LV13959B - Adenylate deaminase containing hidrogel and use thereof for the treatment of dermal wounds (ulcers) - Google Patents

Abstract

This invention relates to pharmacology, particularly to drug production and compositions of medical means which contains components of natural microbiological origin, which promote healing of dermal wounds (ulcers). The object of this invention is use of Penicillium lanoso-viride adenylate deaminase (AMP aminohydrolase, E.C. 3.5.4.6.) in manufacturing drug, especially gel for treatment of dermal wounds (ulcers), as well as manufacturing adenylate deaminase containing hydrogel composition. Offered hydrogel contain from 0,3 till 3,0 (U)/ml units of adenylate deaminase. Offered hydrogel promote healing of wounds, this is experimentally proved comparing with one of in Latvia registered gels for the treatment of wounds and ulcers which is set on base of Carbopol polymer.

Description

Hydrogen Gel Containing ADENYL DAMAMINASE AND ITS USE FOR THE TREATMENT OF SKIN WINES

Technical field

The invention relates to pharmacology, in particular to the manufacture of medicaments and to pharmaceutical compositions containing natural components of microbiological origin which promote the healing of skin wounds (ulcers).

State of the art

Wound healing is a dynamic process involving soluble mediators, blood cells, extracellular matrix and parenchymal cells. Healing has three phases of inflammation, tissue formation and tissue remodeling; phases overlap (Singer AM, Clark RAF. Cutaneous wound healing. New Engl J Med 1999, 341: 738-746). At the cellular level, each phase is driven by a coordinated interaction of several types of cells, including inflammatory cells, fibroblasts, keratinocytes, and endothelial cells. At the molecular level, cytokines regulate inflammatory cells in the early stages of wound healing, and natural growth factors of polypeptides play a key role in skin cell proliferation and differentiation, as well as in the synthesis of extracellular matrix (Martin P. Wound, 276: 75- 81; Goldman R. Growth Factors and Chronic Wound Healing: Past, Present, and Future. Adv Skin Wound Care 2004, 17: 24-35). Various growth factors, cytokines and haemokines, which are involved in skin healing processes have been identified (Wemer S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003, 83: 835-870). Platelets, neutrophils, macrophages, endothelium, fibroblasts and epithelium express and secrete epidermal growth factors (TGF), platelet-derived growth factor (PDGF), interleukins (IL), proteases, matrix proteins and other compounds (Parenteau N, Hardin-Young J. The biological mechanisms behind injury and inflammation: how they can affect treatment strategy, product performance, and healing. Wounds 2007, 19: 87-96). The regulation of growth factor expression over time and space is very important for normal healing (Wemer S, Grose R. Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev 2003, 83: 835-870).

Many different studies have been conducted worldwide to determine the pharmacological potential of growth factors and other mediators. Unfortunately, the overall experience with using these agents to promote wound healing is not encouraging. This is not surprising because healing is complex and interacts with a variety of soluble cytokines, blood cells, extracellular matrix and cells (Singer AM, Clark RAF. Cutaneous wound healing. New Engl J Med 1999, 341: 738-746; Pierce GF, Mustoe TA: Pharmacologic enhancement of wound healing, Annu Rev Med 1995, 46: 467-481; Bello YM, Phillips TJ. The new therapeutic agents currently under investigation can be divided into the following groups: growth factors, skin substitutes, extracellular matrix proteins, stem cell therapy, gene therapy, protease inhibitors, angiogenesis stimulators, nitric oxide releasing agents, adenosine agonists, immunostimulants, N, Edmonds M. Emerging Drugs for Diabetic Foot Ulcer Expert Opin Emerg Drugs 2006, 11: 709-724).

The microscopic fungus Penicillium lanoso-viride non-specific adenylate deaminase (AMP aminohydrolase, EC 3.5.4.6; AMPD) is a glycoprotein (gM 210 kD) synthesized by the fungus during the conidia formation stage (Revelina V, Muiznieks I, Shafranskea A. viride 8D, Some properties of the enzyme In: Maurina H, Editor Utilization and transformation of purine and pyrimidine compounds in microorganisms (Riga: Latvian University Press, 1981, pp. 88-96). In vivo studies in experimental animals have shown that purified AMPD has immunomodulatory properties and affects both cellular and humoral immune responses. Injections of AMPD stimulate the production of antibody-producing cells in the spleen, promote lymphocyte proliferation in vitro, activate mouse peritoneal macrophages (Nikolayev V, Eze D, Petrina Z, Muiznieks I. 1999, 53: 12-15), stimulates natural killer and tumor cell adhesion, restricts the growth of multiple tumors (Zak M, Novak F, Muiznieks IO, Nikolaev VR, Kamradze AA, et al.) Immunopotentiating and antitumour activity of mold glycoprotein A. Sbomik Lekarsky 1986, 88: 139-145), enhances the body's resistance to infections (Nikolaev V, Eze D, Kamradze A, Indulena M, Muiznieks I. Protective effect of adenylate deaminase (from Penicillium lanoso-viride) against acute infections in mice. Immunopharmacology 1996, 35: 163-169) and inhibits experimental autoimmune encephalomyelitis (Nikolaev V, Eze D, Petrina Z,

Landlord I. Treatment of experimental autoimmune encephalomyelitis with adenylate deaminase from Penicillium lanoso-viride. J Autoimmunity 2000, 14: 1075 113). AMPD has no direct antibacterial activity (Nikolaev V, Eze D, Kamradze A,

Indulena M, Souvenir I. Protective effect of adenylate deaminase (from Penicillium lanoso-viride) against acute infections in mice. Immunopharmacology 1996, 35: 163169). Based on our previous studies on the effects of AMPD on the systemic immune response, it was hypothesized that AMPD also beneficially affects local immune processes, and AMPD was found to promote healing of wounds (ulcers).

Disclosure of Invention

The present invention relates to the use of adenylate deaminase (AMP aminohydrolase, E.C. 3.5.4.6.) For the manufacture of a medicament for the manufacture of a gel for the treatment of skin wounds (ulcers), and to the development of a hydrogel composition containing adenylate deaminase. The new hydrogel accelerates wound healing, which has been experimentally proven compared to Sanagel, a Latvian-based gel for the treatment of wounds and ulcers, which contains Carbopol polymer based active ingredients methyluracil and lidocaine hydrochloride.

a brief description of the drawings

FIG. 1 is a graph of the effect of AMPD on skin ulcer closure (V), FIG. 2 is a graph of the percentage of epithelialized ulcers at day 21 of the experiment, FIG. Fig. 3 is a diagram of the epithelialization process expressed by the regeneration factor (Rkoef), fig. 4 - Diagram of the epithelialization process expressed as Regression Regression Index (Rr _eg ).

The AMPD used was obtained in the Applicant Laboratory of the present invention according to a previously developed method (Revelin V, Muiznieks I, Shafransky A. Adenylate deaminase from Penicillium lanoso-viride 8D. In: Maurina H, editor. Utilization and transformation of purine and pyrimidine compounds in microorganisms. Riga: Latvian University Press, 1981, pp. 88-96). Methods of obtaining 8D-adenylate deaminase producers and adenylate deaminase from Penicillium lanosoviride strains are described in patents LV 5056, LV 5058, and LV 5059. Ampd is assayed per unit of AMPD activity (1 U) to deactivate 1.0 pmole of 5'AMP per 5'-IMP. per minute at 37 ° C, pH 6.0.

The specific activity of the AMPD used was 5-15 (mean - 10) U / mg protein. Carbopol 940 and triethanolamine from PharmaZell GmbH (Germany) were used to prepare the gel.

Developed hydrogel composition: 1% Carbopol 940, 1.35% triethanolamine, sterile 0.9% NaCl and sterile AMPD. Two gel formulations with different concentrations of AMPD in the gel were prepared for experiments: 3.0 U / ml and 0.3 U / ml. The prepared gel was stored at 4 ± 2 ° C.

Hydrogel is used in pharmacy mainly as an inert carrier for various peptides and proteins. It has good biocompatibility with tissues. It maintains a moist environment, which in turn promotes wound healing. The hydrophilic, delicate structure of the hydrogel does not irritate the tissues, and cells and proteins do not show a tendency to adhere to the surface of the hydrogel (Van Tomme SR, Hennink WE. Biodegradable dextran hydrogels for protein delivery applications. Expert Rev Med Devices 2007, 4: 147-164; Chu JS, Yu DM, Amidon GL, Weiner ND, Goldberg AH Viscoelastic properties of polyacrylic acid gels in mixed solvents (Pharm Res 1992, 9: 1659-1663). One of the most common hydrogel carriers is the pharmaceutically acceptable Carbopol polymer, which is neutralized with triethanolamine.

The invention was tested at the University of Latvia using Wistar rats (123-169 g) according to the experimental protocol approved by the Animal Ethics Committee of the Food and Veterinary Service of the Republic of Latvia. In rats, skin ulcers were induced by administering 0.5 ml of 9% acetic acid under the skin in mild ether narcosis and 6% dextran administered intraperitoneally at a dose of 300 mg / kg. Skin necrosis developed within three days. The animals were divided into four groups of 6-8 in each group. In group 1 rats, ulcers were treated with Carbopol gel containing AMPD 3.0 U / ml. Group 2 rats were treated with Carbopol gel containing 0.3 U / ml AMPD. Groups 3 and 4 were controls. Group 3 received Carbopol gel without AMPD, whereas Group 4 rats remained untreated. The wounds were topically treated with gels once daily starting on day 4 of the experiment.

Animals were weighed on days 4, 7, 14 and 21. The rate of ulcer closure, time of epithelialization, and sensation of pain were determined. Measurements were taken at the beginning of the ulcer treatment on day 4 and on days 7, 14 and 21. The wound surface percent closure was calculated using the formula (Kirker KR, Luo Y, Nielson JH, Shelby J, Prestwich GD.

Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing. Biomaterials 2002, 23: 3661-3671): V = (Ao - A _t ) / A _o x 100, where Ao is the initial area of the ulcer and A _t is the area of the ulcer on the day of measurement. The epithelialization process is expressed as the recovery coefficient (Rk _0e f) and the regeneration regression index (R _reg ): Rkoef ⁼ initial ulcer area (mm) / ulcer area on the day of measurement (mm);

Rreg ⁼ Rkoef Experimental Group / Rkoef Control Group.

The sensation of pain was assessed by lightly touching the affected area of skin with a wooden stylus. On day 14 and 21 of the experiment, rat blood tests were performed to determine white blood cells, erythrocytes and platelets, hemoglobin, hematocrit, leukocyte formula,

ALT, AST, albumin, alkaline phosphatase, cholesterol and proteins. On the same days, damaged skin samples were taken for histological analysis.

We found that AMPD-containing gels significantly (p <0.05) accelerated the healing of skin ulcers

On days 14 and 21 compared with both Carbopol and the untreated control group (Figs.

1). The lowest dose (0.3 U / ml) showed a significant (p <0.05) reduction in ulcer surface area as early as day 7, i.e., only after three administrations. The efficacy of the higher dose (3.0 U / ml) was significantly increased the following week. At week 3, the rate of closure declined but remained significantly higher in both AMPD groups than in the control groups.

Epithelialization time corresponded to wound closure time. Complete epithelialization on day 21 was observed in all animals in the AMPD 3.0 U / ml group and 60% in the AMPD 0.3 U / ml group (Fig. 2). At the same time, none of the animals in both control groups had complete epithelialization. Rk _oe f (Fig. 3) and R _reg (Fig. 4) confirm that AMPD improves wound healing and that healing is dose dependent.

All measured blood cell and biochemical parameters were within the normal range and did not significantly differ between groups (p> 0.05). The animals had good health, appetite, and gained weight evenly across all groups (p> 0.05). The assessment of the sensation of pain showed no difference between the animals in the different groups. Histologic examination showed reepithelialization, granulation, and connective tissue formation of varying degrees and intensity in all samples.

Claims (7)

Use of Penicillium lanoso-viride adenylate deaminase (AMP aminohydrolase, E.C. 3.5.4.6) for the preparation of a medicament. Use of Penicillium lanoso-viride adenylate deaminase according to claim 1, characterized in that the medicament is used for external use. Use of Penicillium lanoso-viride adenylate deaminase according to claim 1 or 2, characterized in that the medicament is used for the treatment of skin wounds (ulcers). Use of Penicillium lanoso-viride adenylate deaminase according to claim 1 for the preparation of a hydrogel for external treatment of skin wounds (ulcers). The hydrogel according to claim 4, characterized in that the concentration of adenylate deaminase in the gel is from 0.3 to 3.0 units (U) / ml. The hydrogel according to any one of claims 4 to 5, characterized in that the neutral carrier is a Carbopol polymer. Hydrogel according to any one of claims 4 to 6, characterized in that it contains saline (0.9% NaCl).