is two times lower than the dose of the components in the preparation of SMD AT to TNF-α and in the preparation of SMD AT to CD4, tested as separate preparations.

Example 3

Evaluation of the antiretroviral activity of the claimed complex drug containing activated-potentiated forms of aqueous dilution of polyclonal affinity purified rabbit antibodies to interferon alpha in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , 100 ³⁰ 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50, the activated-potentiated form aqueous dilution of affinity-purified rabbit polyclonal antibodies to inte Fearon gamma in ultralow doses (CMD) received 100 ^12, 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50, the activated-potentiated form aqueous sverhrazvedeniem initial matrix solution (2.5 mg / ml) dilution of polyclonal affinity purified rabbit antibodies to the CD4 receptor in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C50 and activated e-potentiated form aqueous dilution of polyclonal rabbit affinity purified antibody to CD8 receptor in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) ¹² 100, 100 ^30, 100 ⁵⁰ times equivalent to centesimal homeopathic mixture dilution of C12, C30, C50 in a ratio of 1: 1: 1: 1 (SMD AT to IFN-α + IFN-γ + CD4 + CD8) was performed using human peripheral blood mononuclear cells infected with an in vitro strain of HIV-1-LAI . Azidothymidine (Sigma-AZ169-100 mg, lot 107 K1578) was used as a comparison drug.

Human peripheral blood mononuclear cells were isolated from the blood of a healthy seronegative donor by centrifugation in a density gradient of ficoll-hypak. Cells were activated for 3 days using 1 μg / ml phytohemagglutin R and 5 IU / ml recombinant human interleukin-2 in RPMI1640 medium (DIFCO) with 10% fetal calf serum (complement was removed by heating for 45 minutes at 56 ° C ), 1% antibiotic solution (Gibco PSN containing 50 μg / ml penicillin, 50 μg / ml streptomycin and 100 μg / ml neomycin).

Cells were infected with the HIV-1-LAI strain, delivering 50 μl of the inoculum of the HIV-1-LAI strain, corresponding to a dose of 100 TCID50 (dose infecting 50% of tissue culture cells).

To evaluate antiretroviral activity, the preparation of SMD AT for IFN-α + IFN-γ + CD4 + CD8 and the comparison drug azidothymidine were introduced into the wells 15-30 minutes after infection of the cells with the HIV-1-LAI strain. On the 7th day after infection of the cells, the supernatant was collected, used to evaluate the effect of the drugs on inhibition of HIV replication.

Before entering into wells containing 150 μl of cell culture, the preparation of SMD AT for IFN-α + IFN-γ + CD4 + CD8 was diluted with RPMI1640 medium (DIFCO) 4 times (dilution 1/4) to achieve a final volume of 50 μl, azidothymidine was diluted with RPMI1640 medium (DIFCO) to a concentration of 8 nM.

Drug efficacy was determined by inhibition of HIV replication, which was assessed by the enzymatic activity of HIV reverse transcriptase in supernatants of human peripheral blood mononuclear cells using the HIV RT RetroSys kit manufactured by INNOVAGEN (lot 10-059C). To calculate% inhibition of HIV replication, a cell supernatant was used as a control to which the SMD AT drug for IFN-α + IFN-γ + CD4 + CD8 and the azidothymidine preparation were not added (Table 5).

Table 5. Antiretroviral activity of the drug SMD AT to IFN-α + IFN-γ + CD4 + CD8 using human peripheral blood mononuclear cells infected with in vitro strain HIV-1-LAI A drug The degree of dilution of the drug in RPMI1640 medium (DIFCO) Inhibition of enzymatic activity of HIV reverse transcriptase (% of control) 7 days SMD AT to IFN-α + IFN-γ + CD4 + CD8, in the ratio 1: 1: 1: 1 1/4 81 ± 11 Azidothymidine (8 nM) - 58 ± 7

Thus, under the conditions of this experimental model, it was shown that the preparation of SMD AT to IFN-α + IFN-γ + CD4 + CD8B in a ratio of 1: 1: 1: 1 has antiretroviral activity.

Example 4

Evaluation of the antiretroviral activity of the claimed complex drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to the CD4 receptor in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , 100 ³⁰ 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 and activated-potentiated form aqueous dilution of affinity purified polyclonal rabbit antibodies interferon gamma in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) 100 ^12, 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 in the ratio of 1: 1 (SMD AT to CD4 + IFN-γ), as well as a drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to CD4 receptor in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent a mixture of hundreds of homeopathic dilutions of C12, C30, C50 (SMD AT to CD4) and a drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to interferon gamma in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2 , 5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C50 (SMD AT to IFN-γ), was carried out using human peripheral blood mononuclear cells infected in v itro strain of HIV-1-LAI. Azidothymidine (Sigma-AZ169-100 mg, lot 107 K1578) was used as a comparison drug.

Human peripheral blood mononuclear cells were isolated from the blood of a healthy seronegative donor by centrifugation in a density gradient of ficoll-hypak. Cells were activated for 3 days using 1 μg / ml phytohemagglutin R and 5 IU / ml recombinant human interleukin-2 in RPMI1640 medium (DIFCO) with 10% fetal calf serum (complement was removed by heating for 45 minutes at 56 ° C ), 1% antibiotic solution (Gibco PSN containing 50 μg / ml penicillin, 50 μg / ml streptomycin and 100 μg / ml neomycin).

Cells were infected with the HIV-1-LAI strain, delivering 50 μl of the inoculum of the HIV-1-LAI strain, corresponding to a dose of 100 TCID50 (dose infecting 50% of tissue culture cells).

To evaluate antiretroviral activity, the preparations were added to the wells 15-30 minutes after infection of the cells with the HIV-1-LAI strain. On the 7th day after infection of the cells, the supernatant was collected, used to evaluate the effect of the drugs on inhibition of HIV replication.

Before entering into wells containing 150 μl of cell culture, the preparations were diluted with RPMI1640 medium (DIFCO) until a final volume of 50 μl was reached. The preparation of SMD AT to CD4 and the preparation of SMD AT to IFN-γ were diluted 8 times in RPMI1640 medium (DIFCO) (1/8 dilution), and the preparation of SMD AT to CD4 + IFN-γ was diluted 4 times in RPMI1640 medium (DIFCO) (dilution 1/4). Thus, the number of components of the preparation of SMD AT to CD4 + IFN-γ introduced into the experimental well during testing is identical to the number of components of the preparation of SMD AT to CD4 and components of the preparation SMD AT to IFN-γ and tested as single drugs, which allows a comparison of the effectiveness of the drug SMD AT to CD4 + IFN-γ with its individual components that make up its composition. The reference drug azidothymidine was diluted with RPMI1640 medium (DIFCO) until a concentration of 8 nM was reached.

Drug efficacy was determined by inhibition of HIV replication, which was assessed by the enzymatic activity of HIV reverse transcriptase in supernatants of human peripheral blood mononuclear cells using the HIV RT RetroSys kit manufactured by INNOVAGEN (lot 10-059C). To calculate% inhibition of HIV replication, the supernatant of cells to which the test drugs or azidothymidine were not added was used as a control (Table 6).

Table 6. Antiretroviral activity of drugs using mononuclear cells of human peripheral blood infected with an in vitro strain of HIV-1-LAI A drug The degree of dilution of the drug in RPMI1640 medium (DIFCO) Inhibition of enzymatic activity of HIV reverse transcriptase (% of control) 7 days SMD AT to CD4 + IFN-γ 1/8 0 ± 5 SMD AT to CD4 1/8 67 ± 22 SMD AT to CD4 + IFN-γ, in the ratio 1: 1 1/4 85 ± 1 Azidothymidine (8 nM) - 58 ± 7

Thus, under the conditions of this experimental model, it was shown that the antiretroviral activity of the drug DMD AT to CD4 + IFN-γ is superior to the antiretroviral activity of the drug DMD AT to CD4 and IFN-γ.

Example 5

Evaluation of the antiretroviral activity of the claimed complex drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to the CD4 receptor in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , 100 ³⁰ 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 and activated-potentiated form aqueous dilution of affinity purified polyclonal rabbit antibodies interferon gamma in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) 100 ^12, 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 in the ratio of 1: 1 (SMD AT to CD4 + IFN-γ), as well as a drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to CD4 receptor in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent a mixture of hundreds of homeopathic dilutions of C12, C30, C50 (SMD AT to CD4) and a drug containing activated-potentiated forms of aqueous dilutions of polyclonal affinity purified rabbit antibodies to interferon gamma in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (concentration 2 , 5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C50 (SMD AT to IFN-γ), was carried out using macrophages obtained from human peripheral blood mononuclear cells eca, and infected in vitro strain of HIV-1-Ba-L. Azidothymidine (Sigma-AZ169-100 mg, lot 107 K1578) was used as a comparison drug.

Human peripheral blood macrophages were obtained from mononuclear cells of human peripheral blood, which were isolated from the blood of two healthy seronegative donors by centrifugation in a density gradient of ficoll-hypak. Human peripheral blood mononuclear cells were grown for 3 days in RPMI1640 medium (DIFCO) supplemented with 10% fetal calf serum (complement was removed by heating for 45 minutes at 56 ° C), 1% antibiotic solution (Gibco PSN containing 50 μg / ml penicillin, 50 μg / ml streptomycin and 100 μg / ml neomycin), 15 ng / ml GM-CSF (granulocyte-macrophage colony stimulating factor). The cells were then placed on culture plates (150,000 cells / well in a 48-well plate), grown for 7 days together with 1 ng / ml GM-CSF and 10 ng / ml M-CSF (macrophage colony stimulating factor) so that the cells could differentiate completely into macrophages.

Cells were infected with HIV-1-Ba-L strain, delivering 100 μl of the inoculum of HIV-1-Ba-L strain, which corresponds to a dose of 1000 TCID50 (dose infecting 50% of tissue culture cells).

To assess antiretroviral activity, the preparations were added to the wells 24 hours before the cells were infected with the HIV-1-Ba-L strain, as well as on days 3, 7, 10, 14, 17 after infection. On the 3rd, 7th, 10th, 14th, 17th day after infection of the cells, the supernatant was collected, which was used to assess the effect of drugs on inhibition of HIV replication.

Before entering into wells containing 750 μl of cell culture, the preparations were diluted with RPMI1640 medium (DIFCO) until a final volume of 250 μl was reached. The preparation of SMD AT to CD4 and the preparation of SMD AT to IFN-γ were diluted in RPMI1640 medium (DIFCO) 8 times (1/8 dilution), and the preparation of SMD AT to CD4 + SMD AT to IFN-γ was diluted in RPMI1640 medium (DIFCO ) 4 times (1/4 dilution rate). Thus, the number of components of each component in the composition of SMD AT to CD4 + SMD AT to IFN-γ, introduced into the experimental well during testing, is identical to the number of components in the composition of SMD AT to CD4 and the components in the composition of SMD AT to IFN-γ tested as monopreparations, which allows a comparison of the effectiveness of the drug SMD AT to CD4 + SMD AT to IFN-γ with its individual components that make up its composition. The reference preparation azidothymidine was diluted with RPMI1640 medium (DIFCO) to a concentration of 8 nM.

Drug efficacy was determined by inhibition of HIV replication, which was evaluated by the enzymatic activity of HIV reverse transcriptase in supernatants of human peripheral blood macrophages using HIV RT RetroSys kit manufactured by INNOVAGEN (lot 10-059C). To calculate% inhibition of HIV replication, a cell supernatant was used as a control for which the test preparation SMD AT to CD4 + SMD AT to IFN-γ, the preparation SMD AT to CD4, the preparation SMD AT to IFN-γ and the azidothymidine comparison drug were used (Tables 7 and 8).

Table 7. Antiretroviral activity of drugs using macrophages of human peripheral blood (donor No. 1) infected with an in vitro strain of HIV-1-Ba-L A drug The degree of dilution of the drug in RPMI1640 medium (DIFCO) Inhibition of the enzymatic activity of HIV reverse transcriptase (% of control) 14 days 17 days 21 days SMD AT to IFN-γ 1/8 24 ± 4 24 ± 4 0 ± 0 SMD AT to CD4 SMD AT to CD4 + 1/8 53 ± 13 37 ± 7 0 ± 0 SMD AT to IFN-γ, in the ratio 1: 1 1/4 69 ± 1 74 ± 9 37 ± 3 Azidothymidine (8 nM) - 97 ± 1 97 ± 0 98 ± 2

Table 8. Antiretroviral activity of drugs using macrophages of human peripheral blood (donor No. 2) infected with an in vitro strain of HIV-1-Ba-L A drug The degree of dilution of the drug in RPMI1640 medium (DIFCO) Inhibition of the enzymatic activity of HIV reverse transcriptase (% of control) 14 days 17 days 21 days SMD AT to IFN-γ 1/8 39 ± 20 0 ± 0 0 ± 0 SMD AT to CD4 1/8 0 ± 0 0 ± 0 0 ± 0 SMD AT to CD4 + SMD AT to IFN-γ, in the ratio 1: 1 1/4 50 ± 5 42 ± 4 30 ± 6 Azidothymidine (8 nM) - 82 ± 2 54 ± 1 41 ± 1

Thus, under the conditions of this experimental model, it was shown that the antiretroviral activity of the drug SMD AT to CD4 + SMD AT to IFN-γ is superior to the antiretroviral activity of the drug SMD AT to IFN-γ and the drug SMD AT to CD4; the antiretroviral activity of the drug SMD AT to CD4 + SMD AT to IFN-γ is maintained throughout the experiment, in contrast to the antiretroviral activity of the drug SMD AT to IFN-γ and the drug SMD AT to CD4; the preparation of SMD AT to CD4 + SMD AT to IFN-γ exerts antiretroviral activity in an in vitro model of infected macrophages of human peripheral blood obtained from different seronegative donors, which indicates a more pronounced antiretroviral effect of the preparation SMD AT to CD4 + SMD AT to IFN-γ , in contrast to the preparation of SMD AT for IFN-γ and the preparation of SMD AT for CD4, the antiretroviral activity of which was detected in an in vitro model of infected macrophages of human peripheral blood obtained from only one seronegative donor.

Example 6

To study the properties of the claimed drugs for the treatment of patients of the active drug group No. 1, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity-purified rabbit antibodies to human gamma interferon (AT to IFN-γ) and CD4 receptor (AT to CD4) in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred homeopathically x dilution of C12, C30, C200 (SMD AT to IFN-γ + AT to CD4); for the treatment of patients of the active drug group No. 2, 300 mg tablets were used, impregnated with a pharmaceutical composition containing aqueous-alcoholic solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (AT to IFN-γ ), CD4 receptor (AT to CD4) and histamine (AT to H) in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C200 ( SMD AT to IFN-γ + AT to CD4 + AT to H); for the treatment of patients of the active drug group No. 3, 300 mg tablets were used, impregnated with an aqueous-alcoholic solution (3 mg / tablet) of an activated-potentiated form of rabbit anti-human gamma interferon polyclonal antibodies purified on antigen in an ultra-low dose obtained by super-dilution of the original matrix solution in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions of C12, C30, C50 (SMD AT to IFN-γ).

Evaluation of the effectiveness of three drugs containing SMD AT to IFN-γ + AT to CD4, SMD AT to IFN-γ + AT to CD4 + AT to H and SMD AT to IFN-γ in the treatment of chronic viral hepatitis C (CHC) was carried out in a comparative study in parallel groups involving 18 patients (14 men and 4 women) aged 27 to 52 years. The diagnosis of HCV was confirmed based on the profile of serum AT markers for HCV and HCV RNA. All patients included in the study had 2 or 3 HCV genotypes, a low-symptom slowly progressive chronic hepatitis C course with low activity (less than a 3-fold increase in serum aminotransferase levels, or <100 U / L), none of the patients had previously received antiviral therapy. The study did not include patients with a positive serological test for HIV, RW, anti-HCA, HBsAg or HBcor Ab, with cirrhosis of the liver, severe concomitant diseases in the acute stage, thalassemia or other hemoglobinopathy, alcohol and / or drug / drug dependence, after organ transplants, constantly taking immunosuppressive drugs, pregnant and lactating women. Patients of the first three groups were prescribed therapy with any studied drug at a dose of 1 tablet 3 times a day for 24 weeks: for patients of group 1 (n = 5) - composition of SMD AT to IFN-γ + AT to CD4; 2 groups (n = 4) - composition of SMD AT to IFN-γ + AT to CD4 + AT to H; 3 groups (n = 4) - SMD AT to IFN-γ The control group (No. 4) consisted of 5 patients with persistent viremia and persistently normal levels of aminotransferases (<20 IU / L) who were not prescribed any therapy. During the study, medical examinations, monitoring of viral load and laboratory parameters were carried out, concomitant therapy was recorded, possible undesirable effects. At the 24th week, the HCV RNA viral load and the activity of alanine aminotransferase (ALT) were evaluated as criteria for the effectiveness of treatment.

The data on the viral load (the number of copies of HCV RNA) presented in table 9 as the median (Me), first and third quartiles (Q1, Q3) indicate its positive dynamics obtained in patients of groups 1-3 by the end of the 24-week therapy. Admission of the drug SMD AT to IFN-γ + AT to CD4 led to a decrease in the number of copies of HCV RNA in 4 out of 5 people of the 1st group, while the average decrease in viral load was 75%. Similar indicators were obtained in patients who took the drug SMD AT to IFN-γ + AT to CD4 + AT to H for 24 weeks: antiviral activity was recorded in all patients (4 out of 4 people in group 2), the average decrease in viral load was 70% Moreover, in 2 patients (one from group 1 and group 2), a complete virological response was obtained at the end of therapy. The antiviral activity of the monocomponent drug SMD AT to IFN-γ was slightly lower, since a decrease in the number of copies of HCV RNA was noted in 3 of 4 patients of group 3, the average decrease in viral load was 55%. Among the participants in the study of the control group, no positive dynamics of the viral load was detected.

Table 9. Dynamics of viral load in the studied groups HCV RNA, copy / ml The average decrease in viral load,% SMD AT to IFN-γ + AT to CD4 (Me [Q1-Q3]) Originally 66200 [450-181400] 75 After 24 weeks of treatment 12500 [50-30560] SMD AT to IFN-γ + AT to CD4 + AT to H (Me [Q1-Q3]) Originally 58900 [600-124500] 70 After 24 weeks of treatment 15600 [50-45700] SMD AT to IFN-γ (Me [Q1-Q3]) Originally 84700 [350-172800] 55 After 24 weeks of treatment 22400 [150-58500] The control group (Me [Q1-Q3]) Originally 79500 [300-155600] - After 24 weeks of treatment 87900 [450-164300]

The antiviral activity of the studied drugs was combined with the positive dynamics of ALT level, which was registered in patients of groups 1-3 by the end of 24 weeks of treatment. Normalization of ALT level was observed in 2 patients of the SMD AT group to IFN-γ + AT to CD4, in 1 patient of the SMD AT group to IFN-γ + AT to CD4 + AT to N, and also in 1 patient of the SMD AT group to IFN-γ . In 1 patient of the control group, against the background of an increase in viral load, after 24 weeks of observation, the ALT indicator exceeded the upper limit of the norm (> 20 U / L).

During the observation, no adverse events associated with the administration of the studied drugs were detected, which indicated their good tolerability. The absence of pathological abnormalities from blood and urine tests, as well as biochemical parameters, including renal and hepatic markers, confirmed the safety of treatment.

Thus, as a result of the study, data were obtained on the efficacy and safety of drugs SMD AT to IFN-γ + AT to CD4, SMD AT to IFN-γ + AT to CD4 + AT to H and SMD AT to IFN-γ, in the treatment of patients with CHC. The most distinct antiviral effect was observed in the preparation of SMD AT to IFN-γ + AT to CD4 and SMD AT to IFN-γ + AT to CD4 + AT to H, which was confirmed by the positive dynamics of the viral load, as well as the complete virological response recorded in 2 patients to end of 24 weeks of therapy. The antiviral efficacy of drugs containing SMD AT to IFN-γ + AT to CD4, SMD AT to IFN-γ + AT to CD4 + AT to H and SMD AT to IFN-γ was combined with a decrease in chronic hepatitis C activity, which was expressed in a decrease and even normalization of ALT level in some patients by the end of a 24-week course of therapy.

Example 7

To study the properties of the claimed drugs for the treatment of patients of the active drug group No. 1, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity-purified rabbit antibodies to human gamma interferon (anti-IFN-γ) and CD4 receptor (anti-CD4) at ultra low doses (RMA) obtained sverhrazvedeniem initial matrix solution 100 ^12, 100 ^30, 100 ^{to 200} times equivalent mixture of centesimal gomeopatiches their dilutions C12, C30, C200 (SMD anti-IFN-γ + anti-CD4); for the treatment of patients of the active drug group No. 2, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (anti-IFN-γ ), to the CD4 receptor (anti-C04) and histamine (anti-H) in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions of C12, C30, C200 (SMD anti-IFN-γ + a ti-CD4 + anti-H). To treat patients of the comparison group, 300 mg tablets were used, impregnated with an aqueous-alcoholic solution (3 mg / tablet) of an activated-potentiated form of rabbit anti-human gamma interferon polyclonal antibodies purified on antigen in an ultra-low dose obtained by super-dilution of the initial matrix solution in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, the equivalent mixture of hundreds of homeopathic dilutions of C12, C30, C50 (SMD anti-IFN-γ).

The antiretroviral activity of the SMD compositions anti-IFN-γ + anti-CD4 and SMD anti-IFN-γ + anti-CD4 + anti-H was evaluated in an open comparative clinical trial in patients infected with human immunodeficiency virus (HIV), which were observed in Territorial Center for the Prevention and Control of AIDS and Infectious Diseases. The study included 65 men and 32 women (a total of 97 patients) aged 18 to 48 years with a viral load of ≥150 copies of HIV-1 RNA in 1 ml of plasma and a CD4-lymphocyte level of at least 250 cells / μl (or ≥0 , 25 × 10 ⁹ / L). 34 of the 97 study participants were observed for asymptomatic infectious status and had not previously received treatment for HIV infection (naive patients). 63 of 97 patients were on antiretroviral therapy (APT) for 1-2 years. The study did not include patients with liver cirrhosis, viral hepatitis C, severe concomitant diseases in the acute stage, pregnant women, as well as persons taking intravenous drugs. The study was conducted in the autumn-winter period, when there was a seasonal increase in the incidence of influenza and acute respiratory infections (ARVI).

75 patients were randomized into three groups, they were prescribed therapy with group 1 and 2 drugs or group 3 comparison drug in a prophylactic (against influenza / SARS) dosage - 1 tablet 1 time per day for 6 weeks, including: patients group 1 (n = 25) was assigned the composition of SMD anti-IFN-γ + anti-CD4 in the form of monotherapy (subgroup 1A: naive patients, n = 12) or in combination with APT (subgroup 1B, n = 13); group 2 patients (n = 23) were prescribed SMD anti-IFN-γ + anti-CD4 + anti-H compositions, including monotherapy (subgroup 2A: naive patients, n = 11) or in combination with APT ( subgroup 2B, n = 12); group 3 patients (n = 27) were prescribed SMD anti-IFN-γ, including monotherapy (subgroup 3A: naive patients, n = 11) or in combination with APT (subgroup 3B, n = 16). The control (fourth) group (n = 22) consisted of patients who continued to receive APT according to the previous scheme without adding any study drug (APT group).

At baseline and after 6 weeks, all patients were assessed for viral load, CD4 and CD8 lymphocyte counts, and CD4 / CD8 immunoregulatory index. To determine the copies of HIV-1 RNA in blood plasma, we used the COBAS AMPLICOR HIV-1 MONITOR Kit, version 1.5 for the automatic PCR analyzer COBAS AMPLICOR (Roche, Switzerland). Phenotyping of circulating peripheral blood lymphocytes was performed on a FACSCount flow cytometer (Becton Dickinson, USA) using standard FACSCount Reagent Kit test systems containing antibodies to CD3, CD4, CD8 labeled with FITC, PE fluorochromes.

Table 10 presents the data on the viral load (the number of copies of HIV RNA) in the form of the median (Me), first and third quartiles (Q1, Q3) in patients of the studied groups in the dynamics of observation. As a result of the study, it was found that a 6-week intake of the SMD anti-IFN-γ + anti-CD4 composition reduced the number of copies of HIV-1 RNA in 58% of naive patients (in 7 of 12 people of subgroup 1A), while the average decrease in viral the load was 16.9%. The combined use of the composition of SMD anti-IFN-γ + anti-CD4 and APT was no less effective, since a decrease in the number of copies of HIV-1 RNA was observed in 62% of the study participants (in 8 out of 13 people of subgroup 1B), and the average viral load reduction was 18.2% of the baseline. Approximately similar indicators were obtained using the composition of SMD anti-IFN-γ + anti-CD4 + anti-H: antiviral activity was recorded in 55% of HIV-infected naive patients (in 6 of 11 people of subgroup 2A) and in 67% of people who received the combination SMD anti-IFN-γ + anti-CD4 + anti-H and APT (in 8 of 12 people of subgroup 2B); the average decrease in viral load was 17.3% and 18.9%, respectively. The antiretroviral activity observed in the first two groups was slightly higher than the treatment results obtained in patients of the comparison group. After 6 weeks, monotherapy with SMD anti-IFN-γ reduced the number of copies of HIV-1 RNA in 36% of naive patients (in 4 of 11 people of subgroup 3A), while the decrease in viral load averaged 9.5%. The combined use of the monocomponent drug SMD anti-IFN-γ and APT increased the effectiveness of therapy, helping to reduce the viral load in 50% of the study participants (in 8 of 16 people of subgroup 3B) by an average of 14.2%. Similar treatment indices in individuals receiving only APT (control group 4) were 32% (in 7 of 22 people in group 4) and 13.3%, respectively.

The analysis of the indicators of the subpopulations of circulating lymphocytes in the dynamics of observation (Table 11) showed a more pronounced than in the control group increase in the number of CD4 lymphocytes after 6 weeks of therapy using SMD anti-IFN-γ + anti-CD4, SMD anti-IFN-γ + anti -CD4 + anti-H and SMD anti-IFN-γ as monotherapy in naive patients (subgroups 1A, 2A and 3A) or in combination with APT (subgroups 1B, 2B and 3B). Moreover, the number of CD8 after 6 weeks of drug administration (mono-or combination therapy) did not change in any of the studied groups. The positive dynamics of CD4 lymphocytes during treatment led to an increase in the immunoregulatory index of CD4 / CD8, the most significant in the subgroups of patients taking SMD compositions anti-IFN-γ + anti-CD4 and anti-IFN-γ + anti-CD4 + anti H (mono - or combination therapy - groups 1 and 2) and SMD anti-IFN-γ in combination with APT (subgroup 3B).

Table 10 Dynamics of viral load depending on the treatment regimen Viral load, copies / ml The average decrease in viral load,% SMD anti-IFN-γ + anti-CD4 (Me [Q1-Q3]) Originally 5769 [368-62584] 16.9 After 6 weeks of treatment 4575 [337-58526] APT and SMD anti-IFN-γ + anti-CD4 (Me [Q1-Q3]) Originally 5238 [385-59695] 18.2 After 6 weeks of treatment 4408 [320-50197] SMD anti-IFN-γ + anti-CD4 + anti-H (Me [Q1-Q3]) Originally 5638 [385-61742] 17.3 After 6 weeks of treatment 4754 [278-57426] APT and SMD anti-IFN-γ + anti-CD4 + anti-H (Me [Q1-Q3]) Originally 5189 [350-59798] 18.9 After 6 weeks of treatment 46108 [269-47987] SMD anti-IFN-γ (Me [Q1-Q3]) Originally 5813 [150-33356] 9.5 After 6 weeks of treatment 5786 [150-38359] APT and SMD anti-IFN-γ (Me [Q1-Q3]) Originally 4680 [274-9838] 14.2 After 6 weeks of treatment 4652 [272-8874] APT (Me [Q1-Q3]) Originally 5547 [385-58996] 13.3 After 6 weeks of treatment 5308 [338-57709]

Table 11 Indicators of subpopulations of circulating lymphocytes in patients of the studied groups in the dynamics of observation Observation period CD4, cells / μl (M ± SE) CD4 / CD8 (M ± SE) SMD ANTI-IFN-γ + ANTI-CD4 (n = 12) Originally 516 ± 33 0.46 ± 0.09 After 6 weeks of treatment 712 ± 24 0.58 ± 0.07 * APT and SMD anti-IFN-γ + anti-CD4 (n = 13) Originally 499 ± 41 0.50 ± 0.08 After 6 weeks of treatment 728 ± 29 0.60 ± 0.06 * SMD anti-IFN-γ + anti-CD4 + anti-H (n = 11) Originally 509 ± 45 0.49 ± 0.06 After 6 weeks of treatment 706 ± 27 0.58 ± 0.08 * APT and SMD anti-IFN-γ + anti-CD4 + anti-H (n = 12) Originally 521 ± 37 0.48 ± 0.09 After 6 weeks of treatment 734 ± 22 0.62 ± 0.10 * SMD anti-IFN-γ (n = 11) Originally 513 ± 98 0.38 ± 0.19 After 6 weeks of treatment 563 ± 26 0.44 ± 0.12 APT and SMD anti-IFN-γ (n = 16) Originally 491 ± 49 0.55 ± 0.06 After 6 weeks of treatment 623 ± 45 0.67 ± 0.05 * APT (n = 22) Originally 510 ± 29 0.44 ± 0.06 After 6 weeks of treatment 595 ± 35 0.50 ± 0.12 * significant differences compared to baseline, p <0.05

The absence in patients during treatment of adverse events associated with the administration of the studied drugs indicated a good tolerance to the drugs. Repeated monitoring of laboratory parameters, including blood and urine tests, biochemical markers, confirmed the safety of treatment.

Thus, the study demonstrated the antiretroviral efficacy of the drug SMD anti-IFN-γ + anti-CD4 and the drug SMD anti-IFN-γ + anti-CD4 + anti-H, apparently due to a change in the functional activity of CD4 receptors, which prevents the penetration of HIV into cells, as well as suppression of HIV replication inside the cell due to activation of transcription processes of antiviral protein mRNA. It was found that the decrease in viral load observed as a result of a 6-week intake of the drug SMD anti-IFN-γ + anti-CD4 and the drug SMD anti-IFN-γ + anti-CD4 + anti-H at a dose of 1 tablet per day exceeds the antiviral the effect that is recorded after 6 weeks of taking the monocomponent drug SMD anti-IFN-γ in the same dose or continuing according to the previous APT regimen. The combination of any drug SMD anti-IFN-γ + anti-CD4, SMD anti-IFN-γ + anti-CD4 + anti-H or SMD anti-IFN with APT slightly enhances the antiviral activity of the latter, which manifests itself in a decrease in the average value of the viral load after 6 weeks in a larger percentage of study participants.

The effect of SMD preparations anti-IFN-γ + anti-CD4 and anti-IFN-γ + anti-CD4 + anti-H on the ratio of CD4 / CD8 lymphocytes in HIV-infected patients (due to a slight increase in the absolute number of CD4 cells) was shown. most pronounced when used with APT. Given the simultaneous decrease in viral load in patients taking SMD anti-IFN-γ + anti-CD4 and SMD anti-IFN-γ + anti-CD4 + anti-H drugs, we can assume that the increase in the number of CD4 cells is associated with an increase in the population of these cells due to healthy (uninfected) cells. The combination of APT with SMD anti-IFN-γ + anti-CD4, SMD anti-IFN-γ + anti-CD4 + anti-H or SMD anti-IFN is more effective in restoring the immunoregulatory index of CD4 / CD8 than continuing APT over the previous scheme.

The antiretroviral activity of SMD anti-IFN-γ + anti-CD4 and SMD anti-IFN-γ + anti-CD4 + anti-H drugs allows them to be used for prophylactic and therapeutic purposes both in naive HIV-infected patients and in patients receiving Apt.

Example 8

For experimental study of the claimed technical solution, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (AT GI), to the CD4 receptor (aT CD4), histamine (aT T) in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution 100 ^12, 100 ^30, 100 ^{to 200} times of each component, equivalent mixture of centesimal homeopathic dilutions 12, C30, C200, deposited on lactose as a water-alcohol mixture of homeopathic dilutions C12, C30, C200 each (AT GOP + AT CD4 + AT T). The placebo group received 300 mg tablets containing lactose monohydrate, microcrystalline cellulose, magnesium stearate.

The ongoing double-blind, placebo-controlled study included patients aged 18-60 years of both sexes, with acute respiratory viral infection with signs of intoxication and catarrhal syndrome. The study includes patients with a body temperature of 37.8 ° C or more (provided that such a temperature is recorded from the moment of the onset of the disease), the duration of the disease is not more than 48 hours before treatment, without severe concomitant diseases. Patients undergo a rapid test for the presence of influenza antigens. Patients with positive test results are not included in the study. All patients give written voluntary consent to participate in the study before starting all study procedures. Patients fill out diaries in which they record body temperature twice a day, concomitant therapy, etc. Patients receive AT GI + AT CD4 + AT G or placebo, depending on the treatment group, 8 tablets on the first day and three tablets per day from the second to fifth days. Patients can take antipyretic drugs if necessary. The use of antiviral, immunomodulatory, antihistamines and antibiotics is not allowed. Before the first dose and at the last visit, patients give blood and urine samples for laboratory tests in order to monitor the safety of the therapy. Treatment lasts 5 days, follow-up - 2 days. Thus, on average, each patient takes part in the study within 7 days.

The effectiveness of therapy was evaluated by the timing of reaching body temperature of 37.0 ° C or less, the number of antipyretic doses was also compared.

At the time of the analysis, 78 patients completed the therapy (40 patients received AT GI + AT CD4 + AT G, 38 received a placebo). The proportion of patients with a body temperature of 37.0 ° C or less is presented in Figure 1. As can be seen from Figure 1, therapy AT GI + AT CD4 + AT G by the end of the second day from the start of therapy allowed to achieve a decrease in body in patients in 17.4% of cases more often than in the placebo group (p <0.05). At the same time, the number of antipyretic drugs in the groups was statistically significantly lower at the same time in the AT GI + AT CD4 + AT G group (3.5 ± 0.25 antipyretic drugs since the start of AT GI + AT CD4 + AT therapy G by the end of the second day of treatment, versus 3.9 ± 0.32 in the placebo group, p <0.005). The superiority of AT GI + AT CD4 + AT G over placebo, which began already on the morning of the second day of treatment, continued throughout the entire treatment period.

The safety analysis included data from all 78 patients who participated in the study and completed treatment within the protocol established deadlines, there were no early drop-out patients. During the entire observation period, AT GI + AT CD4 + AT G was well tolerated. Adverse events associated with taking AT GI + AT CD4 + AT G were not observed. A blood test at the beginning and end of the treatment period did not reveal pathological abnormalities. A general urinalysis on the first and final day of the study also did not reveal any pathology in any patient.

When comparing the data with the results of a 2005 double-blind, placebo-controlled, randomized study of the clinical efficacy and safety of the therapeutic use of a drug containing activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (AT GI) in ultra-low doses (SMD) obtained by ultra-dilution the initial matrix solution is 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C200 (SMD AT to GI) in influenza and other acute respiratory viral infections (State Research Institute of Influenza RAMS, St. Petersburg, 2005), it was found that the drug AT GI + AT CD4 + AT G is more effective than the SMD AT and GI lower the temperature (Table 12, Table 13 and Figure 1).

Table 12. The proportion of patients with a body temperature of 37.0 ° C or less while taking AT GI + AT CD + AT G / placebo 1 day, morning 1 day, evening 2 days, morning 2 days, evening 3 days, morning 3 days, evening 4 days, morning 4 days, evening 5 days, morning 5 days, evening 6 days, morning 6 days, evening 7 days, morning AT GI + AT CD4 + AT G, t = 40 Total number of patients 40 40 40 40 40 40 40 40 40 40 40 40 40 Normal temperature number 19 twenty 24 28 27 thirty 31 33 36 38 40 40 40 Share with normal temperature,% 47.5 50.0 60.0 70.0 67.5 75.0 77.5 82.5 90.0 95.0 100.0 100.0 100.0 Placebo, t = 38 Total number of patients 38 38 38 38 38 38 38 38 38 38 38 38 38 Normal temperature number eighteen 17 19 twenty 23 24 27 24 29th thirty 33 37 38 Share with normal temperature,% 47.4 44.7 50.0 52.6 60.5 63.2 71.1 63.2 76.3 78.9 86.8 97.4 100.0 AT GI *, n = 30 Total number of patients thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty Normal temperature number 0 3 fourteen 12 eighteen 19 25 29th 29th thirty 29th thirty 29th Share with normal temperature,% 0 10.0 46.7 40.0 60.0 63.3 83.3 96.7 96.7 one hundred 96.7 one hundred 96.7 Placebo *, n = 30 Total number of patients thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty Normal temperature number 0 0 10 7 16 fifteen 28 28 28 thirty thirty thirty thirty Share with normal temperature,% 0 0 33.3 23.3 53.3 50.0 93.3 93.3 93.3 one hundred one hundred one hundred one hundred * According to a double-blind, placebo-controlled randomized study of the clinical efficacy and safety of the therapeutic use of the drug SMD AT for GI in influenza and other acute respiratory viral infections (GU Research Institute of Influenza RAMS, St. Petersburg, 2005).

Table 13. The average body temperature in patients depending on the treatment group, ° C, M ± SD 1 day, morning 1 day, evening 2 days, morning 2 days, evening 3 days, morning 3 days, evening 4 days, morning 4 days, evening 5 days, morning 5 days, evening 6 days, morning 6 days, evening 7 days, morning CD4 + AT G, n = 40 37.5 ± 0.54 37.7 ± 0.56 37.2 ± 0.67 37.1 ± 0.53 36.8 ± 0.43 36.8 ± 0.49 36.7 ± 0.31 36.6 ± 0.33 36.6 ± 0.25 36.6 ± 0.23 36.6 ± 0.22 36.6 ± 0.15 36.6 ± 0.18 Placebo, n = 38 37.6 ± 0.71 37.6 ± 0.63 37.2 ± 0.48 37.1 ± 0.49 36.9 ± 0.41 36.9 ± 0.36 36.8 ± 0.49 36.7 ± 0.37 36.6 ± 0.32 36.6 ± 0.21 36.6 ± 0.28 36.5 ± 0.18 36.5 ± 0.18 AT GI *, n = 30 38.1 ± 0.62 38.0 ± 0.58 37.4 ± 0.80 37.3 ± 0.61 37.1 ± 0.50 37.0 ± 0.47 36.8 ± 0.35 36.6 ± 0.32 36.6 ± 0.21 36.5 ± 0.26 36.6 ± 0.21 36.6 ± 0.26 36.6 ± 0.26 Placebo, * n = 30 38.0 ± 0.48 38.0 ± 0.50 37.4 ± 0.60 37.4 ± 0.47 37.0 ± 0.37 37.0 ± 0.42 36.8 ± 0.23 36.6 ± 0.34 36.6 ± 0.28 36.6 ± 5.42 36.6 ± 0.21 36.5 ± 0.24 36.6 ± 0.18 * According to a double-blind, placebo-controlled randomized study of the clinical efficacy and safety of the therapeutic use of the drug SMD AT for GI in influenza and other acute respiratory viral infections (GU Research Institute of Influenza RAMS, St. Petersburg, 2005).

Example 9

For experimental study of the claimed technical solution, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (AT GI), to the CD4 receptor molecule of T-lymphocytes (AT CD4), to histamine (AT G), in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times of each component, equivalent to a mixture of hundred homeopaths Atomic dilutions of C12, C30, C200 (AT GI + AT CD4 + AT G). As a comparison drug used the drug Tamiflu® (the active substance is oseltamivir, F. Hoffmann-La Roche Ltd.-Switzerland) (Oseltamivir).

The ongoing open comparative controlled clinical trial of AT GI + AT CD4 + AT G and Tamiflu® includes patients aged 18-60 years of both sexes, with influenza A or B with signs of intoxication and catarrhal syndrome. The study includes patients with a body temperature of 37.8 ° C or more (provided that such a temperature is recorded from the moment of the onset of the disease), the duration of the disease is not more than 48 hours before treatment, without severe concomitant diseases. Patients undergo a rapid test for the presence of influenza antigens. Patients with negative test results are not included in the study. All patients give written voluntary consent to participate in the study before starting all study procedures. Patients fill out diaries in which they record body temperature twice a day, concomitant therapy, etc. Patients receive AT GI + AT CD4 + AT G, 8 tablets on the first day and three tablets a day from the second to the fifth days, or Tamiflu® 75 mg twice daily, according to manufacturer's instructions. Patients can take antipyretic drugs if necessary. The use of antiviral, immunomodulatory, antihistamines and antibiotics is not allowed. Before the first dose and at the last visit, patients give blood and urine samples for laboratory tests in order to monitor the safety of the therapy. Treatment lasts 5 days, follow-up - 2 days. Thus, on average, each patient takes part in the study within 7 days.

The effectiveness of therapy was evaluated by the timing of reaching body temperature of 37.0 ° C or less, the number of antipyretic doses was also compared.

At the time of the analysis, the study completed 17 patients (6 patients in the AT GI + AT CD4 + AT G group and 11 patients in the Tamiflu® group).

The proportion of patients with a body temperature of 37.0 ° C or less in the groups did not statistically differ among themselves throughout the treatment period. By the beginning of 4 days of treatment in both groups was achieved almost complete recovery of patients (Figure 2). By the beginning of the second day of treatment in both groups, a third of patients had normalized body temperature. The average number of antipyretic doses in the groups also did not have statistically significant differences and at the beginning of the 4th day of therapy was 7.6 ± 0.84 in the AT GI + AT CD4 + AT G group and 7.4 ± 0.90 in the group taking Tamiflu®, respectively.

The safety analysis included data from all 17 patients participating in the study and completing treatment within the protocol established terms; there were no early drop-out patients. During the entire observation period, the drug was well tolerated, adverse events were not observed. A blood test at the beginning and end of the treatment period did not reveal pathological abnormalities. A general urinalysis on the first and final day of the study also did not reveal any pathology in any patient.

When comparing the data with the results of a 2005 double-blind, placebo-controlled, randomized study of the clinical efficacy and safety of the therapeutic use of a drug containing activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (AT GI) in ultra-low doses (SMD) obtained by ultra-dilution initial matrix solution 100 ^12, 100 ^30, 100 ^{to 200} times equivalent mixture of centesimal homeopathic dilutions C12, C30, C200 (SMD GR aT) n and influenza and other acute respiratory viral infections (State Research Institute of Influenza RAMP, St. Petersburg, 2005), it was found that the drug AT GI + AT CD4 + AT G is more effective than the SMD AT GI reduces the temperature (see Table 14, Table 15 and FIG. 2).

Table 14. The proportion of patients with a body temperature of 37.0 ° C or less while taking AT GI + AT CD4 + AT G / Tamiflu® morning day 1 evening day 1 morning day 2 evening day 2 morning day 3 evening day 3 morning day 4 evening day 4 morning day 5 evening day 5 morning day 6 evening, day 6 morning day 7 evening, day 7 morning day 8 AT GI + AT CD4 + AT G, n = 60 AT GI + AT CD4 + AT G, n = 6 Total number of patients 6 6 6 6 6 6 6 6 6 6 5 5 four four n / a Normal temperature number 0 0 2 3 four four 6 5 6 6 5 5 four four n / a Share with normal temperature,% 0 0 33.3 50.0 66.7 66.7 100.0 83.3 100.0 100.0 100.0 100.0 100.0 100.0 n / a AT GI *, Oseltamivir, n = 11 Total number of patients eleven eleven eleven eleven eleven eleven eleven 10 10 10 9 7 5 four 3 Normal temperature number 0 one four 5 5 6 10 8 9 9 9 7 5 four 3 Share with normal temperature,% 0 9.1 36.4 45.5 45.5 54.5 90.9 80.0 90.0 90.0 100.0 100.0 100.0 100.0 100.0 Total number of patients thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty Normal temperature number 0 3 fourteen 12 eighteen 19 25 29th 29th thirty 29th thirty 29th thirty thirty Share with normal temperature,% 0 10.0 46.7 40.0 60.0 63.3 83.3 96.7 96.7 one hundred 96.7 one hundred 96.7 one hundred one hundred Placebo *, n = 30 Total number of patients thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty thirty Normal temperature number 0 0 10 7 16 fifteen 28 28 28 thirty thirty thirty thirty thirty thirty Share with normal temperature,% 0 0 33.3 23.3 53.3 50.0 93.3 93.3 93.3 one hundred one hundred one hundred one hundred one hundred one hundred * According to a double-blind, placebo-controlled randomized study of the clinical efficacy and safety of the therapeutic use of the drug SMD AT GI for influenza and other acute respiratory viral infections (GU Research Institute of Influenza RAMS, St. Petersburg, 2005).

Table 15. The average body temperature in patients depending on the treatment group, ° C, M ± SD morning day 1 evening day 1 morning day 2 evening day 2 morning day 3 evening day 3 morning day 4 evening day 4 morning day 5 evening day 5 morning day 6 evening, day 6 morning day 7 evening, day 7 morning day 8 AT GI + AT CD4 + AT G, n = 6 38.5 ± 0.49 38.1 ± 0.62 37.2 ± 1.01 37.2 ± 0.67 36.5 ± 0.61 36.8 ± 0.47 36.5 ± 0.37 36.6 ± 0.46 36.5 ± 0.26 36.6 ± 0.28 36.4 ± 0.35 36.5 ± 0.20 36.4 ± 0.26 36.5 ± 0.22 n / a Oseltamivir, n = 11 38. ± 0.82 37.3 ± 0.71 37.3 ± 0.72 36.9 ± 0.53 36.9 ± 0.47 36.7 ± 0. 46 36.8 ± 0.37 36.5 ± 0.38 36.7 ± 0.25 36.5 ± 0.21 36.6 ± 0.21 36.3 ± 0.19 36.5 ± 0. 10 36.6 ± 0.12 36.5 ± 0.14 AT GI *, n = 30 38.1 ± 0.62 38.0 ± 0.58 37.4 ± 0.80 37.3 ± 0.61 37.1 ± 0.50 37.0 ± 0.47 36.8 ± 0.35 36.6 ± 0.32 36.6 ± 0.21 36.5 ± 0.26 36.6 ± 0.21 36.6 ± 0.26 36.63 ± 0.26 36.5 ± 0.27 36.5 ± 0.23 Placebo, * n = 30 38.0 ± 0.48 38.0 ± 0.50 37.4 ± 0.60 37.4 ± 0.47 37.0 ± 0.37 37.0 ± 0.42 36.8 ± 0.23 36.6 ± 0.34 36.6 ± 0.28 36.6 ± 5.42 36.6 ± 0.21 36.5 ± 0.24 36.6 ± 0. eighteen 36.5 ± 0.19 36.4 ± 0.21 * According to a double-blind, placebo-controlled randomized study of the clinical efficacy and safety of the therapeutic use of the drug SMD AT GI for influenza and other acute respiratory viral infections (GU Research Institute of Influenza RAMS, St. Petersburg, 2005).

Example 10

For experimental study of the claimed technical solution, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated forms of polyclonal affinity purified rabbit antibodies to human gamma interferon (AT GI), to the CD4 receptor molecule of T-lymphocytes (AT CD4), to histamine (AT G), in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times of each component, equivalent to a mixture of hundred homeopaths Atomic dilutions of C12, C30, C200 (AT GI + AT CD4 + AT G). As the reference drug using the drug containing activated-potentiated form of polyclonal affinity-purified rabbit antibody to gamma interferon, human (AT GOP) in ultralow doses (RMA) obtained sverhrazvedeniem initial matrix solution 100 ^12, 100 ^30, 100 ^{to 200} times equivalent mixture hundredths of homeopathic dilutions of C12, C30, C200 (SMD AT GI). The placebo group received 300 mg tablets containing lactose monohydrate, microcrystalline cellulose, magnesium stearate.

In the ongoing double-blind, placebo-controlled study of the efficacy and safety of the drug AT GI + AT CD4 + AT G in SARS (see Example 8) and in the ongoing open comparative controlled study of the efficacy and safety of the drug AT GI + AT CD4 + AT G in influenza (see Example 9) we additionally evaluated the number of complications that developed against the background of an acute infectious process, including bacterial ones (bacterial pneumonia, tracheitis, otitis media, glomerulonephritis, etc.).

In cases where the defense system is perfect, the infectious process may be interrupted or, remaining localized, not accompanied by the development of severe clinical symptoms, i.e. the adequacy of protective reactions leads to rapid inactivation of the pathogen, the restoration of impaired body functions and recovery. A different picture arises in an organism highly susceptible to an infectious agent and lacking the perfect mechanism of specific and nonspecific protection (immunocompromised patients). In such cases, pathogens reproduced in an increasing number and the products of their interaction with epithelial and immune cells, as well as the destroyed cells themselves enter the blood, causing the development of severe forms of the course of the disease, the formation of complications and a possible adverse outcome.

The use of the drug AT GI + AT CD4 + AT G both in influenza and in acute respiratory viral infections contributes to a significant reduction in the frequency of bacterial complications compared with placebo (Table 16), and, as a consequence, to a decrease in the need for antibiotic therapy. The drug AT GI + AT CD4 + AT G prevents the development of secondary immunodeficiency at the stage of convalescence, providing an immunomodulating effect and increasing the body's natural defenses. The ability of AT GI + AT CD4 + AT G to reduce the incidence of bacterial complications was greater than that of SMI AT GI.

Table 16. The incidence of bacterial complications A drug Patient count Bacterial complications Otitis count /% Tracheitis number /% Pneumonia qty /% Total Qty /% AT GI + AT CD4 + AT G (ARVI) 40 0/0 1 / 2.5 0/0 1 / 2.5 Placebo (ARVI) 38 3 / 7.9 7 / 18.4 0/0 10 / 26.3 AT GI + AT CD4 + AT G (Flu) 6 0/0 0/0 0/0 0/0 Tamiflu® (Flu) eleven 0/0 2 / 18.2 1/9.1 3 / 27.2 SMD AT GI (Influenza and SARS) * thirty 1 / 3.3 2 / 6.7 0/0 3/10.0 Placebo (Flu & ARVI) * thirty 4 / 13.3 5 / 16.7 0/0 9/30 * - According to a double-blind, placebo-controlled randomized study of the clinical efficacy and safety of the therapeutic use of the drug SMD AT GI for influenza and other acute respiratory viral infections (GU Research Institute of Influenza RAMS, St. Petersburg, 2005).

Example 11

Study of the effect of a complex drug, which includes activated-potentiated forms of polyclonal affinity-purified rabbit antibodies of anti-CD4 antibodies (a mixture of ultra-low doses of aqueous homeopathic dilutions of C12 + C30 + C50) and activated-potentiated forms of polyclonal affinity-purified rabbit antibodies of anti-gamma interferon ( a mixture of ultra-low doses of aqueous homeopathic dilution C12 + C30 + C50) in a ratio of 1: 1 (hereinafter the complex preparation), as well as the components included in its composition (activated potentiated forms of polyclonal affinity-purified rabbit antibodies of anti-CD4 antibodies (a mixture of ultra-low doses of aqueous homeopathic dilutions C12 + C30 + C50) (hereinafter referred to as SMD AT to CD4) and activated-potentiated forms of polyclonal affinity-purified rabbit antibodies of an antibody to interferon gamma (a mixture of ultra-low dose-finding homeopathic dilution of C12 + C30 + C50) (hereinafter referred to as SMD AT to IFN gamma)) in vitro for binding of the standard [ ³ H] pentazocine ligand to the recombinant human sigma 1 receptor was evaluated by the radioligand method. As a control of the tested drugs, a potentiated-activated form of distilled water (a mixture of homeopathic dilutions C12 + C30 + C50) was tested (hereinafter potentiated water).

Sigma-1 receptor is an intracellular receptor localized in the cells of the central nervous system, the cells of most peripheral tissues and immunocompetent cells. This receptor, by controlling intracellular calcium homeostasis, regulates intracellular signaling cascades leading to activation of the corresponding transcription factors and transcription of a whole family of genes encoding, in particular, resistance factors to infectious agents and cytokines. In this regard, the ability of drugs to influence the effectiveness of the interaction of ligands with the sigma-1 receptor indicates the presence of antiviral and immunomodulating components in the spectrum of their pharmacological activity, which allows us to consider these drugs as effective drugs for the treatment and prevention of various infectious diseases.

In the experiment (for measuring total binding), 20 μl of the complex preparation or 10 μl of SMD AT to CD4 or SMD AT to IFN gamma were added to the incubation medium. Thus, the number of SMD AT to CD4 and SMD AT to IFN gamma introduced into the experimental well when testing the complex preparation was identical to the number of SMD AT to CD4 and SMD AT to IFN gamma tested as single drugs, which allows a comparison of the effectiveness of the complex preparation with its individual components that make up its composition. Potentiated water was introduced into the incubation medium in a volume of 20 μl and 10 μl.

Then 160 μl (~ 200 μg protein) of the homogenate of the cell membranes of the Jurkat line (human leukemic T-lymphocyte line), and lastly 20 μl of the tritium-labeled [ ³ H] pentazocine radioligand (15 nM), were added.

To measure nonspecific binding, instead of preparations or potentized water, 20 μl of unlabeled ligand, haloperidol (10 μM), was added to the incubation medium.

Radioactivity was measured on a scintillation counter (Topcount, Packard) using a scintillation mixture (Microscint 0, Packard) after incubation for 120 minutes at 22 ° C in 50 mM Tris-HCl buffer (pH = 7.4) and filtration on glass fiber filters (GF / B, Packard). Specific binding (in experiment or control) was calculated as the difference between total (in experiment or control) and non-specific binding.

The results are presented as percent inhibition of specific binding in the control (distilled water was used as the control) (see Table 17).

Table 17. The effect of drugs and potentiated water on the binding of a standard radioligand [ ³ H] pentazocine to a recombinant human sigma 1 receptor Experimental group The amount introduced into the experimental well % of specific radioligand binding in the control % inhibition of radioligand binding in the control 1st dimension 2nd dimension Average value Complex drug 20 μl 50.8 49.1 49.9 50.1 SMD AT to CD4 10 μl 74.0 76,2 75.1 24.9 SMD AT to IFN gamma 10 μl 158.9 149.8 154.3 -54.3 Potentized water 20 μl 98.1 75.8 86.9 13.1 Potentized water 10 μl 140.1 106,2 123,2 -23.2 Note:% specific binding in the control = (specific binding in the experiment / specific binding in the control) * 100%; % inhibition of specific binding in the control = 100% - (specific binding in the experiment / specific binding in the control) * 100%).

- Results reflecting inhibitions above 50% represent significant effects of the test compounds.

- Results reflecting inhibitions of 25% to 50% indicate mild to moderate effects.

- Results reflecting an inhibition of less than 25% are considered minor effects of the test compound and are within the background level.

Thus, in the conditions of this experimental model it is shown that:

1. The complex preparation is more effective than its individual components (SMD AT to CD4 and SMD AT to IFN gamma) inhibits the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor.

2. SMD AT to CD4, introduced into the experimental well in a volume of 10 μl, inhibits the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor, but the severity of the effect is less than the severity of the effect of the complex drug.

3. SMD AT for IFN gamma, introduced into the experimental well in a volume of 10 μl, did not affect the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor.

4. Potentiated water introduced into the experimental well in a volume of 10 μl or 20 μl did not affect the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor.

Claims (29)

1. A comprehensive drug for the prevention and treatment of infectious viral diseases, characterized in that it contains a mixture of several components in the form of activated-potentiated forms of antibodies to the dissolved antigen and antigen associated with the outer membrane of the cells of the immune system. 2. The complex drug according to claim 1, characterized in that the activated-potentiated form of antibodies to the antigen associated with the outer membrane of the cells of the immune system is an activated-potentiated form of antibodies to receptors of immunocompetent cells. 3. The complex drug according to claim 1, characterized in that the activated-potentiated form of antibodies to the dissolved antigen is an activated-potentiated form of antibodies to cytokines. 4. The complex drug according to claim 2, characterized in that an activated-potentiated form of antibodies to differentiation clusters is used. 5. The complex drug according to claim 1, characterized in that the activated-potentiated form of antibodies to the dissolved antigen and the activated-potentiated form of antibodies to the antigen associated with the outer membrane of the cells of the immune system are used in the form of an activated-potentiated aqueous or hydroalcoholic solution, the activity of which is due to the process of sequential multiple dilution of the matrix - initial - solution of antibodies in an aqueous or aqueous-alcoholic solvent in combination with an external their mechanical action - shaking each dilution. 6. The complex drug according to claim 1, characterized in that it is made in solid dosage form and contains the technologically necessary amount of a neutral carrier saturated with a mixture of aqueous or aqueous-alcoholic solutions of an activated - potentiated form of antibodies to a dissolved antigen and an activated-potentiated form of antibodies to an antigen bound to the outer membrane of cells of the immune system; and pharmaceutically acceptable additives. 7. The complex drug according to claim 1, characterized in that the aqueous or aqueous-alcoholic solutions of the activated-potentiated forms of antibodies to the dissolved antigen and to the antigen associated with the outer membrane of the cells of the immune system are obtained by repeated serial dilution of the matrix - initial - solution antibodies to the antigen associated with the outer membrane of the cells of the immune system, and / or antibodies to the dissolved antigen in combination with an external mechanical action - shaking of each dilution, the concentration of the matrix solution is 0.5 ÷ 5.0 mg / ml. 8. The complex drug according to claim 1, characterized in that each of the components based on ultra-low doses of antibodies is used in the form of a mixture of various, mainly hundred, dilutions prepared by homeopathic technology. 9. The complex drug according to claim 6, characterized in that the pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate. 10. A method for the prevention and treatment of infectious viral diseases, characterized in that they use a mixture of several components in the form of activated-potentiated forms of antibodies to the dissolved antigen and antigen associated with the outer membrane of the cells of the immune system. 11. The method according to claim 10, characterized in that as the antigen associated with the outer membrane of the cells of the immune system, receptors of immunocompetent cells are used. 12. The method according to claim 11, characterized in that differentiation clusters are used as receptors for immunocompetent cells. 13. The method according to claim 10, characterized in that cytokines are used as dissolved antigens. 14. The method according to claim 10, characterized in that the mixture of activated-potentiated forms of antibodies is used in the form of an activated-potentiated aqueous or aqueous-alcoholic solution of each component, the activity of which is due to the process of multiple dilution of the matrix - initial - solution of antibodies in aqueous or aqueous alcohol solvent in combination with external mechanical action - shaking each dilution. 15. The treatment method according to 14, characterized in that use is made of a mixture of different dilutions of antibodies to a dissolved antigen in combination with a mixture of different dilutions of antibodies to an antigen associated with the outer membrane of immune cells prepared in the form of a single drug by homeopathic technology. 16. The method according to 14, characterized in that the aqueous or aqueous-alcoholic solutions of the activated-potentiated forms of antibodies to the dissolved antigen and to the antigen associated with the outer membrane of the cells of the immune system are obtained by repeated serial dilution of the matrix - initial - solution of antibodies to an antigen associated with the outer membrane of the cells of the immune system, and / or antibodies to dissolved antigens in combination with an external mechanical action - shaking of each dilution, while the concentration of the matrix the total solution is 0.5 ÷ 5.0 mg / ml. 17. The method according to claim 10, characterized in that the infectious viral disease is influenza. 18. The method according to claim 10, characterized in that the infectious viral disease is a disease caused by the human immunodeficiency virus. 19. The method according to claim 10, characterized in that the infectious viral disease is chronic hepatitis C. 20. The method according to claim 10, characterized in that the infectious viral disease is an acute respiratory viral infection. 21. A pharmaceutical composition having antiretroviral activity, characterized in that it contains a mixture of several components in the form of activated-potentiated forms of antibodies to the dissolved antigen and antigen associated with the outer membrane of cells of the immune system. 22. The pharmaceutical composition according to item 21, characterized in that the activated-potentiated form of antibodies to the antigen associated with the outer membrane of the cells of the immune system is an activated-potentiated form of antibodies to receptors of immunocompetent cells. 23. The pharmaceutical composition according to claim 21, characterized in that the activated-potentiated form of antibodies to the dissolved antigen is an activated-potentiated form of antibodies to cytokines. 24. The pharmaceutical composition according to item 22, characterized in that use the activated-potentiated form of antibodies to differentiation clusters. 25. The pharmaceutical composition according to item 21, characterized in that the activated-potentiated form of antibodies to the dissolved antigen and the activated-potentiated form of antibodies to the antigen associated with the outer membrane of the cells of the immune system are used in the form of an activated-potentiated aqueous or aqueous-alcoholic solution whose activity is due to the process of successive multiple dilutions of the matrix - initial - solution of antibodies in an aqueous or aqueous-alcoholic solvent in combination with an external anicheskim influence - shaking each dilution. 26. The pharmaceutical composition according to item 21, characterized in that it is made in solid dosage form and contains the technologically necessary amount of a neutral carrier saturated with a mixture of aqueous or aqueous-alcoholic solutions of an activated-potentiated form of antibodies to a dissolved antigen and an activated-potentiated form of antibodies to an antigen associated with the outer membrane of cells of the immune system, and pharmaceutically acceptable additives. 27. The pharmaceutical composition according to item 21, characterized in that the aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to the dissolved antigen and to the antigen associated with the outer membrane of the cells of the immune system are obtained by repeated serial dilution of the matrix - initial - antibody solution to the antigen associated with the outer membrane of the cells of the immune system, and / or antibodies to the dissolved antigen in combination with an external mechanical effect - shaking of each dilution, when th matrix solution concentration is 0.5 ÷ 5.0 mg / ml. 28. The pharmaceutical composition according to item 21, characterized in that each of the components based on ultra-low doses of antibodies is used as a mixture of various, mainly hundred, dilutions prepared by homeopathic technology. 29. The pharmaceutical composition according to p. 26, characterized in that the pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate.

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