RU2536230C2 - Therapeutic agent for treating neurological behaviour disorders and method of treating neurological behaviour development disorders - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to treating neurological behaviour development disorders, including attention deficit syndrome (ADS), attention deficit/hyperactivity syndrome (ADHS). A therapeutic agent presented in the form of a pharmaceutical composition 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 the above pathology by the synergetic neurotropic action of the components of the therapeutic agent.

14 cl, 2 ex, 4 tbl, 1 dwg

Description

The invention relates to medicine and can be used to treat neurological-behavioral developmental disorders, including attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD).

Neurological-behavioral developmental disorders are manifested by symptoms such as difficulty concentrating, hyperactivity and poorly controlled impulsivity, which can occur individually (attention deficit disorder without hyperactivity disorder (ADD), hyperactivity without attention deficit disorder), and in combination (deficiency syndrome attention and hyperactivity disorder (ADHD)). Attention Deficit Disorder (ADD) is a common childhood illness that is usually characterized by serious and long-lasting symptoms, such as reduced ability to concentrate for a long time. The main causes of ADD are: the presence of a genetic predisposition; brain damage due to trauma, such as prolonged labor; toxic damage to the central nervous system, for example, bacterial or viral toxins, alcohol (if the mother used it during pregnancy). Hyperactivity without attention deficit disorder is characterized by a condition in which the activity and excitability of a person exceeds the norm. Hyperactivity causes can be infections, birth injuries, premature or difficult births, lead poisoning, poor or unhealthy diets. Hyperactivity is often combined with attention deficit disorder and is called Attention Deficit Hyperactivity Disorder (ADHD). Attention Deficit Hyperactivity Disorder (ADHD, synonymous with Attention Deficit Hyperactivity Disorder (ADHD)) is one of the most common neurological and behavioral disorders in children, which occurs in 4-10% of children. In approximately 50% of patients diagnosed with ADHD, the symptoms of this disease persist in adulthood. The clinical manifestations of ADHD include: motor anxiety, impulsive behavior and thought, lack of attention, inability to concentrate, talkativeness, distraction, etc.

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). However, this drug is not in all cases of treatment of neurological-behavioral disorders, including attention deficit hyperactivity disorder, provides sufficient therapeutic efficacy.

The invention is aimed at improving the treatment of neurological-behavioral developmental disorders, including attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD).

The solution to this problem is provided by the fact that the drug for the treatment of neurological-behavioral development disorders, containing the activated-potentiated form of antibodies to the brain-specific protein S-100, according to the invention, is made in the form of a pharmaceutical composition and contains an activated-potentiated form as an additional - enhancing component antibodies to endothelial NO synthase.

Moreover, a pharmaceutical composition containing an activated-potentiated form of antibodies to the brain-specific protein S-100 and an activated-potentiated form of antibodies to endothelial NO synthase is used to treat attention deficit disorder.

In addition, a pharmaceutical composition comprising an activated-potentiated form of antibodies to the brain-specific protein S-100 and an activated-potentiated form of antibodies to endothelial NO synthase is used to treat attention deficit hyperactivity disorder.

In addition, a pharmaceutical composition comprising an activated-potentiated form of antibodies to the brain-specific protein S-100 and an activated-potentiated form of antibodies to endothelial NO synthase is used to treat hyperactivity (without attention deficit disorder).

Moreover, 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 multiple sequential dilutions in an aqueous or aqueous-alcoholic 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 granules of a neutral carrier 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 repeated serial 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.

In addition, each of the components based on the activated-potentiated form of antibodies is used as a mixture of various, mainly hundred, dilutions obtained by homeopathic technology.

The solution to this problem is also provided by the fact that in the method of treating neurological-behavioral development disorders 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 simultaneously and simultaneously administered. .

Moreover, 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 to treat attention deficit disorder.

In addition, 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 to treat attention deficit hyperactivity disorder.

In addition, 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 to treat hyperactivity (without attention deficit disorder).

Preferably, 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, the activity of which is due to the process of repeated dilution in aqueous or aqueous-alcoholic solvent in combination with external mechanical action - vertical shaking of each dilution.

In this case, a mixture of various dilutions of antibodies to the brain-specific protein S-100 in combination with a mixture of various dilutions of antibodies to endothelial NO synthase prepared by homeopathic technology is used prepared in the form of a single drug - one dosage form.

In addition, the drug for the treatment of neurological-behavioral developmental disorders contains active ingredients in a volume ratio of 1: 1, each component being used as a mixture of three appropriate matrix solutions, diluted 100, ¹² , 100, ³⁰ and 100, ²⁰⁰ times, respectively, which is equivalent hundred dilutions of C12, C30, C200 prepared according to homeopathic technology.

The claimed pharmaceutical composition is recommended to be taken preferably 1-2 tablets 2-4 times a day.

In the treatment of neurological-behavioral development disorders, 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 activated the 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 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 dilutions in combination with external mechanical action - vertical shaking of each dilution (see, for example, V. Schwabe "Homeopathic medicines", M., 1967, p.14-29), which possess The activity due to the technology of potentiation in pharmacological models and / or clinical methods of treating attention deficit disorder and hyperactivity disorder) provides an unexpected synergistic therapeutic effect, confirmed by adequate (valid) experimental models and clinical studies, which consists in increasing the effectiveness of treatment of attention deficit disorder and hyperactivity disorder (ADHD). 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, increased resistance brain toxic effects, which improves integrative activity and restores interhemispheric connections of the brain, contributes to roubleshooting 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 consequently enhances the effectiveness of the treatment of attention deficit hyperactivity disorder.

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

In addition, the claimed drug expands the arsenal of drugs intended for the treatment and prevention of attention deficit hyperactivity disorder.

The graph shows a decrease in the severity of ADHD symptoms (total score on the ADHDRS-IV-Home Version scale) after 2, 4, 6, 8, 12 weeks of therapy compared to the initial value.

The drug is prepared mainly as follows.

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

Monoclonal antibodies are obtained, for example, using hybridoma technology. Moreover, the initial stage of the process includes immunization, based on the principles already developed in the preparation of polyclonal antisera. Further stages of the work include obtaining 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 antigen substances required in accordance with 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 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 100 ¹² , 100 ³⁰ and 100 ²⁰⁰ times, respectively, which corresponds to hundred dilutions of 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.

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 "Neuro-specific protein S-100. Successes in modern biology", 1977 ., t. 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:

- 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 injected subcutaneously to a laboratory animal - a rabbit - in the back area in an 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.

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 cycle of immunization of rabbits for 30 days, they restore health and re-immunization, including 1-3 intravenous injections. To obtain antisera from immunized rabbits, blood is collected in a 50 ml centrifuge tube. Using a wooden spatula, the formed clots are removed from the walls of the tube and the wand is placed in the clot formed in the center of the tube. Blood is placed in a refrigerator (temperature 4 ° C) at night. The next day, the clot attached to the spatula is removed and the remaining liquid is centrifuged at 13000g for 10 minutes. The supernatant (supernatant) is an antiserum. The resulting antiserum should be yellow. Add to the antiserum 20% (weight concentration) NaN ₃ to a final concentration of 0.02% and store until use in a frozen state at a temperature of -20 ° C (or without adding NaN ₃ at a temperature of -70 ° C). To isolate antibodies to 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, 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 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.

The activated-potentiated form of each component is prepared by uniformly decreasing the concentration as a result of sequential 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 (at least 10 times) vertical shaking (“dynamization”) of each dilution obtained and the use of separate containers for each subsequent dilution to Acquiring the required potency - the multiplicity of homeopathic dilution method (see, eg, W. 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 15% 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 component (for example, an activated-potentiated form of antibodies to the brain-specific protein S-100), mixtures of different, mainly hundred, dilutions of each dilution of the component (for example, C12, C30, C200) are prepared separately according to the technology described above to their last but one dilution ( respectively, until C11, C29, C199 is obtained) and then one part of each penultimate dilution obtained is introduced into one container and mixed with the required amount of solvent (respectively, with 97 parts for hundred th breeding). 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 hundred-percent dilutions of C 12, C30 and C200 prepared by homeopathic technology.

It is possible to use the active substance 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 D10, C30, M100, etc.), the effectiveness of which is 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 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 an activated-potentized form antibodies to endothelial NO synthase, and pharmaceutically acceptable additives, including mainly lactose, microcrystalline cellulose and magnesium with tearate.

To obtain a solid oral form of the claimed medicinal product, a fluidized bed is installed (for example, Huttlin Pilotlab type manufactured by Hüttlin GmbH) irrigation until saturation of particles of a neutral substance - lactose (milk sugar) introduced into the fluidized bed - with a particle size of 100 ÷ 300 microns, 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 1 kg of antibody solution to 5 or 10 kg of lactose (1: 5-1: 10) with simultaneous drying in a heated air stream supplied under the grate at a temperature of not higher than 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 / tablet) with an 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 in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ , respectively, which is equivalent to a mixture hundredth dilutions C 12, C30 and C200, Prig tovlennyh 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 2-4 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 components included in its composition - activated potenti bath forms of polyclonal rabbit antibodies to brain-specific protein S-100, purified on antigen, in an ultra-low dose (SMD anti-S100) obtained by super-dilution of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30 , C200 and an 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 cell All homeopathic dilutions of C12, C30, C200 were performed in vitro to bind the standard [ ³ H] pentazocine ligand to the recombinant human sigma-1 receptor using 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, mental and affective disorders, stroke, and is involved in the learning and memory processes. 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 used as a control.

In the experiment (for measuring total binding), 20 μl of the complex preparation of SMD anti-S100 + anti-eNOS or 10 μl of SMD AT to S100 or 10 μl of SMD AT to NOS 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 AT to S100 and SMD AT to NOS tested as single drugs, which allows us to compare the effectiveness of the complex preparation 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 of protein) of the homogenate of the membranes of the Jurkat cell line (human leukemic T-lymphocyte line), and lastly 20 μl of the tritium [ ³ H] pentazocine-labeled radioligand (15 nM), were added.

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

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

The 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% indicates the effects of mild to moderate; inhibition of less than 25% is considered minor effects of the test compound and is 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 with recombinant human 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

To study the properties of the claimed drug for the treatment of patients with attention deficit hyperactivity disorder (ADHD) of the active drug group, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated-potentiated affinity polyclonal forms antigen-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 (with a concentration of 2.5 mg / ml) 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions C12, C30, C200 (SMD anti-S100 + anti-eNOS). For patients of the comparison group, 300 mg tablets were used, impregnated with an aqueous-alcoholic solution (3 mg / tablet) of an activated-potentiated form of polyclonal rabbit antibodies to brain-specific protein S-100, purified on antigen, in an ultra-low dose (SMD anti-8100) obtained by super-dilution of the initial matrix solution in 100 ¹² , 100 ³⁰ , 100 ⁵⁰ times, the equivalent mixture of hundred-percent homeopathic dilutions C12, C30, C50. For patients of the control group (placebo), 300 mg tablets were used containing auxiliary substances (lactose monohydrate - 267 mg, microcrystalline cellulose - 30 mg, magnesium stearate - 3 mg).

The effectiveness of the active drug SMD anti-S100 + anti-eNOS in the treatment of patients with attention deficit hyperactivity disorder (ADHD) was evaluated in a comparative double-blind, placebo-controlled study involving 146 children aged 6 to 12 years (mean age 9.3 ± 0.24 years), which were randomized into three groups depending on the prescribed therapy. For 12 weeks, patients of the active drug group No. 1 (n = 46) received a composition of SMD anti-S100 + anti-eNOS 2 tablets 2 times a day; participants in comparison group No. 2 (n = 50) received 2-daily 2-day SMD anti-S100; patients of the control group No. 3 (n = 50) received placebo 2 tablets 2 times a day. All patients included in the study had clinically pronounced manifestations of ADHD, which was confirmed by high scores on the ADHDRS-IV-Home Version rating scale for symptoms of ADHD: 33.8 ± 0.92 in group No. 1; 32.5 ± 1.14 in group No. 2 and 33.6 ± 0.91 in group No. 3. Most children were characterized by moderate severity of ADHD according to the CGI-ADHD-Severity questionnaire. The total score on this scale was 4.0 ± 0.02 points in group No. 1, 4.0 ± 0.03 points in group No. 2, 4.0 ± 0.00 points in group No. 3. Thus, initially the patients of the three groups had comparable indicators of the severity of ADHD. According to the results of neurological, clinical, laboratory and instrumental examination at the time of inclusion in the study, patients did not register any pathological abnormalities. During the treatment, patients paid the doctor 6 visits for 12 weeks, during which the researcher recorded the dynamics of the severity of the clinical manifestations of ADHD (total score according to the ADHDRS-IV-Home Version scale) and the severity of the disease (according to the CGI-ADHD-Severity scale), supervised the prescribed and concomitant therapy, evaluated the safety of the treatment.

Analysis of the effectiveness of 12-week therapy in three groups showed a decrease of more than 25% of the initial total score on the ADHDRS-IV-Home Version scale in 75% (n = 36) of children who took the composition of SMD anti-S100 + anti-eNOS; in 66% (n = 33) of patients who took anti-S100 SMD, and in 56% (n = 28) of children who took placebo. Differences in effectiveness between the groups, established during a more detailed assessment, taking into account a three-level gradation of improvement (lowering the total score on the ADHDRS-IV scale by <25%; 25-49.9% or ≥50% of the initial value) are presented in Table 2. Significant improvement with a decrease in the total score by 50 or more percent of the initial value was observed in 52% of children of group No. 1 taking SMD anti-S100 + anti-eNOS, and in 34% of children of group No. 2 taking SMD anti-S100 (versus 8% group 3 patients taking a placebo).

The dynamics of the reduction in the severity of ADHD symptoms during the treatment period (the total score on the ADHDRS-IV-Home Version scale at each of the six visits) is presented in the graph and table 3. A significant decrease (p <0.001) in the clinical manifestations of ADHD compared to the initial state occurred after 2 weeks of therapy in all three observation groups. The positive dynamics was more significant in patients No. 1 and No. 2 groups, because they revealed significant differences in the total score of the ADHDRS-IV-Home Version, not only when compared with the screening visit, but also when compared with the placebo group No. 3. In the following weeks of therapy, the effectiveness of treatment with the SMD composition anti-S100 + anti-eNOS and the monocomponent drug SMD anti-S100 increased, most significantly in the active drug group (p <0.05). The total decrease in the total score according to the ADHDRS-IV-Home Version scale in children of group No. 1 SMD anti-S100 + anti-eNOS was 16.5 points, in patients of group No. 2 SMD anti-S100 - 12.4 points (versus 6.3 points in the placebo group No. 3). As a result of the 12-week treatment, the severity of the clinical manifestations of ADHD in children treated with the anti-S100 + anti-eNOS SMD composition decreased by almost half (-48.8%), and in patients treated with the anti-S100 SMD more than a third (-38.2%), compared with the initial state.

Reception of the composition of SMD anti-S100 + anti-eNOS or SMD anti-8100 had an effect on both clusters of ADHD symptoms, which was confirmed by the dynamics of estimates on two sections of the scale with the ADHDRS-IV-Home Version (Table 3). Moreover, treatment with the anti-S100 + anti-eNOS SMD composition significantly exceeded the effectiveness of the anti-S100 SMD treatment with the degree of effect on the severity of manifestations and attention deficit hyperactivity / impulsivity.

The positive therapeutic effect of the active drug SMD anti-S100 + anti-eNOS and the drug comparison SMD anti-S100 was also demonstrated by evaluating the results of treatment of patients on the scale for assessing the severity of ADHD (CGI-ADHR-Severity) (Table 4). In almost a quarter of patients with SMD anti-S100 + anti-eNOS, the severity of the disease decreased from moderate to mild and even minimal, which was confirmed by a 15% decrease in the average CGI-ADHR-Severity score after 3 months of therapy (from 4.0 ± 0.02 to 3.4 ± 0.06; p <0.001). The effect of monotherapy with SMD anti-S100 was slightly less and amounted to 10% on the CGI-ADHR-Severity scale for 3 months (versus 5% in the placebo group).

The safety analysis included data from all patients participating in the study. Throughout the observation period, tolerance of both the active anti-S100 + anti-eNOS SMD preparation and the anti-S100 SMD comparison drug was good, comparable to placebo. Adverse events were recorded in one patient of the SMD anti-8100 group (headaches that were transient during the fourth week of the study) and in one patient in the placebo group (convergence during the second month of observation). The listed adverse events were not associated with the therapy. In addition, during the treatment there were isolated cases of acute respiratory diseases, also not related to the therapy. All patients of the study groups completed the treatment within the time period established by the study protocol; there were no prematurely retired patients. The absence of pathological changes according to the physical examination of patients and during repeated analyzes of laboratory parameters confirmed the safety of the studied therapy.

According to the results of the physical examination (heart rate, SBP, DBP, body temperature), patients did not register any pathological changes during treatment. Differences in the analyzed indicators for visits and in the compared groups did not reach statistical significance and did not go beyond physiologically acceptable deviations. High levels of adherence to therapy additionally testified both to the effectiveness and safety of the studied drugs. By the end of the 3rd month, adherence to treatment was 99.8 ± 1.15% and 98.8 ± 2.25% in groups No. 1 of SMD anti-S100 + anti-eNOS and No. 2 of SMD anti-S100, respectively (versus 74 , 6 ± 2.54% in the placebo group No. 3).

Thus, the study demonstrated the efficacy and safety of the anti-S100 + anti-eNOS SMD composition and the anti-S100 single-component SMD preparation in the treatment of children with ADHD. The most pronounced therapeutic effect as a result of a 12-week course was observed in the complex drug (SMD anti-S100 + anti-eNOS), which was manifested by the positive dynamics of clinical symptoms in the majority (75%) of children. The composition of SMD anti-S100 + anti-eNOS had a corrective effect on both clusters of ADHD symptoms, resulting in a significant reduction in attention disorders and manifestations of hyperactivity in patients with ADHD.

table 2 The dynamics of the total score on the scale of ADHDRS-IV-Home Version by the end of 12 weeks of therapy Patient groups The proportion of patients with a decrease in the total score on the scale ADHDRS-IV-Home Version less than 25.0% of the original 25.0-49.9% of the original 50.0% or more of the original SMD anti-S100 + anti-eNOS, n = 48 12 (25%) 11 (23%) 25 (52%) ^## SMD anti-S100, n = 50 17 (34%) 16 (32%) 17 (34%) ^## Placebo, n = 50 22 (44%) 24 (48%) 4 (8%) Significance of differences compared with the placebo group: ^## p <0.01.

Table 3 The dynamics of the severity of the clinical manifestations of ADHD according to the ADHDRS-IV-Home Version scale Treatment stage SMD anti-S100 + anti-eNOS, n = 48 SMD anti-S100, n = 50 Placebo, n = 50 Value (M ± SE) Δ from the original Value (M ± SE) Δ from the original Value (M ± SE) Δ from the original Total score Screening 33.8 ± 0.96 32.5 ± 1.14 33.6 ± 0.91 2 weeks 24.1 ± 0.97 *** ^# -28.7% 25.1 ± 1.03 *** ^# -22.8% 28.8 ± 1.26 *** -14.3% 4 weeks 22.6 ± 0.98 *** ^## -33.1% 22.7 ± 1.23 *** ^## -30.2% 29.9 ± 1.06 *** -11.0% 6 weeks 19.4 ± 0.95 *** ^## -42.6% 20.8 ± 1.06 *** ^## -36.0% 29.0 ± 1.25 *** -13.7% 8 weeks 18.9 ± 0.94 *** ^{# ##} -44.1% 20.9 ± 1.30 *** ^### -35.7% 27.6 ± 1.35 *** -17.9% 12 weeks 17.3 ± 0.96 *** ^{# ## &} -48.8% 20.1 ± 1.21 *** ^### -38.2% 27.3 ± 1.48 *** -18.8% Attention Disorders Screening 18.4 ± 0.55 17.4 ± 0.57 18.4 ± 0.43 2 weeks 12.8 ± 0.57 *** ^# -30.4% 13.7 ± 0.68 *** ^# -21.3% 16.1 ± 0.66 *** -12.5% 4 weeks 11.6 ± 0.56 *** ^{# ##} -37.0% 12.9 ± 0.79 *** ^### -25.9% 16.4 ± 0.57 *** -10.9% 6 weeks 10.7 ± 0.54 *** ^### -41.8% 11.9 ± 0.64 *** ^### -31.6% 16.0 ± 0.70 *** -13.0% 8 weeks 10.3 ± 0.53 *** ^### -44.0% 11.5 ± 0.70 *** ^### -33.9% 15.1 ± 0.76 *** -17.9% 12 weeks 9.7 ± 0.55 *** ^{## &} -47.3% 11.4 ± 0.68 *** ^## -34.5% 14.9 ± 0.78 *** -19.0% Hyperactivity / impulsivity

Continuation of table 3 Hyperactivity / impulsivity Screening 15.4 ± 0.61 15.1 ± 0.77 15.2 ± 0.62 2 weeks 11.3 ± 0.63 *** -26.6% 11.4 ± 0.61 -24.5% 12.7 ± 0.74 *** -16.4% 4 weeks 11.0 ± 0.62 + *** ^### -28.6% 9.8 ± 0.64 *** ^### -35.1% 13.5 ± 0.67 ** -11.2% 6 weeks 8.7 ± 0.59 *** ^## -43.5% 8.9 ± 0.64 *** ^### -41.1% 12.9 ± 0.73 ** -15.1% 8 weeks 8.6 ± 0.60 *** ^## -44.2% 9.5 ± 0.76 *** ^## -37.1% 12.5 ± 0.81 *** -17.8% 12 weeks 7.6 ± 0.57 *** ^{### &} -50.6% 8.7 ± 0.70 *** ^### -42.4% 12.5 ± 0.82 *** -17.8% Note. Significance of differences compared with the initial indicator: * p <0.05, ** p <0.01, *** p <0.001.
Significance of differences compared with the placebo group: ^# p <0.05, ^## p <0.01, ^### p <0.001.
Significance of differences compared with the anti-S100 SMD group: ^& p <0.05.

Table 4 CGI-ADHD-Severity ADHD Severity Dynamics Indicator SMD anti-8100 + anti-eNOS, n = 48 SMD anti-S100, n = 50 Placebo, n = 50 Screening 4 weeks 12 weeks Screening 4 weeks 12 weeks Screening 4 weeks 12 weeks Severity of ADHD M ± SE 4.0 ± 0.02 3.6 ± 0.02 ** 3.4 ± 0.06 *** 4.0 ± 0.03 3.8 ± 0.06 ** 3.6 ± 0.08 *** 4.0 ± 0.01 3.9 ± 0.05 3.8 ± 0.06 *** Δ from ref. -10% -fifteen% -5% -10% -2.5% -5% Significance of differences compared with the initial indicator: ** p <0.01, *** p <0.001.

During the study, the following methods were used and the following state parameters were determined:

Diagnostic and Statistical Handbook of Mental Disorders (DSM-IV) (to be completed by the researcher)

BUT 1. six (or more) of the following symptoms of inattention were observed for at least 6 months and are so pronounced that they indicate insufficient adaptation and discrepancy with normal age characteristics: Inattention (circle the correct statement) (a) often unable to maintain attention to detail; due to negligence, frivolity, makes mistakes in school assignments, in the work performed and other activities; (b) it is usually difficult to maintain attention when completing tasks or during games; (c) often it seems that the child is not listening to the speech addressed to him; (d) often it is not able to adhere to the proposed instructions and cope to the end with the implementation of lessons, homework or duties at the workplace (which is in no way connected with negative or protest behavior, inability to understand the task); (e) often has difficulty organizing independent tasks and other activities; (e) usually avoids engaging in assignments that require continued retention of mental stress (e.g., school assignments, homework); (g) often loses things needed at school and at home (for example, toys, school supplies, pencils, books, work tools); (h) easily distracted by extraneous stimuli; (i) often shows forgetfulness in everyday situations. 2. six (or more) of the following symptoms of hyperactivity / impulsivity were observed for at least 6 months with a degree of intensity that is unacceptable and does not correspond to the level of development: Hyperactivity (circle the correct statement) (a) restless movements in the hands and feet are often observed; fidgets a chair; (b) often leaves his place in the classroom and in other situations where it is required to remain in his place; (c) often exhibits aimless motor activity: runs, spins, tries to climb somewhere, and in situations where this is unacceptable;

(d) usually can’t play quietly or calmly or do anything at leisure; (e) is often in motion or acts as if "with a motor" (e) often speaks too much Impulsivity (circle the correct statement) (g) often begins to answer without hearing the question to the end; (h) it is usually difficult to wait for their turn in various situations; (i) often interferes with others, pestering others (for example, intervenes in conversations or games) B. Some symptoms of impulsiveness, hyperactivity, and inattention begin to cause concern for those around the age of a child under seven years of age. C. Problems caused by the above symptoms occur in two or more types of environments (for example, at school and at home). D. There is convincing evidence of clinically significant impairment in social contact or schooling. E. Symptoms occur not only as a manifestation of pervasive developmental disorder (PDD), schizophrenia, or other psychotic disorders and cannot be clearly explained by other mental disorders (e.g., mood disorder, anxiety disorder, dissociative disorder, or personality disorder).

ADHDRS-IV-Home Version (DuPaul G., et al. 1998)

(filled out by the researcher according to the legal representatives / parents)

Total score

Inattention (the sum of all odd points)

Hyperactivity / Impulsivity (the sum of all even points)

CGI-ADHD-Severity (Guy W., et al. 1976)

(filled out by the researcher)

Score Description Severity of ADHD one A child is adequate in any environment Normal, healthy No external structure required No different from other children 2 Minor difficulties in any one environment Minimum disease severity If an external structure is required, then only a little Border degradation 3 Some degradation in any one environment Mild disease severity four Some degradation in at least two types of environment Moderate severity of disease Significant difficulties at school and / or control of their behavior 5 Deterioration in all types of environments Severe disease severity Significant stress for others Disrupted social connections 6 Significant degradation constantly in all types of environments Seriously ill Insolvency in study / social environment Daily surveillance is required throughout the day or most of it. 7 The most severe form of the disease One of the hardest patients Complete failure even with constant supervision Note: Circle the score for the severity of ADHD.

Claims (14)

1. A drug for the treatment of neurological-behavioral development disorders, 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 as an additional reinforcing component synthase. 2. The drug according to claim 1, characterized in that the pharmaceutical composition containing the activated-potentiated form of antibodies to the brain-specific protein S-100 and the activated-potentiated form of antibodies to endothelial NO synthase is used to treat attention deficit disorder 3. The drug according to claim 1, characterized in that the pharmaceutical composition containing the activated-potentiated form of antibodies to the brain-specific protein S-100 and the activated-potentiated form of antibodies to endothelial NO synthase is used to treat attention deficit hyperactivity disorder. 4. The drug 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, the activity of which due to the process of successive multiple dilutions in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - vertical shaking of each dilution. 5. The drug according to claim 1 or 4, 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 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. 6. The drug according to claim 1 or 4, characterized in that the aqueous or aqueous-alcoholic solutions of the 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 each dilution from matrix solutions of affinity purified antibodies to brain-specific protein S-100 and to endothelial NO synthase with a concentration of 0.5 ÷ 5.0 mg / ml. 7. The drug according to claim 1 or 4, characterized in that each of the components based on the activated-potentiated form of antibodies is used as a mixture of various, mainly hundred, dilutions obtained by homeopathic technology. 8. The drug according to claim 5, characterized in that the pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate. 9. A method of treating neurological-behavioral developmental disorders 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. 10. The treatment method according to claim 9, 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 to treat attention deficit disorder. 11. The treatment method according to claim 9, 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 to treat attention deficit hyperactivity disorder. 12. The treatment method according to claim 9, 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 to treat hyperactivity. 13. The method according to claim 9, 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 repeated dilution in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - vertical shaking of each dilution. 14. The treatment method according to claim 9, characterized in that use is made of 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 one of a single drug - one dosage form homeopathic technology.

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