RU2595809C2 - Therapeutic agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present group of inventions concerns treating multiple sclerosis in experiment. That is a therapeutic agent representing an activated potentiated antibodies to brain-specific protein S-100.

EFFECT: injection drug experimental animals removed time of occurrence of signs of autoimmune encephalomyelitis and reduced severity of clinical manifestations of the disease.

9 cl, 4 dwg, 2 ex

Description

The invention relates to medicine and can be used for the effective treatment of multiple sclerosis.

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 a medicine for the treatment of multiple sclerosis.

The solution to this problem is provided by the fact that the drug is made in the form of a pharmaceutical composition for the treatment of multiple sclerosis and according to the invention contains an activated-potentiated form of antibodies to the brain-specific protein S-100.

In this case, the activated-potentiated form of antibodies to the brain-specific protein S-100 is used in the form of an activated-potentiated aqueous or hydroalcoholic solution obtained in the process of serial multiple dilutions in an aqueous or hydroalcoholic solvent and an intermediate external mechanical action - shaking.

In addition, the pharmaceutical composition 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 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 the brain-specific protein S-100 are obtained by repeated serial dilution and intermediate external exposure from matrix solutions of affinity-purified antibodies to the brain-specific protein S-100 with a concentration of 0.5 ÷ 5.0 mg / ml Preferably used in the form of a mixture of various, mainly hundred, homeopathic dilutions.

Preferably, the pharmaceutical composition contains 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 hundred-percent homeopathic dilutions C12, C30 and C50.

You can 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.

In this case, the activated-potentiated form of antibodies to the brain-specific protein S-100 is used in the form of an activated-potentiated aqueous or hydroalcoholic solution obtained in the process of repeated dilution in an aqueous or hydroalcoholic solvent and an intermediate external mechanical action - vertical shaking.

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 pathological syndrome, 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-potentiated form of ultra-low doses of affinity-purified antibodies to the brain-specific protein S-100.

The proposed drug contains activated-potentiated forms of antibodies to the brain-specific protein S-100 in the pharmaceutical composition, prepared according to the homeopathic technology of potentiation by repeated serial dilution and additional intermediate external action - vertical shaking, which are active in pharmacological models and / or clinical methods of treating disseminated sclerosis, provides an unexpected therapeutic effect, experiment mentally confirmed on an adequate model, which consists in a significant improvement in the effectiveness of the treatment of multiple sclerosis. In addition, the claimed pharmaceutical composition 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 pharmaceutical composition is prepared predominantly as follows.

To prepare a homeopathically activated-potentiated form of the active ingredient, 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. Frimel. 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. For this, according to a specially developed scheme, animals are given a series of injections of the substance required in accordance with the invention, the brain-specific protein S-100. 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 pharmaceutical composition is the use of polyclonal antibodies to the brain-specific protein S-100, which can be obtained by immunization of rabbits as follows.

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. Starostina, S.M. Sviridov. Neurospecific protein S-100 // Successes in modern biology, 1977, v.5, p.170-178; the book of MB 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 Da.

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 nervous tissue, but does not react with extracts of heterologous organs, and forms a single precipitation peak with both pure S-100 protein and nervous tissue extract, which indicates the monospecificity of the obtained antiserum.

Thus obtained buffer solution of antibodies to the brain-specific protein S-100 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 activated potentiated forms of the pharmaceutical composition.

The activated-potentiated form 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 - dilution ratio by homeopathic 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 a 12th hundred-fold dilution of C12, one part of the aforementioned matrix solution of antibodies to the brain-specific protein S-100 with a concentration of 3.0 mg / ml is diluted in 99 parts of a neutral aqueous or aqueous-alcoholic solvent (mainly 15% ethyl alcohol) and repeatedly (10 or more times) vertically shake - potentiate the obtained 1st hundredth C1 dilution. From the 1st hundredth dilution C1, the second hundredth dilution C2 is prepared. 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 in different containers 12 times of the 1st part of the initial matrix solution of antibodies to the brain-specific protein S-100 with a concentration of 3.0 mg / ml in 99 parts of a neutral solvent , i.e. the solution obtained by diluting the matrix solution in 100 to ¹² times. Similar operations with the appropriate dilution ratio are carried out to obtain dilutions of C30 and C50.

When using a mixture of various homeopathic, mainly hundred, dilutions (for example, C12, C30, C50) as the active substances of each component, 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 introduced into one container and mixed with the required amount of solvent (respectively, with 97 parts for hundredth dilution). In this case, an activated-potentiated form of antibodies to the 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 a mixture of hundred-percent 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.

The claimed pharmaceutical composition can be used in solid dosage form, which contains an effective amount of granules of a neutral carrier - lactose, saturated by impregnation to saturation with a mixture prepared by the above technology, aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to the brain-specific protein S- 100, and pharmaceutically acceptable additives, including predominantly lactose, microcrystalline cellulose and magnesium stearate.

To obtain a solid oral form of the claimed pharmaceutical composition, a fluidized bed is installed (for example, the Hüttlin Pilotlab type manufactured by Hüttlin GmbH) until the granules of a neutral substance — lactose (milk sugar) introduced into the fluidized bed — are saturated with a particle size of 100–300 microns, a pre-prepared aqueous or aqueous-alcoholic solution of activated-potentiated forms of antibodies to the brain-specific protein S-100, 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 a stream of heated air supplied under the grate at a temperature not exceeding 40 ° C. The estimated amount of 10 ÷ 34 wt. parts of dried granules saturated with the activated-potentiated form of antibodies are loaded into the mixer and mixed with 25 ÷ 85 wt. 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 wt. parts of the total mass of the load and microcrystalline cellulose in an amount of 3 ÷ 10 wt. 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 / tablet) with the activated-potentiated form of antibodies 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 hundred-percent homeopathic dilutions C12, C30 and C200 .

It is preferable to use tablets weighing 250 ÷ 300 mg, which include 3.0 ÷ 6.0 mg / table. activated-potentized form of water-alcohol dilutions of a substance - the active substance of polyclonal rabbit antibodies to brain-specific protein S-100, purified on antigen, in an ultra-low dose obtained by diluting the matrix solution 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundred 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 pharmaceutical composition 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 pharmaceutical composition 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 ( a mixture of 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, the binding of a standard [ ³ H] pentazocine ligand to a recombinant human sigma-1 receptor was evaluated by the radioligand method.

Sigma-1 receptor is an intracellular receptor localized in cells of the central nervous system, 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 (pharmaceutical compositions) 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 (for measuring total binding), 20 μl of the complex preparation or 10 μl of DMD AT to S100 or SMD AT to IFN-γ were added to the incubation medium. Thus, the amount 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 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 was added 160 l (~ 200 protein μg) membrane homogenate Jurkat cell line (a line of human leukemic T-lymphocytes) and, lastly, 20 ul of radioligand tritiated ^[3 H] Pentazocine (15 nM).

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 filtering 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 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 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 10 μl 158.9 149.8 154.3 -54.3 IFN-γ 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%).

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

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 model of MS 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 increased 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 carried out on the effectiveness of the claimed pharmaceutical composition containing the 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 ¹² , 100 ³⁰ , 100 ⁵⁰ times, the equivalent mixture of hundreds of homeopathic dilutions C12, C30, C50, and the activated-potentiated form of antibodies to the brain-specific protein S-100, o purified on antigen, in an ultra-low dose obtained by super-dilution of the initial matrix solution (concentration 2.5 mg / ml) 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, equivalent to a mixture of hundred-percent 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 - the 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 ¹² , 100 ³⁰ , 100 ⁵⁰ 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-p tentsirovannuyu 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 preparation, which was administered intramuscularly to female Wistar rats (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) delayed 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, in animals injected with the complex preparation (SMD AT to IFN-γ + SMD AT to S100), a tendency was observed to shorten the timing of the appearance of clinical signs of multiple sclerosis compared to 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 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 EAE pathogenesis 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 the decrease in the average score for the manifestation of clinical signs of the disease in the clinical manifestation stage and in the recovery stage, 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 lesion of animals at the stage of clinical signs diseases and at the stage of recovery.

It should be noted that in some animals, after the clinical signs of the disease completely disappeared, 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 high severity of the pathological process, no development of relapse was detected in any animal.

The model of experimental autoimmune encephalomyelitis (EAE) shows 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 components (SMD AT to S100) 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 pharmaceutical composition (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 the brain-specific protein S-100. 2. The drug according to claim 1, characterized in that the activated-potentiated form of antibodies to the brain-specific protein S-100 is used in the form of an activated-potentiated aqueous or aqueous-alcoholic solution obtained by sequential multiple dilution of a matrix solution of the corresponding antibodies in aqueous or water-alcohol solvent and an intermediate external mechanical impact - shaking each dilution. 3. The drug according to claim 1, characterized in that the pharmaceutical composition is in solid dosage form and contains an effective amount of an activated-potentiated form of antibodies to the brain-specific protein S-100 granules of a neutral carrier and pharmaceutically acceptable additives. 4. The drug according to claim 2, characterized in that the aqueous or aqueous-alcoholic solutions of the activated-potentiated forms of antibodies to the brain-specific protein S-100 are obtained by repeated serial dilution and intermediate external exposure from matrix solutions of affinity-purified antibodies to the 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 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 an activated-potentiated form of antibodies to the brain-specific protein S-100 is introduced into the body. 8. The treatment method according to claim 7, characterized in that use is made of a mixture of various homeopathic dilutions of antibodies to the brain-specific protein S-100 prepared in the form of a single drug. 9. The method according to claim 7 or 8, characterized in that the activated - potentiated form of antibodies to the brain-specific protein S-100 is used in the form of an activated-potentiated aqueous or aqueous-alcoholic solution obtained in the process of sequential multiple dilutions in aqueous or aqueous-alcoholic solvent and intermediate external mechanical action - shaking each dilution.

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