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Title:
REGENERATION ANTI-INFLAMMATORY COMPOSITION AND METHOD FOR TREATING A RETINAL DYSTROPHY USING SAME
Document Type and Number:
WIPO Patent Application WO/2009/042708
Kind Code:
A1
Abstract:
A regenerative anti-inflammatory composition is described to include 1 to 2 percent of comenic acid and 0.55 to 1.1 percent of sodium hydrocarbonate in a water solution, with an optional additive of up to 2.5 percent of benzyl-comenic acid. A method of treating a retinal dystrophy by injecting this composition parabulbarly or intravenously in appropriate amounts is also described. A non-peptide nature of the compound allows reducing side effects commonly associated with peptide-based compounds presently used for treatment of such dystrophy. Water-based solution of the composition is stable and amenable for storage in ampoules ready for injection thus obviating a need to make a solution from a powder, which is a common step with current treatment compounds.

Inventors:
LOPATINA EKATERINA V (RU)
KARETSKIY ANDREY V (RU)
KRYLOV BORIS VLADIMIROVICH (RU)
Application Number:
US2008/077555
Publication Date:
April 02, 2009
Filing Date:
September 24, 2008
Export Citation:
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Assignee:
TECHNOLOGY COMMERCIALIZATION (US)
International Classes:
C07D315/00
Foreign References:
US20070004795A12007-01-04
US5411985A1995-05-02
US20050054586A12005-03-10
US4486440A1984-12-04
Other References:
DERBENEV ET AL.: "Effects of meconic and comenic acids on slow sodium channels of secondary neurons", MEMBR. CELL BIOL., vol. 13, no. 3, 2000, pages 379 - 387, Retrieved from the Internet [retrieved on 20081117]
AYTEMIR ET AL.: "Synthesis of new antimicrobial agents; Amide derivatives of pyranones and pyridinones", TURK. J. CHEM., vol. 27, 2003, pages 445 - 452
Attorney, Agent or Firm:
LESCHINSKY, Boris (Waldwick, NJ, US)
Download PDF:
Claims:

WE CLAIM:

1. A regenerative anti-inflammatory composition comprising about 1 to about 2 percent by volume of comenic acid and about 0.55 to about 1.1 percent by volume of sodium hydrocarbonate in a water solution.

2. The composition as in claim 1 further including up to 2.5 percent by volume of benzyl-comenic acid.

3. A method of treating a retinal dystrophy comprising a step of injecting a regenerative anti-inflammatory composition, said composition comprising about 1 to about 2 percent by volume of comenic acid and about 0.55 to about 1.1 percent by volume of sodium hydrocarbonate in a water solution.

4. The method as in claim 3, wherein said injection is done parabulbarly.

5. The method as in claim 4, wherein the total amount of comenic acid per injection is about 10 mg to 20 mg.

6. The method as in claim 5, wherein said injections are repeated once a day for 10 days.

7. The method as in claim 6, wherein said 10-day course is repeated two to three times per year.

8. The method as in claim 4, wherein said injection is done into both eyes.

9. The method as in claim 3, wherein said injection is done intravenously.

10. The method as in claim 9, wherein the total amount of comenic acid per injection is between 20 and 350 mg.

1 1. The method as in claim 9, wherein the total daily dose of said comenic acid does not exceed 5 mg per 1 kg of body weight.

12. The method as in claim 1 1 , wherein said injections are repeated once a day for 10 days.

13. The method as in claim 12, wherein said 10-day course is repeated one to three times per year.

14. The method as in claim 12, wherein said 10-day course is repeated after an interval of about three to six months.

Description:

REGENERATION ANTI-INFLAMMATORY COMPOSITION AND METHOD FOR TREATING A RETINAL DYSTROPHY USING SAME

This application claims priority from a Russian Patent Application No. 2007135967/15 (039320) filed September 27, 2007 incorporated herein in its entirety by reference.

This invention relates to pharmaceutics and composition, specifically to a comenic acid-based regenerative composition and its use to treat retinal dystrophies including a chorioretinal dystrophy.

This invention can be used to produce highly efficient synthetic, non-peptide regenerative and anti-inflammatory compositions that subsequently can be applied in composition and veterinary science to treat chorioretinal dystrophies of varied genesis including of tuberculosis origin.

Regenerative substances include compositions from various pharmacological groups capable to directly affect processes of proliferation, cell regeneration and metabolic rate.

Normal biological activity is based on a continuous process of replacement of dying cells and tissues known as physiological regeneration. Various tissues differ in their ability to restore/regenerate; the higher this ability, the more important role physiological regeneration plays in tissue structure and functioning. Blood cells and cells of mucous membranes, gastrointestinal tract, integumentary epithelium, etc. regenerate promptly; thus, tissues they form are characterized by a high regenerative potential. On the contrary, in neurons and muscular cells the regenerative potential is minimal (near zero). Due to aging, contracted diseases, toxic and ecological factors, and radiation the process of physiological regeneration can slow down. Certain compositions, such as immunodpressants and antitumorogenic compositions, corticosteroids, some antibiotics, non steroid antiinflammatory substances, etc. have a similar effect. Inhibition of the physiological regeneration process is accompanied by alterations in metabolic processes, development of leyco-and thrombocytopenia, anemia, and deterioration of the mucous membranes, including membranes of gastrointestinal tract. Medicinal substances that can accelerate and intensify physiological regeneration became known as regenerative stimulants or regenerants.

Regeneration of areas of tissues and organs that died due to injuries, damages or dystrophy (intoxication, hypoxia, infection, etc.) is known as a process of reparative regeneration. Thus, compositions capable of stimulating reparative regeneration are called reparants. (Mashkovsky, M. D., Compositions, Ed. 14, Moscow, Novaya Volna, 2003, VoU , p. 145). As a result of this reparation, replacement of necrotic foci by specific and/or connective tissues takes place.

Common mechanisms of regenerative action include increases in biosynthesis in purine and pyrimidine bases, RNA, functional and fermentative elements of cells, including membrane phospholipids, as well as stimulation of DNA reduplication and cell division. It must be pointed out that biosynthesis during both physiological and reparative regeneration requires significant substratal supplies such as essential amino- and fatty acids, microelements and vitamins. Besides, biosynthesis of proteins and phospholipids is characterized by high energy consumption, and its stimulation requires an adequate power supply (energy materials). Among the compositions, that substratally and energetically ensure regeneration process, are actovegin, solkoseril, etc. It is often difficult to differentiate between the effects of these compositions and the results of regeneration itself.

According to the localization of action (and goals of pharmacotherapy), the regenerative and reparative stimulators are conditionally subdivided into generic (universal) and tissue specific. Generic stimulators produce effect on any regenerative tissue and include anabolic steroids and nonsteroid anabolics, such as sodium desoxy-ribonucleate (Derinat), methyluracil, inosine and others, as vitamins of plastic exchange. Tissue specific stimulators of the regeneration process are substances with different mechanism of action united into subgroups based on their selective action on one or another tissue, or system of organs (Mashkovsky, M. D., Compositions, Ed. 14, Moscow, Novaya Volna, 2003, Vol. 1 , Vol. 2). The most common compositions used to treat chorioretinal dystrophies are glutoksim, cortexin, fezam and emoksipin. Their application is limited due to allergic reactions.

Certain diseases, especially in ophthalmology, result in a large number of patients that become handicapped, while their treatment remains purely symptomatic. One of the main goals of modern ophthalmology is to increase the effectiveness of treatment in patients with retinal pathology which requires research and development of new drugs. In the past years, considerable attention was paid to bioregulatory therapy characterized by organotrophism and pathogenic direction of

action. Properties of this group of compositions are based on the peptides that participate in protein synthesis and cell metabolism regulation during peptide processing (splitting of certain amino-acid sequences). Multiple regulatory systems of the organism imply the presence of universal mediators, known as regulatory peptides. They are necessary to transmit informational signals between cells, ensuring structural and functional homeostasis in cell populations. Peptide cascade principle is fundamental to biological regulation. One of the most effective compositions of this group is Retinalamine which is a regenerant and reparant of retina (registered in 1999). Retinalamine represents a complex of polypeptide fractions obtained from eye retina of large livestock or pigs, capable of causing a tissue specific regenerative effect on the eye retina (Maksimov, LB, Moshetova, L.K, Savostyanova, S.A. Use of Retinalamine in complex treatment of involutional central chorioretinal dystrophies. St. Petersburg, 2006, p. 96). A known in the art composition was selected as a closest analog of the claimed regenerative composition. It represents a complex of peptides of para- and autocrinic nature (size of about 1000 - 10 000 dalton) functioning as intra- and intercellular messenger, and belongs to a citomedine group. It is produced in a form of lyophilized powder in bottles of 5 mg of Retinalamine each. Retinalamine is used in dissolved form as a transparent, colorless water solution for parabulbar and intramuscular injections. Retinalamine's complex composition does not allow for a regular pharmacokinetic analysis of its individual components. Thus, pharmacokinetic study of Retinalamine was not performed, presenting an obvious disadvantage.

Specific action of the known drug is directed to stimulate retinal photoreceptors and cell elements, improve functional interaction of pigment epithelium and external segments of photoreceptors, and accelerate restoration of light sensitivity in retina. A nonspecific action of Retinalamine is manifested in normalization of vessel permeability, reduction in various inflammatory reactions, and stimulation of reparative processes in the eye retina. Retinalamine possesses metabolic and retinoprotective effects rendering a bioregulatory influence on the eye and a tissue specific action on the eye retina. Individual intolerance and pregnancy are contraindication for use of this composition. No side effects were found during treatments at recommended dosages. Medical interaction of Retinalamine with other drugs is not registered. Retinalamine is stored in a dry and light-protected place at temperatures of 2° - 20° C, and has two-year expiration. As solution, this drug is not

stable; it is stored in a powder form and dissolved immediately prior to injections. This represents a substantial limitation of this drug. It is sold by a doctor's prescription only.

Treatment method using Retinalamine is selected as a closest analog method to the claimed method of the invention. Retinalamine is used to treat central chorioretinal dystrophy of the eye retina, central chorioretinal dystrophy, diabetic retinopathy, and tapetoretinal abiotrophy. During postoperative period, Retinalamine it used to treat patients with scaling of retina, during laser hypercoagulation, and patients with involutional central chorioretinal dystrophy. It is recommended in treatments of thrombosis of the central vein of retina, as preventive maintenance of retrombosis, and chorioretinitis of different etiology. When used in adults, Retinolamine is injected (as yet, it is not recommended for children) parabulbarly or intramuscularly once (contents of bottle is dissolved in 1 -2 ml of 0.5% solution of Novocain or water for injections, or isotonic solution of sodium chloride) in amount of 5-10 mg in a 24 hour period over 5-10 days (if needed this treatment can be repeated in 3-6 months.). A slightly deferred result of this therapy and its durability are based on the peptide cascade effect. After conducting special experiments, the inventors established that this composition does not interfere with effectiveness of complex antituberculosis therapy when treating chorioretinitis of tuberculosis nature.

A common drawback of the known treatment methods is that they produce many side effects related to cardiovascular, immune, respiratory and reproductive systems, and affect liver and kidneys of the patients.

Based on the analysis given above, it can be concluded that due to complexity of the regenerative mechanism in the organism and non-obvious nature of the regulatory action of the components of the known compositions (mostly peptide compositions), and side effects caused by them, the task of developing a universal synthetic regenerative compositions remains urgent.

The goal of the present invention was to create a highly effective synthetic regenerative composition of non-peptide nature without negative pharmacological side effects that are common in the known analogs, and develop a method to treat a chorioretinal dystrophy of diverse etiology based on this composition.

This problem is solved by the claimed group of two inventions linked by common inventive concept: the regenerative composition based on the comenic acid and the method of treatment of chorioretinal dystrophy using this composition.

The claimed composition is characterized as follows:

1. The regenerative anti-inflammatory composition comprising about 1 to about 2 percent by volume of comenic acid in a water solution, preferably 1 or 2% solution, the solution further including about 0.55 to about 1.1 percent of sodium hydrocarbonate.

2. Regenerative anti-inflammatory composition as per item 1 , further containing benzyl-comenic acid in a quantity not exceeding 2.5 percent of the total solution and such 2.5% is taken from the water portion in the resulting 100 percent of solution.

The totality of essential features of the claimed invention achieves desired result: this novel regenerative anti-inflammatory composition is intended to stimulate humoral immunity, increasing phagocytic activity of macrophages when exposed to emotional stress; it manifests anti-inflammatory and regenerative properties, increases effectiveness of standard antituberculous therapy, shows neuroretinoprotective and retina stimulating properties, and blocks toxic effects of heart glycoside group substances; the neurotrophic and trophic properties of the composition are tissue specific. According to data of preclinical research and expert evaluation, the composition does not have negative side effects. In therapeutic dosages, the claimed composition does not affect cardiovascular system, blood ingredients, respiratory system or embryogenesis; it does not stimulate terratogenesis, provoke allergic reaction, affect reproductive function, or stimulate tumor growth. The claimed composition does not have any neurotoxic effects. The high values of such indices as clearance (450-500 ml/kg/hr) and stationary volume of distribution (2.5 I/kg) show that the claimed composition is very actively distributed within the system blood flow. Large part of the composition is not eliminated, but undergoes metabolism and is utilized within the organism itself. This allows using the claimed composition to treat patients with kidney and liver insufficiency. Pharmacokinetic study of the claimed composition makes the use of the claimed composition more preferable, since Retinalamine analog pharmacokinetic analysis was not performed. Specific pharmacological properties of the claimed composition can be observed in 5-7 min after intravenous or parabulbular injection (when treating retinopathies of varied genesis). The active substance, which is comenic acid is easily accessible. A study of acute and sub acute toxicity showed that the composition (medicinal form of 1 and 2% solutions for injections in ampoules of 1 , 2 and 5 ml) belongs to class V of practically nontoxic chemical compounds. The

claimed composition does not cause any addiction or dependency, and penetrates through hematoencephalitic barrier. The claimed composition is stable in form of solution for injections. When stored under natural conditions, its decomposition rate is about 1 % a year. Therefore, this composition has an expiration period of 2 years.

Creation of this composition became possible due to proprietary studies of the delicate mechanisms of action for functioning substances capable of forming chelate complexes with calcium and magnesium ions that manifest anti-inflammatory properties, tissue-specific trophic and neurotrophic properties, and directly regulate processes of coding of the nociceptive information in the central nervous system without any side effects on vital functions of the organism. Furthermore, it was possible due to a thorough research of the membrane signaling pathway through which Na/K- AT Phase is activated as a signal transducer during regulation of synthetic processes in different types of tissues (Lopatina, E. V., Penniyaynen, V.A., Zayka, A.A., Research of Na + , K + - AT Phase role in growth regulation of cardiac tissue explants in organotypic culture. Bulletin of Experimental Biology and Composition, 2005, Vol. 140, No. 8, p. 150-153; Penniyaynen, V.A., Lopatina E.V. Study of Na + , K + -ATPhase role in growth regulation of neurites of sensory neurons. Bulletin of Experimental Biology and Composition, 2005, Vol. 139, No. 2, p. 147-160; Lopatina E. V., Penniyaynen, V.A., Tsyrlin, V.A. Comparative analysis of cardiac glycosides effect on cardiac tissue explants growth. Sechenov Physiol. Jour., 2005, Vol. 91 , No. 1 1 , p. 1299-1304; Karetsky A.V; Lopatina E. V; Penniyaynen V.A. α3- izoform of Na + , K + -AT Phase modulates process of cells growth in the chicken retina. Humboldt-Kolleg conference "Technologies of the 21 st century: biological, physical, informational and social aspects ", St-Petersburg, 2005, p. 30). Analogous function of a transducer, Na/K- AT Phase carries out during coding modulation of nociceptive signal (Krylov B. V., Derbenev A.V., Podzorova, S.A., Lyudyno, M. I., Kuzmin, A. V., Izvarina, N. L. Morphine decreases sensitivity potential of slow sodium channels, Russian Physiol. Jour., 1999, Vol. 85, No. 2, p. 225-236). As a result of this study, it was proposed that comenic acid can be used as an active ingredient in production of the anti-inflammatory, synthetic and regenerative composition that exhibits immunomodulator, antioxidant and other effects described above. The claimed composition shows these properties due to its ability to form chelate complexes with ions of calcium and magnesium with subsequent activation of a specific molecular

membrane signaling mechanism that includes alpha three isoform of Na/K- AT Phase as a signal transducer.

The claimed composition differs from the closest analog by a number of essential features and first of all, by its qualitative composition (as its uses comenic acid, which is a different active ingredient), corresponding quantitative characteristics, and by the fact that a ready-for-injections solution is achievable. The proposed composition is different as it is a synthetic composition. Being different from the closest analog and other known analogs, it is a non-peptide compound with medicinal effect which is difficult to predict. The composition is produced in a form of a finished, stable and sterile solution ready for injections.

Analysis of the known in the art compositions did not show any solution that completely duplicates its essential properties with the claimed composition. Earlier, the inventors demonstrated that comenic acid possesses sedative action in dosages exceeding the values of the claimed invention (patent of the Russian Federation No. 2209062 entitled "Substance with sedative action", filed March 19, 2002, published July 27, 2003 incorporated herein in its entirety by reference).

Only the totality of essential properties of the claimed regenerative composition allows for the described result. The ability of its unique composition to demonstrate a stable regenerative activity was unexpectedly discovered while studying the role of Na/K-AT Phase as a signal transducer during regulation of synthetic processes in tissues of various types.

It should be noted that until now there was no regenerative compositions without negative side effects that can be compared by their regenerative effectiveness with the anabolics.

The claimed method of treatment is characterized as follows:

The method of treatment of a chorioretinal dystrophy of diverse etiology including a step of parabulbarly injecting the subject with a regenerative composition, as described above, so that the total amount of injected comenic acid is between about 10 to about 20 mg, such injections are done once every 24 hours over a 10 day period. This course may be repeated 2 - 3 times a year.

The method as above further including injection of the composition parabulbarly into both eyes of the patient.

During comprehensive antituberculous therapy, the method of treatment differs in that the regenerative composition is injected intravenously such that the total

amount of the active ingredient, comenic acid, ranges from about 20 to about 350 mg over 24 hrs, such injections repeated for 10 days. This course of injections is carried out 1 - 3 times a year with 3-6 months break between the treatments. Maximum daily dose of comenic acid should not exceed 5 mg per 1 kg of body weight of the patient.

The claimed composition does not cause dependence or addiction, thus the dosage used to treat the chorioretinal dystrophy and during complex antituberculous therapy remains constant during the entire treatment no matter how prolonged it is.

The parabulbar injection of the claimed regenerative composition in an amount in excess of 20 mg in 24 hrs (calculated relative to the active ingredient which is comenic acid) is unnecessary since there is no further increase in regenerative and anti-inflammatory action. The intravenous injection of the claimed composition in an amount over 350 mg over 24 hrs (5 mg/kg) of comenic acid used in antituberculous therapy is unnecessary since there is no further increase in anti-inflammatory and regenerative action.

The totality of essential properties of the proposed method of treatment makes it possible to reach the desired result allowing to effectively treat retinopathies, including those of tuberculosis origin, improve effectiveness of the integrated tuberculosis treatment of a number of degenerative diseases ensuring effective therapy while treating main disease and essential improvement in the quality of life of the patients. During treatment, no addiction to the composition is developed allowing for a steady effect during prolonged treatment without change in medication.

Compared to the method of treatment with the use of Retinalamine closest analog, the proposed method of treatment does not possess any side effects normally associated with peptide-based compositions, such as allergic reactions and vertigo. The claimed composition has a very specific mechanism of action. This has value especially in the case of ophthalmologic surgery. The claimed method of treatment, therefore, is considered to be of a mild interference. The method of treatment using the claimed composition is more convenient for medical personnel as it does not require any additional preparation (dissolution), shortening the work time with each patient.

The claimed method of treatment differs from the method that uses Retinalamine closest analog by the following specific properties:

It uses a different active ingredient that is the claimed regenerative antiinflammatory composition based on comenic acid with dosages and periods of treatment as described above. Comparing to the claimed method, the closest analog method includes a preliminary stage of Retinalamine solution preparations. While Retinalamine is injected intramuscularly and parabulbarly, the claimed composition can be injected parabulbarly and intravenously.

Only the totality of essential features of the claimed treatment method makes it possible to reach the technical result, which is: carrying out an effective complex pathogenetic antituberculous therapy by increasing humoral immunity and phagocytic activity of macrophages, directed modulation of retina cell and cardiomyocytes proliferation process, and stimulation of neurite growth. Until now, the method of complex pathogenetic treatment of tuberculosis by direct stimulation of humoral immunity resulting in increase of phagocytic macrophages activity, and improved action of compositions used in standard antituberculous therapy, has not been known. During treatment of retinal dystrophy due to ocular tuberculosis complication, it was discovered that there was an obvious reduction in the area of fibrous foci when a complex therapy using the claimed composition was administered. This is due to the fact that this composition realizes its properties by formation of chelate complexes with ions of calcium (magnesium) and activates the function of Na/K-ATPhase as a signal transducer stimulating the process of proliferation without intoxication, addiction or relapses. Special studies showed that while the claimed composition possesses powerful tissue-specific trophic, retinoprotective and neurotrophic properties, it does not stimulate generation and growth of new formations. The claimed composition can be recommended to patients with nephritic and hepatic insufficiency since it is not processed by liver and kidneys, but metabolized in the organism itself.

For better understanding of the essence of this invention, the following examples (controls) of its specific use are provided.

Retinalamine closest analog used as off-the-shelf composition (Gerofarm Company).

Reagents used to prepare the claimed regenerative composition include:

- Comenic acid (5-hydroxy-4-oxo-4H-pyran-2-carbon acid) is synthesized using the disclosed method (Garkusha, Zh.A., Magazine of General Chemistry, 1961 , Vol.

31 , page 2573). Melting point is 261 - 267 DC; spectral analysis corresponds to available registered data.

- As a component of the claimed composition (GOST. 4201 -79; FS 42-0324- 4716-03), sodium hydrocarbonate is added to create an optimal value of pH to facilitate comenic acid dissociation and increase its solubility.

- Water for injections is per FS 42-2620-97 requirements. Sterilization of this composition is achieved by membrane filtration under aseptic conditions. The filtrate is poured into ampoules of neutral glass and soldered.

The positive effect of the claimed composition is detected when used in a 1 -2% water solution of comenic acid. The preferred form of the claimed composition (medicinal form) is a 1% or 2% water solution ready for injections in ampoules of 1 , 2 and 5 ml. Comenic acid at concentrations of 10 mg/1 ml (1 % solution), or 20 mg/1 ml (2% solution) serves as the active ingredient . The content of the claimed composition (for medicinal purposes), calculated with respect to 100% of substance, includes: sodium hydrocarbonate 5.5 g (1 % solution), or 1 1 g (2% solution), water for injections up to 1 liter (or 1 -2 g of comenic acid, 0.55-1.1 g of sodium hydrocarbonate and water up to 100 ml). The claimed composition is a transparent liquid with yellowish to yellow color (10 mg/ml), or yellowish- greenish to yellowish-green color (20 mg/ml) with no smell, and pH 4.1 - 5.6. Benzyl-comenic acid, a byproduct of comenic acid synthesis, was identified among impurities. The standard for the total content of impurities is established based on the storage data and permissible levels of additives in the comenic acid-based compositions, and is not to exceed 5% of the amount of comenic acid. The claimed composition represents a stable compound. Expiration of the sterile claimed composition stored in a light protected place with temperatures no higher than +25 degrees C is 2 years. The composition is of nonpyrogenic nature. Test: 10 mg dosage per 1 kg of body weight of the animal, in volumes of 0.5 ml and 1 ml, respectively, for claimed compositions at concentrations of 20 mg/ml and 10 mg/ml. The composition is nontoxic, belongs to Class V which is practically nontoxic medicinal substances (in our case, comenic acid). (H. Hodge et al., Clinical Toxicology of Commercial Compositions. Acute Poisoning. Ed. IV, Baltimore, 1975, p.427).

Example 1. Pharmacokinetics of the Claimed Composition.

When the claimed composition is injected intravenously in dogs (preferably, 1 % and 2% solutions for injections), pharmacokinetics of its active ingredient (comenic acid) in blood plasma can be described as a 3-phase sequence. The first phase of distribution occurs rapidly with standard time of 1.5 min. During the first 10 min, about 95% of comenic acid leaves the blood plasma, and its concentration is reduced from 50-60 g/kg to 3 g/kg, or almost 20 times. The second phase, starts on the 10th minute and lasts until the beginning of the 2nd hour, during which time the concentration of comenic acid is further reduced from 3 to 0.6 mg/ml, or by 5 times. After the 2nd hour, the phase of true elimination takes place with time T 1/2 of about 5.5 hrs. Average time of this composition being present in the organism (MRT index) is 5.5-6 hrs.

High values of clearance rate (450-500 ml/kg/hr) and the stationary volume of distribution (2.5 I/kg) show that the claimed composition is very actively eliminated from the blood flow system. This is unexpected since comenic acid belongs to a hydrophilic compounds group. Thus, it can be concluded that the large part of this composition may not be eliminated, but is rather metabolized and utilized by the organism itself. This allows recommending the use of the claimed composition in patients with the kidney and liver insufficiencies.

After the first parabulbar injection of the claimed regenerative composition that possesses anti-inflammatory action, positive dynamics can be observed within 10-14 days from the beginning of its use. To ensure positive results, if needed, this treatment can be repeated up to 3 times a year.

With inclusion of this composition into standard complex antituberculous therapy, the positive effect using a 10-day course of intravenous injections is observed in 14 days from the beginning of its use with a durable positive effect. Provided there is a need, this treatment can be repeated up to 3 times a year.

The claimed composition does not cause drug dependency and addiction, and allows for a treatment with a constant dosage of the claimed composition necessary to achieve regenerative effect even during prolonged administration. In therapeutic dosage, this composition does not affect blood formula, cardiovascular and respiratory systems, neither causes allergic reactions, not provoke terratogenesis, nor does it affect reproductive function and embryogenesis.

Example 2. Specific Pharmacological Activity.

2.1. Proposed Mechanism of Action. Neurotrophic and Trophic Activity. Experiments in-vitro

The organotypic tissue cultivation method was used to study neurotrophic and trophic properties of the claimed composition. The experiments were performed on the explants of cardiac and retinal tissues, and spinal ganglia neurons, all taken from 10-12 day old chicken embryos. A total of 400 cardiac tissue explants, 500 retinal tissue explants and 400 spinal ganglia explants were studied. To cultivate explants, a nutrient medium with pH of 7.2 with the following composition was used: Hanks' solution (40%), Eagle's medium (40%), bovine embryonic serum (15%), and chicken embryo extract (5%); with addition of glucose (0.6%), insulin (0.5 units/ml), gentamicin (100 units/ml), and glutamine (0.35%). The explants were cultured in a nutrient medium on the collagen-coated cover slips placed in Petri dishes at 37 0 C in a CO 2 incubator (Sanyo) with concentrations of O 2 (95%) and CO 2 (5%). (Penniyaynen, V.A., Lopatina, E. V. Study of the role of Na + , K + - ATPhase in growth regulation of the sensory neurons neurites. Experimental Biology and Composition Bulletin. 2005. Vol. 139, No. 2, p. 147-160; Lopatina, E.V., Penniyaynen. V.A., Tsyrlin, V.A. Comparative analysis of cardiac glycosides effect on cardiac tissue explants growth. Sechenov Physiolog. Journal, 2005, Vol. 91 , No. 1 1 , p. 1299-1304. Karetsky A. V., Lopatina E. V., Penniyaynen V.A. A α3-izoform of Na + , K + -ATPase modulates process of cells growth in chicken retina. Humboldt-Kolleg Conference Technologies of the 21 st century: biological, physical, informational and social aspects', Saint-Petersburg, 2005, p. 30).

Explants cultured solely in the nutrient medium were used as a control The claimed composition was added to nutrient medium at concentrations of 10 "6 M, 10 "7 M, 10 "8 M and 10 "9 M (calculated relative to the active ingredient - comenic acid). Control value of SI was taken as 100%, and confidence level of SI variances was estimated using Student t-criterion. As part of the experiments, ouabain {Sigma), a cardiac glycoside and blocker of Na/K-ATPhase, was added to the nutrient medium. Ouabain at 10 "8 M concentration was added. At this concentration, this composition blocks signal transducer functions of Na/K-ATPhase while completely inhibiting the growth of cardiac, retinal and spinal ganglia tissue explants [Idem].

These studies showed that the claimed composition possesses neurotrophic and trophic properties. The effect of this composition depends on the dosage and type of tissue.

After addition of the claimed composition to the nutrient medium at 10 "6 M concentration (calculated relative to the active ingredient - comenic acid) a confident up to 24% inhibition of retinal tissue explant growth was observed, when compared to the control results. This concentration caused a similar effect on neurite growth with SI 20% lower than that of the control. At the same time, the effect of the claimed composition at 10 "6 M concentration on the cardiac tissue explant growth proved to be opposite. At this concentration, addition of the composition resulted in a confident up to 40% stimulation in cardiac tissue explant growth compared to the control value. The extent of growth inhibition in the retinal tissue explant and sensory neuron neurites was considerably lower at 10 "7 M concentration of the composition with SI of retinal tissue explants of 17.5%, which is lower than the control. The SI of sensory neurons neurites was lower than the control value by 5%. The SI value of cardiac tissue explants under similar culturing conditions was higher than the control value by 23%. Addition of this composition at 10 "8 M concentration resulted in a confident stimulation of retinal and sensory neuron neurites tissue explant growth of up to 18% and 24%, respectively. At similar concentrations, this composition did not affect the cardiac tissue explant growth: The data did not differ from the control values. The confident stimulation of growth of retinal tissue explants was detected upon addition of this composition at 10 "9 M concentration to the nutrient medium. SI was 50% higher than the control value. The growth of sensory neurons neurites was 60% higher than the control value. At this concentration, the composition did not affect the growth of cardiac tissue explants.

As part of the experiments, ouabain at 10 "8 M concentration was added to the nutrient medium, and the claimed composition was used at effective tissue-specific concentration. At this concentration, ouabain completely inhibited growth of retinal tissue explants, neurites of sensory neurons and cardiac tissue explants. Culturing of retinal tissue explants in medium containing the claimed composition at 10 "9 M concentration and ouabain at 10 "8 M concentration combined showed that SI of experimental explants did not differ from the control value. Comenic acid at 10 "8 M concentration removed the blocking effect of ouabain on the neurites of sensory neurons, and SI of experimental explants matched the control value. At 10 "6 M

concentration, the claimed composition removed the toxic effect of ouabain on the cardiac tissue explants. While culturing cardiac tissue explants in the medium containing the claimed composition (10 "6 M) and ouabain (10 "8 M), the extent of growth was the same as the control value, i.e. the SI of cardiac tissue explants cultured in nutrient medium only. Therefore, the tissue-specific neurotrophic and trophic action of the claimed composition is achieved due to its direct action on Na/K- ATPase, most likely to its alpha-3 isoform. In this case, increase in growth and proliferation processes in tissues of various types is linked to the activation of Na/K- ATPase function as a signal transducer. As such, Na/K-ATPase can participate in regulation of synthetic processes (proliferation and growth) in various types of tissues [Idem].

Cytological studies of retinal tissue composition containing hematoxilineosin revealed that in the growth zone of the control and experimental explants cultured for 3 days in nutrient medium containing the claimed composition (10 "9 M), the cells of retinal epithelium pigment, rods, cones and ganglionic cells were present. Registered hematoxilineosin containing composition for cardiac tissue revealed that the zone of growth of the control explants and explants cultured in medium containing the claimed composition (10 "6 M), showed cardiomyocytes and small quantity of fibroblasts. In the growth zone of the silver colored sensory neurons explants, the neurites were found according to Bilshovsky-Gross in both control and preliminary cultivation of explant in nutrient medium containing the claimed composition (10 "8 M). Thus, an additional confirmation that the claimed composition stimulates the natural cycle of proliferation and growth was obtained.

The composition of the claimed composition does not contain ions of calcium and magnesium, although they were present in the culturing nutrient medium in sufficient quantity. The nutrient medium in in-vitro experiments duplicates the internal medium composition of the organism (blood, interstitial tissue liquid, etc).

Experiments to study the sub acute and chronic toxicity of the claimed composition (medicinal form of 1 % and 2% solutions for injections) were performed in-vivo on rats and dogs, and confirmed that the composition can be present in the system blood flow in a form of chelate complex with the ions of calcium and magnesium. It should be noted that the activity of Na/K-ATPases, standard and pathological, depends on the concentration of precisely these ions in the near

membrane cell space (Schonner Endogenous cardiac glycosides, a new class of steroid hormones, Eur. J. Biochem. 2002, Vol. 269, p. 2440-2448).

Study of the biochemical blood analysis of male and female rats in the experimental groups that were injected with the claimed composition at dosages of 100 and 300 mg/kg (calculated relative to the active ingredient - comenic acid) showed a confident decrease in calcium level at the maximum dosage of 300 mg/kg after 90 days of continuous treatment. In 2-3 weeks after discontinuing the composition, this index returned to the normal level. Experiments on dogs showed that the confident decrease in calcium level, that occurred in the rats at maximum dosage of the claimed solution of comenic acid (300 mg/kg), when compared to the dogs injected with the same composition at maximum dosage of 50 mg/kg, was of uncertain tendency. The difference in the maximum dosages of this composition tested depended on animal species due to the fact that it is technically impossible to intravenously inject the dogs with a large volume of the composition.

The proposed composition and method of treatment discovered by the inventors are based on a deep understanding of the physiological role of the molecular mechanism capable of the proliferation process modulation by activating Na/K-ATPhase as a signal transducer, and the ability of the active ingredient of comenic acid-based composition to form chelate complexes with ions of calcium and magnesium. [Idem]

G-proteins are not part of this mechanism (given that the claimed composition does not build tolerance at molecular or cellular levels, nor does it stimulate growth of new tumor formations). The experiments in vivo on mice and rats of grafting Ehrlich carcinoma and Pleece sarcoma tumors showed that the composition does not stimulate tumor growth. No data exists on the direct correlation between G-proteins and Na/K-ATPhase functioning as a signal transducer. The substances that activate G- proteins do not remove the toxic effects of compositions of the cardiac glycoside group, nor do they increase effectiveness of the compositions used in the complex antituberculous therapy.

The known analogs used to treat chorioretinal dystrophies, such as retinalamine, glutoksim, cortexin, fezam and emoksipin are not capable of manifesting comenic acid (Anoceptin) medicinal properties as they do not contain comenic acid, cannot create chelate complexes with bivalent ions of calcium and, possibly, magnesium, nor initiate Na/K-ATPhase mechanism as a signal transducer.

The ability of the claimed composition to create chelate complexes, when injected at therapeutic dosages, does not affect vital constants of the organism.

In summary, the idea of a new activation path which is fundamentally different from the classical way of triggering metabolic processes in the organism lies at the basis of the present invention. The existence of this mechanism confirms the theory that human and animal organisms have several parallel paths to protect its integrity from excessive damaging factors.

There is no addiction to the claimed composition at the molecular level.

2.2. Study of Neurotrophic and Anti-inflammatory Properties in Experiments in-

VNO.

2.2.1. Methodology of Determination of Virulence of Tuberculosis Mycobacteria. Prior to each infection of rabbits, the virulence of tuberculosis mycobacteria (MBT) was determined by injecting 0.001 mg of culture into inguinal area of guinea pigs. Calculation was done based on the longevity of guinea pigs. For these purposes a total of 16 guinea pigs were used. Their survival period after infection was 4-5 months (MBT J1MXT-320 culture, low virulence).

2.2.2. Study of Antimicrobial Action on M. Tuberculosis. The study of the antimicrobial action of the claimed composition employed the standard method of double series culturing using H37 RwM . Tuberculosis cultures. The M. Tuberculosis suspensions were prepared based on the 5 U turbidity standard (which corresponds to five hundred million microbial bodies in 1 ml of suspension) and dissolved by 10 times (same as for the medicinal stability determination), and injected in a volume of 0.2 ml (10 million microbial bodies) by surface layering method.

A modified Soton liquid medium with addition of 0.35% of nutrient agar (0.35%) and horse serum (0.25%) was used as a nutrient medium.

The initial concentration of the claimed composition was 200 mkg (calculated relative to the active ingredient - comenic acid) per 1 ml of medium (i.e. 1 - 200mkg/ml; 2-100 mkg/ml; 3-50 mkg/ml; 4-25 mkg/ml; 5-12.5 mkg/ ml; 6-6.25 mkg/ml; 7-3.125 mkg/ml; and 8-1.56 mkg/ml).

Simultaneously, seeding of the utilized M. Tuberculosis strains was conducted (as a culture control without application of the studied composition to the modified Soton agarized medium, and as a medium control to the solid Levenshteyn-Jensen medium).

The incubation of seedings was conducted at 37° C over the period of 7 days, and the results were registered visually. Upon expiration of this period, all test tubes demonstrated an identical increase in M. Tuberculosis cultures (in Levenshteyn- Jensen medium - initial growth). This shows that the claimed composition at the tested concentrations does not possess antimicrobial action on M. Tuberculosis.

2.2.3. Modeling of Focal Tuberculosis Chorioretinitis

Neurotrophic retinoprotetive and anti-inflammatory properties of the claimed composition were investigated by modeling focal tuberculosis chorioretinitis. The tuberculosis chorioretinitis modeling procedure was carried out using 40 eyes of 20 mature males Chinchilla rabbit with body weight of 3.3 - 3.8 kg. This model was selected as it represents a sufficiently rigid experimental model allowing for formation of secondary dystrophic changes in the retinal tissue.

Effect of a 2% solution of the claimed composition was studied when it was parabulbarly injected to mitigate toxic effect on the ocular tissue of 6 healthy eyes of 3 Chinchilla rabbits. The ophthalmologic study of the eye grounds in rabbits, and subsequent study of the eye + parabulbar cellulose tissue macro-preparation showed that no changes in the eye grounds and Para orbital cellulose tissue were discovered. Thus, it is possible to conclude that the injection of the composition by itself does not have a toxic effect on the healthy ocular tissues nor does it stimulate the development of pathologic processes.

Modeling of focal tuberculosis chorioretinitis was performed using methods developed by Bellendir, A.N. at al. [Bellendir, A.N, Sukonschikova, A.A., Nakonechny, G. D., Peschanskaya, I.N. Effect of nonspecific and specific sensitization of organism on the development of experimental ocular tuberculosis. Problems of Tuberculosis, 1972, Vol.7, p. 68-73; Peschanskay, I.N. Experimental modeling of tuberculosis ocular damages. Problems of Tuberculosis, 1973, Vol.12, pages 65-70; Peschanskaya, I.N. Evaluation and comparative analysis of ocular tuberculosis modeling methods. Thesis, L. 1977, page 17.

Upon formation of the chorioretinal foci, an antibacterial therapy (ABT) was carried out prior to the process of stabilization according to the standard procedure.

Method of Standard Treatment of Active Stage Tuberculosis Chorioretinitis To treat the active stage of tuberculosis chorioretinitis the following antituberculous compositions were used:

• parabulbarly - .05 ml of 3% tubazide solution

• intramuscularly - 10% tubazide solution calculated 10 mg per 1 kg of body weight

• intramuscularly - 40 000 U of streptomycin calculated per 1 kg of animal body weight once daily

Stabilization process implied the absence of clinical dynamics in the inflammatory foci and perifocal zone during ABT over a period of 7-10 days. Prior to the experiment, the animals were divided into experimental and control groups. Each group consisted of 10 rabbits (20 eyes) each.

Chorioretinal foci took the form of rounded yellowish granuloma with indistinct boundaries, which prominent into vitreous body with extension into perifocal area.

Degree of manifestation of exudative changes in the chorioretinal foci zone was conditionally expressed in grades (Prospects of studying laser therapeutic effect using tuberculosis chorioiretinits experimental model. Hokkanen, V.M., Bellendir, A. N., Balashevich, Ll., Ustinova, E.I. Experimental pathology and therapy of pulmonary and extra pulmonary tuberculosis. Moscow, 1985, p. 60-65, 1990).

0 - absence of exudation signs; 1 - exudative changes were located only in foci area; 2 - exudative changes were noted in inflammation foci area and adjacent layers of vitreous body; 3- exudative changes were noted in inflammation foci area and in all layers of vitreous body.

In the majority of cases (65.0%), the degree of manifestation of inflammation was insignificant. In 14 eyes (35%), the level of gravity of focal tuberculosis chorioretinitis was average with inflammatory process in middle and rear layers of vitreous body.

The size of chorioretinal foci was correlated with disc nervous opticus (DNO) size, and maximum sizes were registered (with DD designation).

The size of chorioretinal foci in their majority (90% - 36 eyes) were of 1 - 3 DNO diameters, in 2 eyes (5%) the size of chorioretinal tuberculosis foci exceeded disk dimensions 3 or more times, and in two cases the size of foci was less than 1 DD. The chorioretinal foci prominence into vitreous body was measured with an ophthalmoscope, evaluated in dioptrics in accordance with their maximum prominence compared to the intact tissue.

In the majority of cases, 21 eyes (77.5%), the prominence level in the experimental animals did not exceed 2 dioptrics, and in 9 eyes (23.4%) it showed 2-3 dioptrics.

To study the effectiveness of treatment of tuberculosis chorioretinitis whereas the claimed composition (2% solution for injections) is incorporated into standard therapy, the rabbits were divided into two experimental and control groups:

1. The animals of experimental group were administered a standard antituberculous therapy combined with the course of parabulbar injections of 0.5 ml of 2% solution of the claimed composition over the period of 10 days.

2. The control group of experimental animals was administered a standard antituberculous therapy.

As far as focal size, the distribution of animals in experimental and control groups was equal; the focal size varied from 1 to 3 disk diameters of optical nerve. As far as prominence level, no confident difference between animals in experimental and control group were found.

This study showed that in exudation parameters the animals in experimental group practically did not differ from the control.

The studies carried out prior to when the claimed composition was added to the complex antituberculous therapy, showed that the animals in experimental and control groups did not have a confident difference in such indices as focal size, prominence and exudation.

During the entire study, the animals of experimental and control groups were administered a standard antituberculous therapy. On the 10th day into the treatment, the animals in the experimental group in addition to the standard therapy started parabulbar 0.5 ml injections of 2% solution of the claimed composition into each eye. The course of injections of the claimed composition was 10 days.

The experimental animals reacted well to the injections of the claimed composition (0.5 ml of 2% solution into each eye); no necrosis areas in the injected places, allergic or toxic reactions, were noted.

During therapy with the claimed composition, the ophthalmoscope picture of eye ground was evaluated in the animals of experimental and control groups every two days. On the second day, there was no change in the ophthalmoscope picture of eye ground in both groups. On the 6th day, strengthening of pigmentation of the

dystrophic foci of the eye ground of the rabbits in the experimental group was detected. The eye ground foci in the rabbits of control group showed no essential dynamics. In the rabbits of experimental group, a partial dissolution of retina hemorrhages was also noted. In the control group, no similar changes were observed. On the 10th day into therapy, a confident reduction in the animal foci area in the experimental group was detected. Reduction in area was due to strengthening of pigmentation from the foci periphery to its centers. No similar ophthalmoscope picture of the eye ground was observed in the animals of control group. Thus, a confident reduction of the area of fibrous foci was registered in the animals of experimental group, which is related to the inclusion of the claimed composition into the complex antituberculous therapy.

None of the animals in the experimental group died before experiment ended. Total time of the experiment was 90-120 days.

To terminate the experimental animals at the end of the experiment, they were treated with intravenous injections of 5.0 ml of thiopental of sodium after 12 weeks into the treatment. The ocular enucleation was performed per traditional ophthalmologic method. Under sterile conditions, the eyeballs of animals were fixated in a 2.5% solution of glutaraldehyde on the phosphate buffer (pH = 7.4). Then the rear wall of the eye was separated and subsequently filled in vacuum with paraffin. Using these paraffin blocks, histotopographical shears with thickness of 8- 10 mkm were made and tinctured with gemotocsylin-eosin.

Histological studies confirmed the data of the ophthalmoscope study in vivo. Such results as an increase in pigment epithelium, hypertrophy of inner nuclear layer, decrease in number and area of lacunas between ganglionic cells were detected in the prepared specimens.

2.2.4. Results of Experimental Study of Tuberculosis Chorioretinitis per Ophthalmoscope Signs.

Ophthalmoscope study of eye ground was carried out in the animals of control and experimental groups every 3-4 days. Data of dynamic observation of the ophthalmologic criteria prior and into 30 days of experiment is presented in Table 1.

Table 1. Dynamics of Ophthalmologic Indices in Experimental Animals during Treatment.

Positive dynamics of tuberculosis chorioretinitis treatment was observed in both groups of animals. However, the treatment results in the animals of experimental group that along with the standard antituberculous therapy were parabulbarly injected 0. 5 ml of 2% solution of the claimed composition over the period of 10 days exceeded the same criteria in the animals of control group (Table 1 ).

Foci size showed a confident 20% decrease compared to the initial level (1.98 ± 0.12 prior and 1.59 ± 0.13 post experiments). The control group also showed positive dynamics; however, the foci size in this series decreased only by 4% (2.03 ± 0.14/1.95 1 0.14).

Prominence grade of chorioretinal foci in the experimental group, in which the claimed composition was injected parabulbarly, was lowered by 66% (1.27 ± 0.14/0.43 ± 0.12) compared with the initial level, when three cases showed no prominence at all. In the control group, where the claimed composition was not used, the level of prominence was reduced by 39% (1.2 ± 0.15/0.73 ± 0.13).

Exudative manifestations showed a greater decrease during parabulbarly injections of the claimed composition (by 70%) compared to the initial parameters. A 63% decrease was observed in the control group, while showing turbidity and infiltration into vitreous body.

Thus, the claimed composition, with respect to this criterion, acted 7% more effectively than the standard therapy compositions.

Therefore, the use of a 2% solution of the claimed composition as part of the complex therapy of tuberculosis chorioretinitis allows to confidently decrease foci size, further reduce the levels of prominence and exudation, and stop development of inflammation of tuberculosis process of the eye ground showing higher qualitative results compared to the control group after only 1 month into treatment. This is evidence of the high efficiency of the claimed composition when it is included with complex antituberculous therapy. Standard evaluation procedure to study pharmacological activity of the compositions with potential to increase effectiveness of antituberculous therapy was used, allowing for recommendation of the claimed composition for incorporation into complex pathogenetic antituberculous therapy.

This experimental model is the model of post-inflammatory retinopathies and is used to research pharmacological activity of the compositions from regenerants and metabolites groups with anti-inflammatory properties. Criteria, selected by the authors, are adequate for this type of studies. They allow for a correct evaluation of the properties of the claimed composition as regenerative and anti-inflammatory composition, since the retinal tissue changes during tuberculosis chorioretinitis are secondary during development of an acute inflammatory process.

2.2.5. Results of Histomorphologic Studies of Eye ground Tissue in Experimental Animals with Tuberculosis Chorioretinitis.

After taking the rabbits out from the experiment, all eyeball shears of experimental animals were examined. Analysis of 224 histomorphologic slides obtained from 40 eyes of both experimental and control groups showed that tuberculosis granuloma was present in the chorioidea of all eyes, located next to the optical nerve disk or myelinic fiber. In 14 cases, the foci were located only in chorioidea (in base layer of middle vessels); in 10 cases, the foci of tuberculosis damage were located in the chorioidea and retina under the pigment epithelium layer. In 2 eyes, the foci, besides chorioidea, imbibed all layers of retina including optical nerve disk; in one case, tuberculosis foci affected a small part of retina while its major part protruded into ocular cavity in a form of encapsulated mushroom. In all shears, a lymphoid infiltration of chorioideal adjacent areas was observed. In the majority of cases, the vessels of chorioidea in perifocal area were drooping due to edema, while in 6 cases, they were enlarged and full-blooded with thickened walls due to leukocyte

infiltration. In all ocular shears, exudation in the perifocal area or above retina was visible.

Changes in retina were examined in all ocular specimens; main changes affected the foci apex where the retina was destroyed completely with the destruction of pigment epithelium (PE). However, 40 % of cases in experimental group of this study showed a normal PE structure next to the foci. In control group, the destruction of pigment epithelium with cell pigment outcrop extended up to the dentate line at the periphery of retina.

2.2.6. Modeling Methodology of Traumatic Dystrophy of Retina in Rabbits

The modeling methodology of traumatic dystrophy of retina in rabbits is developed by inventor Karetsky, A.V. Experiments were carried out on 20 Chinchilla rabbits. Males with 3- 3.5 kg body weight were used. To simulate traumatic dystrophy of retina, ocular microsurgery instrumentation was used. Animals were anesthetized with a 1% solution of Promedol (1 -2 ml depending on body mass). Promedol was injected intramuscularly 10-15 minutes prior to surgical procedure.

Eyelids were stitched through with silk and separated, in the upper area of the eyeball, along the limb, a cut of conjunctiva was made, which then was separated out, the seam was superimposed on the upper straight muscle and the eyeball was rotated downwards at its maximum. Towards inside the upper straight muscle, the sclera was notched in layers up to the vascular shell. In the suprachorioidal spatium in the direction toward the rear and outside the needle with a grind away end was injected and used to mechanically inflict traumatic damages, after that needle was extracted. Sclera and conjunctiva were not stitched. The formation of traumatic dystrophy of retina was evaluated according to data obtained after ophthalmoscope inspection of eye ground with the aid of the manual specular ophthalmoscope OR-2 (Russia). On the 2 nd to 3 rd day after operation on the eye ground of rabbits, the presence of flat 0.5-1.5 PD (diameter of the nervous opticus) size white color dystrophic foci with hemorrhages into retina were observed, and on the 7th day the treatment using the claimed composition was started. A 0.5 ml dosage of 2% solution of this composition was parabulbarly injected in each eye of animals in the experimental group once every 24 hours. The course of treatment was 10 days. The animals of control group were parabulbarly injected 0.5 ml of physiological solution into each eye over the period of10 days. This model is adequate to study the claimed

composition in treatment of post-traumatic dystrophies of retina (due to injury, surgery complication, etc).

2.2.7. Treatment Results of Experimental Post-Traumatic Dystrophies.

To study experimental materials, general clinical observations (such as behavior, appetite and body weight change), along with standard ophthalmoscope dynamic research methods were used, such as: study in transmitted light, tonometry and eye ground ophthalmo-biomicroscopy. During experiment on animals with body weight of 400-500 grams, no changes in their behavior and appetite were detected. 0.5 ml of claimed composition was parabulbarly injected into each eye of animals in the experimental group, starting on the 5 day into experiment and over a period of 10 days. Physiological solution, following the same procedure, was used in the control group.

Over the course of this therapy using the claimed composition, the ophthalmoscope picture of the eye ground of animals in the experimental and control group was evaluated every two days. On the second day, the ophthalmoscope picture of the eye grounds in both groups showed no changes. On the 6th day, a strengthening in pigmentation of dystrophic foci in the eye grounds of the rabbits in experimental group was observed. The eye ground foci in the rabbits of control group were without essential dynamics. The rabbits in experimental group also showed a partial resolution of retinal hemorrhages. In the control group, no similar changes were observed. On the 10th day into therapy, a confident reduction in area of foci in the experimental group of animals was noted. Reduction in area was due to strengthening of pigmentation from the periphery of foci to its center. In the animals of control group, no similar ophthalmoscope eye ground picture was present (Table. 2). Final results in animals of both groups were evaluated on the 10 th day after treatment was completed.

Foci size in the experimental group showed a confident decrease of 19% compared to the initial level (1.19 ±0.40 prior and 0.96±0.1 1 post experiment). Positive dynamics were also noted in the control group, however, the foci size in this series decreased by only 3% (1.225±0.30/1.2±0.13) (Table 2).

Experimental animals were terminated at the end of the experiment by 5.0 ml intravenous injection of thiopental of sodium after 12 weeks into the treatment. The ocular enucleation was performed according to traditional ophthalmologic methodology. Under sterile conditions the eyeballs of animals were fixated in 2.5%

solution of glutaraldehyde on the phosphate buffer (pH = 7.4). Then the rear wall of eye was separated being later filled in vacuum with paraffin. The obtained paraffin blocks were used to make histotopographical shears with 8-10 m thickness and colored with gemotocsylin-eosin. Dynamics of ophthalmologic parameters in experimental animals during post-traumatic retinal dystrophy treatment is shown in Table 2. Histological studies confirmed the results of experiments in vivo. The animals that were administered comenic acid (Anoceptin) therapy showed a build-up of pigment epithelium roller and decrease in vacuolization.

Table 2. Dynamics of Ophthalmologic Indices in Experimental Animals Observed during Post-Traumatic Retinal Dystrophy Therapy.

The hypothesis of effectiveness of the claimed composition when used in tuberculosis chorioretinitis therapy (model of post-inflammatory retinal dystrophy) and post-traumatic retinal dystrophies was verified statistically. Three tests were used: Student t- test, sign test and sign rank test. When a sample was extracted from the normal general population, t-test, based on Student distribution, showed the highest effectiveness. In our case, Student t- test verifies the experimental hypothesis that the mathematical expectation is equal to zero (zero effectiveness of treatment) against the alternate hypothesis that the effectiveness is confirmed with the confidence coefficient of ninety five percent.

Sign test and sign rank test verify the hypothesis that the median is equal to zero (zero effectiveness) against the alternative that the effectiveness is positive. Sign test and sign rank test are effective even in those cases when anomalous observations appear in the sample. Sign test is based on the calculation of the number of observations in the sample, which are located above and below hypothetical median (zero in our case). Accordingly, sign rank test is based on the calculation of the average number of ranks that are larger and smaller than the hypothetical median.

As statistic calculations showed for all four parameters: size of foci, prominence, exudation in the case of tuberculosis chorioretinitis, and the size of foci in case of post-traumatic retinal dystrophy, in a total of three tests, the calculated level of significance did not exceed the value of 0.001. This means that the presence of positive effect of the therapeutic treatment carried out by the authors is confirmed for all four parameters with use of both experimental models with the confidence coefficient no less than 99.9%.

2.3. Study of the Effects on the Immune System. Experiments in-vivo A 0.1 % solution of the claimed composition that did not directly affect the antibody producing cells (APC) number in the spleen of mice, hypersensitivity reaction of the delayed type (HRRT), transplant-against-host reaction (TAHR) and phagocytosis, but causing an insignificant stimulation in antibodies production (index of action 1 , 2), was prepared. Experimental animal were C57BI/6, CBA and Fl (CBAxC57BI) mice.

In this research, the following methods to study the effects of the claimed composition on the immune system of animals were used:

1. Determination of antibody producing cells (APC) number in the spleen of mice on the 7 day after immunization with optimal (1 x10 8 kl) and suboptimal (2x10 7 kl) dosages of Ef? (erythrocytes of ram) (Jerne N. -K., Nordin A.A. Plague formation in agar antibody-producing cells. Science, 1963, v. 140, N 3565, p. 405)

2. The determination of GA titers (hemagglutination) and ER (erythrocytes of ram) conducted on the 7 and 14 days after immunization with optimal and suboptimal dosages of antigen in 96-hole round-bottom planchettes. Results were studied macroscopically. Diagrams of injection of the claimed composition are analogous to the diagrams used for APC number determination (Ziegel, E.

Hemagglutination Reaction. In the book: Immunological methods (edited by Ch. Frimel). M., 1979, p.108-1 12).

3. Determination of GZT reaction was performed by intravenous immunization of mice with Ef? dosage of 2x10 5 kl. An effective dose of ER (1 x10 8 kl in 0.04 ml of physiological solution) was injected into the cushion of rear paw on the 7 day after immunization. Local inflammatory reaction was measured in 24 hours based on the difference in mass in experimental (Rexp) and control (Rcont) paws.

4. The index of reaction (IR) was calculated for each mouse per formula (Kimatura, K. A food weight assay method to evaluate DTH in the mice. J. Immunol. Meth., 1980, Vol. 39, N 2, p. 277-283):

Rexp- Rcont

IR = x 100%,

Rcont

5. Determination of the transplant against host reaction (TAHR). Development of TAHR was provoked by injection into the cushion of the right rear paw of the Fl (CBAxC57BI) mice hypodermically of 2x10 6 kl of the lymph nodes of parental genotype (CBA). Syngenic lymphocytes were injected into the left paw. The number of cells in 5 ml of homogenate of left (control) and right (experiment) lymph nodes was determined on the 9 days.

6. The phagocytic activity of the peritoneal macrophages of mice was evaluated, using as an object of phagocytosis the opsonized JgGER (Argyris B. F. Role of macrophages in antibody production. J. Immunol., 1967, Vol. 99, N 4, p.744- 750). Macrophages were drawn to the abdominal cavity by the hydrolyzed starch solution. The dosage of 0.5 ml of 5% ER suspension was hypodermically injected on the 3-4 day. A wash out of cells suspension was done. The percentage of phagocytizing cells was determined in the claimed composition colored according to Romanovsky-Giemza. To determine the phagocytic index (Phh), the number of seized Ef? was calculated spectrophotometrically based on hemoglobin concentration and using calibration curve. After 3 hours of incubation at 37 0 C, the phagocytic index (PhI 2 ) was determined again. The index of completion of phagocytosis (ICPh]) was determined per formula:

PhI=E 1 - PhI 2

ICPh = x 100%.

Phh

7. During the determination of phagocytic activity in vivo, the mice were intravenously injected 0.5 ml of 25% of body solution, then in the course of 15 minutes every 3 minutes the blood from the orbital sine (0.02 ml) was taken, and 1 ml of 3% acetic acid was injected. After taking blood, the animals were put to death, and weight of body, liver and spleen was determined. The optical density of the hemolyzed blood was determined using Cφ-26 with wavelength of 610 nm, and f (D) graph was built. The points Ig 0' (zero time) and Ig 10' (per graph) were found. The constant (K) of phagocytosis was calculated per formula:

Ig 0' - Ig 10' To = ,

10 After that, the true index of phagocytosis (α) was calculated per formula: body weight α = x K liver weight + spleen weight

8. The complement activity was determined by titration method using for direct activation method the erythrocytes of rabbit (Erabit) processed by the hyperimmune antiserum of guinea pig (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 the alternative way of activation, Erabit processed with solution of iodide of potassium (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, Vol. 85, p. 233-243). Activity was expressed in units of 50% lysis per 1 ml of serum {CH50).

9. Statistical analysis of the results was done using SW tools package. The Student criterion and Wilcockson-Mann-Whitney method were used. Logarithmic indices were used to process APC number and GA titer determination results.

As part of the experiments, experimental emotional stress was caused by congestion (40 mice in cage of 17x25x8 cm) over the period of 10 days. During experiment, the animals in control group were daily intravenously injected a 1.1 % of sodium solution, and the animals of experimental group were injected a 50 mg/kg dosage of the claimed composition.

2.3.1 Effect on Humoral Immune Response Development Results of studying the effects of the claimed composition on formation of antibody-producing cells (APC) in the spleen of mice and of hemagglutinin titers (GA) in the serum of different mice species which were immunized by optimal and suboptimal dosages of erythrocytes of ram (Eram) are shown in Tables 30 and 31. As it can be seen from the Tables, when injected intravenously, the claimed composition stimulates approximately twofold the APC formation in spleen and one titer (i.e. 2 times) in GA level in serum of C57BI/6 mice, and showed a weak reaction to Eram when immunized with both optimal and suboptimal antigen dosages. Stimulation of APC formation in mice with strong Fl response when immunized with suboptimal Eram dosage produced by the claimed composition at dosages of 5 and 50 mg/kg, and GA titer increase when injected 50 mg/kg of the claimed composition, testifies to the presence of adjuvant properties.

On the 7 th day after intravenous injection of 50 mg/kg dosage of the claimed composition, an increase in APC number in spleen of C57BI/6 mice, which were on optimal and suboptimal immunization regime, was observed, while GA titers showed increase only when an antigen optimal dosage was injected. The fact of dosage- dependent inhibition of APC formation in spleen of Fl mice, when injected with optimal Eram dosage and the reduction in GA titers in serum when intravenously injected with the claimed composition at 50mg/kg dosage is a topic of interest. This phenomenon can be connected to the fact that when there is a simultaneous injection of significant quantity of Eram and comenic acid, an activation of both of chain of respiratory ferments (Blandova Z.K., Dushkin V.A., Malashenko A.M., Schmidt E. F. Lines of laboratory animals for biomedical studies. M., "Science", 1983, p. 190), and of monooxygenase, specifically, R -450 cytochrome (Ozherelkov, S. V., Vargin, V. V., Semenov, B. F. Effect of experimental informational and emotional stress on the primary immune response in mice of different lines. Jour, of Microbiol., Epidemiol, and Immunobiology, 1985, No. 8, p. 43-46) takes place. This latter fact, related to the functional coupling of immune system with metabolism, leads to an expedited degradation of erythrocytes in phagocytizing cells and decrease in Eram antigenic properties, and, therefore, to a decrease in manifestation of humoral immune response in the intact animals with proper physiological parameters.

Reduction in GA titers is of a phase nature; by the 14 day after immunization the indices in experimental and control groups do not show any confident difference.

Thus, the claimed composition can insignificantly stimulate the development of humoral immune response, possesses adjuvant properties and modulates manifestation of humoral immune response in the lines of mice with strong Eram response.

2.3.2. Effect on Phagocytic Activity of Macrophages

Research results of the effect of the claimed composition on the body Indian ink clearance and peritoneal macrophage phagocytic activity showed that the claimed composition with a single intravenous injection does not change either the number of phagocytizing cells in the peritoneal exudate, or the capability of phagocytes of capturing opsonizised Eram; however, the digestive ability of macrophages increases 1.5-2 times. With intravenous injection at 50 mg/kg dosage the claimed composition increases the content of phagocytizing cells in exudate, but does not change phagocytic index (PhI), nor index of completion of phagocytosis (ICPh). With intravenous injection the claimed composition does not change the capture rate of inert particles in the body and does not affect the rate of blood purification.

2.3.3. Effect on Immune System in Stressed Animals

Studies of the effect of the claimed composition on the presence of state of immunodeficiency, provoked by emotional stress, were performed per standard procedure. Congestion of animals over the period of 10 days lead to suppression of humoral immune response (APC number and GA titer), inhibition of phagocytosis completion and affects the rate of blood purification from the inert flesh particles. All listed alterations, with exception of the latter, are induced by stress. A 10-day intravenous injection of the claimed composition at 50 mg/kg dosage prevents these changes.

Thus, intravenous injections of the claimed composition protect immune system from depressive effects caused by emotional stress.

Table.3. Effects on Immune System in Stressed Mice

Ri - confident difference in indices of intact animals R 2 - confident difference in indices of stressed animals

Thus, the study of possible anti-inflammatory properties of the claimed composition allowed for detecting of the following:

1. Claimed composition stimulates development of the humoral immune response in C57B1/6 mice with weak response at 5 and 50 mg/kg dosages, and in F1(CBAχC57B1) mice with strong response immunized with suboptimal antigen dosage. At 5 and 50 mg/kg dosages when intravenously injected in F1(CBAχC57B1)

mice immunized with optimal antigen dosage, the claimed composition inhibited the humoral immune response development.

2. Claimed composition with a single intravenous injection does not affect GZT reaction; however, when intravenously injected over a period of 7 days at 50mg/kg dosage inhibits GZT development in C57B 1/6 mice with strong response.

3. Claimed composition does not affect the development of the TAHR reaction.

4. Ability of the claimed composition to stimulate completion of phagocytosis by peritoneal macrophages with a single intravenous injection at 5 and 50 mg/kg dosages, and with intravenous injection at 50 mg/kg dosage over the period of 7 days, i.e. ability to increase the number of phagocytizing cells in the peritoneal exudate.

5. Claimed composition does not have any effect on the rate of blood purification from flesh particles or activity of the mice blood serum complement.

6. Claimed composition when it is intravenously injected at 50mg/kg dosage over the period of 10 days prevents reduction in the humoral immune response and alteration in macrophage function caused by emotional stress.

Example 4. Study on Safety of Usage of the Claimed Composition

4.1. Study of Pyrogenic Effects. Pyrogenic effect of the claimed composition was studied in rabbits with body mass of 2.0-3.5 kg according to GF Xl requirements, iss. 2, p. 183. When injected at 10 mg/1 kg dosage of animal's body mass, no temperature increase exceeding 0.3 C was observed. These studies showed that the claimed composition (medicinal form of 1 % and 2% solution for injections) is nonpyrogenic.

4.2. Study of Acute Toxicity on Rodents

To determine the indices of acute toxicity, the claimed composition was intravenously injected into white mice and rats of both gender into the tail vein at the increasing dosages per Litchfield- Wilcockson. The calculations of mean lethal dosages were conducted per Prozorovsky, V.B. (Prozorovsky, 1978). To achieve larger dosages of the claimed composition, the injections were administered repeatedly with the interval of 20-30 min over the period of 4 hrs. Control animals were injected similar volumes of solvent, i.e. 1.1 % solution of sodium hydrocarbonate (sodium).

To investigate each dosage of the claimed composition, groups of 5-6 animal of the same gender were used. Furthermore, there were similar in number groups of control animals of each gender, which were injected the equivalent volumes of solvent using the same methods.

The animals were randomly distributed by groups using randomization methodology. The absence of external signs of diseases and homogeneity of body weight (± 10%) in groups were considered as acceptable randomization criteria.

Dosage rate depended on the amount of the active ingredient. Observation period was 14 days. Recorded indices were: lethality, time of death, symptomatology of poisoning, daily observation of general state and behavior, weighing prior to injections on the 7 th and 14 th days into observation, volumes of consumption of food and water (for these purposes, the animals were placed into manufactured in Italy Techno last exchange cages, a dissection and a macroscopic study of all perished and survived animals at the end of the study; euthanasia in rodents was done by an overdosage of ether), and determination of mass coefficients of internal organs.

The results of toxicometry, observational data on the experimental animals during post-intoxication period of acute poisoning, and also necropsy data, allowed to attribute the medicinal form of the claimed composition to class V of practically nontoxic medicinal substances (H. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed. IV, Baltimore, 1975, p. 427). LD 50 of the claimed composition is 1300 mg/kg.

The state of those animals that survived acute intoxication testifies to a good tolerance and harmlessness of the claimed composition at the dosages exceeding maximum therapeutic dosages by one hundred times for humans.

Experimental data on dogs, confirmed that the claimed composition during intensive injections practically does not have any toxic effect. The maximum therapeutic dosage of the claimed composition for a human being with average weight of 70 kg is about 5 mg/kg of body weight, i.e., 350 mg over a 24 hour period.

Preclinical studies of comenic acid (Anoceptin, medical form as a solution for injections) on mice, rats, dogs and guinea pigs showed that the claimed composition does not cause allergic reactions, affect reproductive function, embryogenesis, stimulate mutagenesis or terratogenesis, or render negative effect on the cardiovascular and nervous systems.

Example 5. Use of Claimed Composition for Post-Tuberculosis Chorioretinal Dystrophy Treatment.

Ten patients with diagnosis of post-tuberculosis chorioretinal dystrophy participated in this study. Prior to the study, all patients signed informed consent. Duration of treatment using the claimed composition was 10 days. All patients were daily injected with the claimed composition parabulbarly at a dosage of 0.5 ml of 1 % or 2% solution (dosage of the acting substance of comenic acid in the first case was of 10 mg per day and in the second of 20 mg per day). Dosage of the claimed composition was selected based on the presence of post-tuberculosis changes. Depending on the localization of dystrophic foci, the patients were divided into groups with central and peripheral localization of foci. According to the experimental data, it was detected that 1 % and 2% solution of the claimed composition had a favorable affect on the pigment epithelium preservation. To estimate effectiveness of the claimed composition prior to and after treatment, the visual acuity and field of sight were studied; electrophysiological study allowed estimating the effect of the claimed composition on the retinal cells functionality. After treatment with the claimed composition the majority of patients noted positive dynamics. Visual acuity increased on average by 10-15%. Evaluation of the sight field was conducted one month after the treatment. The field of sight in patients, who were injected with the claimed composition, increased on average by 4%. Changes in the field of sight occurred at both quantitative and qualitative levels. Improvement in visual functions corresponded to the dynamics of electrophysiological study indices. The use of the claimed composition during treatment of retinal dystrophy of different genesis allows for improvement or normalization of the electrophysiological activity in retina cells both in amplitude and time of impulse transmittal in the receptor layers, which contributes to visual function improvement. Recommended dosages of the claimed composition are 10 mg or 20 mg parabulbarly a day (in conversion to the acting substance which is comenic acid) depending on severity of the disease. Treatment has to be repeated twice a year.

Effectiveness of the claimed composition to treat post-tuberculosis chorioretinal dystrophies allows recommending it for treating chorioretinal dystrophies of different etiology, as this research model includes both dystrophic retina changes of post-inflammatory nature and diseases due to complications after traumatic phenomena (ischemia, surgery, injury of eye). An increase in dosage of the claimed composition above 20 mg parabulbarly into each eye is unnecessary.

The anti-inflammatory properties of the claimed composition are manifested during intravenous injections in amount from 20 mg to 350 mg in a 24 hour period; further increase of dosage does not strengthen anti-inflammatory effect, and the claimed composition starts manifesting soft sedative properties.

Application of the claimed invention is not limited only to the above examples.

Thus, the experimental results shown prove that the claimed composition possesses an obvious regenerative action and powerful anti-inflammatory properties. The discovered properties of the claimed composition allow for attributing it to a broad class of reparants, regenerants and metabolites that include compositions of different origin and type of action. The mechanism of action of the claimed composition in its principle differs from the known analogs, since it is not peptide and not of a plant origin. The claimed composition does not have allergic reactions or drug tolerance. The claimed composition is convenient in its use, since comparing to its closest analog it is a finished solution ready for injections. The claimed method of treatment using this composition is intended for therapy of chorioretinal dystrophies of various etiologies, and is more effective and mild in comparison to the known in the art. This composition can be a part of integrated antituberculous therapy to increase the effectiveness of the used compositions.

Going below the low level of the claimed range of active ingredient does not allow for proper efficacy of this invention. Exceeding the upper level is not advisable both as it is technically impossible and does not increase regenerative potential.