RU2362554C2 - Regenerating resolvent and ways of treatment by means of this agent - Google Patents

Abstract

FIELD: medicine; pharmaceutics. ^ SUBSTANCE: regenerating resolvent represents an aqueous solution of comenic acid and auxiliary substance - sodium bicarbonate at a following parity of components, wt %: comenic acid - 1-2, sodium bicarbonate - 0.55-1.1, water for injections - up to 100. The way of treatment of chorioretinal dystrophias of various etiology consists that the regenerating agent is administered to the patient parabulbary in a dose from 10 to 20 mg (in recalculation on comenic acid) once a day within 10 days. At including in complex antituberculous therapy the way of treatment consists that the regenerating agent is administered to the patient intravenously in a dose to 350 mg a day (in recalculation on comenic acid) within 10 days. ^ EFFECT: agent does not cause dependence and addiction, intoxication. ^ 7 cl, 5 ex, 3 tbl

Description

The claimed invention relates to pharmaceuticals and medicine, specifically to a regenerating agent based on comenic acid and to a method for treating chorioretinal dystrophies using this agent.

The claimed invention can be used to produce highly effective synthetic non-peptide nature regenerative anti-inflammatory drugs, which, in turn, can be used in medicine and veterinary medicine for the treatment of chorioretinal dystrophies of various origins, including tuberculosis.

Regenerating agents combine drugs of various pharmacological groups that can directionally affect the proliferation, regeneration and metabolic rate.

The normal functioning of the body is accompanied by a continuous process of replacing dying cells and tissues, called physiological regeneration. Different tissues differ in their ability to restore (regenerate), which is the higher, the greater the role played by physiological regeneration in the structure and functioning of the tissue. Blood cells, mucous membranes of the gastrointestinal tract, integumentary epithelium of the skin, etc. are rapidly renewed and therefore the tissues that they form are characterized by a high regeneration potential. On the contrary, in neurons and muscle cells, the regeneration potential is minimal (approaching zero). Under the influence of age, concomitant diseases, toxic and environmental factors, radiation, the process of physiological regeneration can be slowed down. Some drugs have a similar effect, including immunosuppressants, antitumor drugs, corticosteroids, some antibiotics and NSAIDs, etc. Inhibition of the physiological regeneration process is accompanied by the development of metabolic disorders, the occurrence of leukemia and thrombocytopenia, anemia, and mucosal lesions, including gastrointestinal tract, etc. Drugs that can increase the speed and intensity of physiological regeneration are called regeneration stimulants, or regenerants.

The restoration of tissue sites and organs that died as a result of injuries, injuries or degeneration (intoxication, hypoxia, infection, etc.) is a process of reparative regeneration. Accordingly, drugs with the ability to stimulate reparative regeneration are reparants (Mashkovsky M.D. Medicines. 14th edition. M: New wave, 2003, v.1, p.145). As a result of the repair, the foci of necrosis are replaced by specific and / or connective (has the highest regeneration potential) tissue.

The general mechanism of regenerative action includes enhancing the biosynthesis of purine and pyrimidine bases, RNA, functional and enzymatic cellular elements, including membrane phospholipids, as well as the stimulation of DNA reduplication and cell division. It should be noted that the process of biosynthesis during both physiological and reparative regeneration needs substrate support (essential amino and fatty acids, trace elements, vitamins). In addition, the biosynthesis of proteins and phospholipids is highly energy intensive and its stimulation requires appropriate energy supply (energy materials). Actovegin, solcoseryl, etc. include such agents that provide substrate and energy for the regeneration processes. The effect of these drugs and the regeneration process itself is often difficult to differentiate.

In accordance with the localization of the action (and the tasks of pharmacotherapy), regeneration and repair stimulators are conditionally divided into general cell (universal) and tissue-specific. Common cell stimulants acting on any regenerating tissue include anabolic steroids, non-steroidal anabolics - sodium deoxyribonucleate (Derinat), methyluracil, inosine, etc. and vitamins of plastic metabolism. Tissue-specific stimulants of the regeneration process are drugs with different mechanisms of action, combined into selective subgroups the effect on one or another tissue or system of organs (Mashkovsky M.D. Medicines. 14th edition. M: New wave, 2003, t.1, t.2). The most famous drugs for the treatment of chorioretinal dystrophies are glutoxim, cortexin, phezam, emoxipin. All of them have a number of limitations in use due to allergic reactions.

A number of diseases, in particular in ophthalmic practice, lead to disability of a large number of patients, and treatment remains purely symptomatic. Improving the treatment of patients with retinal pathology is one of the main tasks of modern ophthalmology and requires the search and development of new medications. In recent years, much attention has been paid to bioregulatory therapy with organotropy and pathogenetic orientation. The properties of this group of drugs are due to the participation of peptides in protein synthesis, the regulation of cellular metabolism in peptide processing (cleavage of the necessary amino acid sequences). The multiplicity of the regulatory systems of the body suggests the presence of universal intermediaries - regulatory peptides. They are necessary for the transmission of information signals between cells and are involved in maintaining the structural and functional homeostasis of cell populations. The functioning of biological regulation is based on the principle of the peptide cascade. One of the most effective drugs in this group is the retinal agent, registered in 1999, and retinal reporter - retinalamine. Retinalamin (a complex of polypeptide fractions from the retina of cattle or pigs) has a tissue-specific regenerating effect on the retina of the eye (IB Maksimov, LK Moshetova, SA Savostyanova. Retinalamine in the complex treatment of involutional central chorioretinal dystrophies. St. Petersburg ., 2006, 96 p.). A well-known drug is selected as a prototype for the inventive regenerating agent. It is a complex of peptides of para- and autocrine nature ranging in size from 1000 to 10000 Da, performing the function of an intracellular and intercellular messenger. It belongs to the group of cytomedines. Available in the form of lyophilized powder in vials of 5 mg retinalamine in each. Diluted retinalamine is used as a clear, colorless aqueous solution for parabulbar and intramuscular injections. The complex composition of retinalamine does not allow the usual pharmacokinetic analysis of individual components. Thus, a pharmacokinetic study of retinalamin has not been carried out, which is a clear drawback of the drug. The specific action of the known drug is aimed at stimulating photoreceptors and cellular elements of the retina, improving the functional interaction of the pigment epithelium and the outer segments of the photoreceptors, accelerating the restoration of light sensitivity of the retina. The non-specific effect of retinalamin is manifested in the normalization of vascular permeability, a decrease in various manifestations of the inflammatory reaction, and stimulation of reparative processes in the retina. Retinalamin has a metabolic and retinoprotective effect, has a bioregulatory effect on the organ of vision and tissue-specific effect on the retina. The drug is contraindicated in case of individual intolerance and pregnancy. When used according to the indications in recommended doses, no side effect was detected. The drug interaction of the drug retinalamin with other drugs is not described. Retinalamine is stored in a dry, dark place at a temperature of 2 ° to 20 ° C. Shelf life is 2 years. The drug is not stable in solution, so it is stored in powder form and diluted immediately before injection. This is a significant disadvantage of the known drug. The drug is dispensed by prescription.

A treatment method using retinalamin is selected as the prototype method. Retinalamin is used to treat central chorioretinal dystrophy of the retina (CCRD), diabetic retinopathy, and tapetoretanal abiotrophy. In the postoperative period, retinalamin is used in patients with retinal detachment, with laser hypercoagulation, in patients with involutional CVD. The use of the drug in the treatment of central retinal vein thrombosis and the prevention of retrombosis, as well as chorioretinitis of various etiologies, is recommended. The drug is administered to adults (for children, recommendations on the use of the drug are not yet available) parabulbularly or intramuscularly once daily (the contents of the vial are dissolved in 1-2 ml of a 0.5% solution of novocaine or water for injection, or isotonic sodium chloride solution) - 5 -10 mg per day for 5-10 days (if necessary, repeat the course after 3-6 months.). The somewhat delayed effect of therapy and the persistence of the result are due to the effect of the peptide cascade. A well-known drug, as the authors of the invention established in the course of specially set experiments, does not affect the effectiveness of complex anti-tuberculosis therapy in the treatment of tuberculous chorioretinitis.

A common disadvantage of the known methods is that they cause a series of side effects in relation to the cardiovascular, immune, respiratory, reproductive systems and adversely affect the liver and kidneys of patients.

From the above analysis it follows that due to the complexity of the mechanism of the regeneration process in the body and the non-obviousness of the regulation of the action of components of known drugs, especially peptide, side effects caused by them, the problem of developing universal synthetic regenerating drugs remains relevant.

The objective of the present invention is to provide a highly effective synthetic regenerative agent, non-peptide in nature, without the negative side pharmacological effects inherent in known analogues, and methods of treating chorioretinal dystrophy of various etiologies using this agent.

This problem is solved by the claimed group of three inventions, united by a single inventive concept, a regenerating agent based on comenic acid and two methods of treatment using this tool.

The inventive composition is characterized by the following set of essential features:

1. A regenerating anti-inflammatory agent, which is an aqueous solution of the active principle - comenic acid and auxiliary substance - sodium bicarbonate, in the following ratio, in wt.%:

comenic acid - 1-2, mainly 1 and 2, sodium bicarbonate - 0.55-1.1

water for injection - up to 100.

2. The regenerating anti-inflammatory agent according to claim 1, characterized in that it additionally contains an admixture of comenic acid, benzylcomenic acid - in an amount of not more than 2.5% in terms of comenic acid.

The set of essential features of the claimed invention provides a technical result: the created regenerative anti-inflammatory agent is intended to stimulate humoral immunity, which increases macrophage phagocytic activity against the background of emotional stress, exhibits anti-inflammatory and antioxidant properties, increases the effectiveness of standard anti-tuberculosis therapy, exhibits neuroretin-protective and retinostimin effects and retinostim prepar Atoms of the group of cardiac glycosides, the neurotrophic and trophic properties of the drug are tissue-specific. According to preclinical studies and expert evaluation, he has no negative side effects. The claimed agent in therapeutic doses does not affect the cardiovascular system, blood count, respiratory system, embryogenesis, does not stimulate terratogenesis, does not cause allergies, does not affect reproductive function, does not stimulate tumor growth. Neurotoxic effects of the claimed drug are absent. High values of such indicators as clearance (450-500 ml / kg / h) and stationary volume of distribution (2.5 l / kg) indicate that the claimed drug is very actively distributed from the systemic circulation. Most of the drug is not excreted, but is metabolized and utilized in the body itself. The latter makes it possible to recommend the use of the claimed drug in patients with renal and hepatic insufficiency. A pharmacokinetic study of the inventive drug, conducted by the authors, makes the use of the inventive agent more preferable, since no pharmacokinetic analysis of the prototype retinalamine has been performed. The specific pharmacological properties of the claimed drug are realized after intravenous or parabulbar (in the treatment of retinopathies of various origins) administration after 5-7 minutes. The active substance - comenic acid - is technologically available. A study of acute and subacute toxicity showed that the drug (dosage form in the form of 1 and 2% solutions for injection in ampoules of 1, 2, 5 ml) belongs to class V of practically non-toxic compounds. The inventive tool is not addictive and addictive, crosses the blood-brain barrier. The inventive tool is stable in the form of a solution for injection. When stored under natural conditions, its decomposition is about 1% per year. Based on these data, the expiration date is set to 2 years.

The creation of the claimed drug became possible due to the study by the inventors of the elements of a subtle mechanism of exposure to substances capable of forming chelate complexes with calcium and magnesium ions, which exhibit anti-inflammatory properties, tissue-specific trophic and neurotrophic properties that directionally regulate the coding of nociceptive information in the central nervous system that do not have side effects on vital functions of the body, and a thorough study of the membrane signal path tion of, through which the activation of Na / K-ATPase as a signal transducer during regulation of synthetic processes in tissues of various types (Lopatin EV Penniyaynen VA AA Hare Research participation Na ^+, K ⁺ -ATPase in the regulation of the growth of explants of heart tissue in organotypic culture. Bulletin of experimental biology and medicine. 2005, v. 140, No. 8, pp. 150-153; Penniyaynen V.A., Lopatina E.V. Study of the role of Na ⁺ , K ⁺ - ATPases in the regulation of neurite growth of sensory neurons. Bulletin of Experimental Biology and Medicine. 2005.T. 139, No. 2. S.147-160; Lopatina E.V., Penniyaynen V.A., Tsyrlin V.A. Comparative analysis of the action of cardiac glycosides on the growth of explants of heart tissue. Physiological magazine them. Sechenova, 2005.V. 91. No. 11. S.1299-1304; Karetsky AV, Lopatina EV, Penniyaynen VA a3-izoform of Na ⁺ , K ⁺ -ATPase modulates process of cells growth in the chicken retina. Humboldt-Kolleg Conference "Technologies of the 21st century: biological, physical, informational and social aspects" Saint-Petersburg. 2005. P.30). A similar function - the function of the transductor Na / K-ATPase performs when modulating the coding of a nociceptive signal (Krylov B.V., Derbenev A.V., Podzorova S.A., Lyudino M.I., Kuzmin A.V., Izvarina N. L. Morphine reduces the potential sensitivity of slow sodium channels // Russian Physiol. Zh. 1999, v. 85, No. 2, pp. 225-236). As a result of this study, the authors of the claimed invention predicted that comenic acid can be used as an active substance in the manufacture of an anti-inflammatory synthetic regenerating agent that exhibits immunomodulatory, antioxidant and other effects described above. The claimed properties are realized by the claimed means due to the ability to form chelate complexes with calcium and magnesium ions, followed by activation of a specific molecular mechanism of membrane signaling, including the alpha three isoform of Na / K-ATPase as a signal transducer. The mechanism for implementing the effects of the claimed drug was discovered by the authors of the claimed invention.

The inventive composition differs from the prototype preparation by a number of essential features: a qualitative composition, primarily using another active substance - comenic acid, corresponding quantitative characteristics, in that a solution ready for injection is claimed. The proposed drug is characterized in that it is a synthetic agent, the drug, in contrast to the prototype drug and known analogues, is not a peptide compound, the drug effect of which is difficult to predict and regulate, the dosage form of the drug is made in the form of a ready-made stable, sterile injection solution.

Analysis of the prior art did not allow to find a solution that completely coincides in the totality of essential features with the claimed. Previously, the inventors have shown that kamenovo acid has a sedative effect, in doses exceeding the values of the claimed invention (RF patent No. 2209062 "A substance with a sedative effect" from 03.19.02, publ. 07.27.03), which does not discredit the novelty of the claimed invention . Thus, we can assume that the claimed tool has novelty.

Only the set of essential features of the claimed regenerating means allows to achieve the specified technical result. The ability of the composition of the substances that form the basis of the claimed agent to exhibit stable regenerative activity was discovered by the inventors, as mentioned above, by chance, when studying the role of Na / K-ATPase as a signal transducer in the regulation of synthetic processes in various types of tissues.

It should be emphasized that so far no regenerating agents have been created that have no negative side effects and are comparable in degree of regeneration with anabolics.

Thus, the proposed regenerative tool may meet the eligibility condition "inventive step" ("non-obviousness").

The claimed methods of treatment are the following set of essential features:

Method 1

1 (3). A method of treating chorioretinal dystrophies of various etiologies, namely, that the patient is administered a regenerating agent according to claims 1 to 2 parabulbar at a dose of 10 to 20 mg (in terms of comenic acid) once a day for 10 days.

2 (4). The treatment method according to claim 3, characterized in that the course of treatment is repeated 2-3 times a year.

3 (5). The treatment method according to claim 3, characterized in that in ophthalmology the patient is administered a regenerating agent in one or both eyes.

Method 2

4 (6). A method of increasing anti-tuberculosis therapy, which consists in the fact that the patient is given a regenerating agent according to claims 1 to 2 intravenously at a dose of up to 350 mg per day (in terms of comenic acid) for 10 days.

5 (7). The treatment method according to claim 4 (6), characterized in that the course of treatment is carried out 1-3 times a year with a break of 3-6 months.

The inventive tool is not addictive and addictive, therefore, the dose of the drug used in the treatment of chorioretinal dystrophies and in complex anti-tuberculosis therapy remains unchanged during treatment even with prolonged use.

The introduction of the inventive regenerating agent parabulbar at a dose greater than 20 mg per day (in terms of the active substance - comenic acid) is impractical, since the regenerating and anti-inflammatory effect does not increase.

The introduction of the claimed drug intravenously at a dose of more than 350 mg per day (5 mg / kg) (in terms of the active substance - comenic acid) when included in complex anti-tuberculosis therapy is not practical, since the anti-inflammatory effect does not increase.

The set of essential features of each of the proposed methods of treatment allows to achieve a technical result consisting in the effective treatment of retinopathies, including tuberculosis in nature, improving the effectiveness of the complex treatment of tuberculosis and a number of degenerative diseases, which allows high-quality treatment to treat the underlying disease and significantly improve the quality of life of patients . During treatment, there is no addiction to the drug, which allows for continuous treatment, without changing the drug, to achieve a sustainable effect.

In contrast to the method of treatment using the prototype retinalamin, the proposed methods of treatment do not have side effects characteristic of peptide preparations, namely, the development of allergic reactions, dizziness. The mechanism of action of the claimed funds is purely selective. This is especially valuable with surgical ophthalmic interventions. Thus, the claimed method of treatment is gentle. Methods of treatment using the inventive tool are more convenient for medical personnel, since it does not require additional manipulations associated with its preparation (dissolution), therefore, the working time of the staff with each patient is reduced.

It should be emphasized that the protinal retinalamine is not used in the treatment of tuberculosis.

The claimed methods of treatment differ from the prototype method using retinalamine with a number of essential features: using another active substance - the claimed regenerating anti-inflammatory agent based on comenic acid in a certain dose and duration of treatment. The prototype method, in contrast to the claimed, includes a preliminary stage of preparation - preparation of a retinalamine solution. Retinalamin is administered intramuscularly and parabulbarno, the claimed drug is parabulbarno and intravenous.

Completely coinciding with the claimed combination of essential features of a method for the treatment of chorioretinal dystrophies was not found, this can serve as proof of the novelty of the claimed methods.

Only the combination of essential features of each of the claimed methods of treatment allows to achieve the specified technical result - the implementation of an effective comprehensive pathogenetic anti-tuberculosis therapy by increasing the humoral immunity and phagocytic activity of macrophages; directed modulation of the proliferation of retinal cells and cardiomyocytes, stimulation of neurite growth. Indeed, the method of complex pathogenetic treatment of tuberculosis is still not known, during the implementation of which there is a directed effect on the stimulation of humoral immunity, as a result of which the macrophage phagocytic activity and potentiation of the drugs used in standard anti-tuberculosis therapy are increased. In the treatment of retinal dystrophy, which arose as a complication of developing tuberculosis of the eye, a significant decrease in the area of fibrous foci was detected when the inventive drug was included in the complex therapy. This is due to the fact that the latter realizes its properties in the formation of chelate complexes with calcium (magnesium) ions and activates the function of Na / K-ATPase as a signal transducer that stimulates the proliferation process without intoxication, addiction and relapse. Special studies have shown that if the claimed agent has powerful tissue-specific trophic, retinoprotective and neurotrophic properties, the drug does not stimulate the onset and growth of tumors. The inventive tool can be recommended for patients with renal and hepatic insufficiency, since it is not excreted by the liver and kidneys, but is metabolized in the body itself, forming glucuronic conjugates or complex compounds with cations of calcium and magnesium metals. All this indicates the non-obviousness of the claimed methods.

Thus, based on the foregoing, the claimed group of inventions as a whole can claim to meet the eligibility criteria of “novelty” and “non-obviousness”.

To confirm the compliance of the proposed solution to the requirement of "industrial applicability", as well as to better understand the essence of the invention, we give examples of its specific implementation.

Prototype

Retanalamine used for sale (Heropharm company).

The reagents used to prepare the inventive regenerating agent:

The active principle of the drug is comenic acid (5-hydroxy-4-oxopyrone-2-carboxylic acid) synthesized according to the procedure (Garkusha, J.A. Zh. General chemistry. 1961, v.31, p.2573). The melting point (261-267 ° C) and spectral analysis are consistent with published data. As an impurity of comenic acid, benzylcomenic acid may be present - in an amount of not more than 2.5% in terms of comenic acid.

As an auxiliary substance, sodium bicarbonate (GOST 4201-79; FS 42-0324-4716-03) is included in the composition of the claimed agent in order to create an optimal pH value that promotes dissociation of comenic acid and increases its solubility.

Water for injection corresponded to the requirements of FS 42-2620-97. Sterilization of the agent is carried out by membrane filtration under aseptic conditions. The filtrate is poured into neutral glass ampoules and sealed.

The composition of the claimed funds

The positive claimed effect of the drug is manifested for a 1-2% aqueous solution. The preferred form of the claimed funds (dosage form) is a 1% or 2% aqueous solution for injection in ampoules of 1, 2 and 5 ml. The active substance is comenic acid, the concentration of which is 10 mg in 1 ml (1% solution of an analgesic), or 20 mg in 1 ml (2% solution). The composition of the dosage form of the claimed drug in terms of 100% substance includes sodium bicarbonate 5.5 g (1% solution), or 11 g (2% solution), water for injection up to 1 l (or 1-2 g of comenic acid, 0 , 55-1.1 g of sodium bicarbonate and water to 100 ml). The inventive tool is a clear liquid from yellowish to yellow (10 mg / ml), from yellowish-greenish to yellow-green (20 mg / ml) odorless, pH is from 4.1 to 5.6 (potentiometric). Among foreign impurities, benzylcomenic acid, which is an intermediate product of the synthesis of comenic acid, has been identified. The standard for the total content of impurities is established on the basis of storage data and taking into account the admissible level of impurities in the substance of comenic acid: not more than 5% (the content of benzylcomenic acid is not more than 2.5%) in terms of comenic acid. The inventive tool is a stable compound. When stored under natural conditions: the degree of decomposition is about 1% per year. The shelf life of a sterile preparation in a dark place at a temperature of no higher than + 25 ° C is 2 years. The agent is pyrogen-free. A test dose of 10 mg per kg of animal weight, in a volume of 0.5 ml and 1 ml, respectively, for preparations with a concentration of 20 mg / ml and 10 mg / ml. The tool is non-toxic, belongs to the V class of practically non-toxic medicinal substances (for comenic acid) (N. Hodge et al .. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed. IV, Baltimore, 1975, 427 p.).

Example 1. Pharmacokinetics of the claimed drug

With the intravenous administration of the claimed drug (mainly in the form of 1 and 2% solution for injection) to dogs, the pharmacokinetics of the active substance of the drug - comenic acid - in the blood plasma is described by a 3-part dependence. The first phase of the distribution proceeds rapidly - the characteristic time in this section is 1.5 minutes. In the first 10 minutes, about 95% of comenic acid (the active substance of the drug) leaves the blood plasma, while its concentration decreases from 50-60 μg / kg to 3 μg / kg, i.e. almost 20 times. The next phase lasts from the 10th minute until the beginning of the 2nd hour; the characteristic time is about 30 minutes, and the concentration of comenic acid decreases from 3 to 0.6 μg / ml, i.e. 5 times. After the 2nd hour, apparently, the phase of true elimination begins with a time T _{1/2 of the} order of 5.5 hours. The average time of the presence of the drug in the body - the MRT indicator - is 5.5-6 hours.

High values of such indicators as clearance (450-500 ml / kg / h) and stationary volume of distribution (2.5 l / kg) indicate that the claimed drug is very actively distributed from the systemic circulation. This fact is unexpected since comenic acid belongs to a number of hydrophilic compounds. It follows that most of the drug is probably not excreted, but metabolized and utilized in the body itself. This makes it possible to recommend the use of the claimed drug in patients with renal and hepatic insufficiency. Considering the chemical nature of comenic acid (i.e., the presence of hydroxyl and carboxyl groups in its structure), glucuronide conjugates or complex compounds with metal cations may be its metabolites.

Positive dynamics after parabulbar intake of the inventive regenerating agent having anti-inflammatory effects, manifests itself in 10-14 days from the start of use. To consolidate a positive result, if necessary, the treatment is repeated up to 3 times a year.

When the drug is included in the standard complex anti-tuberculosis therapy, a positive effect of a 10-day course of intravenous injection is observed 14 days after the start of use of the drug. The beneficial effect is persistent. If necessary, the treatment with the claimed drug is repeated up to 3 times a year.

The inventive tool does not cause dependence and addiction, therefore, the dose of the drug necessary to achieve a regenerating effect remains unchanged during treatment, even with prolonged use. In a therapeutic dose, the drug does not affect the blood formula, cardiovascular and respiratory systems, does not cause allergic reactions, does not provoke terratogenesis, does not affect reproductive function and embryogenesis.

Example 2. Specific pharmacological activity

2.1. Proposed mechanism of action. Neurotrophic and trophic activity. In vitro experiments

The neurotrophic and trophic properties of the claimed drug were investigated using the method of organotypic tissue cultivation. The experiments were carried out on explants of heart tissue, retina and neurons of the spinal ganglia of 10-12 day old chicken embryos. 400 heart tissue explants, 500 retinal tissue explants, and 400 spinal ganglia explants were examined. The culture medium in which the explants were cultured had a pH of 7.2 and the following composition: 40% - Hanks solution; 40% - Wednesday Needle; 15% - fetal cow serum; 5% - chicken embryonic extract; with the addition of glucose (0.6%), insulin (0.5 units / ml), gentamicin (100 units / ml), glutamine (0.35%). The explants were placed on a collagen substrate and cultured in a nutrient medium in Petri dishes at a temperature of 37 ° C in a CO ₂ incubator (Sanyo), at a concentration of O _{2 of} 95% and CO _{2 of} 5% (Penniyaynen V.A., Lopatina E.V. Research Roles of Na ⁺ , K ⁺ -ATPase in Regulation of the Growth of Sensory Neuron Neurites Bulletin of Experimental Biology and Medicine. 2005, v. 139, No. 2, pp. 147-160; Lopatina E.V., Penniyaynen V.A., Tsyrlin V .A. Comparative analysis of the action of cardiac glycosides on the growth of heart tissue explants. Sechenov Physiological Journal, 2005, Vol. 91, No. 11, pp. 1299-1304. Karetsky AV, Lopatina EV, Penn iyaynen VA α3-izoform of Na ⁺ , K ⁺ -ATPase modulates process of cells growth in the chicken retina. Humboldt-Kolleg Conference "Technologies of the 21st century: biological, physical, informational and social aspects" Saint-Petersburg, 2005, p .thirty).

Controls were explants cultured only in a nutrient medium. The inventive tool was added to the nutrient medium in concentrations: 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M (in terms of the active substance - comenic acid). The control value of PI was taken as 100%, the significance of differences in PI was evaluated using Student's t-test. In some experiments, cardiac glycoside, Na / K-ATPase blocker ouabain (Sigma) was added to the nutrient medium. Ouabain was added at a concentration of 10 ^-8 M; at this concentration, the drug blocks both Na / K-ATPase functions — pump function and signal transducer function, and completely blocks the growth of explants in heart tissue, retina, and spinal ganglia [ibid.].

Studies have shown that the claimed tool has neurotrophic and trophic properties. The effect of the drug is dose-dependent and tissue-specific.

When introduced into the nutrient medium of the claimed agent in a concentration of 10 ^-6 M (in terms of the active substance - comenic acid), a significant inhibition of retinal tissue explant growth was observed by 24% compared with the control. This concentration had a similar effect on neurite growth; PI was 20% lower than the control value. While the effect of the claimed drug in a dose of 10 ^-6 M on the growth of explants of heart tissue turned out to be the opposite. At this concentration, the drug significantly stimulated the growth of heart tissue explants by 40% in relation to the control. At a concentration of 10 ^-7 M, the degree of inhibition of the growth of explants of retinal tissue and neurites of sensory neurons by the drug was significantly less. The retinal tissue explant PI was 17.5% lower than the control value. The neuritis IP of sensory neurons was lower than the control value by 5%. The IP value of heart tissue explants under similar cultivation conditions was 23% higher than the control value. Adding the drug at a concentration of 10 ^-8 M led to significant stimulation of the growth of retinal tissue explants and sensory neuron neurites by 18% and 24%, respectively. The drug in this concentration did not affect the growth of heart tissue explants. The data did not differ from the control. When the drug was introduced into the culture medium at a concentration of 10 ^-9 M, significant stimulation of the growth of retinal tissue explants was observed. PI was above the control value by 50%. The growth of neurites of sensory neurons remained at the same level. The drug in this concentration did not affect the growth of heart tissue explants.

In some experiments, ouabain at a concentration of 10 ^-8 M was added to the nutrient medium together with the claimed agent in an effective concentration for each type of tissue. It was at this concentration that ouabain completely inhibited the growth of retinal tissue explants, sensory neuron neurites, and heart tissue explants. When co-culturing retinal tissue explants in a medium containing the inventive agent at a concentration of 10 ^-9 M and ouabain at a concentration of 10 ^-8 M, it turned out that the PI of the experimental explants was no different from the control value. Anoceptin at a concentration of 10 ^-8 M eliminated the blocking effect of ouabain in relation to sensory neuron neurites; the PI of experimental explants did not differ from the control value. The toxic effect of ouabain in relation to heart tissue explants was eliminated by the claimed agent at a concentration of 10 ^-6 M. The growth rate of heart tissue explants cultured in a medium containing the claimed agent (10 ^-6 M) and ouabain (10 ^-8 M) corresponded to the control value IP explants of heart tissue cultured only in a nutrient medium. Thus, the tissue-specific neurotrophic and trophic effect that the claimed agent exerts is realized due to its direct effect on the Na / K-ATPase, most likely its alpha three isoform. The enhancement of growth and proliferation processes in tissues of various types in this case is associated with the activation of the function of Na / K-ATPase as a signal transducer. In this capacity, Na / K-ATPase can take part in the regulation of synthetic processes (proliferation and growth) in tissues of various types [ibid.]

Cytological studies of retinal tissue preparations stained with hematoxylineosine revealed that in the growth zone of the control and experimental explants cultured for 3 days in a nutrient medium containing the inventive agent (10 ^-9 M), retinal pigment epithelial cells, rods, cones, ganglion cells. On fixed preparations of heart tissue stained with hematokilineosin, it was found that the growth zone of the control explants and explants cultured in a medium containing the claimed agent (10 ^-6 M) contained cardiomyocytes and a small amount of fibroblasts. In the growth zone of the explants of sensory neurons stained with silver according to Bilshovsky-Gross, there were neurites both in the control and in the case of preliminary cultivation of the explants in a nutrient medium containing the claimed agent (10 ^-8 M). Thus, received additional confirmation of the fact that the claimed tool stimulates the natural cycle of proliferation and growth.

The inventive tool in its composition does not contain calcium and magnesium ions, but they were present in sufficient quantities in the nutrient medium in which the cultivation was carried out. The nutrient medium in experiments in vitro reproduces the composition of the internal environment of the body (blood, interstitial fluid, etc.).

In vivo experiments on the subacute and chronic toxicity of the claimed drug (dosage form in the form of 1 and 2% solution for injection) in rats and dogs confirmed that the drug in the systemic circulation can be in the form of a chelate complex with calcium and magnesium ions. It should be noted that Na / K-ATPase activity in normal and pathological conditions depends on the concentration of these ions in the near-membrane cell space (Schonner Endogenous cardiac glycosides, a new class steroid hormones, Eur. J. Biochem. 2002. V.269. 2440 -2448).

A study of a biochemical blood test in males and females of experimental groups receiving the claimed drug at a dose of 100 and 300 mg / kg (in terms of the active substance comenic acid) revealed a significant decrease in calcium levels at the maximum dose (300 mg / kg) after 90 days continuous use of the drug. 2-3 weeks after discontinuation of the drug, this indicator returned to normal levels. In experiments on dogs, it was found that a significant decrease in the level of calcium observed in rats at the maximum dose of the claimed drug (300 mg / kg) was observed in dogs that received the drug at a maximum dose of 50 mg / kg, which had the character of an unreliable trend. The difference in the tested maximum doses of the drug for different species groups of animals is explained by the fact that it is technically not possible for dogs to administer the drug in such a large volume intravenously.

The proposed drug and method of treatment are based on an in-depth understanding of the physiological role of the molecular mechanism for modulating the proliferation process discovered by the inventors due to the activation of Na / K-ATPase as a signal transducer and the ability of the active substance of the comenic acid preparation to form chelate complexes with calcium and magnesium ions [ibid] .

G-proteins are not involved in this mechanism (which is why the claimed drug is not addictive already at the molecular and cellular levels and does not stimulate the growth of neoplasms). In in vivo experiments on mice and rats with perivalent tumors, Erlich carcinoma and Plis sarcoma showed that the drug does not stimulate tumor growth. There is no evidence of a direct relationship between G-proteins and the activity of Na / K-ATPase as a transducer. Substances that activate G-proteins do not eliminate the toxic effects of drugs of the cardiac glycoside group and do not increase the effectiveness of drugs used in complex anti-tuberculosis therapy.

Well-known analogues used in the treatment of chorioretinal dystrophies - retinalamin, glutoxim, cortexin, phezam, emoxipin, are objectively unable to exhibit the properties of the claimed drug, since they do not contain comenic acid, do not create chelate complexes with divalent ions of calcium and possibly magnesium and do not activate a mechanism that triggers Na / K-ATPase as a signal transducer.

The ability of the drug to form chelate complexes when used in a therapeutic dose does not affect the vital constants of the body.

As a result, the basis of the present invention is the idea of activating a fundamentally different than the classical way of activating metabolic processes in the body. The existence of this mechanism only confirms the theory of the presence in the human and animal body of several parallel ways of protecting their integrity from super-powerful damaging stimuli.

Addiction to the claimed drug at the molecular level is absent.

2.2. The study of neurotrophic and anti-inflammatory properties in in vivo experiments.

2.2.1. The method for determining the virulence of mycobacterium tuberculosis

Mycobacterium tuberculosis (MBT) virulence was determined before each rabbit infection by introducing 0.001 mg of culture into the inguinal region of guinea pigs. Accounting was made according to the life expectancy of the pigs. A total of 16 guinea pigs were used for these purposes, the life expectancy of which after infection was 4-5 months (MVT LIHT-320 culture, low virulence).

2.2.2. Research of antimicrobic action on M. Tuberculosis

The study of the antimicrobial action of the claimed funds was carried out by the standard method of double serial dilution on cultures of H37 RwM and LIHT-98 M. Tuberculosis. Suspensions of M. Tuberculosis were prepared according to the standard turbidity of 5 units (which corresponds to five hundred million microbial bodies in 1 ml of suspension) and diluted 10 times, as for drug resistance. Inoculated in a volume of 0.2 ml (10 million microbial bodies) by surface layering.

As a nutrient medium used modified Soton liquid medium with the addition of 0.35% nutrient agar and 0.25% horse serum.

The initial concentration of the claimed drug was 200 μg (in terms of the active substance comenic acid) per 1 ml of medium (i.e. 1-200 μg / ml; 2-100 μg / ml; 3-50 μg / ml; 4-25 μg / ml; 5-12.5 μg / ml; 6-6.25 μg / ml; 7-3.125 μg / ml; 8-1.56 μg / ml).

At the same time, the used strains of M. tuberculosis were inoculated. As a control culture without the use of the studied drug on a modified agarized Soton medium and to control the medium on a dense Levenshtein-Jensen medium.

Crop incubation was carried out at 37 ° C for 7 days. The result was taken into account visually. At the end of this period, identical growth of M. tuberculosis cultures was observed in all test tubes (on the Levenshtein-Jensen medium, initial growth). This suggests that the claimed tool does not have an antimicrobial effect on M. Tuberculosis in the studied concentrations.

2.2.3. Modeling of tuberculous focal chorioretinitis

Neurotrophic retinoprotective and anti-inflammatory properties of the claimed drugs were investigated on the model of tuberculous focal chorioretinitis. The operation of modeling tuberculous chorioretinitis was performed on 40 eyes of 20 sexually mature male Chinchilla rabbits weighing 3.3-3.8 kg. This model was chosen because it is a rather rigid experimental model for the formation of secondary dystrophic changes in retinal tissue.

Previously, on 6 healthy eyes of 3 rabbits of the Chinchilla breed, the effect of a 2% solution of the claimed drug was studied with its parabulbar administration in order to exclude toxic effects on eye tissue. An ophthalmological examination of the fundus of rabbits, and then the study of a macro product - “eye + parabulbar fiber” showed that no changes were found in the fundus and in the paraorbital tissue. Thus, we can conclude that the administration of the drug alone does not have a toxic effect on the tissues of the healthy eye and does not stimulate the development of pathological processes.

Modeling of tuberculous focal chorioretinitis was carried out according to the method developed by E. N. Bellendir et al. (E. N. Bellendir, A. A. Sukonshchikova, G. D. Nakonechny, I. N. Peschanskaya. Effect of nonspecific and specific sensitization of the body on development of experimental eye tuberculosis. Problems of tuberculosis, 1972, T.7, S.68-73; I.N. Peschanskaya. Experimental modeling of tuberculous eye lesions. Problems of tuberculosis, 1973, t, 12, p.65-70; I.N . Peschanskaya. Assessment and comparative description of methods for modeling tuberculosis hl for. Avtoref.kand.dis. L. 1977, p.17).

After the formation of the chorioretinal lesion, antibacterial therapy (ABT) was performed until the process was stabilized according to the standard scheme.

2.2.4. The method of standard treatment of tuberculous chorioretinitis in the active stage

In the active stage of treatment of tuberculous chorioretinitis, anti-TB drugs were used:

- parabulbarno (3% solution of tubazide - 0.5 ml),

- intramuscularly 10% solution of tubazide at the rate of 10 mg per 1 kg of mass,

- intramuscularly for 40,000 IU of streptomycin per 1 kg of animal weight once a day.

By stabilization of the process was meant the absence of dynamics of clinical changes in the focus of inflammation and the perifocal zone on the background of ABT for 7-10 days. Before the experiment, the animals were divided into the main and control groups. The main group included 10 rabbits (20 eyes), the control group consisted of 10 rabbits (20 eyes).

The chorioretinal foci were rounded granulomas of a yellowish color, with fuzzy borders that were promoted to the vitreous, with exudation in the perifocal region.

The severity of exudative changes in the area of chorioretinal foci was conditionally expressed in points (On the prospects of studying the therapeutic effect of the laser using an experimental model of tuberculous choroiditis. Hokkanen V.M., Bellendir E.N., Balashevich L.I., Ustinova E.I. Experimental pathology and therapy of pulmonary and extrapulmonary tuberculosis. - M., 1985, S. 60-65. 1990).

0 - no signs of exudation, 1 - exudative changes were only in the area of the focus, 2 - exudative changes were noted in the area of the inflammation site and in the adjacent layers of the vitreous body, 3 - exudative changes were noted in the area of the focus of inflammation and in all layers of the vitreous body.

In most cases (65.0%), the severity of inflammation was negligible. In 14 eyes (35%), the course of focal tuberculous chorioretinitis was moderate, involving the middle and posterior vitreous layers in the inflammatory process.

The size of the chorioretinal foci was correlated with the size of the optic disc and the largest size was recorded (designation DD)

Most of the chorioretinal foci (90% - 36 eyes) were from 1 to 3 diameters of the optic disc, in 2 eyes (5%) the size of the chorioretinal tuberculous foci was 3 or more times the size of the disc, and in two cases the size of the focus was less than 1 DD. The vitreous humor (prominence) of the chorioretinal foci was measured with skyscraper rulers, evaluated in diopters by the maximum distance compared to intact tissue.

In most cases, in 21 eyes (77.5%), the level of prominence in experimental animals did not exceed 2 diopters, in 9 eyes (23.4%) it was 2-3 diopters.

To study the effectiveness of treatment of tuberculous chorioretinitis with the inclusion of the claimed drug (2% solution for injection) in standard therapy, rabbits were divided into 2 groups of control and main:

1. The main group included experimental animals, which, against the background of standard anti-tuberculosis therapy, underwent a course of parabulbar administration of 0.5 ml of a 2% solution of the claimed drug for 10 days

2. The control group included experimental animals that underwent standard anti-tuberculosis therapy.

The distribution of animals of the main and control groups by the size of the lesions was equal, the size of the lesion ranged from 1 to 3 diameters of the optic nerve head. No significant differences were found between the animals of the main and control groups according to the level of prominence.

Studies have shown that experimental parameters of the main group did not differ from the control animals in terms of exudation parameters.

Studies conducted before the inclusion of the claimed drug in complex anti-tuberculosis therapy showed that the experimental animals of the main group and the control group did not significantly differ in such indicators as the size of the lesion, the prominence and exudation.

Throughout the study, experimental animals of the main and control groups received standard anti-tuberculosis therapy. On the 10th day from the start of treatment, the animals of the main group, in addition to standard therapy, began to receive 0.5 ml of a 2% solution of the claimed drug in each eye parabulbar. The injection course of the claimed drug was 10 days.

Injections of the claimed drug (0.5 ml of a 2% solution in each eye) were well tolerated by the experimental animals, there were no areas of necrosis at the injection site, allergic and toxic reactions.

During therapy with the claimed agent in animals of the main and control groups, an ophthalmoscopic fundus image was evaluated every two days. On the second day, the ophthalmoscopic picture of the fundus in both groups was unchanged. On the 6th day, an increase in pigmentation of dystrophic foci of the fundus of rabbits of the main group was found. The foci in the fundus of the rabbits of the control group were without significant dynamics. In rabbits of the main group, partial resorption of retinal hemorrhages was observed in parallel. In the control group, no such changes were observed. On the 10th day of therapy, a significant decrease in the area of foci in the main group of animals was noted. The decrease in area occurred due to increased pigmentation from the periphery of the focus to its center. In animals of the control group, a similar ophthalmoscopic picture was not observed on the fundus. Thus, in animals of the main group, a significant decrease in the area of fibrous foci was recorded, which is associated with the inclusion of the claimed drug in the complex anti-tuberculosis therapy.

Not a single animal of the main group died before the end of the experiment. The total experiment time is 90-120 days.

Experimental animals were withdrawn from the experiment by intravenous administration of 5.0 ml of thiopental sodium 12 weeks after the start of treatment. Eye enucleation was carried out according to a technique traditional in ophthalmology. Under sterile conditions, the eyeballs of animals were fixed in a 2.5% glutaraldehyde solution on phosphate buffer (pH = 7.4). Then, the posterior wall of the eye was isolated, which was subsequently filled with paraffin under vacuum. Histotopographic sections 8–10 μm thick were made from paraffin blocks and stained with hemotoxylin-eosin.

Histological studies confirmed in vivo ophthalmoscopic findings. The preparations showed an increase in pigment epithelium, hypertrophy of the inner nuclear layer, a decrease in the number and area of lacunae between ganglion cells.

2.2.5. The results of an experimental study of tuberculous chorioretinitis on ophthalmoscopic signs.

Animals of the control and main groups underwent ophthalmoscopic examination of the fundus every 3-4 days. The data of dynamic observation of ophthalmic criteria before and after 30 days from the start of the experiment are presented in table 1.

In both groups of animals, a positive trend was observed in the treatment of tuberculous chorioretinitis. However, the results of treatment of experimental animals of the main group, which along with standard anti-tuberculosis therapy received within 10 days 0, 5 ml of a 2% solution of the claimed drug parabulbar surpassed those in animals of the control group (table 1).

The size of the lesion significantly decreased by 20% compared with the initial level (1.98 ± 0.12 before and 1.59 ± 0.13 after the experiment). In the control group, we also noted positive dynamics, however, the size of the lesion in this series decreased only by 4% (2.03 ± 0.14 / 1.95 ± 0.14).

The level of prominence of chorioretinal foci in the main group in which the inventive agent was administered parabulbarly decreased by 66% - 1.27 ± 0.14 / 0.43 ± 0.12 compared with the initial level, and in three cases there were no prominences at all. In the control group, where the claimed drug was not used, the level of prominence decreased by 39% - 1.2 ± 0.15 / 0.73 ± 0.13.

Exudative manifestations significantly decreased to a greater extent with parabulbar administration of the claimed drug (70%) compared with the original figures. In the control group, 63%, while turbidity and infiltration in the vitreous remained.

Thus, the claimed tool in relation to this criterion acted 7% more effective than standard therapy drugs.

Thus, the use of a 2% solution of the claimed drug in the complex treatment of tuberculous chorioretinitis can significantly reduce foci in size, reduce the level of promenade and exudation in a larger volume and after 1 month to stop the development of inflammation of the tuberculous process in the fundus is qualitatively better compared to the control group. The latter is evidence of the high effectiveness of the proposed drug when it is included in a comprehensive anti-tuberculosis therapy. The research scheme is classical for studying the pharmacological activity of drugs that potentiate the effectiveness of anti-TB treatment, and allows you to recommend the claimed drug for inclusion in a comprehensive pathogenetic anti-TB treatment.

This experimental model is a model of post-inflammatory retinopathies and is used to study the pharmacological activity of drugs from the group of regenerants and metabolites with anti-inflammatory properties. The criteria selected by the authors are adequate for this kind of research. They allow you to correctly assess the properties of the claimed funds as regenerative and anti-inflammatory, since changes in retinal tissue during the development of tuberculous chorioretinitis occur as secondary in the development of acute inflammatory process.

2.2.6. Results of histomorphological studies of fundus tissues of experimental animals with tuberculous chorioretinitis

After removing the rabbits from the experiment, we examined all sections of the eyeball of the experimental animals. Analysis of 224 histomorphological sections obtained from 40 eyes of 2 experimental groups (experimental and control) showed that all eyes had tuberculous granulomas in the choroid, which were located next to the optic disc or myelin fiber. In 14 cases, the foci were located only in the choroid (mainly in the layer of the middle vessels), in 10 cases, the foci of tuberculosis were located in the choroid and retina under the layer of pigment epithelium. In 2 eyes, the foci, in addition to the choroid, imbibed all layers of the retina, with the involvement of the optic nerve disk, in one case, the tuberculous focus affected a small part of the retina, and the main mass appeared in the eye cavity in the form of a mushroom in a capsule. In all sections, lymphoid infiltration of nearby choroid sites was determined. In most cases, in the perifocal region, the choroid vessels were in a collapsed state due to edema, in 6 cases they were dilated and full-blooded, with thickened walls due to leukocyte infiltration. On all sections of the eyes, exudation was visible in the perifocal region or above the retina.

Retinal changes were seen on all eye preparations; the main changes concerned the top of the focus, where the retina was completely destroyed, with destruction of the pigment epithelium (PE). However, in 40% of cases in the study study group, PE had its normal structure near the outbreak. In the control group, destruction of the pigment epithelium with the release of pigment from the cells extended up to the dentate line located on the periphery of the retina.

2.2.7. Methods of modeling traumatic retinal dystrophy in rabbits

The technique for modeling traumatic retinal dystrophy in rabbits was developed by the author A. Karetsky The experiments were carried out on 20 chinchilla rabbits. We used males weighing 3–3.5 kg. To simulate traumatic dystrophy of the retina, instruments for eye microsurgery were used. Animals were anesthetized with a 1% solution of promedol (1-2 ml depending on body weight). Promedol was administered intramuscularly 10-15 minutes before surgery.

The eyelids were stitched with silk and parted, in the upper half of the eyeball along the limb, a conjunctiva was cut, which was then separated, a suture was placed on the upper rectus muscle and the eyeball was rotated as far down as possible. Inner from the superior rectus muscle, the sclera was cut into layers to the choroid. A needle with a polished end was inserted into the suprachoroidal space in the direction posteriorly and outward, which mechanically inflicted traumatic injuries, then the needle was removed. No sutures were made on the sclera or on the conjunctiva. The formation of traumatic dystrophy of the retina was evaluated according to an ophthalmoscopic examination of the fundus using an OR-2 manual specular ophthalmoscope (manufactured in Russia). On the 2-3rd day after the operation on the fundus of the rabbits, the presence of flat dystrophic foci of white color with an area of 0.5-1.5 PD with hemorrhages in the retina of the eye was detected. On the 7th day after surgery, treatment with the claimed drug was started. The drug in the form of a 2% injection solution for animals of the main group was administered parabulbarno 0.5 ml in each eye once a day. The course of treatment was 10 days. Animals of the control group were parabulbally injected with 0.5 ml saline in each eye for 10 days. This model is adequate for studying the effects of drugs used in the treatment of post-traumatic retinal dystrophies (injuries, complications after surgical interventions, etc.).

2.2.8. The results of the treatment of experimental post-traumatic dystrophy.

To study the experimental material, we used general clinical observations (behavior, appetite, changes in weight), standard ophthalmoscopic methods of research, which were carried out in dynamics and included: transmission in transmitted light, tonometry, ophthalmobiomicroscopy of the fundus. During the experiment, the animals gained 400-500 grams in weight; no changes in behavior and appetite disorders were detected. The inventive tool was administered to animals of the main group of 0.5 ml in each eye parabulbularly, starting from the 5th day of the experiment for 10 days. The animals of the control group were injected with saline according to the same scheme.

During therapy with the claimed agent in animals of the main and control groups, an ophthalmoscopic fundus image was evaluated every two days. On the second day, the ophthalmoscopic picture of the fundus in both groups was unchanged. On the 6th day, an increase in pigmentation of dystrophic foci of the fundus of rabbits of the main group was found. The foci in the fundus of the rabbits of the control group were without significant dynamics. In rabbits of the main group, partial resorption of retinal hemorrhages was observed in parallel. In the control group, no such changes were observed. On the 10th day of therapy, a significant decrease in the area of foci in the main group of animals was noted. The decrease in area occurred due to increased pigmentation from the periphery of the focus to its center. In animals of the control group, a similar ophthalmoscopic picture was absent on the fundus (Table 2). The final results in both groups of animals were evaluated 10 days after completion of treatment.

The size of the lesion in the main group significantly decreased by 19% compared with the initial level (1.19 ± 0.40 before and 0.96 ± 0.11 after the experiment). In the control group, we also noted positive dynamics, however, the size of the lesion in this series decreased only by 3% (1.225 ± 0.30 / 1.2 ± 0.13) (Table 2).

Experimental animals were withdrawn from the experiment by intravenous administration of 5.0 ml of thiopental sodium 12 weeks after the start of treatment. Eye enucleation was carried out according to a technique traditional in ophthalmology. Under sterile conditions, the eyeballs of animals were fixed in a 2.5% glutaraldehyde solution on phosphate buffer (pH = 7.4). Then, the posterior wall of the eye was isolated, which was subsequently filled with paraffin under vacuum. Histotopographic sections 8–10 μm thick were made from paraffin blocks and stained with hemotoxylin-eosin. The dynamics of ophthalmic parameters in experimental animals during the treatment of post-traumatic retinal dystrophies is shown in table 2

Histological studies confirmed the results of in vivo experiments. In animals treated with the claimed drug, an increase in the roller of pigment epithelium was observed, a decrease in vacuumization.

Table number 2 The dynamics of ophthalmic parameters in experimental animals in the treatment of post-traumatic retinal dystrophies Series No. Experience Conditions
Number of eyes Ophthalmic indicators Outbreak size in DD
M ± m Before treatment After treatment one The main group. The claimed tool parabulbarno N = 20 1.19 ± 0.40 0.96 ± 0.11 2 Control group. Fiz. solution N = 20 1.225 ± 0.30 1.2 ± 0.13

The hypothesis that treatment using the claimed agent of tuberculous chorioretinitis (a model of post-inflammatory retinal dystrophy) and post-traumatic retinal dystrophy is effectively tested using the StatGraphics package. Three tests were used: Student's t-test, sign test and sign rank test. The t-test is based on the distribution of Student and has the greatest power in the case when the sample is extracted from the normal population. In our case, Student's t-test verifies the main hypothesis that the mathematical expectation is zero (zero treatment effectiveness) against the alternative hypothesis, that the effectiveness is confirmed with a confidence probability of 95%.

The sign test and the sign rank test test the hypothesis that the median is zero (zero efficiency) versus the alternative that the effectiveness is positive. The sign test and the sign rank test are effective even in cases when there are abnormal observations in the sample. The sign test is based on counting the number of observations in the sample that are located above and below the hypothetical median (zero in our case). Accordingly, the sign rank test is based on calculating the average number of ranks of the larger and smaller hypothetical medians.

As the calculations in the StatGraphics package showed, for all four parameters — the size of the lesion, the prominence, the exudation in the case of tuberculous chorioretinitis, and the lesion size in the case of post-traumatic retinal dystrophy for all three tests, the calculated significance level did not exceed 0.001. This means that for all four parameters when using both experimental models with a confidence probability of at least 99.9%, the presence of positive effectiveness from the treatment measures carried out by the authors is confirmed.

2.3. Studying the effects on the immune system. In vivo experiments

A 0.1% solution was prepared from the inventive drug, which did not have a directed effect on the number of AOKs in the spleen of mice, HRT reaction, GVHD (graft versus host reaction), phagocytosis, but caused slight stimulation of antibody production (action index 1, 2). The experimental animals are mice of the C57BI / 6, CBA and FI line (CBAxC57 B1).

The following methods were used to study the effect of the claimed drug on the immune system of animals.

1. Determination of the number of AOKs in the spleen of mice on the 7th day after immunization with optimal (1 × 10 ⁸ cells) and suboptimal (2 × 10 ⁷ cells) doses of EB (sheep erythrocytes) (Jeme N. - K .., Nordin AA Plague formation in agar antibody-producting cells / - Science, 1963, v. 140, N 3565, p. 405).

2. Determination of titers of HA (hemagglutination) and EB (sheep erythrocytes) was performed on days 7 and 14 after immunization with optimal and suboptimal doses of antigen in 96-well round-bottom plates. The results were taken into account macroscopically. Schemes for administering the drug are similar to those for determining the number of AOKs (Zigl E. Hemagglutination reaction. - In:

Immunological methods (under the editorship of X. Fremel). M., 1979, 108-112 p.).

3. Determination of the HRT reaction was carried out using intravenous immunization of EB mice at a dose of 2 × 10 ⁵ cells, a resolving dose of EB (1 × 10 ⁸ cells in 0.04 ml of physiological saline) was injected into the paw pad on the 7th day after immunization. The local inflammatory response was taken into account after 24 hours according to the difference in the mass of the experimental (Po) and control (Pk) paws.

4. The reaction index (IR) was calculated for each mouse according to the formula [Kimatura K. A food weight assay method to avaluate DTH in the mice. - J. Immmunol. Method., 1980, v. 39, N2, p.277-283]:

5. Determination of the reaction "transplant against the host." The development of GVHD was caused by subcutaneous injection of 2 × 10 ⁶ cells of the parental genotype lymph nodes (CBA) into the pads of the right hind paw of FI mice (CBAxC57 B1). Syngenic lymphocytes were introduced into the left paw. On day 9, the number of cells in 5 ml of homogenate of the left (control) and right (experience) lymph node was determined.

6. The phagocytic activity of mouse peritoneal macrophages was evaluated using opsonized JgG3B as an object of phagocytosis (Argyris BF Role of macrophages in antibody production, J. Immmunol., 1967, v. 99, N 4, p. 744-750). Macrophages were drawn into the abdominal cavity with a solution of hydrolyzed starch. For 3-4 days, 0.5 ml of a 5% suspension of EB was subcutaneously injected. Washed suspension of cells. In the Romanovsky-Giemsa stained preparation, the percentage of phagocytic cells was determined. To determine the phagocytic index (full name, the number of captured EBs was calculated spectrophotometrically from the hemoglobin concentration using a calibration curve. After 3 hours of incubation at 37 ° C, the phagocytic index (PH ₂ ) was again determined. The phagocytosis completion index (PPI) was determined by the formula:

7. When determining the phagocytic activity in vivo, mice were injected intravenously with a 25% carcass solution in a volume of 0.5 ml, then blood was taken from the orbital sinus (0.02 ml) every 15 minutes and introduced into 3 ml of 3% acetic acid . After taking the blood, the animal was killed, the weight of the body, liver and spleen was determined. The optical density of hemolyzed blood was determined on SF-26 at a wavelength of 610 nm and plotted f (D). We found the point lg 0 ′ (zero time) and lg 10 ′ (according to the schedule). The constant (K) of phagocytosis was calculated by the formula:

and then calculated the true rate of phagocytosis (α) by the formula:

8. Complement activity was determined by titration using a rabbit erythrocyte (EC) red blood cell treated direct pathway of guinea pig hyperimmune antiserum (Tanaka S., Suzuki T., Nishioka K. Assay of classical and alternative pathway activities of murine complement using antibody sensitized rabbit erythrocytes. J. Immunol. Meth., 1986, v. 86, p. 161-170), and for an alternative activation pathway, ECs treated with potassium iodide solution (Van Dijk H., Pietenel M., Klerx J., Willers J Study of the optimal reaction condition for assay of the mouse alternative complement pathway activities. J. Immunol. Meth., 1985, v. 85, p. 233-243). Activity was expressed in units of 50% lysis per ml of serum (CH50).

9. Statistical processing of research results was carried out using a software package. The student criterion and the Wilcoxon-Mann-Whitney method were used. When processing the results of determining the number of AOK and GA titers, we operated with logarithmic indicators.

In some experiments, experimental emotional stress was caused by crowding (40 mice in a cage measuring 17 × 25 × 8 cm) for 10 days. Control animals during the experiment received daily iv 1.1% soda solution, experimental - the claimed drug in a dose of 50 mg / kg

2.3.1 Effect on the development of a humoral immune response

The results of a study of the effect of the claimed drug on the formation of antibody-forming cells (AOKs) in the spleen of mice and titers of hemagglutinins (GA) in the serum of various mouse lines immunized with an optimal and suboptimal dose of ram erythrocytes (EB) showed the following. With the on / in the introduction of the claimed drug it stimulates approximately 2 times the formation of AOK in the spleen and per titer (i.e. 2 times) the level of HA in the serum of C57BI / 6 mice, poorly responding to EB, both during optimal and suboptimal immunization dose of antigen. Stimulation of AOK formation in highly responsive FI mice upon immunization with a suboptimal dose of EB caused by the studied drug at doses of 5 and 50 mg / kg, and an increase in HA titer with the introduction of 50 mg / kg of the claimed drug, indicate the presence of adjuvant properties.

With a 7-day iv administration of the drug at a dose of 50 mg / kg, an increase in the number of AOKs in the spleen of C57BI / 6 mice was observed with an optimal and suboptimal immunization regimen, and GA titers increased only with the introduction of the optimal dose of antigen.

Of interest is the fact of dose-dependent inhibition of AOK formation in the spleen of FI mice when an optimal dose of EB is introduced and a decrease in serum HA titer with iv administration of the drug at a dose of 50 mg / kg. This phenomenon may be due to the fact that, with the simultaneous administration of a significant amount of EB and the Anoceptin analgesic, activation occurs as a chain of respiratory enzymes (Blandova Z.K., Dushkin V.A., Malashenko AM, Schmidt E.F. Lines of laboratory animals for biomedical research. - M .: Nauka, 1983, 190 pp.), as well as the activity of monooxygenases, in particular, cytochrome P-450 (Ozherelkov S.V., Vargin V.V., Semenov B.F. Influence experimental informational and emotional stress on the primary immune response of mice of different lines. - J. Micro IOL., epidemiology. and Immunobiology 1985, №8, s.43-46). The latter circumstance associated with the functional conjugation of the immune system with metabolism leads to an acceleration of the erythrocyte degradation in phagocytic cells, a decrease in the antigenic properties of EBs and, as a result, a decrease in the severity of the humoral immune response in intact animals that are full in all physiological parameters.

The decrease in HA titers is of a phase nature, and by day 14 after immunization, the indices of the experimental and control groups do not significantly differ.

Thus, the claimed agent slightly stimulates the development of a humoral immune response, has adjuvant properties and modulates the severity of the humoral immune response in mouse EBs that are highly responsive to EB.

2.3.2. Effect on the phagocytic activity of macrophages

The results of a study of the effect of the claimed drug on the clearance of the carcass and the phagocytic activity of peritoneal macrophages showed that the claimed drug with a single iv administration does not change either the number of phagocytic cells in the peritoneal exudate or the ability of phagocytes to capture opsonized EBs, but it increases the digestion factor by a factor of 1.5-2 ability of macrophages. With the on / in the introduction of a dose of 50 mg / kg, the drug increases the content of phagocytic cells in the exudate, but does not change the phagocytic index (PI) and the index of completion of phagocytosis (IZF). With the on / in the introduction of the inventive tool does not change the capture rate of inert particles of the carcass and does not affect the rate of blood purification.

2.3.3. Effect on the immune system of stressed animals

Studies of the impact of the proposed drug on the severity of the immunodeficiency state induced by emotional stress, was carried out according to the standard scheme. The crowding of animals for 10 days leads to the suppression of the humoral immune response (the number of AOKs and the titer of HA), the suppression of the completeness of phagocytosis and the speed of blood purification from inert carcass particles. All of these disorders, with the exception of the latter, are induced by stress. A 10-day iv administration of the inventive drug in a dose of 50 mg / kg prevents these disorders (Table 3).

Thus, intravenous administration of the claimed drug protects the immune system from the depressive effects of emotional stress.

Example 3. The study of the anti-inflammatory properties of the claimed drug

The inventive tool stimulates the development of a humoral immune response in weakly responding C57 B1 / 6 mice at doses of 5 and 50 mg / kg and in highly responsive F1 mice (CBA × C57B1) immunized with a suboptimal dose of antigen. At doses of 5 and 50 mg / kg with iv administration in F1 mice (CBA × C57B1) immunized with the optimal dose of antigen, the drug inhibits the development of a humoral immune response.

The inventive tool with a single intravenous administration does not affect the severity of the HRT reaction, and with a 7-day intravenous administration at a dose of 50 mg / kg, it inhibits the development of HRT in the highly responsive C57 B1 / 6 mouse line.

The drug does not affect the development of GVHD reaction.

The ability of the drug to stimulate the completion of phagocytosis of peritoneal macrophages with a single intravenous administration at doses of 5 and 50 mg / kg was revealed, and with an intravenous administration at a dose of 50 mg / kg for 7 days, the ability to increase the number of phagocytic cells in peritoneal exudate.

The inventive tool does not affect the rate of blood purification from carcass particles and the activity of complement serum of mice.

The drug with a 10-day iv administration at a dose of 50 mg / kg prevents a decrease in the humoral immune response and impaired macrophage function caused by emotional stress.

Example 4. The safety study of the use of the claimed funds

4.1. Pyrogenicity Study

The pyrogenic effect of the drug was studied in rabbits weighing 2.0-3.5 kg in accordance with the requirements of Global Fund XI, issue 2, p. 183. When the drug was administered at a dose of 10 mg per 1 kg of animal body weight, an increase in temperature in animals of more than 0.3 C was not observed. Studies have shown that the inventive tool (dosage form in the form of 1% and 2% solution for injection) is pyrogen-free.

4.2. Research on acute toxicity in rodents

To determine the indicators of acute toxicity, the claimed drug was administered to white mice and rats of both sexes intravenously (iv) in the tail vein in increasing doses according to Litchfield-Wilcoxon. Calculations of average lethal doses were carried out according to V. B. Prozorovsky (Prozorovsky, 1978). To achieve large doses of the drug, administration was repeated at intervals of 20-30 minutes for 4 hours. Control animals received similar solvent volumes — a 1.1% solution of sodium bicarbonate (soda).

To study each dose of the drug, groups of 5-6 animals of the same sex were used. In addition, there were similar in number groups of control animals of each sex, which were injected with the same methods equivalent volumes of solvent.

Animals were randomly assigned to groups using randomization. The criteria for the acceptability of randomization were considered to be the absence of external signs of diseases and the homogeneity of groups by body weight (± 10%).

Dosing was carried out on the content of the active principle. The observation period was 14 days. Recorded indicators: mortality, time of death, symptoms of poisoning, daily observation of the general condition and behavior, weighing before administration, on days 7 and 14 of observation, volumes of feed and water consumption (for this the animals were placed in metabolic cages of the Italian company Texnoplast), autopsy and a macroscopic study of all dead and surviving animals at the end of the study (euthanasia in rodents was carried out by an overdose of ether), determination of mass coefficients of internal organs.

The results of toxicometry, observations of experimental animals in the post-toxicity period of acute poisoning, as well as necropsy data, made it possible to classify the dosage form of the claimed drug into class V practically non-toxic drugs (H. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed. IV , Baltimore, 1975, 427 p.). LD _{50 of the} proposed drug is 1300 mg / kg

The condition of animals that have survived acute intoxication indicates good tolerance and harmlessness of the drug in doses exceeding the maximum therapeutic for humans by hundreds of times.

The data of experiments on dogs confirmed that the drug with acute administration has almost no toxic effect. The maximum therapeutic dose for a person is about 5 mg / kg body weight, i.e. 350 mg per day with an average weight of 70 kg.

Preclinical studies of the drug Anoceptin (dosage form in the form of a solution for injection) in mice, rats, dogs, guinea pigs showed that the drug does not cause allergic reactions, does not affect reproductive function, embryogenesis, does not stimulate mutagenesis and terratogenesis, does not negatively affect cardiovascular and nervous systems.

Example 5. The use of the claimed funds in the treatment of ostuberculous chorioretinal dystrophies

The study involved 10 patients with a diagnosis of post-tuberculous chorioretinal dystrophy. Prior to the study, all patients signed an informed consent form. The course of treatment of the claimed agent was 10 days. All patients received daily injections of the drug parabulbarno in 0.5 ml of 1% or 2% solution (the dose of the active substance of comenic acid in the first case was 10 mg per day in the second - 20 mg per day). In ophthalmology, the drug was administered in one or both eyes. The dose of the drug was selected depending on the severity of post-tuberculous changes. Depending on the localization of the dystrophic focus, the patients were divided into groups with central and peripheral localization of the focus. According to an experimental study, it was found that a 1% and 2% solution of the claimed drug positively affects the preservation of pigment epithelium. To assess the effectiveness of the drug before and after treatment, visual acuity and a visual field were studied, electrophysiological study (EFI) allowed us to assess the effect of the drug on the functional activity of retinal cells. After the course of treatment with the drug, most patients noted a positive trend. Visual acuity increased on average by 10-15%. Assessment of the visual field was performed one month after the end of the course of treatment. The field of view in patients receiving injections of the claimed drug, on average increased by 4%. Changes in the field of view occurred at the quantitative and qualitative levels. The improvement of visual functions corresponded to the dynamics of EFI indicators. The use of the claimed agent in the treatment of retinal dystrophy of various origins allows us to improve or normalize the electrophysiological activity of retinal cells both in amplitude and in time of the pulse in the receptor layers, which helps to improve visual functions. Recommended doses of the drug are 10 mg or 20 mg (in terms of the active substance comenic acid) per day parabulbarno, depending on the severity of the disease. The course of treatment must be repeated twice a year.

The effectiveness of the drug in the treatment of post-tuberculous chorioretinal dystrophies allows us to recommend it for the treatment of chorioretinal dystrophies of various etiologies, since this “research model” combines dystrophic changes in post-inflammatory retinal tissue and diseases that arise as complications after traumatic events (ischemia, surgical interventions, eye injuries). An increase in the dose of the drug over 20 mg parabulbarly in each eye is technically impossible and inappropriate.

The regenerating and anti-inflammatory properties of the drug appear when administered intravenously at a dose of up to 350 mg per day, a further increase in the dose does not cause an increase in the anti-inflammatory effect, the drug begins to exhibit mild sedative properties.

The implementation of the claimed invention is not limited to the above examples.

Thus, the experimental results shown in the examples indicate that the inventive tool has a pronounced regenerative effect, has powerful anti-inflammatory properties. The discovered properties of the drug allow it to be attributed to a wide class of reparants, regenerants, metabolites, combining drugs of various origin and type of exposure. The mechanism of action of the claimed funds is fundamentally different from the known analogues, because it is not peptide and does not have plant origin. The inventive tool does not give allergic reactions and is not addictive. The inventive tool is convenient to use, because, unlike the prototype drug, it is a ready-made solution for injection. The inventive method of treatment using the specified funds is intended for the treatment of chorioretinal dystrophies of various etiologies, is more effective and gentle compared to the known ones. The tool can be included in complex anti-TB therapy to increase the effectiveness of the drugs used in it.

Going beyond the stated interval parameters in the lower boundary makes it impossible to implement the invention, in the upper boundary it is impractical, because technically not possible and does not increase regenerative activity.

Table 1 Dynamics of ophthalmological parameters in experimental animals during treatment No.
series Test conditions Number of eyes Ophthalmic indicators Outbreak size in DD M ± m The focal area in diopters M ± m Exudation in ST in points M ± m Before treatment After treatment Before treatment After treatment Before treatment After treatment one Main group
Standard HT
+ declared Wed
parabulbar
N = 20 1.98 ± 0.12 1.59 ± 0.13 1.27 ± 0.14 0.43 ± 0.12 0.91 ± 0.2 0.27 ± 0.12 2 Control
Group
Standard
chemotherapy
N = 20 2.03 ± 0.14 1.95 ± 0.14 1.2 ± 0.15 0.73 ± 0.13 0.94 ± 0.15 0.34 ± 0.16

Table 3 Effect on the immune system of stressed mice Immunoreactivity indicators, units Intact animals Stressed animals The control P ₁ < Experience (declared. Means, 50 mg / kg) P ₂ < The number of AOK per 10 ⁶ cells per spleen (M ± m) 4.8 (5.4 ÷ 4.3) 1000
(1122 ÷ 324) 2.4 (2.8 ÷ 2.0)
398 (490 ÷ 324) 0.01 6.5 (6.6 ÷ 6.3)
891 (933 ÷ 851) 0.001 HA titer on day 14 (M + m) 5.4 ± 0.2 4.3 ± 0.1 0.001 5.0 ± 0.4 0.01 HRT (M ± m reaction index) 20.4 ± 1.9 24.5 ± 3.33 0.001 25.3 ± 2.8 GVHD (M ± m reaction index) 2.06 ± 0.12 1.96 ± 0.14 2.08 ± 0.11 Phagocytosis, phagocytosis index (M ± m) 13.8 ± 3.6 16.4 ± 3.2 16.0 ± 2.0 Index of completion of phagocytosis,% (M ± m) 37.3 ± 7.4 16.8 ± 3.5 0.05 34.6 ± 5.1 0.01 Carcass clearance, index (M ± m) 4.32 ± 0.1 3.02 ± 0.23 0.01 3.60 ± 0.10 0.01 P ₁ - significance of differences with indicators of intact animals
P ₂ - significance of differences with indicators of stressed animals

Claims (7)

1. A regenerating anti-inflammatory agent, which is an aqueous solution of the active principle - comenic acid and auxiliary substance - sodium bicarbonate, in the following ratio, wt.%:
comenic acid 1-2, mainly 1 and 2 sodium bicarbonate 0.55-1.1 water for injections up to 100 2. The regenerating anti-inflammatory agent according to claim 1, characterized in that it additionally contains an admixture of comenic acid - benzylcomenic acid - in an amount of not more than 2.5% in terms of comenic acid. 3. A method of treating chorioretinal dystrophies of various etiologies, namely, that the patient is administered a regenerating agent according to claims 1 and 2 parabulbar at a dose of 10 to 20 mg (in terms of comenic acid) once a day for 10 days. 4. The treatment method according to claim 3, characterized in that the course of treatment is repeated 2-3 times a year. 5. The treatment method according to claim 3, characterized in that the patient is administered a regenerating agent in one or both eyes. 6. A way to increase the effectiveness of anti-tuberculosis therapy, which consists in the fact that the patient is administered a regenerating agent according to claims 1 and 2 intravenously in a dose of up to 350 mg per day (in terms of comenic acid) for 10 days. 7. The treatment method according to claim 6, characterized in that the course of treatment is carried out 1-3 times a year with a break of 3-6 months.