RU2709513C1 - Method for producing colored collagen and determining activity of collagenase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to enzymology, biotechnology and medical industry, specifically to a method of producing colored collagen in order to use it as a substrate for determining collagenase enzyme activity, comprising collagen milling to particle size of not more than 2 mm with moisture content of not more than 8.0 %, followed by treatment with a RO16 dye solution, characterized by absorption spectrum maxima at 210±3; 288±2; 360±2; 480±2 nm, with pH of suspension in range of 6.3–7.5.

EFFECT: present invention provides obtaining a substrate for determining specific activity of collagenase-collagen, chemically bound to RO16 dye, suitable for determining specific activity of drugs based on collagenase from Clostridium histolyticum.

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Description

The invention relates to enzymology, biotechnology and the medical industry, in particular, to a method for producing colored collagen as a specific substrate and a method for controlling the quality of drugs containing the collagenase enzyme produced by the Clostridium histolyticum culture as an active ingredient.

Description of the Related Art

Collagenase is a proteolytic enzyme produced by a culture of Clostridium histolyticum. As concomitant enzymes representing minor impurities, the culture produces some other proteases. Under the action of drugs containing collagenase as an active substance, softening and resorption of scars, scars, contractures, as well as cleansing of wounds, which contributes to the more rapid development of granulation tissue, occurs. The drug does not act on intact epithelium, adipose and muscle tissue.

Collagenase is an endopeptidase that cleaves the triple helix of an insoluble collagen protein macromolecule (Experience in the use of collagenase Clostridium histolyticum in the management of Peyronie's disease: current data and future prospects / MA Egui Rojo, I. Moncada Iribarren, J. Carballido Rodriguez, JI Martinez Salamanca // Therapeutic Advances in Urology. 2014. V. 6, N 5. P. 192-197. Minimally invasive options in Dupuytren's contracture: aponeurotomy, enzymes, stretching, and fat grafting / A. Murphy, DH Lalonde, C. Eaton , K. Denkler, SE Hovius, AA Smith, A. Martin, A. Biswas, C. Van Nieuwenhoven // Plastic and Reconstructive Surgery. 2014. V. 134, N 5. P. 822-829. Schulze, SM, Postapproval clinical experience in the treatment of Dupuytren's contracture with collagenase clostridium histolyticum (CCH): the first 1,000 days / SM Schulze, JP Tursi // Hand (NY). 2014. V. 9, No. 4. P. 447-458. Van Doren, S. R. Matrix metalloproteinase interactions with collagen and elastin / S.R. Van Doren, // Matrix Biology. 2015. V. 44-46. P. 224-231).

Collagen has a special macrostructure formed by a triple helix, which mainly consists of the Gly-Pro-X tripeptide (X is the remainder of proline or hydroxyproline). Due to such a complex structure, collagen as a substrate is available only for the highly specific collagenase enzyme.

Collagenase can be widely used in ophthalmology, cosmetology, surgery for the prevention and treatment of scarring, adhesive capsulitis, keloid scars, hypertrophic scars, postoperative adhesions, wrinkles, cellulite (Pat. WO 2007089851 A2 Compositions and methods for treating collagen-mediated diseases / GL Sabatino, T.Jr. BJ Del, PJ Bassett, HA Tharia, AG Hitchcock. Aug 9, 2007).

Particular attention is paid to the potential use of collagenase in the treatment of Dupuytren's contracture - hand disease associated with Cicatricial degeneration and shortening of the palmar tendons (Multiple concurrent collagenase Clostridium histolyticum injections to Dupuytren's cords: an exploratory study / Coleman S., Gilpin D., Tursi J., Tursi J. G., Jones N., Cohen B. // BMC Musculoskeletal Disorders. 2012. V. 13, N61. Doi: 10.1186 / 1471-2474-13-61. URL: http: //www.ncbi.nlm.nih. gov / pmc / articles / PMC3409044 / pdf / 1471-2474-13-61.pdf. (accessed July 1, 2015). Schulze, SM, Postapproval clinical experience in the treatment of Dupuytren's contracture with collagenase clostridium histolyticum (CCH): the first 1,000 days / SM Schulze, JP Tursi // Hand (NY). 2014. V. 9, N 4. P. 447-458. Van Doren, SR Matri x metalloproteinase interactions with collagen and elastin / S.R. Van Doren, // Matrix Biology. 2015. V. 44-46. P. 224-231).

A. Lauritzson, E. Ahlgren, J. Waldau,

// Acta Orthopaedica. 2015. V. 86, N 3. P. 310-315).Injections of collagenase into the healed fascia covering the tendon lead to its disintegration and, as a result, to the disappearance of the scar. The use of collagenase in the treatment of Dupuytren's contracture allows you to abandon inpatient surgery and exclude long-term postoperative recovery. According to modern literature, most studies in this direction are at the stage of clinical trials (Efficacy and safety of collagenase Clostridium histolyticum in the treatment of proximal interphalangeal joints in dupuytren contracture: combined analysis of 4 phase 3 clinical trials / MA Badalamente, LC Hurst, P Benhaim, BM Cohen // Journal of Hand Surgery. 2015. V. 40, N 5. 975-983). For example, the possibility of simultaneous treatment of two or more contractures in one patient's arm is being studied (The efficacy and safety of concurrent collagenase Clostridium histolyticum Injections for 2 Dupuytren contractures in the same hand: a prospective, multicenter study / RG Gaston, SE Larsen, GM Pess, S Coleman, B. Dean, BM Cohen, GJ Kaufman, JP Tursi, LC Hurst // Journal of Hand Surgery. 2015. [Epub ahead of print]), Collagenase treatment of Dupuytren's contracture using a modified injection method: a prospective cohort study of skin tears in 164 hands, including short-term outcome / I. Atroshi,

A. Lauritzson, E. Ahlgren, J. Waldau,

// Acta Orthopedic. 2015. V. 86, N 3. P. 310-315).

In addition, positive results were obtained with the use of clostridial collagenase for the treatment of Peyronie’s disease (Experience in the use of collagenase Clostridium histolyticum in the management of Peyronie's disease: current data and future prospects / MA Egui Rojo, I. Moncada Iribarren, J. Carballido Rodriguez, JI Martinez-Salamanca // Therapeutic Advances in Urology. 2014. V. 6, N 5. P. 192-197), capsular fibrosis caused by silicone implants (animal studies) (The collagenase of the bacterium Clostridium histolyticum for the treatment of capsular fibrosis after silicon implants / S. Fischer, T. Hirsch, Y. Diehm, J. Kiefer, EM Bueno, M. Kueckelhaus, T. Kremer, C. Hirche, U. Kneser, B. Pomahac // Plastic and Reconstructive Surgery. 2015. [Epub ahead of print]), transplant and pancreatic islets, which is an important step in the treatment of type I diabetes (Characterization of collagenase blend enzymes for human islet transplantation / B. Antonioli, I. Fermo, S. Cainarca, S. Marzorati, R. Nano, M. Baldissera, A Bachi, R. Paroni, C. Ricordi, F. Bertuzzi // Transplantation. 2007. V. 84. P. 1568-1575). Collagenase can be used for diagnostic and laboratory purposes, for example, for in vitro tissue disintegration (Pat. US 8715985 B2 Clostridium histolyticum recombinant collagenases and method for the manufacture thereof / F. Bertuzzi, A. Cuttitta, G. Ghersi, S. Mazzola, M. Salamone, G. Seidita. 6 May 2014).

Given the promise of using collagenase as part of medical preparations, it is urgent to develop a reliable method for determining the collagenase activity of the enzyme produced by Clostridium histolyticum.

Known methods for determining collagenase activity involving the use of both native collagen and a synthetic substrate FALGPA (N- [3- (2-Furyl) acryloyl] -Leu-Gly-Pro-Ala) (Isolation and Characterization of Proteinase and Collagenase from C. histolyticum [Text] / I. Mandl, JD MacLennan, EL Howes, RH DeBellis, A. Sohler, // Journal of Clinical Investigation. - 1953. - V. 32. - P. 1323-1329. Van Wart, HE, Steinbrink , DR A continuous Spectrophotometric Assay for Clostridium histolyticum Collagenase. Anal. Biochem. 113, 356 (1981)). Collagen is a natural substrate for collagenase (Von Hippel, R.N., et. Al., J. Am. Chem. Soc., 82, 2774 (1960)). Concomitant minor impurities, usually present in clostridial collagenase and representing a neutral protease, clostridiopeptidase B, with proteolytic activity, is evaluated using casein and a synthetic low molecular weight substrate benzoyl-arginine ethyl ester (BAEE).

The traditional method for determining collagenase activity using collagen from bovine tendons has significant drawbacks. The activity is evaluated by the number of hydrolysis products formed, and the ninhydrin indicator is used for their quantitative determination. This is one of the most sensitive colorimetric methods, but the reaction with ninhydrin takes place at 100 ° C (in a water bath) using organic reagents such as methyl and ethyl cellosolve and depends on the presence of oxygen, so it is carried out in a stream of nitrogen. The total analysis time is 4-6 hours.

In the case of determining the enzymatic activity of collagenase, which is the active substance of a drug, the use of a low molecular weight substrate (FALGPA) is impractical, since it is the high molecular weight substrate, which is collagen, that allows collagenase to be characterized under physiological conditions.

The closest analogue of the proposed technical solution is a method for determining the activity of neutral collagenase (Pat. 2034029 Russian Federation, IPC C12Q 1/34, C12N 9/50, C12N 9/52, C12N 9/64 / Method for determining the activity of neutral collagenase / TI Zamyslova , L.V. Kukhareva, B.I. Feigelman; applicant and patentee of the Leningrad Scientific Research Institute of Vaccines and Serums - No. 4934907/13; application 08.05.1991; publ. 04.30.1995) using collagen from the dermis as a substrate cattle (produced by Olaine), which is processed dye solution of active orange VT (according to TU 6-14-625-85) at pH 8.0 for 2 hours at 40 ° C, then washed from excess dye with water and dried at room temperature. The ratio of dye / collagen from 20 to 200 wt / wt.

When determining the activity of collagenase in this way, the mass ratio of the enzyme and substrate should be 1: 100. Additionally determine the concentration of protein in the sample and the optical density of the filtrate with complete dissolution of collagen, which is carried out with an excess of the enzyme or a corresponding increase in the duration of the hydrolysis reaction.

The disadvantage of this method is the low accuracy and reproducibility of the method. Currently, the active orange VT dye is discontinued due to its inefficiency in the textile industry due to the weak strength of the bond formed between the dye and the substrate.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide a new substrate for determining the specific activity of collagenase - collagen chemically bound to the dye reactive (active) orange RO16, suitable for determining the specific activity of drugs based on collagenase from Clostridium histolyticum.

The problem was solved by the use of a dye, the molecules of which are capable of forming covalent chemical bonds with hydroxyl and amino groups of protein macromolecules. This property of the dye was used to obtain colored collagen by reacting reactive dye groups of reactive orange RO16 with the amino groups of amino acid residues forming collagen fibers.

Vinyl sulfonic active dyes, to which RO16 belongs, contain a vinyl sulfonyl group —SO ₂ CH = CH ₂ or a group that turns into a vinyl sulfonyl group during coloring, usually a β-sulfatoethyl sulfonyl group —SO ₂ (CH ₂ ) ₂ OSO ₃ Na. Under the action of alkali, the remainder of sulfuric acid is split off from this group and a vinyl sulfonyl group is formed, which interacts with the colorant, in our case, collagen

Dye - SO ₂ (CH ₂ ) ₂ OSO ₃ Na → Cr - SO ₂ - CH = CH ₂ + Collagen - NH ₂ → Collagen - NH - (CH ₂ ) ₂ SO ₂ - Dye (Fig. 1).

This principle is described in the book of Stepanov B.I. (Stepanov, B.I. Introduction to chemistry and technology of organic dyes [Text] / B.I. Stepanov // Textbook for universities. - 3rd ed., Revised and enlarged. - M .: Chemistry, 1984. - 592 p.). Obtaining collagen stained with dye RO16

Bovine tendon collagen (Sigma, Cat. No. C9879) is crushed using a knife mill for solid products like DM-6 manufactured by NT Machinery (Japan-Taiwan) or similar. For staining, crushed collagen is placed in a flask, purified water is added, mixed and left to swell and obtain a homogeneous suspension. Then a dye solution in water is prepared and added to the swollen collagen. The reaction mixture is stirred on an orbital shaker at 40 ° C and 130 rpm, and then a 6% sodium chloride solution is added portionwise with an interval of several minutes with continuous stirring, after which a 2% sodium carbonate solution is added with continuous stirring on a shaker and held for end of the reaction within a few hours.

The colored collagen is separated from the solution and repeatedly washed with water, controlling the degree of washing potentiometrically and by the magnitude of the optical density of the wash water. The colored collagen is lyophilized and subsequently milled in a mill for 5 minutes at 1100 rpm.

The amount of bound dye (μmol / mg of colored collagen) is determined spectrophotometrically at a wavelength of 480 nm after acid hydrolysis of the sample against a sample obtained by similar processing of the original collagen.

Description of the process of obtaining colored collagen

Mill preparation:

The working surface of the mill is treated with a lint-free cloth moistened with 70% alcohol.

Preparation of 6% sodium chloride solution:

6.0 g of sodium chloride is placed in a 100 ml volumetric flask, 80 ml of purified water are added and stirred until the sample is completely dissolved, adjusted to the mark with purified water and mixed.

The solution is used freshly prepared.

Preparation of 2% sodium carbonate solution:

2.0 g of sodium carbonate is placed in a 100 ml volumetric flask, 80 ml of purified water are added and stirred until the sample is completely dissolved, adjusted to the mark with purified water and mixed.

The solution is used freshly prepared.

Preparation of dye solution RO16:

0.200 g of reactive orange dye RO16 is placed in a 100-ml beaker, 50 ml of purified water is added, heated to (70 ± 10) ° С and stirred on a magnetic stirrer until the dye is completely dissolved. The resulting dye solution is cooled to 40 ° C in a water bath.

Collagen Milling:

Collagen is ground using a mill at room temperature for 5 minutes. Particle size no more than 2 mm.

Collagen staining:

5 g of crushed collagen are placed in a conical flask with a capacity of 250-500 ml, 80 ml of purified water is added and mixed until a homogeneous suspension is obtained. Stand for 10 minutes and add 50 ml of dye solution RO16. Rinse the glass with purified water in a volume of 20 ml and add to the total reaction mixture.

The reaction mixture was stirred on an orbital shaker at 40 ° C. at 130 rpm. After 15 minutes, 50 ml of a 6% sodium chloride solution are added twice with an interval of 5 minutes with continuous stirring. After 25 minutes, 37.5 ml of a 2% sodium carbonate solution was added with continuous stirring at 130 rpm and kept at (40 ± 3) ° C for 2 hours.

Collagen washing:

The reaction mixture is placed on a sieve with a pore size of 1 mm and washed with purified water in a volume of 3 l. The degree of washing is controlled by the pH of the wash water and by optical density at 490 nm.

The reaction mass is additionally washed with another 700 ml of purified water, the excess water is allowed to drain, placed on a Petri dish and frozen at minus (40 ± 3) ° С for 12 hours for subsequent lyophilization.

Lyophilization:

The frozen mass is placed in a lyophilizer at a temperature of minus (50 ± 3) ° С. Lyophilization is carried out for 8 hours at a temperature of minus (50 ± 3) ° С and a residual pressure of 8 MPa.

Shredding:

The dried mass is placed in a mill and ground for 1 minute at 1100 rpm.

The crushed product is removed from the mill, weighed and placed in a glass bottle with a capacity of 50 ml, corked with a stopper and rolled up with an aluminum cap.

The finished product is stored in a dry, dark place at a temperature of (5 ± 3) ° C.

Quantitation:

Collagen stained with dye reactive orange RO16 according to the proposed method contains from 40 to 60 μmol of dye RO16 / g of colored collagen.

In 3 heat-resistant test tubes with a capacity of 20 ml, 25 mg each (accurately weighed) of collagen stained with dye RO16 are added, 2 ml of hydrochloric acid, diluted twice with purified water, are added, mixed thoroughly. In the 4th test tube, 25 mg (exact weight) of collagen from bovine tendons, 2 ml of hydrochloric acid diluted twice with purified water (control solution) are placed. All tubes are covered with foil and incubated at 130 ° C for 3 hours in a sand bath. The tubes are removed from the sand bath and cooled in air for 15 minutes. The contents of all tubes are quantitatively transferred to 50 ml volumetric flasks, the volume is adjusted to the mark with purified water and mixed. The optical density of the solutions was measured at a wavelength of 480 nm using a control solution as a comparison solution.

The calibration graph determines the amount of micromole of dye in solution.

The amount of micromoles of dye RO16 in 1 g of colored collagen is determined by the formula:

where N _K is the content of dye RO16 in the test solution, determined according to the calibration graph, µmol / l;

V is the volume of the reaction mixture, 50 ml;

m is the mass of the sample, mg.

Building a calibration graph:

0.050 g (accurately weighed) of the dye of reactive orange RO16 is placed in a 100 ml volumetric flask, 70 ml of purified water is added and stirred until the sample is completely dissolved, the volume of the solution is adjusted to the mark with purified water and mixed. In heat-resistant tubes with a capacity of 20 ml, a dye solution containing 50 is placed; 100; 150; 200; 250; 300 μg of dye and 2 ml of hydrochloric acid previously diluted with purified water twice, stirred and mineralized at 130 ° C in a sand bath for 3 hours. After completion of exposure, the tubes are removed from the sand bath and cooled in air for 15 minutes. The contents of all tubes are quantitatively transferred into 10 ml volumetric flasks, the solution volume is adjusted to the mark with purified water and mixed. The optical density of the solutions is measured at a wavelength of 480 nm. The measurement results are presented in Table 1. Based on these results, a calibration graph is constructed of the dependence of the optical density of the solution on the dye content in the stained collagen sample (Fig. 2).

The resulting substrate - colored collagen is used to determine the specific activity of collagenase and measure the products of enzymatic hydrolysis in solution. Based on the obtained experimental data, the specific collagenase activity is calculated, which is expressed in units of collagenase activity.

The amount of collagenase taken as a unit of collagenase activity (IU), leading to an increase in optical density at a wavelength of 490 nm, is caused by the enzymatic cleavage of 1 mg of collagen stained with reactive orange dye RO16 under standard reaction conditions.

Example 1

Bovine tendon collagen is crushed in a DM-6 mill, 5 g of product are taken and placed in a 500 ml flask, 80 ml of purified water is added. There is added 50 ml of a solution containing 0.1 g of dye RO16.

The mixture is stirred on a shaker at 40 ° C at 130 rpm, after 15 minutes 50 ml of a 6% sodium chloride solution are added twice, after another 25 minutes 37.5 ml of a 2% sodium carbonate solution are added and the mixture is kept for 2 hours. Then the reaction mixture is placed on a sieve and washed with 3 l of water. After removing excess water, the mixture is placed on a Petri dish, frozen at a temperature of minus 40 ° C and lyophilized. The dried mixture is ground in a mill for 20 minutes at 1100 rpm.

The resulting substrate is a cotton-like fibers of orange color; pH of a suspension of colored collagen in purified water 7.07; loss in weight on drying 5.8%; quantitative determination of dye in 1 g of colored collagen 58 μmol; particle size no more than 2 mm. The absorption spectrum of the acid hydrolyzate of a colored collagen sample has a maximum at 213 nm, 288 nm, 361 nm and 479 nm.

Example 2

Bovine tendon collagen is crushed in a DM-6 mill, 5 g of product are taken and placed in a 500 ml flask, 80 ml of purified water is added. There is added 50 ml of a solution containing 0.1 g of dye RO16.

The mixture is stirred on a shaker at 40 ° C at 130 rpm, after 15 minutes 50 ml of a 6% sodium chloride solution are added twice, after another 25 minutes 37.5 ml of a 2% sodium carbonate solution are added and the mixture is kept for 2 hours. Then the reaction mixture is placed on a sieve and washed with 3 l of water. After removing excess water, the mixture is placed on a Petri dish, frozen at a temperature of minus 40 ° C and lyophilized. The dried mixture is ground in a mill for 20 minutes at 1100 rpm.

The resulting substrate is a cotton-like fibers of orange color; pH of a suspension of colored collagen in purified water 7.19; loss in mass upon drying of 6.1%; quantitative determination of dye in 1 g of colored collagen 52 μmol; particle size no more than 2 mm. The absorption spectrum of an acid hydrolyzate of a colored collagen sample has a maximum at 209 nm, 287 nm, 360 nm and 480 nm.

Example 3

Bovine tendon collagen is crushed in a DM-6 mill, 5 g of product are taken and placed in a 500 ml flask, 80 ml of purified water is added. There is added 50 ml of a solution containing 0.1 g of dye RO16.

The mixture is stirred on a shaker at 40 ° C at 130 rpm, after 15 minutes 50 ml of a 6% sodium chloride solution are added twice, after another 25 minutes 37.5 ml of a 2% sodium carbonate solution are added and the mixture is kept for 2 hours. Then the reaction mixture is placed on a sieve and washed with 3 l of water. After removing excess water, the mixture is placed on a Petri dish, frozen at a temperature of minus 40 ° C and lyophilized. The dried mixture is ground in a mill for 20 minutes at 1100 rpm.

The resulting substrate is a cotton-like fibers of orange color; pH of a suspension of colored collagen in purified water 6.56; loss in mass upon drying of 6.2%; quantitative determination of dye in 1 g of colored collagen 58 μmol; particle size no more than 2 mm. The absorption spectrum of an acid hydrolyzate of a colored collagen sample has a maximum at 209 nm, 287 nm, 360 nm and 480 nm.

Example 4

Bovine tendon collagen is crushed in a DM-6 mill, 5 g of product are taken and placed in a 500 ml flask, 80 ml of purified water is added. There is added 50 ml of a solution containing 0.1 g of dye RO16.

The mixture is stirred on a shaker at 40 ° C at 130 rpm, after 15 minutes 50 ml of a 6% sodium chloride solution are added twice, after another 25 minutes 37.5 ml of a 2% sodium carbonate solution are added and the mixture is kept for 2 hours. Then the reaction mixture is placed on a sieve and washed with 3 l of water. After removing excess water, the mixture is placed on a Petri dish, frozen at a temperature of minus 40 ° C and lyophilized. The dried mixture is ground in a mill for 20 minutes at 1100 rpm.

The resulting substrate is a cotton-like fibers of orange color; the pH of the suspension of colored collagen in purified water is 7.9; loss in mass upon drying of 7.9%; quantitative determination of dye in 1 g of colored collagen 60 μmol; particle size no more than 2 mm. The absorption spectrum of the acid hydrolyzate of a colored collagen sample has a maximum at 212 nm, 288 nm, 361 nm and 480 nm.

As a result of the analysis of the obtained samples of colored collagen, the main characteristics of the product can be formulated: it should be orange-colored cotton-like fibers. the pH of the suspension of colored collagen in purified water is from 6.6 to 7.3, the loss in mass during drying is no more than 8%, the quantitative content of the dye should be in the range from 40 to 60 μmol per 1 g of colored collagen, particle size no more than 2 mm , absorption spectra of acid hydrolyzate should have maximums at 210 ± 3 nm, 288 ± 2 nm; 360 ± 2 nm; 480 ± 2 nm, the full characteristics are presented in table 2.

The present invention is a method of obtaining a standardized substrate - colored collagen, which reliably determines the specific activity of collagenase-based drugs using an original technique.

Claims (1)

A method of obtaining colored collagen with the aim of using it as a substrate for determining the activity of the collagenase enzyme, including grinding collagen to a particle size of not more than 2 mm with a moisture content of not more than 8.0%, followed by treatment with a solution of dye RO16, characterized by maxima of the absorption spectrum at 210 ± 3; 288 ± 2; 360 ± 2; 480 ± 2 nm, with a pH of the suspension in the range of 6.3-7.5.

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