WO2009002209A1 - Substance for dermatological medicinal agents based on microbiotic collagenase 'ultra-lisin' - Google Patents

Abstract

The invention relates to a substance for dermatological medicinal agents based on microbiotic collagenase in the form of an ultrafiltrate extracted from the cultural liquid of a Streptomyces lavandulae VPKM S-910 strain exhibiting collagenolytic 1800 - 2500 CEA/ml activity and 120 - 200 PE/ml proteolytic activity.

Description

 Substance for dermatological medicines based on collagenase of microbial origin "Ultralizin"

The invention relates to biotechnology, in particular to the creation of a substance for medicines on the basis of collagenase of microbial origin.

A group of microbial collagenases is known, which is widely used in biotechnology and for laboratory purposes, for example, collagenases synthesized by the bacterium Clostridium histoluticum .. Collagenase producers are also insects Nuroderma lipeatum and Kamchatka crabs [1].

Known analogues of the invention may be preparations obtained from bacteria Clostridium histoluticum and crab collagenase [1]. The bacterium Clostridium histoluticum is a causative agent of gas gangrene, a strict anaerobic, requiring special growing conditions. The technology for producing the drug from this bacterium is complex and expensive. Crab collagenase is inferior in bacterial properties, as it does not completely neutralize collagen. In addition, the inherent fibrenolytic and thrombolytic properties of the drug can cause unwanted effects when applied to the skin.

The closest prototype of the proposed biologically active ultrafiltrate is collagenase isolated from the culture fluid of the strain Stromatosis strain VKPM S-910 [2].

The disadvantage of this invention is the low collagenolytic activity, a more complex and expensive way to obtain the named enzyme, as well as a limited area of use in medical biotechnology.

SUBSTITUTE SHEET (RULE 26) The technical task of this invention is to obtain a highly effective biologically active ultrafiltrate containing collagenase and protease of microbial origin, intended for use as a substance for drugs in various wound healing preparations, preparations for the lysis of postoperative or post-traumatic hemothorax, for the lysis of postoperative adhesions, in cosmetology.

The technical problem is solved by the fact that the claimed biologically active substance contains collagenase and protease from Stromatosis lavepdulae VKPM S-910 with the following enzymatic activities:

- collagenolytic activity 1800 - 2500 KE A / ml

- proteolytic activity of 120 - 200 PE / ml.

To obtain the claimed substance-ultrafiltrate, the strain strainomes lavepdulae VKPM S-910 is used, which is grown on solid nutrient medium - on potato agar, and then on a fermentation nutrient medium of the following composition (g / l): corn flour - 20; peptone - 0.5; dry fodder yeast - 0.05; MgSOdLNgO - 0.4; gelatin - 0.5; tap water pH 6.4 - 6.8. Cultivation is carried out in Erlenmeyer flasks with a volume of 250 ml on a rocking chair (200 rpm) at a temperature of 28 degrees Celsius for 72 - 96 hours. The mycelium is separated by centrifugation. The procedure for obtaining biologically active ultrafiltrate from the filtrate of the culture fluid begins with its concentration while separating low molecular weight substances and protein fractions at a temperature of 15 - 20 degrees Celsius and a pressure of 760 - 800 mm Hg. The filtrate of the culture fluid is placed in an ultrafiltration laboratory unit with a working surface of 2 square decimeters. The receiving tank is filled with filtrate. Ultrafiltration of the native solution is carried out under a pressure of 0.4 + 0.02 MPa. For biologically active

SUBSTITUTE SHEET (RULE 26) ultrafiltrate in the work using a membrane with a low permeability of the protease. The obtained biologically active ultrafiltrate is stored at a temperature of +1 - +20 degrees Celsius in the presence of a stabilizer.

Ultralizin, a substance for dermatological medicines, meets the requirements of the developed technical specifications TU 9336-001-11684510-03, effective from 07.07.2003, in compliance with sanitary norms and rules in force at the enterprises of the microbiological industry of the Russian Federation.

According to its physical and chemical characteristics, Ultralizin corresponds to the following indicators:

- the solution has colors from light brown to dark brown;

- has a specific smell;

- soluble in water, 95 percent alcohol, chloroform;

- the pH of the aqueous solution is 5.0 - 7.0;

- collagenolytic activity is from 1800 to 2500 collagenolytic units of activity per 1 ml;

- total proteolytic activity is from 150 to 200 proteolytic units per 1 ml;

- non-toxic, non-allergenic;

- in microbiological purity meets the requirements of Ha XI, no. 2, p. 193.

Collagenolytic activity is determined by the method of Rosen [4]. Proteolytic activity was determined according to Kunitz [3].

Determination of collagenolytic activity.

SUBSTITUTE SHEET (RULE 26) A unit of collagenolytic activity (KEA) is the amount of the enzyme that exposes the hydrolysis products equivalent to 1 μg of L-lycine under standard experimental conditions to the collagen.

In 5 test tubes with a capacity of 20 ml each, add a pipette of 2 ml of a suspension of collagen in a phosphate buffer pH 7.4. In 1, 2 and 3 test tubes (experimental) add 2 ml of the test solution of ultralizine (before analysis, carefully shake the contents of the ampoule or vial). In tubes 4 and 5 (control) add 2 ml of water. All tubes are closed with stoppers and placed in a thermostat at 37 + 0.5 degrees Celsius for 18 hours. Cool.

From each test and control tubes, 1 ml of solution is taken, introduced into 5 conical tubes with a capacity of 10 ml, and 1 ml of a 2% solution of ninhedrin in acetone is added. The tubes are placed for 10 minutes in a water bath at 50 degrees Celsius. It is cooled and after evaporation of acetone (determined by the absence of a smell of acetone), the volume of the solution is adjusted with water to 10 ml.

The absorbance values are determined on a spectrophotometer at a wavelength of 600 nm in cuvettes with a layer thickness of 10 mm. As a comparison solution, water is used. According to the calibration graph, the L-lycine concentration values are found in the experimental and control tubes, and arithmetic mean values are calculated. From the value of the experimental concentration, the value of the control is subtracted. This value is used to calculate the activity of ultralysin. The activity of the substance (Ak) in collagenolytic units (KEA) is calculated by the formula: a x 4 x 10 AK = = a x 10,

2 x 1 x 2

SUBSTITUTE SHEET (RULE 26) where a is the amount of L-lycine in mg, found according to the schedule.

Kunitz determination of proteolytic activity

Preparation of a two percent casein solution: reagent A 0.2 M Na 'H ₂ PO ₄ H ₂ O - 27.8 g / l.

Reagent B: 0.2 M Na ₂ HPO ₄ "7 H ₂ O - 53.65 g / l. Take 81 ml of reagent B.

Pour 81 ml of B. reagent to 4 g of casein. Hold for 5 minutes at room temperature, transfer to a boiling water bath, cool. Add 19 ml of reagent A and 100 ml of distilled water.

Experimental and control tubes containing 2 ml of two percent casein are thermostated for 10 minutes at a temperature of 30 degrees Celsius. 2.5 ml of trichloroacetic acid (TCA) are added to the control. 0.5 ml of ultrafiltrate is added to the experimental and control tubes. Incubate for 10 minutes. Then, 2.5 ml of TCA are added to the experiment, after 30 seconds, into each tube. The optical density (OD) of the ultrafiltrate is determined at 280 nm. The resulting indicator is multiplied by dilution.

The enzymatic activity of the ultraconcentrate varied in the following ranges:

Example 1. The ultraconcentrate had the following characteristics: Collagenolytic activity of 1800 KEA / ml

The proteolytic activity is 120 PE / ml.

Example 2. collagenolytic activity - 2000 KEA / ml; proteolytic activity - 150 PE / ml.

SUBSTITUTE SHEET (RULE 26) Example 3. collagenolytic activity - 2500 KEA / ml; proteolytic activity - 200 PE / ml.

The study of acute toxicity of the substance was carried out on white mice (males and females with a body weight of 19-21 g).

The experiments were carried out in accordance with the "Methodological recommendations for the study of the general toxic effects of pharmacological agents", 1997 [5].

Ultralizin was administered to mice intravenously, once, in a volume of 0.2-0.5 ml, at a rate of OD ml / s.

The duration of observation of mice was 10-15 days. During the experiment, we monitored the behavior, appearance, motor activity, and the reaction of animals to external stimuli. In cases of death, the clinical picture was recorded and an autopsy was performed of dead mice.

With the intravenous administration of the drug to mice in doses up to 3500-4000 U / kg, no signs of intoxication were observed. Animals remained motile, reacted normally to the environment. With a further increase in the dose of the administered substance, the death of individual animals was noted. Medium lethal doses (LD ₅₀ ) were established for mice with a single intravenous administration of a substance: LD ₅₀ = 12400 ± 2500 U / kg.

Maximum tolerated dose for intravenous administration

administration to mice was ^and 4500 U / kg. In these cases, when observing animals for 2 weeks, death was not observed.

Studies have also been conducted on the microbiological purity and pyrogenicity of the claimed composition.

SUBSTITUTE SHEET (RULE 26) Studies have been conducted for the presence of pyrogenic impurities in the drug. The test was carried out on chinchilla rabbits, kept on a standard diet of vivarium.

Since this drug is used only externally as an ointment, it is difficult to establish a clear therapeutic dose. Therefore, to select a test dose for a pyrogenicity test, we used a dose for a collagenase preparation of 50 U / kg.

It has been established that this drug has a test dose above 5000 IU per kg of animal weight - pyrogenic.

A tendency toward a decrease in the pyrogenic reaction with a decrease in the input volume was noted.

The establishment of a clear therapeutic dose will make it possible to more reliably select a test dose for pyrogenicity. Conducting additional tests with new samples of biologically active ultrafiltrate, in a minimum diluent volume, in a reasonable test dose, will probably allow to obtain results indicating the absence of pyrogenicity.

Thus, a biologically active ultrafiltrate based on collagenase isolated from the strain of Stertomas lavepdulae VKPM S-910 with collagenolytic activity of 1800 - 2500 KEA / ml and proteolytic activity of 120 - 200 PE / ml is a highly effective, non-toxic, non-allergenic drug. Having in its composition collagenase and a protease enhancing its action, the proposed biologically active ultrafiltrate can be used in various wound healing preparations and dermatology.

SUBSTITUTE SHEET (RULE 26) Information sources:

1. Demina H.C., Lysenko CB. Collagenolytic enzymes synthesized by microorganisms. The Journal of Microbiology, 1996, Volume 65, JChO 3, pp. 293 - 304.

2. Demina H.C., Lysenko CB. Pharmaceutical composition based on collagenase of microbial origin. RF patent No 2075219, publ. 10/03/1997

3. Kunits J / Theme оf оf defеstiop рroteolutis epzume. J. Gep. Phusiol, 1947-v. 30, .NM p. 291-310.

4. Rozep H. Amodifiin ninhydryn colorimetriс apalysis for amipo asids. Arsh. Вiohem. Biorhus., 1957, v. 67, p 10-15.

5. "Guidelines for the study of the general toxic effects of pharmacological agents", 1997.

SUBSTITUTE SHEET (RULE 26)

Claims

 CLAIM A substance for dermatological medicines based on collagenase of microbial origin, which is an ultrafiltrate isolated from the culture fluid of the strain Stromatous strain VKPM S-910 with a collagenolytic activity of 1800 - 2500 KEA / ml and a proteolytic activity of 120 - 200 PE / ml. SUBSTITUTE SHEET (RULE 26)