RU2700941C1 - Method for preventing development of corneal opacity in case of mechanical injuries - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ophthalmology, and aims at preventing development of corneal opacity in case of mechanical injuries. To prevent development of corneal opacity after its mechanical injuries subconjunctival injections and external applications in the form of drops on the corneal wound of the solution are carried out. Above solution is prepared using oxyethyl ammonium methylphenoxy acetate powder and 0.9 % sodium chloride solution. Components are used in claimed amounts. Said components are mixed ex tempore until complete dissolution of oxyethyl ammonium methylphenoxy acetate powder. Subconjunctival injections are 0.5 ml once a day daily, applications – 2 drops 2 times a day, 1 time in morning and 1 time in the evening, course of 7 days.

EFFECT: use of the invention provides prevention of corneal opacity in case of mechanical injuries in all cases of its use.

1 cl, 6 dwg, 1 ex

Description

The invention relates to pharmacology and can be used to prevent corneal disorders arising from its mechanical damage.

Corneal diseases account for at least 25% of all ocular pathology. The constant presence of microflora in the conjunctival cavity is often dangerous even with minimal damage to the cornea. The first place (up to 70-80%) belongs to viral keratitis. Keratitis is more common in immunocompromised patients (regardless of age and gender).

The consequences of corneal diseases: up to 50% of persistent visual impairment (which requires surgical treatment to restore the optical function of the cornea) and even blindness (Clinical recommendations. Ophthalmology, n / a L.K. Moshetova et al., 12.05.2006, p. 45- 71).

For the prevention of complications, including clouding of the cornea, it is necessary to timely contact a specialist, rejection of the uncontrolled use of drugs that contribute to the spread of the pathogen, and observance of the rules of personal and public hygiene.

A known method for correcting regeneration processes during thermal skin burns. This is achieved by the fact that for the stimulation of proliferative-reparative processes, the one developed under the guidance of M.G. Voronkov at the Irkutsk Institute of Chemistry SB RAS [see Voronkov M.G., Dyakov V.M., Semenova N.V. and others A.S. No. 515742 of the USSR.] The drug trekrezan, the active substance of which is hydroxyethylammonium methyl phenoxyacetate, and its analogues [see Rasulov M.M., Motorina I.G., Yushkov G.G., et al. - J. Pathogenesis, 2017; Rasulov P.M., Motorina I.G., Demanova I.F., et al. - Sat: Science today: Theor. and prakt. Aspects. Vologda, 2015; Yakhkind M.I., Rasulov M.M., Abzaeva K.A., et al. - RF Patent No. 2645115].

The above literature data allowed us to hypothesize that hydroxyethylammonium methylphenoxyacetate can, in principle, stimulate reparative processes in traumatic lesions of the cornea, preventing its clouding, despite very large differences in the structure and function of the skin and cornea. In particular, the cornea is a thinner tissue, which, unlike the skin, has greater light transmission, other blood and lymph supply (its capillaries do not "pass" red blood cells), more pronounced and sensitive innervation, a different structure of the protein skeleton (the prevalence of glycosaminoglycans) and etc. These factors create fundamentally different conditions for the normal healing of the cornea compared with the skin during mechanical injuries.

Earlier, such studies of the effects of hydroxyethylammonium methylphenoxyacetate have not been carried out, despite the urgency of the problem of stimulating proliferative and reparative processes and preventing complications in ophthalmic practice.

Accordingly, a positive result of the study would allow the use of the specified substance for a new purpose - to prevent the development of opacification with mechanical injuries of the cornea.

Oxyethylammonium methylphenoxyacetate is the basis of the well-known drug trekrezan used in the form of tablets. Another name for this compound is tris- (2-hydroxyethyl) -ammonium- o- cresoxyacetate.

A method of producing trekrezan in the form of a crystalline form is known, for example, from SU 515742, 06/22/1976.

The use of trekrezan in medicine in the form of a powder and other acceptable dosage forms is known from RU 2429832 C1, 09.27.2011, Rasulov M.M., p. 4, lines 38-42.

In addition, aqueous solutions of trekrezan prepared by ex tempore are known for intramuscular injection in animal experiments (Bobkova S.N. Use of hydroxyethylammonium methylphenoxyacetate (trekrezan) for the correction and control of the course of hyperlipoproteinemia and coronary heart disease, diss. MD, M., 2015, pp. 6 and 31), as well as intraperitoneally - 1 ml of 0.5% aqueous solution (M. G. Voronkov, M. M. Asulov. Trekrezan - the founder of a new class of adaptogens and immunomodulators (review) // Chemical and Pharmaceutical Journal, 2007, v.41, No. 1, pp. 3-7).

In the State Register of Medicines, registration number FS 001854 dated January 29, 2019 also contains hydroxyethylammonium methylphenoxyacetate, which is produced as a powder substance in jars - pure for analysis (PSA). The registration certificate was issued by Pharmcorp LLC, and the substance is produced by the Federal State Unitary Enterprise Special Design and Technological Bureau Technologist (FSUE SKTB Technolog) in St. Petersburg (http://grls.rosminzdrav.ru/Grls_viewFS_v2.aspx?routingGuid = 731a3fb9-bdc6-4668-9a04-8d81e0141c4 & t = circulation date 05/31/2019).

As the closest analogue of the invention, a method for the prevention of complications, in particular, clouding of the cornea with its mechanical injuries, is considered. A complex of drugs is used, in particular, for installations (applications) and subconjunctival administration (Clinical recommendations. Ophthalmology, p / r L.K. Moshetova et al., 12.05.2006, p. 45-52). Namely, in the specified prototype, solutions of antibiotics, antiseptics (sulfacetamide 10-20% solution, chloramphenicol 0.25%, etc.) are used, in particular, for application (installation) to the corneal wound (3-4 times a day) and subconjunctival injection. ), agents that contribute to the regeneration of the cornea (methylethylpyridinol 1% solution of 1 ml 1 time per day), sometimes glucocorticosteroids in the form of eye drops. At the same time, these measures, even against the background of the use of systemic treatment with the introduction of detoxification agents, antibiotics, with a long course of drug administration from 14 to 30 days (2-4 weeks), do not provide complete healing of the cornea, in particular, prevention of its clouding. Complications, in particular in the form of clouding of the cornea, range from 1/3 to 1/2 of the total number of cases. In addition, the use of a whole range of medicines makes such treatment and prevention of complications more expensive.

The task was to find out the effectiveness of the use of hydroxyethylammonium methylphenoxyacetate in a new form, suitable for subconjunctival injections and eye drops for applications to prevent the development of corneal opacity due to mechanical injuries.

The technical result is the prevention of clouding of the cornea with its mechanical injuries in all cases with the introduction of funds for 7 days.

This is achieved by the fact that in case of mechanical injury to the cornea, hydroxyethylammonium methylphenoxyacetate (the basis of the drug trekrezan) is used in the form of a pure for analysis (PSA) powder, which is dissolved ex tempore in official physiological saline (0.9% NaCl). The resulting solution is administered subconjunctively and by local application in the form of drops on the corneal wound.

Thus, a method for preventing the development of clouding of the cornea after its mechanical injuries is proposed, which consists in conducting subconjunctival injections and external applications in the form of drops on the corneal wound of the solution. For the manufacture of the solution using the following ingredients, mass%:

oxyethylammonium methylphenoxyacetate powder - 5

0.9% sodium chloride solution - 95;

which are mixed ex tempore until the hydroxyethylammonium methylphenoxyacetate powder is completely dissolved, with subconjunctival injections being 0.5 ml once a day, applications - 2 drops 2 times a day - 1 time in the morning and 1 time in the evening, for 7 days.

Oxyethylammonium methylphenoxyacetate - a white crystalline substance, readily soluble in water with a _{mp of} 80-81 ° C, has the formula:

[2 - CH ₃ C ₆ H ₄ OCH ₂ COO] ^- [NH (CH ₂ CH ₂ OH) ₃ ] ⁺

and the following view:

Methods of synthesis of a substance and its physicochemical properties are described in detail in the monograph [see Rasulov M.M., Storozhenko P.A., Zhigacheva I.V. - Bassin, 2018, 276 pp.].

To obtain the claimed funds in the form of a solution, a PSA powder of hydroxyethylammonium methylphenoxyacetate and a 0.9% sodium chloride solution are used. The composition is prepared ex tempore, mixing the ingredients until the powder is completely dissolved in the ratio, mass%:

oxyethylammonium powder methylphenoxyacetate - 5,

official sterile saline solution (0.9% NaCl solution) -95.

The resulting solution is used in the form of subconjunctival injections and external applications in the form of drops on the corneal wound for 7 days. Injections are carried out 1 time per day, 0.5 ml each solution, applications - 2 drops 2 times a day: 1 time in the morning and 1 time in the evening.

Confirmation of the effectiveness of the use of hydroxyethylammonium methylphenoxyacetate in the practice of ophthalmology to prevent the development of corneal opacities during its mechanical injuries, in comparison with the prototype, demonstrate the following data.

The object of the study is male Chinchilla rabbits with a body weight of 2.5-3 kg. The experiments are carried out on 40 rabbits (80 eyes) in accordance with the "Rules of Laboratory Practice in the Russian Federation" approved by order of the Ministry of Health of the Russian Federation dated 06.06.2003 No. 267 (Rules of laboratory practice in the Russian Federation Ministry of Health of the Russian Federation Order No. 267 of June 19, 2003 http: //www.kodeks.ru (April 24, 2010)). Animals are kept in accordance with the rules of the European Convention for the Protection of Vertebrate Animals, used for experimental and other scientific purposes. At the end of the experiment, animals are euthanized with a cervical dislocation of 5 vertebrae, following the rules of the “European Convention for the Protection of Vertebrate Animals that are Used for Experimental and Other Scientific Purposes” (Strasbourg, 1986). Animals are divided into the following groups:

A - control group, 20 individuals, which are injected daily under the conjunctiva of 0.5 ml of physiological saline;

B - a group of experiment, 20 individuals, who are injected daily with 0.5 ml of the cornea under the conjunctiva of the claimed ex tempore solution, followed by instillation of the same solution on the cornea 2 drops twice a day - 1 time in the morning and 1 time in the evening. The exposure is carried out in a course of 7 days.

It should be noted that previously, the authors conducted studies of dose effects (solution concentration from 0.5% to 10%, the number of applications for 2 drops of the solution varied from 1 to 4 per day, the volume of the solution with subconjunctival administration varied from 0.1 to 0.5 ml, for 5 to 10 days) of the use of a solution of hydroxyethylammonium methylphenoxyacetate for mechanical injuries of the cornea. A greater number of subconjunctival injections was not performed, due to the extreme pain and limited administration area.

As a result, it was found that to solve the problem, the optimal concentration of the inventive hydroxyethylammonium methylphenoxyacetate solution is 5%, the volume of the solution with subconjunctival administration of 0.5 ml, the number of applications of the same solution is 2 times a day (1 time in the morning and 1 time in the evening) course 7 days

Statistical data processing is carried out by the student method. The data are presented as mean and standard error values - M and m, respectively.

Reliable are considered differences at p≤0.05 (see Petri A., Sabin K, 2009].

Example.

At the beginning of the experiment, animals from groups A and B after a 3-fold irrigation of the eye with a 0.5% aqueous solution of dicaine are mechanically injured in the central part of the cornea at half its thickness with a trepan with an opening diameter of 4 mm. The material for histological examination is taken after slaughtering the animals on the 1st, 7th, 15th, 25th and 60th days, 4 heads (8 eyes) for each period and treated with conventional histological methods. Stained sections with hematoxylin-eosin.

A morphological study of the slices reveals that in 100% of the rabbits of the control group (group A), the day after removal of half the thickness of the cornea, development of both necrotic destructive and inflammatory-compensatory processes is observed. The bottom of the wound is filled with an inflammatory protein-mucous effusion with an admixture of various pathogenic cells, mainly segmented neutrophils, located mainly on the border of the exudate and the bottom of the wound cavity. Collagen bundles of the cornea’s own substance are edematous and homogenized; single leukocytes are found between them. Part (55%) of keratocytes degenerates. Among the remaining 45% of undisturbed keratocytes, active cells with figures of a prophase coil are visible; There are up to 10 such active cells in the field of view, which is about 15% of the total number of undisturbed keratocytes. Up to 88% of posterior epithelial cells from the periphery to the center of the injury zone gradually degenerate. The anterior epithelium of the cornea, as it approaches the edge of the wound defect, gradually thins, its cells flattening, elongate, the number of cell layers of the epithelium decreases more and more and in the form of a single-layer epithelial tongue covers the surface of the side walls of the wound defect under the wound scab.

In rabbits of the experimental group (group B), the integrity of the posterior epithelium was not violated anywhere, including opposite the wound defect, the day after the injury. The gaps between the collagen bundles of the main substance of the cornea’s own membrane in the injury zone, despite the presence of edema, are well defined. Otherwise, the integrity of the posterior epithelium in these animals is 100%. Intensive growth of anterior epithelial regenerate is observed along the edges of the side walls of the wound defect. Mitoses among keratocytes both in the area of injury and in the neighboring zone are found among 52.3 ± 1.6 cells, i.e. five to six times more often than in control.

On the 7th day of observation in rabbits of the control group (group A), which were injected with saline, wound epithelization is in the initial stage. The surface of the epithelium in the central parts of the injury becomes wavy-serrated with intensively keratinized cells of the upper layers. However, in some places (up to 16% of the surface), the defect is covered with stratified squamous epithelium with a violation of vertical anisomorphy. All basal epithelial cells and cells of the second layer of the epithelial regenerate are strongly vacuolated. The connection of the epithelium with the underlying tissue is greatly loosened. Due to the edema of the fibers of the own membrane of the neighboring and peripheral zones of the injured area, the interfiber cracks are poorly expressed, the collagen bundles are sparse, there are many fibroblasts, and single hematogenous cells and blood vessels are found. Compared to the previous period, fibrillogenesis is enhanced, but collagen fibers are randomly located (Figure 1), which does not show healing.

In animals of the experimental group (group B) that received the claimed solution, on the 7th day of regeneration of the epithelium of the peripheral zone of the injured area, it differs from the undamaged zones only in a slightly lesser thickness, and the regenerating underlying shell itself consists of relatively thin but already ordered bundles. Inter-beam and intra-beam edema is practically absent, newcomers are not detected, but fibroblasts and keratoblasts are found in slightly larger quantities than in intact areas. The central zones of injury are also covered by a stratified squamous, slightly keratinized epithelium. In these areas, the fibrillogenesis of the regenerating intrinsic membrane proceeds more intensively, and the newly formed collagen fibers and their bundles, in comparison with the control, are more ordered. The surface layers of the regenerating own membrane, where intensive fibrillogenesis proceeds, are rich in fibroblasts (up to 57% of cases), keratoblasts (up to 41% of cases) and are poor in hematogenous cells and necrotic structures. In these areas, sometimes (up to 15% of cases) blood vessels are already detected (Figure 2).

On the 15th day after injury in rabbits of the control group (group A) treated with saline, corneal epithelization was still incomplete. So, the thickness of the regenerated epithelial layer is not the same throughout the damage zone. Closer to the edge of the injury, the epithelium thickening, demonstrates submersible growth in the slightly differentiated underlying tissue of its own membrane. Here mitoses are detected more often. It was established that by the middle of the injured area, the epithelial layer becomes thinner, and in the center of the injury it becomes bilayered. In this area, the surface layers of its own membrane are rich in elements of the fibroblastic and phagocytic series. In some places, the remains of necrotic structures are found, in some places the epithelium degenerates, desquamates. Fibroblast mitoses are more common. The surface of the entire epithelial layer is wavy-serrated, and its own membrane is rich in capillaries (Figure 3).

In animals that received the claimed solution, on the 15th day after the operation, the entire regenerating cornea is already completely rebuilt and has a structure that is practically no different from normal. Only in the central parts of the own membrane sometimes some swelling of the fibrous structures is preserved (Figure 4).

On the 25th day after the operation, the rabbits of the control group (group A), although epithelialization of the injured surface is observed, however, the epithelial layer of unequal thickness: the thinned sections alternate with areas of considerable thickness, with some submersible growth in a slightly differentiated regenerating own membrane in which mitoses fibroblasts and fibrillogenesis continue quite intensively. In these areas, young collagen fibers and their bundles are randomly located. In some places, the connection of the epithelium with the underlying connective tissue of its own membrane is fragile and easily peels off when preparing sections. The surface of the epithelial regenerate is serrated. By this observation period, restoration processes and restructuring of the tissue components of the cornea are still ongoing (Figure 5), in 9 individuals (about 50%) there are initial signs of corneal opacification, which persisted even in long-term observation periods (up to 60 days). New complications do not arise.

In animals of the experimental group (group B), which received the claimed 5% solution of hydroxyethylammonium methylphenoxyacetate in saline, in contrast to the control group, by the 25th day of observation, the processes of regeneration and reconstruction of the regenerated structures were completely completed. By this time, collagen fibers and their bundles are ordered, and the regenerated epithelial layer is thin with a smooth surface (Figure 6). There are no signs of turbidity. In the long-term follow-up (up to 60 days) in this group of rabbits, no complications were noted.

Statistical analysis reveals that the morphological changes identified above are reliable.

A positive effect - subconjunctival injection and instillation of the claimed solution after applying a mechanical injury to the cornea for 7 days activate the processes of early elimination of necrotic tissue at the site of the defect. The claimed solution containing hydroxyethylammonium methylphenoxyacetate limits the spread of the pathological process, accelerates the epithelization of the wound by 13-14 days after injury during treatment for the specified period, ensuring the histotypicity of the epithelial layer, leads to an ordered arrangement of the fibers of the cornea's own substance in the area of the wound defect. At the same time, there are no signs of corneal opacity even in the long term after its healing.

Given that in the prototype discussed above, the healing of the damaged cornea proceeded with complications in 1 / 3-1 / 2 of cases, even when using a complex of drugs of various kinds (antibiotics, hormones, regenerating agents, etc.), the advantages of using hydroxyethylammonium methylphenoxyacetate in the form of the proposed A 5% solution for preventing the development of corneal opacities during mechanical injury in all cases of its use are considered proven.

Thus, it becomes clear that hydroxyethylammonium methylphenoxyacetate (the active ingredient of the drug trekrezan), when injected under the conjunctiva, together with its applications in the form of eye drops of the inventive solution, reliably prevents the development of corneal opacity during mechanical damage in rabbits when used for 7 days. This is the result of effective stimulation of proliferative and reparative processes in the cornea with its mechanical lesions and indicates the fundamental possibility of its use in ophthalmic practice, expanding in the future the scope of hydroxyethylammonium methylphenoxyacetate. The use of a solution of hydroxyethylammonium methylphenoxyacetate in clinical practice is possible only after conducting studies established in accordance with orders of the Ministry of Health of the Russian Federation.

Claims (4)

A way to prevent the development of clouding of the cornea after mechanical injuries, which consists in conducting subconjunctival injections and external applications in the form of drops on the corneal wound of a solution, characterized in that the following ingredients are used for the preparation of the solution, wt.%: oxyethylammonium powder methylphenoxyacetate - 5, 0.9% sodium chloride solution - 95, which are mixed ex tempore until the hydroxyethylammonium methylphenoxyacetate powder is completely dissolved, with subconjunctival injections being 0.5 ml once a day, applications - 2 drops 2 times a day, 1 time in the morning and 1 time in the evening, for 7 days.

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