RU2519196C2 - Therapeutic agent for treating pathological syndrome and method of treating multiple sclerosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present group of inventions refers to medicine, namely neurology, and concerns treating pathological syndrome specific for multiple sclerosis. That is ensured by administering a pharmaceutical composition containing an activated-potentiated form of human gamma-interferon (IFN-γ) antibodies and an activated-potentiated form of brain-specific protein S-100 antibodies.

EFFECT: therapeutic agent provides treating multiple sclerosis ensured by the synergetic action of the ingredients of the composition.

9 cl, 2 ex, 1 tbl, 4 dwg

Description

The invention relates to medicine and can be used for the effective treatment of pathological syndrome, in particular for the treatment of multiple sclerosis.

Multiple sclerosis is a chronic progressive recurrent autoimmune disease in which the myelin sheath of the nerve fibers of the brain and spinal cord is affected.

The etiology of the development of multiple sclerosis is still much unclear, however, it is obvious that multiple sclerosis occurs in genetically predisposed individuals in the presence of certain external factors leading to the development of an autoimmune response and inflammation, demyelination, neurodegeneration, and impaired neuron repair.

The prior art is known for the treatment of inflammatory diseases or autoimmune diseases, including multiple sclerosis, with anti-CD 40 antibodies (RU 2008121899 A, 10.12. 2009). However, this drug in the treatment of multiple sclerosis may not provide the necessary therapeutic efficacy.

The invention is directed to the creation of an effective drug for the treatment of multiple sclerosis.

The solution to this problem is provided by the fact that the drug for the treatment of pathological syndrome according to the invention is made in the form of a pharmaceutical composition and contains an activated - potentiated form of antibodies to human gamma-interferon (IFN-γ) and an activated - potentiated form of antibodies to the brain-specific protein S-100.

Preferably, a pharmaceutical composition comprising an activated — potentiated form of antibodies to human gamma-interferon (IFN-γ) and an activated — potentiated form of antibodies to the brain-specific protein S-100 is used to treat multiple sclerosis.

In this case, the activated — potentiated form of antibodies to human gamma-interferon (IFN-γ) and the activated — potentiated form of antibodies to the brain-specific protein S-100 are used in the form of an activated — potentiated aqueous or hydroalcoholic solution obtained in the process of serial multiple dilutions in aqueous or a water-alcohol solvent and an intermediate external mechanical effect - vertical shaking.

In addition, the drug can be made in solid dosage form, which contains an effective amount of granules of a neutral carrier, saturated with a mixture of the activated - potentiated form of antibodies to human gamma-interferon (IFN-γ), and the activated - potentiated form of antibodies to the brain-specific protein S- 100, and pharmaceutically acceptable additives, which may include lactose, microcrystalline cellulose, and magnesium stearate.

In this case, aqueous or aqueous-alcoholic solutions of activated - potentiated forms of antibodies to human gamma-interferon (IFN-γ) and to the brain-specific protein S-100 were obtained by repeated serial dilution and intermediate external exposure from matrix solutions of affinity-purified antibodies to human gamma-interferon (IFN-γ) and brain-specific protein S-100 with a concentration of 0.5 ÷ 5.0 mg / ml

Preferably, each of the components is used as a mixture of various, mainly hundred, homeopathic dilutions.

Preferably, the drug contains an activated - potentiated form of antibodies to human gamma-interferon (IFN-γ) and an activated - potentiated form of antibodies to the brain-specific protein S-100 in an ultra-low dose obtained by diluting the matrix solution 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, an equivalent mixture of hundreds of homeopathic dilutions of C12, C30 and C50.

It is possible to use a mixture of hundreds of homeopathic dilutions of C12, C30 and C200 or, respectively, dilutions of the matrix solution in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times.

The solution to this problem is also provided by the fact that in the method of treating multiple sclerosis by introducing a drug into the body, according to the invention, said drug is made in the form of a pharmaceutical composition and contains an activated - potentiated form of antibodies to human gamma-interferon (IFN-γ) and activated - potentiated form of antibodies to the brain-specific protein S-100.

In this case, the activated — potentiated form of antibodies to human gamma-interferon (IFN-γ) and the activated — potentiated form of antibodies to the brain-specific protein S-100 are used in the form of an activated — potentiated aqueous or hydroalcoholic solution obtained in the process of serial multiple dilutions in aqueous or a water-alcohol solvent and an intermediate external mechanical effect - vertical shaking.

In addition, the drug contains active ingredients in a 1: 1 ratio, each component being used as a mixture of three appropriate matrix solutions diluted 100 ¹² , 100 ³⁰ and 100 ⁵⁰ times, which is equivalent to hundreds of homeopathic dilutions C12, C30, C50.

The claimed pharmaceutical composition is recommended to be taken preferably 1 ÷ 2 tablets 2 ÷ 6 times a day, mainly 2 ÷ 4 times a day.

In the treatment of multiple sclerosis, separate use is possible in the form of two separately prepared preparations, both in the form of solutions and in solid dosage forms (tablets), each of which contains an activated - potentized form of ultra-low doses of affinity-purified antibodies to human gamma-interferon (IFN-γ ) and, accordingly, the activated - potentiated form of ultra-low doses of affinity-purified antibodies to the brain-specific protein S-100.

The proposed combination of activated - potentiated forms of antibodies to human gamma-interferon (IFN-γ) and to brain-specific protein S-100 in a pharmaceutical composition (i.e., forms of antibodies to human gamma-interferon (IFN-γ) and to brain-specific protein S- 100, prepared according to the homeopathic technology of potentiation by repeated serial dilution and additional intermediate external influence - vertical shaking, which are active in pharmacological models and / or clinical methods treatment of multiple sclerosis) provides an unexpected synergistic therapeutic effect, experimentally confirmed in an adequate model, which consists in a significant improvement in the effectiveness of the treatment of multiple sclerosis. The claimed technical result is due to the fact that in the proposed pharmaceutical composition, the activated - potentiated form of antibodies to the brain-specific protein S-100 enhances the effectiveness of the activated - potentiated form of antibodies to gamma interferon, which consists in a neuroprotective effect, normalization of neurohumoral regulation in case of interferon-dependent diseases of the nervous system, strengthening balanced effect on interferon-sensitive neurodysregulatory processes. The synergistic effect of the claimed solution is due to the manifestation of affinity for the intracellular sigma-1 receptor, localized in the central nervous system, most peripheral tissues, immunocytes, which regulates intracellular signaling cascades through monitoring the concentration of intracellular calcium, which effectively normalizes the regulation of the process of myelination-demyelination of nerve fibers.

In addition, the claimed drug expands the arsenal of drugs intended for the treatment of multiple sclerosis.

Figure 1 presents a graph showing the timing of the onset of clinical symptoms of the disease; figure 2 - the severity of the clinical symptoms of EAE; figure 3 - the severity of the manifestations of EAE depending on the phase of the disease; figure 4 - the incidence of relapse (return of symptoms of EAE).

The drug is prepared mainly as follows.

To prepare a homeopathically activated potentiated form of the active components, monoclonal or predominantly polyclonal antibodies are used, which can be obtained by known technologies - the techniques described, for example, in the book: Immunological methods, ed. G. Fremel. M .: Medicine, 1987, p. 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.

For experimental studies, antibodies prepared by order of a specialized pharmaceutical company can be used.

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 hybrid cells producing clones of antibodies of the same specificity. Their isolation in an individual form is carried out by the same methods as in the case of polyclonal antisera.

Polyclonal antibodies can be obtained by active immunization of animals. To do this, 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: gamma-human interferon (IFN-γ) and brain-specific protein S-100. As a result of such a procedure, a monospecific antiserum with a high content of antibodies is obtained, which is used to obtain an 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 drug is the use of polyclonal antibodies to human gamma-interferon and brain-specific protein S-100, 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:

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:

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 soluble 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 13000g for 10 minutes. The supernatant (supernatant) is an antiserum. The resulting antiserum should be yellow. Add to the antiserum 20% (weight concentration) NaN ₃ 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 ° C). To isolate antibodies to human gamma-interferon from antiserum, solid phase absorption is performed in the following sequence:

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.

The antibodies are then purified by affinity chromatography by attaching the resulting antibodies to human gamma interferon, which is located on an insoluble matrix, followed by elution 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 ( primary) solution for the subsequent preparation of the activated - potentiated form.

To obtain polyclonal antibodies to the brain-specific protein S-100, the brain-specific protein S-100 is used, the physicochemical properties of which are described in the article by M.V. Starostin, S. M. Sviridov. Neurospecific protein S-100. Advances in Modern Biology, 1977, v. 5, p. 170-178; book of M.B.Stark. Brain-specific proteins / antigens / and neuron functions. M .: Medicine, 1985; p.12-14, isolated from tissue of the brain of a bull by the following method:

- the bull brain tissue frozen in liquid nitrogen is ground into powder in a special mill;

- the extraction of proteins is carried out in a ratio of 1: 3 (weight / volume) in the extraction buffer during homogenization;

- the homogenate is heated for 10 min at 60 ° C and cooled to 4 ° C in an ice bath;

- thermolabile proteins are removed by centrifugation;

- carry out stepwise fractionation with ammonium sulfate, followed by removal of precipitated impurity proteins;

- the S-100 protein-containing fraction is precipitated with 100% saturated ammonium sulfate when the pH is reduced to 4.0 and collected by centrifugation;

- the precipitate is dissolved in a minimum volume of a buffer containing EDTA and mercaptoethanol, dialyzed against deionized water and freeze-dried;

- fractionation of acidic proteins is continued by chromatography on ion-exchange media — DEAE cellulose DE-52 and then DEAE-Sephadex A-50;

- collected and dialyzed fractions containing S-100 protein are separated by molecular weight by gel filtration on Sephadex G-100;

- purified S-100 protein is dialyzed and freeze-dried.

The molecular weight of the purified brain-specific protein S-100 is 21,000 D.

Due to the high content of aspartic and glutamic acids, the brain-specific protein S-100 is strongly acidic and occupies the extreme anode position during electrophoresis in a discontinuous buffer system in a polyacrylamide gel, which facilitates its identification.

To obtain a brain-specific antiserum, a purified S-100 protein (antigen) is prepared in combination with methylated bovine serum albumin as a carrier with Freund's complete adjuvant, which is administered subcutaneously to a laboratory rabbit animal in the amount of 1- to a selected brain-specific protein S-100. 2 ml On the 8th, 15th day, re-immunization is carried out. Blood is taken (for example, from an ear vein) on the 26th and 28th day.

The resulting antiserum has a titer of 1: 500 - 1: 1000, forms a single precipitation band with extract from the nervous tissue, but does not react with extracts of heterologous organs, and forms a single precipitation peak with both pure S-100 protein and the extract of nervous tissue, which indicates the monospecificity of the obtained antiserum.

Thus obtained buffer solutions of each component of polyclonal antibodies to human gamma-interferon (IFN-γ) and brain-specific protein S-100 with a concentration of 0.5 ÷ 5.0 mg / ml, preferably 2.0 ÷ 3.0 mg / ml, used as matrix (primary) solutions for the subsequent preparation of activated - potentiated 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 the matrix solution in 9 parts (for decimal dilution) or in 99 parts (for hundredth dilution) or in 999 parts (for thousandth dilution) of a neutral solvent with multiple vertical shaking ("dynamization") of each dilution obtained and using separate containers for each subsequent dilution to obtain the desired potency - to breeding rates according to the homeopathic method (see, for example, V. Schwabe "Homeopathic medicines", M., 1967, p.14-29).

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

For example, for the preparation of the 12th hundredth C12 dilution, one part of the aforementioned matrix solution of antibodies to human gamma-interferon (IFN-γ) (or antibodies to the brain-specific protein S-100) with a concentration of 3.0 mg / ml is diluted in 99 parts of neutral aqueous or aqueous-alcoholic solvent (mainly 15% ethyl alcohol) and repeatedly (10 times or more) vertically shake - potentiate the obtained 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 sequentially in different containers 12 times of the first part of the initial matrix solution of antibodies to human gamma-interferon (IFN-γ) with a concentration of 3.0 mg / ml in 99- te parts of a neutral solvent, i.e. the solution obtained by diluting the matrix solution in 100 ¹² times. Similar operations with the appropriate dilution ratio are carried out to obtain a dilution of C30 and C50.

When using as active substances of each component a mixture of various homeopathic, mainly hundred, dilutions (for example, C12, C30, C50), the active substance of each component is prepared separately according to the technology described above, before their last but one dilution (respectively, to obtain C11, C29, C49) 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 (respectively, 97 parts for a hundred-percent dilution). In this case, an activated - potentiated form of antibodies of each of the components (to human gamma-interferon (IFN-γ) and brain-specific protein S-100) is obtained in an ultra-low dose obtained by diluting the matrix solution 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to mixtures of hundredth homeopathic dilutions C12, C30, C50.

It is possible to use the active substance in the form of a mixture of other various homeopathic dilutions, for example decimal, and / or hundredths and / or thousandths (C12, C30, C200; D20, C30, C100 or D20, C30, M100, etc.), effectiveness which are determined experimentally.

When potentiating instead of shaking in the process of decreasing the concentration, it is also possible to exert external influence by ultrasound, electromagnetic or other physical effect.

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

The claimed medicinal product can be used in solid dosage form, which contains an effective amount of granules of a neutral carrier - lactose, saturated by soaking until saturated with a mixture of activated or potentiated forms of antibodies to human gamma-interferon prepared by the above technology (IFN) -γ) and to the brain-specific protein S-100, and pharmaceutically acceptable additives, including mainly lactose, microcrystalline cellulose and magnesium ste arat

To obtain a solid oral form of the claimed drug, a fluidized bed is installed (for example, Huttlin Pilotlab type manufactured by Huttlin GmbH) irrigation until saturation of the granules of a neutral substance - lactose (milk sugar) with a particle size of 100 ÷ 300 microns, pre-prepared aqueous or aqueous-alcoholic solution of activated - potentiated forms of antibodies to human gamma-interferon (IFN-γ) and to brain-specific protein S-100 mainly in the ratio of 1 kg of solution antibodies for 5 or 10 kg of lactose (1: 5 - 1:10) with simultaneous drying in a stream of heated air supplied under the grate at a temperature not exceeding 40 ° C. The estimated amount of 10 ÷ 34 mass. parts of dried granules saturated with the activated - potentiated form of antibodies are loaded into the mixer and mixed with 25 ÷ 85 mass. parts of the total load mass of “unsaturated” pure lactose (to reduce the cost and some simplification and acceleration of the process without reducing the effectiveness of the therapeutic effect). Then, magnesium stearate is added to this mixture in an amount of 0.1 ÷ 1 mass. parts of the total mass of the load and microcrystalline cellulose in an amount of 3 ÷ 10 mass. parts of the total mass of the load. The resulting tablet mass is uniformly mixed and tabletted by direct dry pressing (for example, in a Korsch tablet press - XL 400) with the formation of round tablets weighing 150 ÷ 500 mg, mainly weighing 300 mg, soaked in an aqueous-alcoholic solution (3.0-6.0 mg / table.) activated - potentiated form of antibodies to human gamma-interferon (IFN-γ) and to the brain-specific protein S-100 in an ultra-low dose of each component prepared from a matrix solution diluted in 100 ¹² , in 100 ³⁰ in 100 ²⁰⁰ , which is equivalent to a mixture of hundreds of homeopathic times knowledge C12, C30 and C200.

It is preferable to use tablets weighing 250 ÷ 300 mg, which include 3.0 ÷ 6.0 mg / table. activated potentiated form of water-alcohol dilutions of a substance - the active substance of polyclonal rabbit antibodies to human gamma interferon (IFN-γ) and to brain-specific protein S-100, purified on antigen, in an ultra-low dose obtained by diluting the matrix solution in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, the equivalent mixture of hundreds of homeopathic dilutions C12, C30, C50.

Glucose, sucrose, maltose, starch, isomaltose, isomalt and other monosaccharides, oligosaccharides and polysaccharides used in the manufacture of pharmaceutical preparations, as well as technological mixtures based on the latter with the addition of pharmaceutically acceptable additives (for example, isomalt, crospovidone, can also be used as a neutral carrier. , sodium cyclamate, sodium saccharin, anhydrous citric acid, etc.), including lubricants, disintegrants, binders and dyes.

Preferably, the claimed drug is recommended to take 1 ÷ 2 tablets 2 ÷ 6 times a day, preferably 2 ÷ 4 times a day.

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

Example 1

Study of the effect of the claimed drug in the form of a complex preparation of SMD AT to IFN-γ + SMD AT to S100, which includes ultra-low doses of antibodies to S100 protein (a mixture of homeopathic dilutions C12 + C30 + C50) and ultra-low doses of antibodies to interferon gamma (mixture homeopathic dilutions C12 + C30 + C50) in a 1: 1 ratio, as well as the components included in its composition (ultra-low doses of antibodies to S100 protein (a mixture of homeopathic dilutions C12 + C30 + C50) (SMD AT to S100) and ultra-low doses of antibodies to interferon gamma (a mixture of homeopathic dilutions C12 + C30 + C5 0) (SMD AT to IFN-γ)) in vitro for binding of the standard [ ³ H] pentazocine ligand to the recombinant human sigma-1 receptor was evaluated by the radioligand method.

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. Sigma-1 receptor is involved in the pathogenesis of various diseases, including infectious and neurodegenerative ones. In this regard, drugs that affect this receptor and the effectiveness of the interaction of ligands with it can be considered as effective drugs for the treatment of neuroinfectious, neurodegenerative and other diseases.

As a control in this experimental model, we used potentiated distilled water (a mixture of homeopathic dilutions C12 + C30 + C50) - (potentized water).

During the study (to measure total binding), 20 μl of the complex preparation or 10 μl of SMD AT to S100 or SMD AT to IFN-γ were added to the incubation medium. Thus, the number of SMD AT to S100 and SMD AT to IFN-γ introduced into the experimental well when testing the complex preparation was identical to the number of SMD AT to S100 and SMD AT to IFN-γ tested as single drugs, which allows a comparison of the effectiveness of complex the drug 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 [ ³ H] pentazocine labeled 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 research results are presented as percent inhibition of specific binding in the control (potentiated water was used as a control) (Table 1).

Table 1 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
total
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
a drug 20 μl 44,2 46.2 45,2 54.8 SMD AT to S100 10 μl 70.9 62.9 66.9 33.1 SMD AT to IFN-γ 10 μl 158.9 149.8 154.3 -54.3 Potentiological
bath water 20 μl 98.1 75.8 86.9 13.1 Potentiological
bath 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%).

Under the conditions of this experimental model, it was shown that a complex preparation (SMD AT to S100 + SMD AT to IFN-γ) is more effective than its individual components (SMD AT to S100) and (SMD AT to IFN-γ) inhibits the binding of a standard radioligand [ ³ H] pentazocine with a recombinant human sigma 1 receptor and can be used as an effective drug for the treatment of neuroinfectious, neurodegenerative and other diseases, in particular for the treatment of multiple sclerosis.

Example 2

The immune response in multiple sclerosis is characterized by a complex, multicomponent cascade of events. The widespread use of experimental models of multiple sclerosis, namely: autoimmune encephalopathy in rodents and knockout mice, has allowed scientists to significantly advance in understanding the pathogenesis of this disease.

Experimental allergic encephalomyelitis (EAE) is a recognized model of multiple sclerosis, a severe autoimmune demyelinating disease that affects people of working age.

EAE can be induced in sensitive animals by a homogenate of homologous (Raine, Traugott, 1984) or heterologous (McCombe et al., 1994) brain tissue, purified myelin or its constituent proteins. Currently, more than 20 myelin proteins have been described (Baumann, Pham-Dinh, 2001), of which they have immunogenic properties and are most often used for the induction of EAE: the main myelin protein is MBP, the proteolipid protein is PLP, glycoproteins: myelin associated glycoprotein is MAG and myelin oligodendrocytic glycoprotein - MTF, specific oligodendrocyte protein - OSB. It was revealed that not only whole proteins, but also certain fragments of these proteins are capable of causing EAE upon administration to animals.

An EAE type of disease (with an inflammatory process) can be reproduced on sensitive animals by CNS proteins that are not part of myelin: calmodulin, S100 protein, glial fibrillar acid protein, arrestin, and transaldolase.

The most appropriate PC model is EAE, caused by the introduction of spinal cord homogenate. In this model, as in the initial disease, an immune response to all components of myelin is carried out: inflammation is induced with subsequent demyelination, degeneration of axons, and then the nerve cells themselves. A pathological chain of events appears with the development of paresis, paralysis and other symptoms of the disease.

The rodent model of experimental autoimmune encephalomyelitis (EAE) is the main model for testing new drugs for the treatment of multiple sclerosis.

A model of experimental autoimmune encephalopathy in rats caused by the introduction of an autologous spinal cord homogenate in Freund's complete adjuvant (PAF) was used at the Scientific Research Institute of Experimental Medicine of the Northwestern RAMS.

In the development of EAE, four stages can be distinguished:

1. Sensitization of T-lymphocytes in the periphery under the action of a brain antigen with PAF and an increase in BBB permeability.

2. Migration of activated T cells to the central nervous system and activation of antigen-presenting cells (astrocytes and microglia) directly in the brain.

The first two stages correspond to a latent (inductive) period in which no clinical manifestations are observed.

3. The development of autoimmune and inflammatory reactions in the brain, leading to demyelination of nerve fibers (clinical period).

4. Suppression of pathological processes and repair of damaged tissues (recovery phase). During this period, in most animals, neurological and motor disorders are smoothed out, and a partial or complete recovery occurs, after which the animals are resistant to repeated induction of EAE.

Similar phases of the development of the pathological process in the central nervous system are observed in multiple sclerosis.

The average duration of EAE is 30 days: the latent stage, the clinical stage, and the recovery stage. The duration of each stage is usually 7-10 days.

In the Scientific Research Institute of Experimental Medicine of the Northwestern RAMS on the model of multiple sclerosis (EAE), studies were conducted on the effectiveness of the claimed drug, made in the form of a pharmaceutical composition - a complex preparation that includes an aqueous solution containing an activated - potentiated form of antibodies to human gamma-interferon purified on antigen , in an ultra-low dose obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times the equivalent mixture of hundred percent homeopathic dilutions of C12, C30, C50, and the activated β-potentiated form of antibodies to the brain-specific protein S-100, purified on antigen, in an ultra-low dose obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) in 10012, 10030, 10050 times equivalent mixture hundredth homeopathic dilutions C12, C30, C50 (SMD AT to IFN - γ + SMD AT to S100). For experimental studies, female Wistar rats (200-220 g) were used, in which experimental autoimmune encephalomyelitis (EAE) was induced by a single inoculation of the encephalitogenic mixture (EHS) at the rate of 100 mg of homologous spinal cord homogenate, 0.2 ml of Freud's complete adjuvant ( PAF) (the content of killed mycobacteria 5 mg / ml) and 0.2 ml of physiological saline per animal. EHS was injected subcutaneously at the base of the tail along the tail vein under light ether anesthesia in a volume of 0.4 ml (0.2 ml on the right and left). Starting from the day of EAE induction, they were administered intragastrically 2 times a day with an interval of 7 hours for 30 days (SMD AT to IFN - γ + SMD AT to S100) (number of mice n = 10, at a dose of 5 ml / kg / day), comparison preparation — activated — potentiated form of antibodies to human gamma-interferon purified on antigen in an ultra-low dose obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) 100, 100, 100 times equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C50 (SMD AT to IFN - γ) (n = 10; 2.5 ml / kg / day), comparison drug - activated - potential ovannuyu form antibodies to brain-specific protein S-100 purified on an antigen in ultra low dose received sverhrazvedeniem initial matrix solution (concentration 2.5 mg / ml) 100 ^12, 100 ^30, 100 ⁵⁰ times equivalent mixture of centesimal homeopathic dilutions C12, C30, C50 (SMD AT to S100) (n = 10, 2.5 ml / kg / day) and distilled water (control; n = 10; 5 ml / kg / day). A copaxone immunomodulator (Teva, Israel) was used as a reference drug, which was administered intramuscularly to female rats of the Wistar strain (n = 10) at a dose of 4 mg / kg, from the 2nd to the 25th day after EAE induction. The research results are presented in Fig.1-4.

The comparison drug (SMD AT to S100) postponed the onset of the onset of clinical symptoms of the disease both in comparison with the negative control (p <0.05) and in comparison with the reference drug copaxone (p> 0.05). The timing of the onset of the disease (Figure 1) in the groups receiving (SMD AT to IFN-γ) and in the group receiving copaxone did not differ from the control. At the same time, animals injected with the complex preparation (SMD AT to IFN - γ + SMD AT to S100) showed a tendency to shorten the appearance of clinical signs of multiple sclerosis in comparison with the control. It should be noted that significant differences were found in the timing of the onset of the disease between the groups (SMD AT to S100) and (SMD AT to IFN - γ + SMD AT to S100).

The severity of neurological disorders was assessed in points by the presence of muscle weakness, tremor (0.5 points), persistent paresis (1 point) and paralysis (1.5 points) in animals. If multiple limbs were affected, scores were summarized to calculate the clinical index (CI). The absence of visible clinical manifestations was evaluated as KI = 0, fatal outcome - KI = 6. When assessing the severity of the disease, the maximum CI was taken into account - the highest value regardless of the duration of the disease. For an integrative assessment of the severity of EAE for each rat, a cumulative index was calculated representing the sum of individual clinical indices for the entire period of the disease.

The proportion of animals with severe disease (> 3 points) (Figure 2) was also lower in the group (SMD AT to S100). In animals treated with (SMD AT to IFN - γ + SMD AT to S100), the percentage of animals with severe course was higher than in other study groups at all stages of the disease.

Due to the fact that different mechanisms of the pathogenesis of EAE are involved in different phases of the development of multiple sclerosis, the influence of the tested drugs on the severity of the disease in the latent period, in the phase of development of clinical signs and in the recovery phase was analyzed (Figure 3). (SMD AT to S100) significantly contributed to lowering the average score for the manifestation of clinical signs of the disease at the stage of clinical manifestations and in the recovery phase, while copaxone and (SMD AT to IFN-γ) significantly reduced the severity of the lesion only in the last phase. The complex preparation (SMD AT to IFN - γ + SMD AT to S100) significantly compared with the control and its constituents (SMD AT to IFN - γ) and (SMD AT to S100) increased the average score of the animals at the stage of clinical signs diseases and at the stage of recovery.

It should be noted that in some animals after the complete disappearance of the clinical signs of the disease, a certain semblance of relapse was noted, that is, the return of the clinical manifestations of EAE (Figure 4). In the group (SMD AT to S100), despite its beneficial effect on the clinical course of EAE (a later onset of the disease, less severe manifestation of clinical signs), the largest number of animals (25%) with relapse was noted. At the same time, in the group (SMD AT to IFN - γ + SMD AT to S100), which was characterized by an early onset of the disease and a greater severity of the pathological process, no development of relapse was detected in any animal.

The model of experimental autoimmune encephalomyelitis (EAE) showed a beneficial effect (SMD AT to S100) on the course of the disease, which delayed the onset of clinical signs of the disease and reduced the severity of the lesion, while (SMD AT to IFN-γ) and the comparison drug copaxone did not during the course of experimental multiple sclerosis, since the timing of the onset of clinical signs of the disease and the severity of symptoms did not differ from the control group (distilled water). The effect of the complex preparation (SMD AT to IFN - γ + SMD AT to S100) differed from the effect of its constituent components (SMD AT to IF100 - γ) and (SMD AT to IFN - γ), while the use of (SMD AT to IFN - γ + SMD AT to S100) led to an increase in the immune response, which was expressed in the earlier onset of the disease, an increase in the proportion of diseased animals and the severity of the disease. However, in the group (SMD AT to IFN - γ + SMD AT to S100) no recurrence was recorded in any animal, while in 25% of the animals of the group (SMD AT to S100) and 15% of the animals of the group (SMD AT to IFN - γ) a relapse was detected during the study period (30 days).

Thus, the claimed drug (SMD AT to IFN - γ + SMD AT to S100) is a promising drug for the treatment of multiple sclerosis, which gives an increase in the immune response at the peak of the disease and can provide more effective rehabilitation and prevention of relapse.

Claims (9)

1. A drug for the treatment of multiple sclerosis, characterized in that it is made in the form of a pharmaceutical composition and includes an activated — potentiated form of antibodies to human gamma-interferon and an activated — potentiated form of antibodies to brain-specific protein S-100. 2. The drug according to claim 1, characterized in that the activated - potentiated form of antibodies to human gamma interferon and the activated - potentiated form of antibodies to brain-specific protein S-100 are used in the form of an activated - potentiated aqueous or aqueous-alcoholic solution obtained in the process of successive multiple dilutions of the matrix solution of the corresponding antibodies in an aqueous or aqueous-alcohol solvent and an intermediate external mechanical action - shaking each breeding. 3. The drug according to claim 2, characterized in that the pharmaceutical composition is in solid dosage form and contains an effective amount of an activated - potentiated form of antibodies to human gamma-interferon and antibodies to the brain-specific protein S-100 of neutral carrier granules and pharmaceutically acceptable additives . 4. The drug according to claim 2, characterized in that aqueous or aqueous-alcoholic solutions of activated potentiated forms of antibodies to human gamma interferon and to brain-specific protein S-100 are obtained by repeated serial dilution and intermediate external exposure from matrix solutions of affinity purified antibodies to human gamma-interferon and brain-specific protein S-100 with a concentration of 0.5 ÷ 5.0 mg / ml. 5. The drug according to claim 2, characterized in that each of the components in the form of an activated - potentiated form of antibodies to human gamma-interferon and antibodies to the brain-specific protein S-100 is used as a mixture of various, mainly hundred, homeopathic dilutions. 6. The drug according to claim 3, characterized in that the pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate. 7. A method of treating multiple sclerosis, characterized in that the activated and potentiated forms of antibodies to human gamma interferon and the activated, potentiated forms of antibodies to brain-specific protein S-100 are simultaneously introduced into the body. 8. The treatment method according to claim 7, characterized in that a mixture of various homeopathic dilutions of antibodies to human gamma-interferon in combination with a mixture of various homeopathic dilutions of antibodies to the brain-specific protein S-100 is prepared in the form of a single drug. 9. The method according to claim 7 or claim 8, characterized in that the activated - potentiated form of antibodies to human gamma-interferon and the activated - potentiated form of antibodies to brain-specific protein S-100 are used in the form of an activated - potentiated aqueous or aqueous-alcoholic solution obtained in the process of successive multiple dilutions in an aqueous or hydroalcoholic solvent and an intermediate external mechanical action - shaking of each dilution.

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