RU2674585C1 - Agent for preservation of donor cornea - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ophthalmology. Agent for the preservation of donor cornea in the mode of organotypic preservation contains 100 ml: medium 199 – 25.0 ml, Ham's medium F-10 – 25.0 ml, chondroitin sulfate A – 0.5 g, dextran-40 – 5.0 g, gentamicin sulfate – 0.00014 g, amphotericin B – 0.00015 g, Dulbecco modified Eagle's medium – the rest is up to 100 ml and the preparation Poludanum is 1,000 IU per 100 ml.

EFFECT: proposed agent for the preservation of donor cornea of the eye provides the ability of keratocytes to produce endogenous interferon using inducers β-interferonogenesis to prevent the replication of herpes viruses and ensure effective preoperative prevention of transmission of herpes virus infection from donor to recipient.

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Description

The invention relates to medicine, namely to ophthalmology, and can be used for viral decontamination, prevention of transmission of herpes viruses from a donor to a recipient, and to improve transplant qualities for end-to-end keratoplasty.

A means for preserving donor corneas of the eye is known (RF Patent No. 2450515), comprising: medium 199, Ham F-10 medium, chondroitin sulfate, dextran-40, gentamicin sulfate, amphotericin B and Dulbecco-Igla medium, additionally contains NeyDIL No. 37 St.III (R / U LRS-008827 / 08-061108) in the following ratio of components, mass. %: Medium 199 25.0, Ham's medium F-10 25.0, Chondroitin sulfate A 0.5, Dextran-40 5.0, Gentamicin sulfate 0.00014, Amphotericin B 0.00015, NeyDIL No. 37 St. III 0.00015, Wednesday Dyulbekko-Igla rest.

The disadvantage of this medium is that the NeyDIL No. 37 St.III regulatory peptides used do not induce β-interferonogenesis, do not contribute to the production of endogenous interferon for the decontamination of herpes viruses, and do not provide effective prevention of the transmission of herpes virus infection from donor to recipient.

The objective of the invention is the creation of a multicomponent agent for preservation of the donor cornea in the regime of organotypic conservation (at a temperature of 37 ° C), contributing to the production of endogenous interferon for the decontamination of herpes viruses.

The technical result is to prevent the replication of herpes viruses and to ensure effective preoperative prevention of transmission of herpes virus infection from a donor to a recipient due to the ability of keratocytes to produce endogenous interferon using β-interferonogenesis inducers.

The technical result is achieved by the fact that this tool for preservation of the donor cornea, in the regime of organotypic preservation, containing medium 199, Ham F-10 medium, chondroitin sulfate, dextran-40, Gentamicin sulfate, amphotericin B and Dulbekko-Igla medium, additionally contains The drug is half-day.

Means for preserving the donor cornea of the human eye contains per 100 ml of solution:

Wednesday 199 25.0 ml ham environment f-10 25.0 ml chondroitin sulfate A 0.5 g dextran 40 5.0 g gentamicin sulfate 0.00014 g amphotericin B 0.00015 g Midday 1000 units Wednesday Dyulbekko-Igla rest.

The environment 199 has the following composition, mg / l: L-alanine 25; L-arginine chloride 70; aspartic acid 30; L-cysteine chloride - 0.0987; L-cystine disodium salt; L glutamic acid 66.82; L-Glutamine 100; glutathione 0.05; glycine 50; L histidine chloride monohydrate 21.88; L hydroxyproline 10; L-isoleucine 20; L-leucine 60; L lysine chloride 70; L-methionine 15; L-phenylalanine 25; L-Proline 40; L-serine 25; L-threonine 30; L-tryptophan 10; L-tyrosine disodium salt - 49.72; L-valine 25; L-ascorbic acid 0.05; biotin 0.01; calciferol 0.1; D-calcium pantothenate 0.01; choline chloride 0.5; folic acid - 0.01; i-inositol 0.05; menaphthone sodium bisulfate three-water 0.019; nicotinic acid 0.025; nicotinamide 0.025; para-amino benzoic acid 0.05; pyridoxal chloride 0.025; pyridoxine chloride 0.025; riboflavin 0.01; thiamine chloride 0.01; D-L - tocopherol phosphate disodium salt of 0.01; Vitamin A-acetate 0.1147; calcium chloride two-water 185.5; iron nitrate, nine-water 0.01; potassium chloride 400; potassium dehydroorthophosphate 60; magnesium sulfate seven-water 200; sodium chloride 8000; sodium hydroorthophosphate 47.5; adenine sulfate 10; 5-AMP 0.2; ATP disodium salt 10; cholesterol 0.2; 2-deoxyribose 0.5; D-glucose 1000; guanine chloride - 0.3; hypoxanthine 0.3; D-ribose 0.5; sodium acetate 36.71; phenol red sodium salt 17; thymine 0.3; twin 80-5; uracil 0.3; xanthine 0.3.

Ham F-10 medium has the following composition, mg / l: L-alanine 8.91; L-arginine chloride 210.7; L-asparagine 1-aqueous - 15.01; L-aspartic acid 13.31; L-cysteine chloride 31.53; L-glutamic acid 14.71; L-glutamine 146.2; glycine 7.51; L-histidine chloride, single-water 20.96; L-isoleucine 2.62; L-leucine 13.12; L-lysine 29.3; L-methionine 4.48; L-phenylalanine 4.96; L-Proline 11.51; L-series 10.5; L-threonine 3.57; L-tryptophan 0.61; L-tyrosine sodium 2.25; L-valine 3.51; Biotin 0.024; D calcium pantothenate 0.715; choline chloride 0.698; folic acid 1.32; i-inositol 0.541; nicotinamide 0.611; pyridoxine chloride 0.206; riboflavin 0.376; thiamine chloride 1.012; Vitamin B12 1.36; calcium chloride two-water 44.1; copper sulfate pentahydrate 0.0025; ferrous sulfate, seven-water 0.8346; potassium chloride - 285; potassium dehydrogen phosphate 83; magnesium sulfate, seven-water 152.7; sodium chloride 7400; sodium hydroorthophosphate 156.2; zinc sulfate seven-water 0.0288; D-glucose 1100; hypoxanthine 4.08; lipoic acid 0.206; phenol red sodium salt 12; sodium pyruvate 110; thymidine 0.727.

Wednesday Dyulbekko-Igla has the following composition, mg / l: L-arginine 84; L-cystine disodium salt 56.78; L-Glutamine 584; glycine 30; L-histidine chloride monohydrate 42; L-isoleucine 104.8; L-leucine 104.8; L-lysine chloride 146.2; L-methionine 30; L-phenylalanine - 66; L-series 42; L-threonine 95.2; L-tryptophan 16; L-tyrosine disodium salt 89.5; L-valine 93.6; D calcium pantothenate 4; choline chloride 4; folic acid 4; i-inositol 7; nicotinamide 4; pyridoxal chloride 4; riboflavin 0.4; thiamine chloride 4; calcium chloride two-water 264.9; ferrous nitrate 0.1; potassium chloride 400; magnesium sulfate seven-water 200; sodium chloride 6400; sodium bicarbonate 3700; sodium dihydroorthophosphate two-water 141.3; D-glucose 4500; phenol red sodium salt 15; sodium pyruvate 110.

Half-Dan (LS-002205), contains polyriboadenylic acid, potassium salt (potassium polyriboadenylate) 0.1 mg, polyriburidylic acid potassium salt (potassium polyriburidylate) 0.107 mg; Excipients: sodium hydrogen phosphate (sodium phosphate 2-substituted) 2.0 mg, potassium dihydrogen phosphate (potassium phosphate 1-substituted anhydrous) 0.408 mg, sodium chloride 8.5 mg.

Each individually taken medium, such as: medium 199 as the most complex in composition, Ham F-10 medium and Dyulbekko-Igla medium as the most universal, supports the high viability of sensitive cells of transplanted lines. However, for organ cultures that remain intact during cultivation and do not belong to transplantable lines, experimental selection of the ratios of several media is required, depending on the histogenetic characteristics of the organ culture. In this regard, for the endothelium of the cadaveric donor cornea, which belongs to the glial tissue, the most optimal is the choice of three environments - Wednesday 199, Ham's medium F-10 and Dulbecco-Eagle's medium, in the stated proportions that mimic the amino acid and metabolic composition of the aqueous humor of the anterior intact chamber eyes.

Chondroitin sulfate A belongs to cytoprotectors - viscoelastics, possessing the corresponding sign and electronic charge, it forms a surface protective film on the cells of the endothelial layer of the cornea. This prevents mechanical damage and desquamation of corneal endothelial cells during the hypothermic preservation of donor material.

Dextran with a molecular weight of 40,000 D refers to high molecular weight oncotic components of the medium and, therefore, selected in the declared concentration creates an oncotic pressure of 320 mlOsm / L in the medium, which prevents the process of swelling of the cells and colloidal substance of the stroma of the donor cornea during prolonged hypothermic preservation .

To prevent the growth of pathogenic microflora added: gentamicin sulfate, which has a bactericidal effect against a wide range of gram-negative and gram-positive microorganisms, and amphotericin B, which has fungicidal and fungistatic effects.

The immunostimulating drug Poludan is a biosynthetic polyribonucleotide complex of polyriboadenyl and polyriburidylic acids, it is an inducer of the synthesis of endogenous interferon. It has a pronounced antiviral and immunomodulatory activity. Approved for use in ophthalmology.

The claimed means for preservation of the donor cornea due to its constituent components in the indicated concentrations has pronounced immunomodulating and antiviral activity, stimulates the function of natural immunocompetent cells.

As a result of this, conditions are created for the viral decontamination of human cadaveric donor corneas at the conservation stage, which contribute to the effective prevention of transmission of herpes virus infection to recipients after keratoplasty.

This is confirmed by the results of a study that used viable human cadaveric corneas obtained from the Eye Bank MNTK “Eye Microsurgery” named after S.N. Fedorov in the amount of 16 corneas from 8 donors, with the presence of herpes simplex virus type I, verified by the method of polymerase chain reaction (NDP). One cornea from each donor was an experimental one, the other, a steam room, served as a control sample.

The corneas from the experimental group underwent short-term organotypic preservation at 33-38 ° C for 24 hours in the claimed agent with Poludan (the drug is approved for use in ophthalmology) for keratocyte induction of β-interferonogenesis. After 24 hours, the corneas underwent hypothermic preservation in the cornea preservation medium obtained by RF patent No. 2069951 at 4 ° C for 2 (n = 4) and 6 (n = 4) days, then they were removed from the experiment for histochemical analysis of endothelial viability and analysis for the presence of viruses by the method of cell cytopathic effect (gold standard). The control group of corneas was subjected to an identical research algorithm, except for the composition of the medium, in which, instead of the drug Poludan added to the experimental group, regulatory peptides were used, which, according to the literature, are potentially capable of stimulating interferonogenesis. Of all the preservation media of 16 corneal samples, aliquots were taken before the start of the experiment and 24 hours after normothermic preservation for analysis of the quantitative content of β-interferon.

The endothelium of all experimental and control corneas, according to immunocytochemical analysis, remained viable during all periods of observation. Its loss after 24 hours of normothermic preservation and after 2 and 6 days of hypothermic preservation was insignificant and did not exceed 1.5%. In all experimental samples, after normothermal preservation during co-cultivation of the corneas on the confluent layer of epithelial cells, a cytopathic effect was not obtained, which confirmed complete viral decontamination. In control corneas from the same donors, when co-cultivated in 6 cases out of 8, a pronounced cellular cytopathic effect was obtained. In all experimental aliquots of the media after hyperthermic preservation, the presence of β-interferon in 8-fold concentration was determined in comparison with aliquots from control corneas, which experimentally showed the inability of the closest analogue of the agent for preservation of donor corneas, due to its regulatory peptides, to produce endogenous keratocytes β-interferon.

The cornea preservation agent is prepared as follows: in a laminar room, two media are poured into a chemically clean and sterile volumetric flask: 25 ml of 199 medium and 25 ml of Ham F-10 medium. Then, 0.00014 g of gentamicin sulfate, 0.00015 g of amphotericin B are successively added. The composition is divided into two equal parts of 25 ml each and dissolved in the first part 5 g of dextran-40, and in the second part 0.5 g of chondroitin sulfate A by slowly applying dry matter to the surface of the liquid mixture. After the introduction of these components, the solutions are left to fully swell for 80-120 minutes, after which they are completely dissolved. Then, both solutions are poured again together into a volumetric flask, Poludan is added in an amount of 1000 PIECES and Dulbecco-Eagle medium is added to the mark of 100 ml. Then, after prefiltration through a Millipor filter with a pore diameter of 0.45 μm, the obtained 100 ml of cornea preservation agent is poured into 20 ml into bottles, with simultaneous "Deep sterilization" through a millipore filter system with a pore diameter of 0.22 μm. The bottles are sealed with sterile caps and placed in a refrigerator for storage at a temperature of + 4 ° C for no more than a day, or are used immediately at a temperature of + 37 ° C.

The tool is used as follows. Under the conditions of a sterile box, a cornea with a rim of sclera with a diameter of 16 mm is cut out and placed in a vial with a previously prepared declared agent, tightly closed and preserved at + 37 ° C for 24 hours, after which it is transferred to the cornea preservation medium obtained by patent No. 2069951, in conditions of hypothermia (4-8 ° C) and stored in it.

The use of the proposed means for preserving the donor cornea in the Eye Bank FGAU MNTK “Eye Microsurgery” made it possible to provide effective preoperative prevention of transmission of herpes virus infection from donor to recipient, due to the ability to produce endogenous interferon using an β-interferonogenesis inducer.

Claims (2)

Means for preserving the donor cornea of the human eye, containing per 100 ml of solution: Wednesday 199 25.0 ml Ham's medium F-10 25.0 ml chondroitin sulfate A 0.5 g dextran 40 5.0 g gentamicin sulfate 0.00014 g amphotericin B 0.00015 g Midday 1000 units Wednesday Dyulbekko-Igla rest