RU2018319C1 - Ferment preparation having proteolytic action for ophthalmology - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: proposed preparation is prepared of protosubtiline with the help of ion-exchange chromatography on carboxymethyl cellulose. It is white powder being soluble in water and in isotonic solution of sodium chloride. Solutions of said preparation have high stability at pH 7.0-8.0 and 5-10 C. Optimal conditions for using preparation are: 50 C, pH 7.0-7.5. Proteolytic activity of preparation at 37 C is 3.5-4.2 proteolytic units per 21 kg of protein. Proposed preparation may be used subconjunctively, as instillations, with the help of electrophoresis and photophoresis. EFFECT: increases activity of preparation. 3 tbl

Description

 The invention relates to the creation of a new proteolytic drug capable of selectively destroying necrotic tissue. The drug may find application, in particular, in ophthalmology.

The purpose of the invention is the use in ophthalmology of the drug protolysin, which can be obtained from the commercial drug protosubtilin according to the following scheme:
- dissolving protosubtilin in a buffer solution or distilled water at a pH of 6.2-7.5, followed by adding 0.1-0.5% CaCl ₂ to the enzyme solution to stabilize the metalloproteinase and coagulation of ballast proteins, separating the precipitate by centrifugation;
- desalting the protosubtilin solution by gel filtration on any suitable sorbent, for example Sephadex G-25;
- ion-exchange chromatography of a desalted enzyme solution on carboxymethyl cellulose with the aim of separating the enzymatic complex of protosubtilin;
- concentration of the proteolytic fraction by ultrafiltration to a proteinase content of 8-11 u / ml in concentrate;
- precipitation with acetone at a pH of 8.2-8.4, separation of the precipitate by centrifugation;
- dissolution of the precipitate in distilled water, taken in the amount necessary to obtain an enzyme solution with an activity of 11-14 u / ml;
- filtration through a sterilizing filter;
- lyophilization.

Protolysin is a white or light beige powder, soluble in water and isotonic sodium chloride solution. Protolizina solutions are most stable at pH 7.0-8.0 and temperature 5 - 10 ° ^C. The optimum conditions protolizina actions: temperature 50 ^{° C,} pH 7.0-7.5. Inhibitors - heavy metal ions, metal chelating agents. The isoelectric point is 8.3, the molecular weight of the active protolysin protein is 30.5-34.0 kDa.

It was found that protolysin catalyzes the hydrolysis of peptide bonds formed by amino groups of hydrophobic amino acids and does not affect peptide bonds formed by carboxyl groups of hydrophobic amino acids. The drug actively breaks down proteins containing aromatic amino acids. Proteolytic activity protolizina not less than 1 CFU / mg of specific activity at ³⁷ ° C is 3.5-4.2 CFU / mg protein. Protolysin breaks down viscous secrets, exudates, dead tissue, fibrinous formations. The drug serves as an effective treatment for patients with intraocular hemorrhage, opacities of the vitreous traumatic and uveal etiology.

 Protolysin is released in hermetically sealed vials of 10 PE. A solution of the drug is prepared immediately before use. Protolysin can be administered subconjunctively, in the form of instillations, using electrophoresis and phonophoresis. Side effects of the drug were not found.

The therapeutic effect of protolysin in the treatment of patients with hemophthalmus was studied in comparison with trypsin (from animal tissue) and fibrinolysin (from human blood). It is known that these drugs are used in ophthalmic practice as a thrombolytic agent for hemorrhages in the anterior chamber of the eye, as well as for diseases of the cornea (Mashkovsky MD Medicines. Pharmacotherapy manual for doctors. - The eleventh stereotypical publication. - M .: Medicine , 1988 .-- Part 2). The drugs were used as subconjunctival injections. For this, freshly prepared 0.25% solutions of the preparations were used. After anesthesia of the conjunctival cavity with a 0.5% solution of dicaine, 0.3 ml of the drug solution was administered via the conjunctiva. Injections were performed after 24-48 hours. The results of the studies are presented in table. 1. The following indicators served as an evaluation criterion: the appearance of a reflex from the fundus, the degree of increase in visual acuity, and the reduction in the duration of clinical recovery. The results of a clinical trial of protolisin in patients with hemophthalmus showed higher efficacy of this drug compared to trypsin and fibrinolysin. Protolysin is more stable in solution. At a temperature of not higher than 10 ^{° C} it retains its therapeutic activity for 4-5 days, and trypsin, and fibrinolysin solutions - only for 12 hours.

Clinical trials of protolisin in the treatment of patients with hemorrhage and opacities of the vitreous body showed the effectiveness of the use of this drug by phonophoresis. The following types of examination of patients were used: determination of visual acuity and field of view, studies in transmitted light, ophthalmoscopy, determination of the threshold of electric sensitivity by phosphene, sonography. The observed patients were divided into 3 groups. The first group consisted of patients (34 people, 35 eyes), in whom phonophoresis of protolisin with an activity of 0.5 PE / ml was used as an independent treatment method. The second group of 23 patients (23 eyes) received phonophoresis of protolysin in combination with other complex therapy. The third group (control) consisted of 21 patients (21 eyes), who were treated in the form of a comprehensive standard therapy without the use of phonophoresis of protolysin. The criterion for evaluating the effectiveness of the treatment was the timing of resorption of hemorrhage or opacification of the vitreous body (in bed days), increased visual acuity, and reduced length of hospital stay. Phonophoresis of protolysin was performed using ultrasound with a frequency of 880 kHz, an intensity of 0.2 W / cm ² , and a duration of 5 minutes. The course consisted of 6-12 daily sessions depending on the condition of the eye.

 Comprehensive conventional treatment included the use of antibiotics, sulfa drugs, osmo, autohemo, tissue and vitamin therapy, specific hypersensitizing therapy.

 Comparison of the treatment results in three groups of patients indicates that the best results were observed when using phonophoresis of protolisin in combination with other types of conventional absorbable therapy (Table 2).

 An increase in visual acuity by 0.2-0.9 after treatment of patients of this group was achieved in 14 of 23 eyes, i.e. in 60.8% (tab. 3).

 The use of protolysin phonophoresis in combination with conventional conservative therapy allowed treatment to be carried out in the shortest possible time - 10.9 ± 0.8 days in patients with vitreous hemorrhage and 17.2 ± 0.4 days in patients with vitreous opacities. The use of protolysin phonophoresis as an independent method of treating patients also leads to a reduction in the treatment time for patients in the case of vitreous hemorrhage from 18.1 ± 0.8 days (for patients in the control group) to 14.4 ± 0.9 days, and in the case of opacities of the vitreous from 22.7 ± 0.6 to 20.9 ± 0.3 days.

 The results of clinical trials allow us to recommend the inclusion of protolysin phonophoresis in the complex therapy of hemorrhage and opacification of the vitreous body of patients who are shown non-surgical treatment in order to increase the effectiveness of treatment.

 Protolysin is well tolerated by patients, does not have an irritating effect, does not destroy tissues at the injection site, which is characteristic of animal proteinases.

 Protolysin has a good latent effect, i.e. when introduced into the eye, it acts only on devitalized tissues (hemophthalmos, adhesions, pus). In contrast to protolysin, trypsin and fibrinolysin can also destroy healthy tissues.

 Protolysin is well compatible with antibiotics and corticosteroids.

Claims (1)

THE ENZYME PROTEOLYTIC ACTION FOR OPHTHALMOLOGY isolated from the Bacilbis subtilis culture, characterized in that it is capable of catalyzing the hydrolysis of peptide bonds formed by hydrophobic amino acid amino groups, exhibits specific activity at pH 7.0 - 7.5 and a temperature of 50 ^o C, is most stable at pH 7.0 - 8.0 and a temperature of no higher than 20 ^o C, has a molecular weight proteinase of 80.5 - 34.0 kDa and an isoelectric point of 8.3, is inhibited by heavy metal ions and metal chelating agents.

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