RU2536232C2 - Therapeutic agent for alzheimer's disease and method of treating alzheimer's disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: therapeutic agent containing an activated-potentiated form of antibodies to the brain-specific S-100 protein, and an activated-potentiated form of antibodies to endothelial NO-synthase as an additional exalting component is administered.

EFFECT: effective treatment of Alzheimer's disease by the synergetic action of the components.

11 cl, 5 tbl, 3 ex

Description

The invention relates to medicine and can be used to treat Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease and is characterized by a decrease in cognitive functions, memory impairment, confusion, changes in the emotional background, dementia (acquired dementia, a persistent decrease in cognitive activity with the loss of previously acquired knowledge and practical skills and difficulty or impossibility acquiring new ones). Although the main reason for the development of this pathology is currently considered to be the accumulation of amyloid beta, leading to the formation of amyloid beta plaques and neurofibrillary tangles in the brain tissue, AD is also accompanied by a deficiency of the cholinergic system. This is the basis for one of the most common methods of modeling AD in animals using an antagonist of the cholinergic scopolamine system. Administration of scopolamine to experimental animals (usually rats or mice) impairs learning ability and leads to memory impairment.

A neurotropic drug based on antiserum to the brain-specific protein S-100 is known in the art (RU 2156621 C1, A61K 39/395, 09/27/2000).

Also known are the results of studies of the drug based on ultra-low doses of antibodies to the brain-specific protein S-100 in violation of cognitive functions, emotional and neurological status in the experimental model of Alzheimer's disease, from which it follows that the drug has the ability to restore impaired cognitive functions, improves emotional state, weakens anxiety and may be promising for the prevention and treatment of Alzheimer's disease (Voronina T.A., Belopolskaya M.V. et al. low doses of antibodies to S-100 in violation of cognitive functions, emotional and neurological status in an experimental model of Alzheimer's disease - Bulletin of Experimental Biology and Medicine, 2009, No. 8, appendix, p.174-176).

The invention aims to create a more effective means for the prevention and treatment of Alzheimer's disease.

The solution of this problem is provided by the fact that in the method of treating Alzheimer's disease by introducing into the body an activated - potentiated form of antibodies to the brain-specific protein S-100, according to the invention, an activated - potentiated form of antibodies to endothelial NO synthase is additionally introduced (simultaneously and combined).

In this case, the activated — potentiated form of antibodies to the brain-specific protein S-100 and the activated — potentiated form of antibodies to endothelial NO synthase are used in the form of an activated — potentiated aqueous or hydroalcoholic solution of each component, the activity of which is due to the process of successive multiple dilutions in aqueous or aqueous -alcohol solvent in combination with external mechanical action - vertical shaking of each dilution.

Preferably, a mixture of different dilutions of antibodies to the brain-specific protein S-100 in combination with a mixture of different dilutions of antibodies to endothelial NO synthase prepared by homeopathic technology is prepared in the form of a single drug - a single dosage form.

The solution of this problem is also ensured by the fact that for the treatment of a pathological syndrome, including Alzheimer's disease, a drug is used according to the invention, which is made in the form of a pharmaceutical composition and contains an activated - potentiated form of antibodies to the brain-specific protein S-100 and as an additional - enhancing component - activated - potentiated form of antibodies to endothelial NO synthase.

In this case, the activated — potentiated form of antibodies to the brain-specific protein S-100 and the activated — potentiated form of antibodies to endothelial NO synthase are used in the form of an activated — potentiated aqueous or hydroalcoholic solution, the activity of which is caused by the process of multiple sequential dilutions in aqueous or hydroalcoholic solvent in combination with external mechanical action - vertical shaking of each dilution.

The pharmaceutical composition may be in solid dosage form and contain an effective amount of a neutral carrier particle saturated with a mixture of aqueous or aqueous-alcoholic solutions of an activated — potentiated form of antibodies to the brain-specific protein S-100 and an activated — potentiated form of antibodies to endothelial NO synthase, and pharmaceutically acceptable additives including lactose, microcrystalline cellulose and magnesium stearate.

Moreover, aqueous or aqueous-alcoholic solutions of activated - potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase are obtained by multiple sequential dilutions in combination with an external action - vertical shaking of each dilution from matrix solutions of affinity-purified antibodies to the brain-specific protein S- 100 and to endothelial NO synthase with a concentration of 0.5 ÷ 5.0 mg / ml.

Preferably, each component of the claimed medicinal product is used in the form of a mixture of various, mainly hundred, dilutions prepared by homeopathic technology.

In addition, the drug for the treatment of pathological syndrome, including Alzheimer's disease, contains active ingredients in a volume ratio of 1: 1, with each component being used as a mixture of three appropriate matrix solutions, diluted, respectively, 100 ¹² , 100 ³⁰ , and ^100,200 times, which is equivalent to hundreds of dilutions of C12, C30, C200 prepared according to homeopathic technology.

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

In the treatment of a pathological syndrome, including Alzheimer's disease, separate but combined and simultaneous use (administration to the body) of the claimed pharmaceutical composition in the form of two separately prepared preparations, both in the form of solutions, and in solid dosage forms (tablets), each of which contains the activated - potentiated form of ultra-low doses of affinity-purified antibodies to the brain-specific protein S-100 and, accordingly, the activated - potentized form of ultra-low doses of affinity purified x antibodies to endothelial NO-synthase.

The proposed combination of activated - potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase in a pharmaceutical composition (i.e., forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase prepared according to the homeopathic technology of potentiation by multiple sequential dilution and external exposure - vertical shaking (see, for example, Schwabe V. "Homeopathic medicines", M., 1967, p.14-29), which have activity due to technology potentiation, in pharmacological models and / or clinical methods of treatment, including treatment of Alzheimer's disease) provides an unexpected synergistic therapeutic effect, confirmed by adequate (valid) experimental models and clinical studies, which consists in increasing the effectiveness of treatment of Alzheimer's disease. The indicated technical result is provided by an increase in the neuroprotective activity of antibodies to the S-100 protein, due to the influence on the efficiency of the interaction of ligands with the sigma-1 receptor, an increase in the vegetative stabilizing effect, normalization of the vegetative status, a synergistic effect of both components on neuronal plasticity, and, consequently, an increase in brain stability to toxic effects, which improves integrative activity and restores interhemispheric connections of the brain, contributes to injury cognitive disorders, stimulates reparative processes and accelerates the recovery of the central nervous system, improves mental performance, restores learning and memory processes, normalizes somatovegetativnye manifestation, increases cerebral blood flow and, therefore, enhances the efficacy of treatment of Alzheimer's disease.

In this case, the claimed drug, as well as its constituent components, do not have a sedative and muscle relaxant effect, do not cause addiction and addiction.

In addition, the claimed drug expands the arsenal of drugs intended for the treatment and prevention of Alzheimer's disease.

The drug is prepared mainly as follows.

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

Monoclonal antibodies are obtained, for example, using hybridoma technology. Moreover, the initial stage of the process includes immunization, based on the principles already developed in the preparation of polyclonal antisera. Further stages of the work include obtaining 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 purpose, according to a specially developed scheme, animals are given a series of injections with the antigens required by the invention: brain-specific protein S-100 and endothelial NO synthase. As a result of this procedure, monospecific antisera with a high content of antibodies are obtained, which are used to obtain activated - potentiated forms of components. 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 and to endothelial NO synthase, which are used as a matrix (primary) solution with a concentration of 0.5 ÷ 5.0 mg / ml for the subsequent preparation of activated - potentiated forms.

Preferred for the preparation of each component is the use of a mixture of three water-alcohol dilutions of the primary matrix solution of antibodies, diluted, respectively, 100 ¹² , 100 ³⁰ , and 100 ²⁰⁰ times, which corresponds to hundreds of dilutions C12, C30 and C200 prepared according to homeopathic technology. When performing the claimed drug in solid dosage form, a mixture of these components is applied to lactose.

Preferred for the preparation of the claimed drug is the use of polyclonal antibodies to the brain-specific protein S-100 and endothelial NO synthase, which can be obtained by immunization of rabbits as follows.

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

Polyclonal antibodies to endothelial NO synthase are prepared using the adjuvant and the whole endothelial NO synthase molecule of the following sequence as immunogen (antigen) for rabbit immunization:

It is possible to obtain polyclonal antibodies to endothelial NO synthase using the following sequence as an immunogen (antigen) of a whole molecule of endothelial NO synthase:

It is possible to obtain polyclonal antibodies to endothelial NO synthase using, as an immunogen (antigen), a synthetic peptide of endothelial NO synthase, selected, for example, from the following amino acid 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 immunization cycle, the rabbits are restored to health for 30 days and re-immunized, 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 endothelial NO synthase 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, add 6.26 g of Na ₂ SO ₄ , mix and incubate 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 removing 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 obtained antibodies to endothelial NO synthase, which is located on an insoluble matrix, followed by elution with concentrated salt solutions.

Thus obtained buffer solution of polyclonal rabbit antibodies to endothelial NO synthase 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, use the brain-specific protein S-100 (the physicochemical properties of which are described in the article: M.V. Starostina, S. M. Sviridov. Neuro-specific protein S-100. Successes in modern biology, 1977 g., t.5, p.170-178; in the book: 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:

- 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.

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 D), or in 99 parts (for hundredth dilution C), or in 999 parts (for thousandth dilution M ) a neutral solvent in combination with multiple vertical (at least 10 times) shaking (“dynamization”) of each dilution obtained and using separate containers for each subsequent dilution to obtain the desired potency - the multiplicity of homeopathic dilution method (see, eg, V. 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, for the preparation of the 12th hundredth C12 dilution, one part of the above matrix solution of antibodies to the brain-specific protein S-100 (or to NO synthase) with a concentration of 2.5 mg / ml is diluted in 99 parts of a neutral aqueous or aqueous-alcoholic 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 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 2.5 mg / ml in 99 parts of a neutral solvent, t .e. a solution prepared by diluting the matrix solution in 100 ¹² times. Similar operations with the appropriate dilution ratio are carried out to obtain dilutions of C30 and C200.

When using as a biologically active liquid component a mixture of various, mainly hundred, dilutions of the active substance obtained by homeopathic technology, each component of the composition (for example, C12, C30, C200) is prepared separately according to the technology described above to their penultimate dilution (respectively, to obtain C11, C29, C199), then bring 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 Autun 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 super-dilution of the matrix solution, respectively, 100 ¹² , 100 ³⁰ and 100 ²⁰⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions C12, C30 and C200.

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

To obtain the claimed pharmaceutical composition, aqueous or aqueous-alcoholic solutions of the active ingredients are mixed, mainly in a volume ratio of 1: 1 and used in liquid dosage form.

The claimed pharmaceutical composition can also be used in solid dosage form, which contains an effective amount of particles of a neutral carrier - lactose, saturated by soaking in a mixture of aqueous or aqueous-alcoholic solutions with an activated - potentiated form of antibodies to the brain-specific protein S-100 and activated - potentized form antibodies to endothelial NO synthase, and pharmaceutically acceptable additives, including mainly lactose, microcrystalline cellulose and magnesium I stearate.

To obtain a solid oral form of the claimed medicinal product, a fluidized bed is installed (for example, Huttlin Pilotlab type manufactured by Huttlin GmbH) irrigation until saturation of particles of a neutral substance - lactose (milk sugar) with a particle size of 100 ÷ 300 is introduced into the fluidized - boiling layer μm, pre-prepared with an aqueous or aqueous-alcoholic solution of activated - potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial synthase of nitric oxide (NO synthase), mainly in the ratio and 1 kg of antibody solution 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 the brain-specific protein S-100 and to NO synthase in an ultra-low dose, each component prepared from a matrix solution diluted, respectively, in 100 ¹² , in 100 ³⁰ in 100 ²⁰⁰ , which is equivalent a mixture of hundred dilutions C12, C30 and C200, pr caught by homeopathic technology.

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.

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

Example 1

Investigation of the effect of a complex preparation, which includes activated - potentiated forms of polyclonal affinity purified rabbit antibodies to brain-specific protein S-100 (anti-S100) and to endothelial NO synthase (anti-eNOS) in ultra-low doses (SMD) obtained by super-dilution of the original matrix solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions C12, C30, C200, in a ratio of 1: 1 (SMD anti-S100 + anti-eNOS), and also the components included in its composition - activated - potent th e form of rabbit polyclonal antibodies to brain-specific proteins S100, purified on an antigen, in ultralow dose (SMD anti-S100), the obtained sverhrazvedeniem initial matrix solution 100 ^12, 100 ^30, 100 ^{to 200} times equivalent mixture of centesimal homeopathic dilutions C12, C30 , C200 and the activated - potentiated form of polyclonal rabbit antibodies to endothelial NO synthase purified on an antigen in an ultra-low dose (SMD anti-eNOS) obtained by super-dilution of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions of C12, C30, C200, was carried out in vitro to bind a standard ligand of [ ³ H] pentazocine with a recombinant human sigma 1 receptor and 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. Receptors demonstrate a unique ability to translocate, which is caused by many psychotropic drugs. The dynamics of sigma-1 receptors is directly related to the various effects carried out by the drugs when they act on sigma-1 receptors. These effects include regulation of activity channels, ecocytosis, signaling, plasma membrane remodeling (raft formation), and lipid transport / metabolism. All this can contribute to the development of plasticity of neurons in the brain. There is evidence that sigma-1 receptors have a modulating effect on all major neurotransmitter systems: noradrenergic, serotonergic, dopaminergic, cholinergic systems and NMDA-regulated glutamate effects. Sigma-1 receptor plays an important role in the pathophysiology of neurodegenerative diseases, including Alzheimer's, and is involved in learning and memory. In this regard, the ability of drugs to influence the effectiveness of the interaction of ligands with the sigma-1 receptor indicates the presence of neuroprotective, anti-ischemic, anxiolytic, antidepressant, anti-asthenic components in the spectrum of their pharmacological activity, which allows us to consider these drugs as effective drugs, including including for the treatment of cerebrovascular disease.

Potentiated distilled water (a mixture of homeopathic dilutions C12 + C30 + C200) was tested as a control of the tested drugs.

In the experiment (for measuring total binding), 20 μl of the complex preparation of SMD anti-S100 + anti-eNOS or 10 μl of SMD anti-S100 or 10 μl of SMD anti-eNOS was added to the incubation medium. Thus, the amount of SMD anti-S100 + anti-eNOS introduced into the experimental well when testing the complex preparation was identical to the number of SMD anti-S100 and SMD anti-eNOS tested as single drugs, which allows us to compare the effectiveness of the complex drug with its individual the components that make up its composition. Potentiated distilled water was introduced into the incubation medium in a volume of 20 μl and 10 μl.

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

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

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

The results are presented as percent inhibition of specific binding in the control (distilled water was used as 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 SMD anti-S100 + anti-eNOS 20 μl 48,4 35.5 42.0 58.0 SMD anti-S100 10 μl 67.3 63.1 65,2 34.8 SMD anti-eNOS 10 μl 147.5 161.1 154.3 -54.3 Potentized water 20 μl 98.1 75.8 86.9 13.1 Potentized water 10 μl 140.1 106,2 123,2 -23.2 Note: % specific binding in the control = (specific binding in the experiment / specific binding in the control) * 100%; % inhibition of specific binding in the control = 100% - (specific binding in the experiment / specific binding in the control) * 100%).

Results reflecting inhibitions above 50% represent significant effects of the test compounds; inhibition of 25% to 50% indicate mild to moderate effects; inhibitions of less than 25% are considered minor effects of the test compound and are within the background level.

Thus, under the conditions of this experimental model, it was shown that: the complex preparation of SMD anti-S100 + anti-eNOS is more effective than its individual components (SMD anti-S100 and SMD anti-eNOS) inhibits the binding of the standard radioligand [ ³ H] pentazocine to human recombinant sigma-1 receptor; SMD anti-S100, introduced into the experimental well in a volume of 10 μl, inhibits the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma-1 receptor, but the severity of the effect is less than that of the complex drug SMD anti-S100 + anti-eNOS; SMD anti-eNOS introduced into the experimental well in a volume of 10 μl did not affect the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma-1 receptor; potentiated water introduced into the experimental well in a volume of 10 μl or 20 μl did not affect the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor.

Example 2

Patients with an established diagnosis of Alzheimer's disease were included in the study.

To study the properties of the claimed drug in the treatment of Alzheimer's disease, 300 mg tablets were used, impregnated with a pharmaceutical composition containing aqueous-alcoholic solutions (6 mg / tablet) of activated - potentiated forms of polyclonal affinity-purified rabbit antibodies to brain-specific protein S-100 (anti -S100) and to endothelial NO-synthase (anti-eNOS) in ultra low doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) 100 ^12, 100 ^30, 100 ^{to 200} times equivalent cm B centesimal homeopathic dilutions C12, C30, C200 (SMD anti-S100 + anti-eNOS).

As a control group, we used a group of patients receiving a drug in the form of tablets weighing 300 mg, impregnated with an aqueous-alcoholic solution (3 mg / tablet) of an activated, potentiated form of polyclonal rabbit antibodies to brain-specific protein S-100, purified on antigen, in ultra-small dose (SMD anti-S100) obtained by over-dilution of the initial matrix solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C200.

The study was an open, randomized, comparative clinical trial of the efficacy and safety of the study therapy (SMD anti-S100 and SMD anti-S100 + anti-eNOS) in two parallel groups in the treatment of patients with mild to moderate Alzheimer's disease.

The study included 6 patients with a diagnosis of Alzheimer's disease of mild to moderate severity, aged 55 to 64 years. The mean age of the patients was 59.0 ± 3.58 years.

The study examined the compliance of patients with the following inclusion and exclusion criteria:

Inclusion Criteria:

1. Patients with mild to moderate Alzheimer's disease, confirmed by medical history, neurological examination and medical documentation.

2. Constancy of concomitant therapy for at least a month before Visit 1.

3. The lack of prerequisites for a change in neurological concomitant therapy throughout the observation period.

4. Lack of prerequisites for the appointment of immunomodulating agents over the next 6 months.

5. The level of education and the degree of understanding of the patient are sufficient to adequately communicate with the researcher and the coordinator of the study.

6. Patients are evaluated by the researcher as reliable, ready to perform all prescribed clinical visits, tests and procedures provided for by the protocol.

7. Patients have a valid home address.

Exclusion Criteria:

1. A history of surgical intervention on the brain.

2. A history of stroke.

3. A history of psychosis, bipolar disorder, or schizoaffective disorder.

4. Major depressive disorder according to the criteria for the depression module of the international neuropsychiatric mini-interview (MINI).

5. Factors / conditions of a medical or other nature that, in the opinion of the researcher, may affect the patient's test results in this study.

6. Answers “2A”, “2B”, “2B” or “3” in the heading “I” of the Beck Depression Questionnaire (active suicidal thinking with some intention to act, without a specific plan or active suicidal thinking with a specific plan and intent).

7. A history of autoimmune disease.

8. Acute liver damage or severe cirrhosis (Child-Pugh class C).

9. Unregulated thyroid dysfunction.

10. An uncompensated history of hypertension.

11. Serious or decompensated cardiovascular disease, liver, kidney disease, metabolic, respiratory or hematological disease, symptomatic peripheral vascular disease, or other medical or psychiatric condition that, in the opinion of the researcher, may affect the patient’s participation in the study or may lead to prolongation of hospitalization or re-hospitalization of the patient during the study.

12. Diseases and conditions that, in the opinion of the researcher, may interfere with patient participation in the study.

13. Taking Impaz (SMD anti-eNOS) or Tenoten (SMD anti-S100) before taking the study.

14. Admission of antidepressants of any group, not excluding herbal and homeopathic medicines.

15. Reception of anxiolytics of any group, not excluding herbal and homeopathic preparations.

16. Reception of immunomodulators, not excluding herbal and homeopathic preparations.

17. Systemic steroid treatment for 1 month or less prior to Visit 1.

18. Vaccinations, including against the flu.

19. Participation in the study of Impaz (SMD anti-eNOS) or Tenoten (SMD anti-S100), if patients have taken at least one dose of the drug.

20. Participation in other clinical trials for 1 month before inclusion in this study.

21. Pregnancy, breast-feeding, the inability to use adequate contraception during the study and within a month after the last administration of the study drug.

22. The presence of allergies / intolerances to any of the components of the drugs used in the treatment, including lactose intolerance.

23. The use of drugs, antipsychotics, alcoholism, mental illness of the patient.

24. Patients are the staff of the center directly related to the study and / or are members of the family of the staff of the research center directly related to the study. The concept of "family members" includes spouse (s), parents, children, brothers (sisters) - related to both biological and legal kinship.

25. Participation in litigation or alleged compensation or participation in litigation, according to the researcher.

After determining whether the patient met the criteria for inclusion and exclusion of the protocol, the patients were randomized into two study groups: the SMD anti-S100 group (3 people, women 100%, men 0%, mean age 59 ± 3.6 years) and the anti-SMD group -S100 + anti-eNOS (3 people, women - 66.66%, men - 33.33%, average age - 59 ± 4.36 years).

During this study, 5 visits were made to the research center. The treatment phase lasted from Visit 1 to Visit 4 for 84 ± 5 days. Visit 4 (Day 84 ± 5) was the first endpoint of the study, after which the follow-up phase began. Follow-up phase continued until Visit 5 (Day 168 ± 5).

The safety analysis included data from all patients participating in the study (n = 6). During the entire period of observation of patients, good tolerability of the studied drugs was noted. Adverse events were absent. All patients of the study groups completed the treatment within the time period established by the study protocol; there were no prematurely retired patients.

When assessing the effect of the drug SMD anti-S100 + anti-eNOS on the main neuropsychiatric manifestations of Alzheimer's disease (NPI neuropsychic questionnaire, severity section), on the severity of concomitant distress of the caregiver of the patient (NPI neuropsychic questionnaire, distress section), the patient's daily life activity (questionnaire ADS-ADL), as well as the patient's cognitive functions (MMSE questionnaire), an improvement was found in the main neuropsychic manifestations of Alzheimer's disease, expressed in a statistically significant decrease in total ba la under "severity" neurodevelopmental questionnaire NPI (from 24.33 ± 4.73 to 12,0 ± 3,46, p <0,05) for visit 4 (Table 2).

A tendency towards a decrease in the distress of the caregiver and an increase in the activity of the patient’s daily life, which, however, did not reach statistically significant values by the end of therapy, which was possibly associated with an insufficient number of patients, were also revealed.

Along with this, a tendency toward improvement in cognitive functions was revealed, which manifested itself in an increase in MMSE score from 23.66 ± 3.21 points to 26.66 ± 1.53 points, which, however, also did not reach statistically significant values at the end of therapy, which may also be due to an insufficient number of patients.

Similar indicators in the group of patients receiving anti-S100 SMD did not show improvement trends, with the exception of a statistically insignificant improvement in MMSE score from 22.66 ± 0.577 points to 23.33 ± 0.577 points.

Moreover, the difference between the groups according to the overall MMSE score by the end of therapy was significant with a probability of p <0.05.

table 2 NPI (severity) NPI (distress) ADS-ADL MMSE SMD anti-S100 + anti-eNOS before treatment 24.33 ± 4.73 9.66 ± 1.53 71.0 ± 6.56 23.66 ± 3.21 SMD anti-S100 + anti-eNOS after treatment 12.0 ± 3.46 * 5.0 ± 3.61 74.33 ± 2.51 26.66 ± 1.53 ** SMD anti-S100 before treatment 35.66 ± 5.50 22.33 ± 5.50 61.66 ± 5.13 22.66 ± 0.577 SMD anti-S100 after treatment 38.33 ± 8.5 23.0 ± 5.0 61.33 ± 5.86 23.33 ± 0.577 * - p from ref. <0.05; ** - p compared with control <0.05.

Thus, a clinical study of the combined preparation of SMD anti-S100 + anti-eNOS showed a positive effect on the main neuropsychic manifestations of Alzheimer's disease and a possible effect on the cognitive functions of patients with Alzheimer's disease. In addition, high tolerance to the drug has been confirmed. Adverse events when taking the drug were absent.

Example 3

The efficacy of drugs for scopolamine amnesia in rats (model of Alzheimer's disease).

Alzheimer's disease (AD) is a neurodegenerative disease and is characterized by a decrease in cognitive functions, memory impairment, confusion, and changes in the emotional background. Although the main reason for the development of this pathology is currently considered to be the accumulation of amyloid beta, leading to the formation of amyloid beta plaques and neurofibrillary tangles in the brain tissue, AD is also accompanied by a deficiency of the cholinergic system. This is the basis for one of the most common methods of modeling AD in animals using an antagonist of the cholinergic scopolamine system. Administration of scopolamine to experimental animals (usually rats or mice) impairs learning ability and leads to memory impairment.

Various methods are used to evaluate the cognitive functions of rats and mice, in particular the Morris water maze. The essence of this test is that in a vessel with opaque water, animals released into the water from different points are forced to look for a hidden fixed platform. The advantage of this method is that it allows you to observe the learning process of the animal (forming an idea of the spatial location of the platform, regardless of where it was lowered into the water), and to evaluate the strength of memorization (for this a test test is carried out, when the platform is taken out).

Example 3 below examined the efficacy in rat scopolamine amnesia of the claimed drug in the form of a composition containing activated - potentiated forms of polyclonal affinity-purified rabbit antibodies to brain-specific protein S-100 (anti-S100) and to endothelial NO synthase (anti- eNOS) in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (with a concentration of 2.5 mg / ml) 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C200 (SMD anti-S10 0 + anti-eNOS).

In a study of the efficacy of SMD anti-S100 + anti-eNOS in scopolamine amnesia in rats (Alzheimer's disease model), 48 male Rj: Wistar (Han) rats were used (weight 180-280 g). For 4 days, rats were injected subcutaneously with saline (n = 12, intact) or scopolamine at a dose of 0.5 mg / kg (n = 36) (scopolamine-induced amnesia). Rats with scopolamine-induced amnesia were divided into 3 groups and, respectively, distilled water (7.5 ml / kg, n = 12, control, group No. 1), SMD anti-S100 (7.5 ml / kg, n = 12, group No. 2) and SMD anti-S100 + anti-eNOS (7.5 ml / kg, n = 12, group No. 3) intragastrically for 9 days (4 days before the start of scopolamine injection, 4 days against the background of administration scopolamine and 1 day after the end of scopolamine administration).

Within 4 days of scopolamine administration, 60 minutes after the administration of the test drugs and 30 minutes after the administration of scopolamine, a training session was conducted in the Morris maze (4 consecutive tests with an interval of 60 seconds). The Morris labyrinth is a round vessel (150 cm in diameter, 45 cm high), 30 cm filled with water (26-28 ° C). 18 cm from the edge of the vessel is a hidden platform (15 cm in diameter), recessed 1.5 cm below the water level. The water is made turbid by adding a non-toxic dye (for example, milk powder), which makes the platform invisible. For each test, the animal was placed in a maze at one of the starting points located at the same distance from the hidden platform, and allowed them to find it. If the animal could not find the platform in 120 seconds, it was placed on the platform for 60 seconds and then a new test was started. During 4 trials in random order, the animals began passing through the maze twice from each starting point. The tests were recorded on videotape, and then the distance covered in search of the platform in each test and the latent period of the search for the platform were analyzed.

On the 5th day, a test was performed: the platform was removed from the maze, and the rat was allowed to swim freely for 60 seconds. The time spent in the place where the platform used to be was recorded.

The introduction of scopolamine significantly impaired the learning ability of animals: in the control group No. 1, the time spent by the animals searching for the platform, and the distance that the animals sailed in search of the platform, increased significantly (Tables 3, 4). The test showed that the memory of the animals of the control group No. 1 deteriorated significantly: in the place where the platform used to be, they spent less time than intact rats (Table 5). The introduction of SMD anti-S100 in group No. 2 did not lead to an improvement in the studied parameters (Tables 3, 4, 5). The introduction of SMD anti-S100 + anti-eNOS in group No. 3 led to some improvement in training, which was expressed in shortening the latent search time of the platform (Table 3) and the distance covered (Table 4) for 4 days of training, and memory improvement, which was expressed in increasing the time spent in the place where the platform was (Table 5).

Table 3 Latent period of platform search, sec Group Training 1 day 2 day 3 day 4 day Intact, n = 12 54.7 ± 6.2 30.8 ± 2.8 26.9 ± 5.1 20.5 ± 3.6 Control, n = 12 100.1 ± 6.8 *** 92.4 ± 9.3 *** 81.4 ± 10.7 *** 77.7 ± 9.4 *** SMD anti-S100, n = 12 106.8 ± 7.0 99.3 ± 7.8 95.6 ± 9.0 80.4 ± 11.1 SMD anti-S100 + anti-eNOS, n = 12 94.4 ± 7.2 90.7 ± 8.2 78.3 ± 8.6 60.1 ± 10.2 *** - significant difference from intact, p <0.05.

Table 4 Distance covered in search of a platform, cm Group Training 1 day 2 day 3 day 4 day Intact, n = 12 1055.7 ± 94.6 659.5 ± 62.2 564.8 ± 119.3 406.1 ± 61.2 Control, n = 12 2587.1 ± 217.2 *** 2559.6 ± 250.5 *** 2397.9 ± 312.6 2366.1 ± 293.8 *** SMD anti-S100, n = 12 2797.2 ± 208.9 2865.2 ± 255.1 2857.01300.8 2457.4 ± 344.4 SMD anti-S100 + anti-eNOS, n = 12 2434,31222,8 2529.9 ± 282.7 2344.2 ± 283.0 1905.1 ± 343.7 *** - significant difference from intact, p <0.05.

Table 5 Time spent in the place where the platform used to be, sec Group Try 0-30 sec 30-60 sec 0-60 sec Intact, n = 12 40.8 ± 4.1 36.8 ± 3.6 38.5 ± 2.6 Control, n = 12 18.4 ± 2.8 *** 18.8 ± 1.9 *** 18.8 ± 1.7 *** SMD anti-S100, n = 12 13.3 ± 2.1 21.5 ± 2.6 17.6 ± 1.3 SMD anti-S100 + anti eNOS, n = 12 19.1 ± 4.8 23.8 ± 2.2 21.2 ± 2.5 *** - significant difference from intact, p <0.05.

Thus, in the model of Alzheimer's disease used, the use of the anti-S100 + anti-eNOS SMD complex was more effective than the isolated administration of anti-S100 SMD.

Claims (11)

1. A method of treating neurodegenerative diseases by introducing into the body an activated - potentiated form of antibodies to the brain-specific protein S-100, characterized in that an activated - potentiated form of antibodies to endothelial NO synthase is additionally simultaneously and simultaneously administered. 2. The method according to claim 1, characterized in that the neurodegenerative disease is Alzheimer's disease. 3. The method according to claim 1, characterized in that the activated — potentiated form of antibodies to the brain-specific protein S-100 and the activated — potentiated form of antibodies to endothelial NO synthase are used in the form of an activated — potentiated aqueous or aqueous-alcoholic solution of each component, activity which is due to the process of multiple dilutions in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - shaking of each dilution. 4. The treatment method according to claim 1, characterized in that a mixture of different dilutions of antibodies to the brain-specific protein S-100 in combination with a mixture of different dilutions of antibodies to endothelial NO synthase prepared according to homeopathic prepared in the form of a single drug is used technologies. 5. A drug for the treatment of neurodegenerative diseases, containing an activated - potentiated form of antibodies to the brain-specific protein S-100, characterized in that it is made in the form of a pharmaceutical composition and contains an activated - potentiated form of antibodies to endothelial NO synthase as an additional - enhancing component. 6. The drug according to claim 5, characterized in that the neurodegenerative disease is Alzheimer's disease. 7. The drug according to claim 5 or 6, characterized in that the activated - potentiated form of antibodies to the brain-specific protein S-100 and the activated - potentiated form of antibodies to endothelial NO synthase are used in the form of an activated - potentiated aqueous or aqueous-alcoholic solution, the activity of which is due to the process of successive multiple dilutions in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - shaking of each dilution. 8. The drug according to claim 5, 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 activated or aqueous-alcoholic solutions of a potentiated form of antibodies to the brain-specific protein S-100 and activated - potentiated forms of antibodies to endothelial NO synthase; and pharmaceutically acceptable additives. 9. The drug according to claim 5, characterized in that aqueous or aqueous-alcoholic solutions of activated - potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase are obtained by repeated serial dilution in combination with an external action - vertical shaking of each dilutions from matrix solutions of affinity-purified antibodies to the brain-specific protein S-100 and to endothelial NO synthase with a concentration of 0.5 ÷ 5.0 mg / ml. 10. The drug according to claim 5, characterized in that each of the components is used in the form of a mixture of various, mainly hundred, dilutions prepared by homeopathic technology. 11. The drug according to claim 6, characterized in that the pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate.