RU2441023C1 - Drug for multiple sclerosis and method for treating multiple sclerosis - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention refers to a drug for multiple sclerosis which is presented in the form of a pharmaceutical composition and contains an activated potentiating form of human gamma-interferon (IFN-) antibodies and an activated potentiating form of brain-specific protein S-100 antibodies as an additive intensifying component. Using the drug in a method for treating multiple sclerosis involves in introduction of the activated potentiating form of human gamma-interferon (IFN-) in combination with the intensifying component in the form of the activated potentiating form of very-low-dose affinity purified brain-specific protein S-100 antibodies. ^ EFFECT: use of the inventions allows higher clinical effectiveness in multiple sclerosis. ^ 9 cl, 1 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 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 multiple sclerosis according to the invention 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 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 aqueous-alcoholic solution obtained by sequential multiple dilution 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.

In this case, aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to human gamma-interferon (IFN-γ) and 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 of the ultra-low doses of affinity-purified antibodies is used as a mixture of various, mainly hundred, homeopathic dilutions.

Preferably, the medicament 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.

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.

Moreover, pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate.

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

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 aqueous-alcoholic solution obtained by sequential multiple dilution 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, with 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 2-4 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-potentiated 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 brain-specific protein S-100 in a pharmaceutical composition (i.e., forms of antibodies to human gamma-interferon (IFN-γ) and 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 echeniya multiple sclerosis) provides an unexpected synergistic therapeutic effect, experimentally confirmed in an adequate model, which consists in a substantial improvement in efficacy of 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 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 through the control of intracellular calcium concentration of the intracellular signaling cascade, which effectively normalizes the regulation of the myelination-demyelination of nerve fibers.

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

The drug is prepared mainly as follows.

To prepare a homeopathically activated-potentiated form of the active components, monoclonal or, mainly, polyclonal antibodies are used, which can be obtained by known technology-methods described, for example, in the book: Immunological methods, ed. G. Fremel, Moscow: 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 with the antigen substance required in accordance with the invention: human gamma interferon (IFN-γ) and 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 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.

Example 1

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

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 13000 g 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. 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. Successes of 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:

- fabric 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 2.5 ÷ 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 the use of separate containers for each subsequent dilution to obtain the desired potency - 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, to prepare the 12th hundredth C12 dilution, one part of the above 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 a water-alcohol solvent (mainly 70% ethyl alcohol) and repeatedly (10 or more times) 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 1st 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 to ¹² times. Similar operations with the appropriate dilution ratio are carried out to obtain dilutions 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 make, in accordance with the composition of the mixture, in one container, one part of each component and mixed with the required amount of solvent (respectively, with 97 parts for hundredth dilution). In this case, an activated-potentiated form of antibodies of each of the components (to human gamma-interferon (IFN-γ) and 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 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 drug can be used in solid dosage form, which contains an effective amount of granules of a neutral carrier - lactose, saturated by soaking to saturation with a mixture prepared by the above technology, aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to human gamma-interferon (IFN-γ) to the brain-specific protein S-100, and pharmaceutically acceptable additives, including mainly lactose, microcrystalline cellulose and magnesium st earat.

To obtain a solid oral form of the claimed drug, a fluidized bed is installed (for example, Hüttlin Pilotlab type manufactured by Hüttlin GmbH) irrigation until saturation of granules of a neutral substance - lactose (milk sugar) with a particle size of 150-300 is introduced into the fluidized - boiling layer μm, a 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 a 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 calculated amount of 0.17 ÷ 0.34 by weight of the solid oral form of the dried granules saturated with the activated-potentiated form of antibodies is loaded into the mixer and mixed with microcrystalline cellulose introduced in an amount of 3-8 wt. parts of the total mass of the load - from the mass of solid oral form. Then 25 ÷ 45 wt. parts of the total mass of the load of "unsaturated" pure lactose (to reduce the cost and some simplification and acceleration of the process without reducing the effectiveness of the therapeutic effect), magnesium stearate in an amount of 0.1 ÷ 0.3 wt. parts of the total mass of the load and microcrystalline cellulose in an amount of 3 ÷ 8 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. After tabletting, 300 mg tablets are obtained, impregnated with an aqueous-alcoholic solution (3.0-6.0 mg / tablet) of the activated-potentiated form of antibodies to human gamma-interferon (IFN-γ) and to the brain-specific protein S-100, in the smallest 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 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 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.

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

Claims (9)

1. A medicine for the treatment of multiple sclerosis, containing an activated-potentiated form of antibodies to human gamma-interferon (IFN-γ), characterized in that it is made in the form of a pharmaceutical composition and includes an activated-potentiated form of antibodies to a brain-specific protein as an additional enhancing component S-100. 2. The drug according to claim 1, characterized in that 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 aqueous alcohol 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 - vertical yahivaniya. 3. The drug according to claim 1 or 2, characterized in that the pharmaceutical composition is made in solid dosage form and contains an effective amount of granules of a neutral carrier saturated with a mixture of the activated-potentiated forms of antibodies to human gamma-interferon (IFN-y) and activated potentiated forms of antibodies to the brain-specific protein S-100; and pharmaceutically acceptable additives. 4. The drug according to claim 1 or 2, characterized in that aqueous or aqueous-alcoholic solutions of activated-potentiated forms of antibodies to human gamma-interferon (IFN-γ) 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 (IFN-γ) and brain-specific protein S-100 with a concentration of 0.5 ÷ 5.0 mg / ml 5. The drug according to claim 1 or 2, characterized in that each of the components of the smallest doses of affinity-purified antibodies 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 by introducing into the body an activated-potentiated form of antibodies to human gamma-interferon (IFN-γ), characterized in that an activated-potentiated form of ultra-low doses of affinity-purified antibodies to brain-specific protein S-100 is additionally simultaneously and simultaneously administered. . 8. The treatment method according to claim 7, characterized in that a mixture of various homeopathic dilutions of antibodies to human gamma-interferon (IFN-γ) is prepared in the form of a single drug - a combination with a mixture of various homeopathic dilutions of antibodies to a brain-specific protein S-100. 9. The method according to claim 7 or 8, characterized in that the activated-potentiated form of antibodies to human gamma-interferon (IFN-γ) 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 -alcohol solution obtained in the process of successive multiple dilutions in an aqueous or aqueous-alcoholic solvent and an intermediate external mechanical action - vertical shaking.