RU2750382C1 - Method for producing immobilized enzyme preparation based on collagenase, hyaluronic acid and polysaccharides modified with vinyl monomers - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry; medicine.

SUBSTANCE: present invention relates to the field of chemical and pharmaceutical industry and medicine, namely, to a method for producing an immobilized enzyme preparation based on collagenase, polysaccharides modified with vinyl monomers, and hyaluronic acid. The method includes the dissolution of collagenase in an aqueous solution of low-molecular-weight hyaluronic acid 300 kDa or medium-molecular-weight hyaluronic acid 500 kDa or high-molecular-weight hyaluronic acid 800 kDa in a ratio of 10 mg of collagenase per 2 ml of an aqueous solution of low-molecular-weight hyaluronic acid 300 kDa or medium-molecular-weight hyaluronic acid 500 kDa or high-molecular-weight hyaluronic acid 800 kDa at a concentration of 1.5%. Mixing is carried out until complete dissolving at room temperature. Collagenase is then immobilized by adding to the resulting mixture of a graft copolymer of carboxymethylcellulose 50-100 kDa (CMC) or chitosan 50-100 kDa (CHTS) with N-vinylimidazole (VI) or N,N-dimethylaminoethylmethacrylate (DMAEMA) in an amount of 100 to 290 mg to obtain a liquid preparation or 300 to 500 mg to obtain a gel. Due to the invention, collagenase solutions can be stored at temperatures from 4 to 25℃ for 21 days, and the enzyme becomes more stable in comparison with the native one and it is able to penetrate into the deep layers of the skin. The introduction of modified polysaccharides into the composition of the preparation allows not only to vary the viscosity and consistency of the samples (from the liquid state to the gel with different fluidity), but also promotes the formation of conjugates with an enzyme with a large number of bonds and interactions between the polysaccharide and the enzyme, compared with unmodified carboxymethylcellulose and chitosan. This further increases the stability of the preparation and the concentration of the active substance in the affected area of the skin, and provides a controlled (portion) release of collagenase, maintaining its necessary concentration for a long time.

EFFECT: technical result is an increase in the stability of the preparation.

1 cl, 3 dwg, 1 ex

Description

The invention relates to biotechnology, namely to a method for stabilizing collagenase with solutions of hyaluronic acid and graft copolymers with a polysaccharide backbone of carboxymethylcellulose (CMC) or chitosan (CTS) with side chains from homopolymers of N-vinylimidazole (VI) or N, N-dimethylamino DMAEMA). The invention can be used in the chemical-pharmaceutical industry, medical practice, cosmetology.

Collagenase (EC 3.4.4.19) is an enzyme with proteolytic activity with a narrowly specific focus on collagen hydrolysis with the release of a free amino acid - oxyproline. Collagenase breaks down collagen fibers, extremely resistant to the action of other proteases, with the formation of high-molecular fragments, performs the most important functions in humans and animals: it participates in the transformations of connective tissue during growth and morphogenesis, as well as in the elimination of some pathological processes in arthritis, tumor metastases, etc. ...

Collagenase is contained in the composition of an anhydrous composition for treating wounds [Patent RU 2585373 C2, IPC A61K 38/48, A61K 9/10, A61K 47/30, A61K 47/44, A61P 17/02, C12N 9/00, publ. 05/27/2016, Bul. No. 15]. An ointment for the healing of burns and infected wounds is described, which contains the collagenase Clostridium histolyticum as a proteolytic enzyme, as an antibiotic - gentamicin sulfate, and as an ointment base - polyethylene oxides with molecular weights of 400 and 1500 Da and glycerin. The invention provides a gentle, bloodless enzymatic cleansing of wounds, stimulates the development of healthy granulations and does not interfere with epithelialization processes [Patent RU 2174389 C2, IPC A61K 9/06, A61K 38/43, A61P 17/02, publ. 10.10.2001, Bul. No. 28]. Known highly effective non-toxic, non-allergenic pharmaceutical composition for healing and cleansing wounds, burns and other skin lesions based on collagenase of microbial origin [Patent RU 2166950 C1, IPC A61K 35/74, C12Q 1/37, publ. 20.05.2001, Bul. No. 14]. Collagenase is used to treat non-cicatricial alopecia [Patent RU 2695364 C1, IPC A61K 35/50, A61K 35/28, A61K 35/51, A61P 17/04, publ. 07/23/2019, Bul. No. 21]. Collagenase is contained in a dressing, which is a complex of three adhesive plasters or wound dressings with different compositions of chitosan pads for the sequential treatment of infected wound lesions [Patent RU 2014113441 A, IPC A61K 9/70, B82 B 1/00, publ. 10/27/2015, Bul. No. 30].

Hyaluronic acid is a naturally occurring linear non-sulfated glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine linked by β-1,3 and β-1,4-glycosidic bonds. Hyaluronic acid is an amphiphilic compound that exhibits hydrophilic (due to the presence of -COOH and -OH groups) and hydrophobic properties (sections of 8 CH units).

An active against acne additive to perfumery and cosmetic products containing hyaluronic acid is described [Patent RU 2678307 C1, IPC A61K 8/00, A61K 8/18, A61K 8/34, A61K 8/36, A61K 8/42, A61K 8/67 , A61K 8/64, A61P 17/10, A61Q 19/00, publ. 01/25/2019, Bul. Number 3]. Known drug for the correction of skin defects, including hyaluronic acid, which provides the production of its own components, namely hyaluronic acid, collagen and elastin, while it has strong antioxidant properties and a pronounced long-term effect of correcting skin defects [Patent RU 2659959 C1, IPC A61K 8 / 73, A61K 8/34, A61K 8/64, A61K 8/96, A61Q 19/08, publ. 07/04/2018, Bul. No. 19]. Hyaluronic acid is a part of compositions for skin rejuvenation [Patent RU 2629593 C1, IPC A61K 8/98, A61K 8/73, A61Q 19/08, publ. 08/30/2017, Bul. No. 25]. Hyaluronic acid with a molecular weight of less than 40 kDa is a transdermal carrier in the composition of a cosmetic cream for skin care [Patent RU 2666602 C1, IPC A61K 8/04, A61K 8/34, A61K 8/73, A61K 8/92, A61K 8 / 97, A61K 8/9767, A61Q 19/08, publ. 09/11/2018, Bul. No. 26].

Graft copolymers of carboxymethyl cellulose or chitosan with side chains of N-vinylimidazole or N, N-dimethylaminoethyl methacrylate, due to the presence of the main chain of a modified natural polysaccharide with a molecular weight in the range of 10-600 kDa, can form solutions with different viscosities, depending on the weight of the initial polysaccharide , and on the amount of introduced graft copolymer [A. Benchabane, K. Bekkour. Rheological properties of carboxymethyl cellulose (CMC) solutions. 2008. Colloid and Polymer Science. Vol. 286. P. 1173-1180.]. In addition, such polymers can be used for biomedical purposes, for example, as a carrier for a broad-spectrum cytostatic antibiotic "Paclitaxel" when administered orally and / or by infusion [V. A. Kuznetsov, A. V. Sorokin, M. S. Lavlinskaya et al. Graft copolymers of carboxymethyl cellulose with N-vinylimidazole: synthesis and application for drug delivery. 2019. Polymer Bulletin. Vol. 76. P. 4929-4949; V. A. Kuznetsov, A. V. Sorokin, M. S. Lavlinskaya. Synthesis of graft copolymers of carboxymethyl cellulose and N, N-dimethylaminoethyl methacrylate and their study as Paclitaxel carriers. 2020. Polymer Bulletin. DOI: 10.1007 / s00289-020-03250-z].

At the moment, there is no information on the complexes of collagenase with hyaluronic acid and polysaccharides modified with vinyl monomers, such as graft copolymers of carboxymethyl cellulose (CMC) or chitosan (ChTZ) with N-vinyl imidazole (VI) or N, N-dimethylaminoethyl methacrylate) (DMA).

A known method of producing a heterogeneous preparation based on collagenase and chitosan [Patent RU 2678435 C2, IPC A61K 31/722, A61K 38/43, A61P 17/02, C12N 11/10, publ. 01/29/2019, Bul. No. 4]. This method involves the immobilization of collagenase in a buffer solution on a matrix of acid-soluble medium molecular weight or high molecular weight chitosan in the ratio of 20 ml of collagenase buffer solution at a concentration of 1 mg / ml per 1 g of the specified matrix, while 0.05 M glycine buffer with pH 8.6 is used as a buffer solution for immobilization. for medium molecular weight and with a pH of 10.5 for high molecular weight chitosan; then incubation is carried out for 5 hours at room temperature with periodic stirring; then the formed precipitate is washed with 50 mM Tris-HCl buffer with pH 7.5 until there is no collagenase in the washings.

The disadvantage of this method is to obtain an enzyme preparation in the solid phase, which does not allow collagenase molecules to penetrate into the deep layers of the skin.

A patented method for stabilizing enzymes - pepsin, chymotrypsin, trypsin, pancreatin, papain and other proteases, by adding polysaccharides to their solutions - guar gum, xanthan gum, locust bean gum, starch, dextran, pullulan, alginic acid, hyaluronan pectin, chitosan and other polysaccharides, as well as cellulose derivatives: carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose [JPH034791A].

A known method of producing a collagenase preparation in a gel based on food-grade chitosan and chitosan succinate, including immobilization of collagenase in a buffer solution on a chitosan matrix, incubation at room temperature with periodic stirring, washing the formed precipitate with a buffer, characterized in that the immobilization is carried out on a matrix of food-grade chitosan with weighing less than 100 kDa or chitosan succinate, 0.05 M acetate buffer with pH 5.8 or 0.05 M glycine buffer with pH 9.0 is used as a buffer, incubation is carried out for 2 hours [Patent RU 2712528, IPC C12N 11/10, A61K 31/722, publ. 01/29/2020 Bul. No. 4].

In contrast to these two methods, the proposed use of polysaccharides (carboxymethylcellulose and chitosan) modified with vinyl monomers (N-vinylimidazole or N, N-dimethylaminoethyl methacrylate) provides a greater number of bonds between the polysaccharide and the enzyme during immobilization, which increases the strength of the complex formed and prolongs release of the enzyme to the area of damaged tissues. In addition, the addition of hyaluronic acid to the preparation will facilitate its deeper penetration into various layers of the skin.

It is known that polymers based on poly-β-glycosides (cellulose and its derivatives, chitosan, hyaluronic acid, etc.) tend to form films that can protect the skin from drying out or provide additional protection on the wound surface. However, the ability to form conjugates in such polymers is somewhat limited by a number of factors: surface charge, type of functional group, etc. Therefore, to impart a number of new properties, it is advisable to carry out chemical modification of natural polysaccharides. For example, the introduction of azole substituents increases the complexing ability of polysaccharides and reduces the surface negative charge of carboxymethylcellulose [V.A. Kuznetsov, A.V. Sorokin, M. S. Lavlinskaya et al. Graft copolymers of carboxymethyl cellulose with N-vinylimidazole: synthesis and application for drug delivery. 2019. Polymer Bulletin. Vol. 76. P. 4929-4949], and the introduction of units of N, N-dimethyaminoethyl methacrylate not only reduces the surface charge, but gives an aqueous solution of the modified polysaccharide stimulus-sensitive properties [V.A. Kuznetsov, A.V. Sorokin, M.S. Lavlinskaya. Synthesis of graft copolymers of carboxymethyl cellulose and N, N-dimethylaminoethyl methacrylate and their study as Paclitaxel carriers. 2020. Polymer Bulletin. DOI: 10.1007 / s00289-020-03250-z].

A topical antimicrobial dermatological composition [Patent RU 2668827 C2, IPC A61K 38/08, A61K 31/728, A61P 31/00, A61P 17/00, A61K 31/722, publ. 02.10.2018, Bul. No. 28], proposed as a drug in medicine and veterinary medicine, comprising a combination of at least one positively charged antimicrobial peptide combined with a lipid and hyaluronic acid with an average molecular weight of 100 kDa to 800 kDa or one of its salts, and the antimicrobial the peptide is a palmitic acid hexapeptide containing disulfide bridges.

Unlike the prototype, we use not an antimicrobial peptide as an active substance, but an enzyme called collagenase, which is capable of selectively hydrolyzing collagen, which is part of the connective tissue of a living organism, splitting collagen fibers extremely resistant to the action of other proteases, and performs the most important functions in humans and animals. , participates in the transformation of connective tissue during growth and morphogenesis, as well as in the elimination of some pathological processes in arthritis, tumor metastases, etc., with purulent wounds, collagenase promotes the rapid removal of non-viable tissues and exudate, the early appearance of granulation tissue, epithelialization, the use of collagenase prevents the development of rough scars, while maintaining the mobility of the skin and soft tissues.

In addition, our addition to the solution of graft copolymers of carboxymethyl cellulose (CMC) or chitosan (CTS) with N-vinylimidazole (VI) or N, N-dimethylaminoethyl methacrylate (DMAEMA) makes it possible by including in a polysaccharide gel (one of the methods of physical immobilization of enzymes) stabilize collagenase and provide it with a prolonged action.

The technical result of the claimed invention is to develop a method for producing an immobilized liquid or gel-like enzyme preparation based on collagenase, polysaccharides modified with vinyl monomers, and hyaluronic acid of low molecular weight (300 kDa), medium molecular weight (500 kDa) or high molecular weight (800 kDa) collagenase solutions. can be stored at temperatures from 4 to 25 ° C for 21 days, while the enzyme becomes more stable in comparison with the native and is able to penetrate into the deep layers of the skin, while the introduction of modified polysaccharides into the preparation allows not only to vary the viscosity and consistency of the samples (from a liquid state to a gel with different fluidity), but also promotes the formation of conjugates with an enzyme with a large number of bonds and interactions between the polysaccharide and the enzyme, compared to unmodified carboxymethylcellulose and chitosan, which further increases the stability of the pre paratha and the concentration of the active substance in the affected area of the skin, and also provides a controlled (portioned) release of collagenase, maintaining its required concentration for a long time. In addition, the ability of modified polysaccharides to form stable films protects the enzyme and the treated area of the skin from drying out, makes it easy to remove the drug along with pus and exudate.

The technical result is achieved by the fact that in the method for producing an immobilized enzyme preparation based on collagenase, polysaccharides modified with vinyl monomers, and hyaluronic acid, which includes dissolving collagenase in an aqueous solution of low molecular weight hyaluronic acid 300 kDa or medium molecular weight hyaluronic acid 500 kDa or 800 kDa high molecular weight hyaluronic acid the ratio of 10 mg collagenase per 2 ml of an aqueous solution of low molecular weight hyaluronic acid 300 kDa or medium molecular weight hyaluronic acid 500 kDa or high molecular weight hyaluronic acid 800 kDa at a concentration of 1.5%, stirring is carried out until complete dissolution at room temperature; then immobilization of collagenase is carried out by adding to the resulting mixture of graft copolymer carboxymethylcellulose 50-100 kDa (CMC) or chitosan 50-100 kDa (ChTS) with N-vinylimidazole (VI) or N, N-dimethylaminoethyl methacrylate (DMAEMA) in an amount from 100 to 290 mg for a liquid preparation or 300 to 500 mg for a gel.

FIG. 1. Diagram of the values of the catalytic activity (A) of collagenase in the presence of hyaluronic acid of various molecular weights after storage at 4 ° C.

FIG. 2. Diagram of the values of the catalytic activity (A) of collagenase in the presence of hyaluronic acid of various molecular weights after storage at 25 ° C.

FIG. 3. Table 1. Viscosity of preparations containing collagenase, hyaluronic acid and polysaccharides modified with vinyl monomers.

An example of the implementation of the method.

Collagenase from Clostridium histolyticum (Sigma-Aldrich) was chosen as the object of the study; azocasein (Sigma-Aldrich) served as a substrate for hydrolysis. As stabilizing agents, we used three types of hyaluronic acid (Laboratory Gialika LLC) with molecular weights of 300 (NMHC), 500 (SMHC), and 800 (HMGC) kDa. Graft copolymers of carboxymethyl cellulose (CMC) or chitosan (CTS) with N-vinylimidazole (VI) or N, N-dimethylaminoethyl methacrylate (DMAEMA) with a molecular weight of 50-100 k Da were used as a matrix for the immobilization of collagenase.

Collagenase was stabilized by dissolving a 10 mg weighed portion in 2 ml of an aqueous solution of hyaluronic acid with a molecular weight of 300 (LMHC), 500 (SMHC), or 800 (HMGC) kDa at a concentration of 1.5%, followed by stirring at room temperature until complete dissolution. Then to the resulting mixture was added for the immobilization of collagenase a graft copolymer of carboxymethyl cellulose (CMC) or chitosan (ChTZ) (with a molecular weight of a polysaccharide of 50-100 kDa) with N-vinylimidazole (VI) or N, N-dimethylaminoethyl methacrylate (from 100 DMAEMA) up to 290 mg for a liquid preparation or from 300 to 500 mg for a gel (Table 1).

The protein content in the immobilized collagenase preparations was determined by the Lowry method [Lowry ON, Rosebrough N.J., Faar A.L., Randall R.J. Protein measurement with folin-phenol reagent // J. Biol. Chem. - 1951. -V.193. - P. 265-275]. The level of proteolytic activity of the enzyme was measured on the substrate azocasein [Garcia-Carreno, FL The digestive proteases of langostilla (Pleuroncodes planipes, Decapoda): their partial characterization and the effect of feed on their composition // Comparative Biochemistry and Physiology Part B: Comparative Biochemistry - 1992. V. 103. P. 575-578]. To 200 μL of the sample, 200 μL of Tris-HCl buffer (pH 7.5), 800 μL of azocasein (0.5% in 50 mM Tris-HCl buffer, pH 7.5) were added and incubated for 2 hours at 37 ° C. Then, 800 μl of trichloroacetic acid (TCA) (5%) was added, incubated for 10 minutes at minus 4 ° C, then centrifuged for 3 min at 13,000 rpm to remove unhydrolyzed azocasein. To 1200 μl of the supernatant, 240 μl of 3% NaOH was added to neutralize the acid, after which the optical density of the experimental sample was measured at 410 nm in a 1 cm cuvette. The control sample contained 800 μl of azocasein, 800 μl of TCA, 200 μl of Tris-HCl buffer and 200 μl of the sample, which was added after incubation of the mixture for 2 hours at 37 ° C. The unit of catalytic activity of collagenase was defined as the amount of the enzyme that, under the experimental conditions, hydrolyzes 1 μM of the substrate in 1 min. The specific proteolytic activity of collagenase was calculated using the formula:

PA = D * 1000/120/200 / C,

where PA is proteolytic activity, μM / min per 1 mg of protein,

D is the optical density of the sample at 410 nm,

С - protein concentration in the sample, mg / ml, measured by the Lowry method,

120 - incubation time in minutes,

200 - sample volume, in μl,

1000 - recalculation in μM.

The results were statistically processed at a 5% significance level using Student's t-test.

Studies have been conducted on the stability of collagenase in hyaluronic acid solutions (300, 500 and 800 kDa) in comparison with the native enzyme. The preparations were incubated at 4 and 25 ° C for 0, 1, 2, 3, 7, 14, and 21 days with further measurement of proteolytic activity. The results are shown in FIG. 12.

In the course of our experiments, it was revealed that hyaluronic acid does not inhibit collagenase.

FIG. 1 shows a diagram of the dependence of the catalytic activity of the enzyme on the time of its storage at 4 ° C. It was found that native collagenase retains 52-53% of its activity after 7-14 days of incubation at 4 ° C, and after 3 weeks of storage it is active by 40% of the original preparation. Collagenase in a solution of low molecular weight hyaluronic acid (300 kDa) on the 14th day of storage retains more than 90% of the catalytic capacity of the non-incubated sample. After 21 days of incubation, the collagenase activity in the low molecular weight hyaluronic acid solution is 69% of the initial one. Collagenase solution with medium molecular weight hyaluronic acid (500 kDa) slightly loses its activity during the entire incubation period and retains 91% of its enzymatic capacity by 21 days of storage. Collagenase in a solution of high molecular weight hyaluronic acid (800 kDa) is gradually inactivated: on the 3rd day, the enzyme activity decreases by 10%, on the 7th day - by 14%, and after 21 days of storage, the catalytic ability of the enzyme is 72% of the initial one.

FIG. 2 shows a diagram of the change in the catalytic activity of collagenase after storage at 25 ° C. On the 7th day of storage at a temperature of 25 ° C, the free enzyme retains 51% activity, the collagenase preparation in a solution of medium molecular weight hyaluronic acid (500 kDa) - 87%, collagenase solution in high molecular weight hyaluronic acid (800 kDa) - 86%, collagenase in a solution of low molecular weight hyaluronic acid acids (300 kDa) - 76%. On the 14th and 21st days of the experiment, native collagenase is active by 45 and 36%, respectively, complexes of the enzyme with medium molecular weight hyaluronic acid - by 71 and 63%, with high molecular weight hyaluronic acid - by 65 and 50%, with low molecular weight hyaluronic acid - 74 and 40%.

It was revealed that the collagenase solution in medium molecular weight hyaluronic acid (500 kDa) after incubation at temperatures of 4 and 25 ° C was the most stable in comparison with other drugs.

Thus, a method was developed for obtaining an immobilized enzyme preparation based on collagenase, low molecular weight (300 kDa), medium molecular weight (500 kDa), and high molecular weight (800 kDa) hyaluronic acid, followed by the addition of carboxymethyl cellulose graft copolymer (CMC) or chitosan N (CTZ) -vinylimidazole (VI) or N, N-dimethylaminoethyl methacrylate (DMAEMA) for collagenase immobilization.

In this case, the preparation obtained by the proposed method can be stored at temperatures from 4 to 25 ° C for 21 days, while collagenase becomes more stable in comparison with native and is able to penetrate into the deep layers of the skin. The introduction of modified polysaccharides into the preparation allows not only to vary the viscosity and consistency of samples (from a liquid state to a gel with different fluidity), but also promotes the formation of conjugates with an enzyme with a large number of bonds and interactions between the polysaccharide and the enzyme, in comparison with unmodified carboxymethylcellulose and chitosan , which additionally increases the stability of the drug and the concentration of the active substance in the affected area of the skin, and also provides a controlled (portioned) release of collagenase, maintaining its required concentration for a long time. In addition, the ability of modified polysaccharides to form stable films protects the enzyme and the treated area of the skin from drying out, makes it easy to remove the drug along with pus and exudate.

Claims (1)

A method of obtaining an immobilized enzyme preparation based on collagenase, polysaccharides modified with vinyl monomers, and hyaluronic acid, including dissolving collagenase in an aqueous solution of low molecular weight hyaluronic acid 300 kDa or medium molecular weight hyaluronic acid 500 kDa or high molecular weight ratio of 10 ml of hyaluronic acid to 800 ml of hyaluronic acid an aqueous solution of low molecular weight hyaluronic acid 300 kDa or medium molecular weight hyaluronic acid 500 kDa or high molecular weight hyaluronic acid 800 kDa at a concentration of 1.5%, while stirring until completely dissolved at room temperature; then immobilization of collagenase is carried out by adding to the resulting mixture of graft copolymer carboxymethylcellulose 50-100 kDa (CMC) or chitosan 50-100 kDa (ChTS) with N-vinylimidazole (VI) or N, N-dimethylaminoethyl methacrylate (DMAEMA) in an amount from 100 to 290 mg for a liquid preparation or 300 to 500 mg for a gel.

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