RU2794958C2 - Wound-healing composition - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: wound-healing composition. Wound-healing composition based on a lipid fraction isolated by pressing from the biomass of Hermetia illucens larvae, Aloe arborescens juice, allatoin and related components: dimethyl sulfoxide (DMSO), carboxymethylcellulose, glycerin, tween-80, talc, benzyl alcohol and water, containing: lipid fraction larvae of Hermetia illucens - 13%, allatoin - 10%, benzyl alcohol - 1%, DMSO - 10%, carboxymethylcellulose - 1%, glycerin - 5%, talc - 33%, Aloe arborescens juice - 10%, tween-80 - 5 % and water - the rest.

EFFECT: pronounced wound-healing effect.

1 cl, 14 dwg

Description

I. Scope

The invention relates to the production of veterinary and pharmaceutical preparations, cosmetics and medical cosmetics, namely the production of creams, ointments and gels, including a lipid complex, aloe juice and allantoin, and can be used in veterinary medicine, medicine and cosmetology.

"In the production of veterinary and medical preparations, as well as cosmetology, various biologically active substances, both of animal and vegetable or mineral origin, are widely used. By activating the metabolism, they improve the physiological condition of the skin when applied externally.

For many years, one of the important problems of veterinary medicine has been the search for effective methods of treating infected wound processes in tissues [Krylova L.S., Remizov E.K., Smirnova K.Yu., Larionova O.S. Indication of peptides from the biomass of insect larvae and the study of their antimicrobial activity // Topical issues of veterinary biology. - 2019. - No. 4 (44). - S. 3-6.]. This is due to the fact that the course of the wound process and the presence of anaerobic, aerobic microflora in the foci of infected inflammation lead to a violation of tissue respiration in the areas of healing, causing disturbances in the surrounding tissues, which ultimately lead to the development of acidosis, hypoxia and metabolic intoxication of the body [Krylova L .S., Remizov E.K., Smirnova K.Yu., Larionova O.S. Indication of peptides from the biomass of insect larvae and the study of their antimicrobial activity // Topical issues of veterinary biology. - 2019. - No. 4 (44). - S. 3-6.; Barsukov N.A. Treatment of infected wounds // Veterinary. - 1986. - No. 8. - S. 68-69; Boateng, J.S.; Matthews, K.N.; Stevens, H. N.; Eccleston, G.M. Wound healing dressings and drug delivery systems: a review. J Pharm Sci 2008, 97, 2892-923; Alekseeva I.V. Development of dosage forms for the treatment of wounds // Pharmacy. - 2003. - No. 2. - S. 43-45; Bulik C.C., Wiskirchen D.E., Shepard A., et al. Tissue Penetration and Pharmacokinetics of Tigecycline in Diabetic Patients with Chronic Wound Infections Described by Using In vivo Microdialysis // Antimicrob. Agents Chemother. - 2010. - Vol. 54. - R. 5209-5213].

Among external medicines, ointments and gels on bases are widely used, which do not injure the damaged surface when applied to the wound, provide wound drainage, and the medicinal substances that make up them provide the necessary therapeutic effect [Barsukov N.A. Treatment of infected wounds // Veterinary. - 1986. - No. 8. - S. 68-69; Boateng, J.S.; Matthews, K. H.; Stevens, H. N.; Eccleston, G.M. Wound healing dressings and drug delivery systems: a review. J Pharm Sci 2008, 97, 2892-923; Alekseeva I.V. Development of dosage forms for the treatment of wounds // Pharmacy. - 2003. - No. 2. - S. 43-45; Bulik C.C., Wiskirchen D.E., Shepard A., et al. Tissue Penetration and Pharmacokinetics of Tigecycline in Diabetic Patients with Chronic Wound Infections Described by Using In vivo Microdialysis // Antimicrob. Agents Chemother. - 2010. - Vol. 54. - P. 5209-5213]. A promising direction is the development and use of ointments of multidirectional action, which would combine such properties as antimicrobial activity, high dehydrating ability, stimulation of tissue regeneration [Karami, A.; Tebyanian, N.; Barkhordari, A.; Motavallian, E.; Soufdoost, R.S.; Nourani, M.R. Healing effects of ointment drug on full-thickness wound. C.R. Acad. Bulg. Sci 2019, 72, 123-129; Blatun L.A. Local medical treatment of wounds. Problems and new opportunities for their solution // Consilium medicum: surgery (app.). - 2007. - No. 1. - S. 9-16; Larionova O.S., Drevko Ya.B., Bannikova A.V., Kovtunova A.S., Mendubaev D.V., Karmeeva Yu.S., Krylova L.S. Method for obtaining chitosan. Patent for invention RU 2615636 C, 04/06/2017. Application No. 2016110254 dated 03/21/2016; Krylova L.S., Amelkina A.A., Drevko Ya.B., Larionova O.S. The study of some biological properties of antimicrobial peptides obtained from the hemolymph of the larvae of Galleria meilonella // In the collection: Actual problems of veterinary medicine, food and biotechnology. Materials of the International scientific-practical conference. - 2017. - S. 263-267; Volenko A.V., Menshikov A.L., Titova G.P., Kuprikov S.V. Prevention of wound infection with immobilized antibacterial drugs // Surgery. - 2004. - No. 10. - S. 54-58; Abaev, Yu.K. Handbook of the surgeon. Wounds and wound infection / Yu.K. Abaev. - Rostov-on-Don: Phoenix, 2006. - 427 p.; Yang, F. Pathogenic alteration in severe burn wounds / F. Yang, X. Bing, D.B Feng [et al.] // J. Burns - 2012. - V. 38. - I.1. - P. 90-94].

II. Prior Art

Known pharmaceutical composition for topical administration based on lipid, described in WO 2010/036947 A2, which contains one or more lipids and one or more other biologically active compounds selected from the group consisting of finasteride, dutasteride, minoxidil, amphotericin B and tacrolimus. In the patent RU 2715103 C2, 02/27/2020 (convention priority 05/03/2013 SE 1300318-1), from 3 to 60% of the phospholipid is used as a pharmaceutical or cosmetic carrier. Fats are also widely distributed as components of wound healing ointments and gels: based on bear fat RF No. 2246310, IPC A61K 35/78, 2002; lamb and badger fat RU 2571487 C9, 12/20/2015; pork fat of the Russian Federation No. 2042357, MPK A61K 35/78, 1993. Vegetable oils are also widely used in ointments and gels for wound healing: aloe RU 2697255 C1, 08/13/2019, castor oil and wild rose RU 2040252 C1, 07/25/1995, fir ( RU 2284809, 10.10.2006), in composition with fat-soluble vitamins A and E (patent No. 2128500, 10.04.1999). Ointments are also known that have both vegetable and animal components in their composition: aloe oil, mandrake root oils and bear fat (RU 2002108231 A 01/27/2004). These recipes show the effectiveness of the use of fat fractions as one of the components of wound healing ointments and gels.

There is a known method for isolating melanin with a mixture of fatty acids from the larvae of Hermetia illucens, this complex can be used to obtain wound healing drugs and biologically active additives in cosmetology, bioengineering, and also in industry to create water-repellent covers (RU 2692260, 06/24/2019). The disadvantages of this invention include the lack of ready-made therapeutic formulations.

Allantoin also finds its application as an active substance in wound healing gels and ointments 11, RU 2567730 С1 , 11/10/2015).

III. The essence of the invention

This invention relates to the production of veterinary and pharmaceutical preparations, cosmetics and medical cosmetics using as the main components of the lipid fraction isolated from the larvae of Hermetia illucens (a complex of esters of fatty acids), Aloe arborescens juice, allantoin and related components: dimethyl sulfoxide, alginate sodium or cellulose carboxymethylate, glycerin, TWEEN-80 and talc.

The use of a lipid fraction isolated from the larvae of Hermetia illucens (a complex of fatty acid esters) will contribute to a more effective restoration of the epithelium, and the therapeutic efficacy of Aloe arborescens juice, which belongs to biogenic stimulants, will consist in a stimulating effect on metabolic processes: improvement of cellular metabolism, trophism and tissue regeneration. This has been confirmed by clinical trials and is used as medicines (Aloe liniment No. LSR-002881/09). Allantoin has an anti-inflammatory effect, stimulates metabolic processes in tissues and promotes cell proliferation (Hepatrombin No. P N013949/01, 2007-12-13). Dimethyl sulfoxide, being a pharmacopoeial solvent, enhances the absorption of active substances, which makes it possible to increase the effectiveness of this formulation.

The purpose of the invention is achieved by using a complex of esters of fatty acids, allantoin, aloe juice with an organic solvent (dimethyl sulfoxide), TWEEN-80 surfactant, sodium alginate or carboxymethyl cellulose, glycerin, talc and water. As a bacteriostatic, benzyl alcohol is introduced.

At a temperature of 50°C and constant stirring, allantoin, a complex of fatty acid esters, tween-80 and benzyl alcohol are added to dimethyl sulfoxide, then after homogenization a solution of water with aloe juice, sodium alginate or carboxymethyl cellulose, benzyl alcohol and talc is added. The resulting mixture is stirred until complete homogenization and left to cool to room temperature.

On the basis of the studies carried out, it can be concluded that using the above method, it is possible to obtain stable compositions containing 9-14% allantoin, 8-36% fatty acid ester complexes, 6-12% aloe juice, using concomitant substances: dimethyl sulfoxide 9-25%, TWEEN-80 4-7%, glycerin 4-7%, talc 0-35%, benzyl alcohol 1%, cellulose carboxymethylate or sodium alginate 0-1%, talc 0-35% - the rest is water.

We have developed a prototype of a wound healing drug based on a complex of fatty acid esters. This sample was obtained by pressing from the biomass of Hermetia illucens larvae. When developing a prototype drug, we obtained 34 samples, the most optimal was sample 6, consisting of a complex of fatty acid esters 13%, allantoin 10%, benzyl alcohol 1%, DMSO 10%, carboxymethylcellulose 1%, glycerin 5%, talc 33% , aloe juice 10%, TWEEN-80 5% and water.

The objects of study in the test for the local irritating effect of the drug were laboratory animals - guinea pigs. To prepare the animals for the test for local irritant action of the drug, the hair on the experimental skin areas was carefully trimmed before the start of the study. The dose injected subcutaneously into each test site is 0.1 ml.

For applications, filter paper or a hygroscopic gauze pad (4-8 cm ² ) were impregnated with the test sample, which were applied to the clipped skin areas and fixed with an occlusive bandage around the animal's body.

Preliminary tests were carried out in order to determine the concentration of the test sample to be used in the main study.

Undiluted extracts when using known solvents do not need a preliminary test.

The possible effect of administering complete Freund's adjuvant (FCA) in combination with the test material during the main test and thus possible confounding of the results should be considered.

According to the application, extreme dilutions were tested on three animals.

After 24 hours, the bandage and samples were removed and the condition of the experimental areas was assessed for the presence of erythema and edema.

For the induction phase in the main test, the highest concentration was chosen that does not cause more than very mild erythema and does not have a general adverse effect on animals.

Research results

Subcutaneous induction phase

For each animal, the clipped skin areas (A, B, and C) in accordance with FIG. 1 carried out paired subcutaneous injections in a volume of 0.1 ml.

Local induction phase

Seven days (±1 day) after the subcutaneous induction phase, skin applications of the test material were started on the injection sites, on the intrascapular region of each animal, using impregnated pieces of gauze with an area of 8 cm ² for this.

In this case, the maximum concentration for site B was used. If the maximum concentration that can be achieved does not cause irritation, the application area was pre-treated with a 10% solution of sodium dodecyl sulfate, massaged into the skin 24 ± 2 hours before application. An occlusive dressing was applied. The bandage was removed after (48±2) hours.

Freshly prepared samples were used. If the samples were prepared earlier than 24 h, then their storage stability was checked.

The procedure with control animals was repeated in the same mode, using only the control solution.

provocative phase

14 days (±1 day) after completion of the local induction phase, all experimental and control animals were challenged with the test material. To do this, the test material was applied to intact skin on the flank of each animal using a hygroscopic gauze pad. A fixing occlusive bandage was applied. The dressing and applications were removed after (24±2) hours.

Animal examination

The surface of the areas where the provocative test of experimental and control animals was carried out was examined after 24; 48 hours after bandage removal. Full spectrum illumination was used to visualize skin reactions.

Described and assessed the degree of skin reactions, including erythema and edema for each site and at each observation time interval.

The description was made without taking into account the preliminary exposure in order to minimize the error in the evaluation of the results.

The study of the wound healing effect of the drug was carried out on laboratory animals.

The objects of study for therapeutic efficacy in burns were laboratory animals - rats. Work was carried out on the formation of experimental and control groups of animals according to the principle of analogues. At the time of the study, laboratory animals (rats) were clinically healthy.

The studies were carried out in accordance with the "Rules of Laboratory Practice in the Russian Federation" (Order of the Ministry of Health of the Russian Federation No. 708n dated August 23, 2010). Animal experiments were carried out in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (ETS 123) Strasbourg, 1986).

Investigations were performed according to an approved written protocol and in accordance with the Investigator's Standard Operating Procedures (SOPs). All animals were subjected to examination, which included a clinical study.

Based on the data obtained in the course of the above studies, groups were formed according to similar physiological indicators.

The study included 30 clinically healthy rats. Experimental animals were kept under the same conditions, optimal for this species. Animals were kept in cages of 10 heads. Before the start of the experiment, all cells were labeled.

The day before the application of the burn, the fur in the back of the experimental animals was removed, with an area of 10 cm ² . On the day of the experiment, rats were anesthetized with isoflurane.

After anesthesia was administered to the animals, they were placed on a table for fixing laboratory animals. Experimental burn wounds were modeled using a metal rod with a surface diameter of 1 ^cm2 , which was heated to 100°C. The rod was applied to the skin of the animal for 20 seconds, resulting in a burn IIIA degree, with a total area of 100-130 mm ² .

After inclusion in the study, animals were assigned to one of three groups:

- 1 experimental group (n=10) - an experimental sample of the cream "Sample No. 6" was applied to the burn wound for 20 days;

- 2 experimental group (n=10) - Panthenol universal cream was applied to the burn wound for 20 days;

- The third group (n=10) of animals was the control, the burn wound remained without treatment.

Experimental groups of animals immediately after the application of the burn and then daily applied the test samples at a dose of 0.5 g.

Every day from the beginning of the experiment, the general condition of the rats was recorded.

The area of experimental burns was measured planimetrically using a caliper. The burn diameter was measured, then its area was calculated using the formula: S=2π ² .

The wound area was measured on the 1st, 5th, 10th and 20th days after the burn wound was applied.

The general condition of the animals was controlled on the basis of behavioral responses.

When studying the local irritating effect after a paired intradermal injection into the corresponding skin areas of substances in a volume of 0.1 ml, each animal was recorded after 24 hours.

In the control group at the injection site, a slight swelling was observed at the injection site, which disappeared after 6-8 hours, the condition of the animals was stable, erythema was not observed (Fig. 2).

In the experimental group of animals, at the injection site of a mixture of Freund's complete adjuvant with the selected solvent in a ratio of 50/50, a slight swelling was observed at the injection site, which disappeared after 6-8 hours, the condition of the animals was stable, slight erythema was observed.

At the site of injection of the test sample with the solvent, a slight swelling was observed, which disappeared after 4-5 hours, the condition of the animals was slightly depressed, but after 1-2 hours it was stable, erythema was not observed (Fig. 3).

After the introduction of the test sample emulsified with complete Freund's adjuvant in the ratio 50/50 and the solvent (50%), a slight swelling was observed at the injection site, which disappeared after 6-8 hours, the condition of the animals was slightly depressed, but after 2-3 hours - stable, erythema was not observed (Fig. 4).

Seven days (±1 day) after the intradermal induction phase, skin applications of the test material were started on the injection sites, on the intrascapular region of each animal, using impregnated pieces of gauze with an area of 8 cm ² for this.

Examined the surface of the skin of animals within 14 days after application of skin applications of the test material in experimental and control animals.

24 hours after removal of the dressing, moderate erythema was observed in all animals, including controls (FIGS. 5, 6, 7).

After 48 hours, a small discrete or focal erythema was observed in the first and second experimental groups of animals (Fig. 8, 9).

After 7 days, there was a slight swelling at the time of injection in all groups of animals (Fig. 10, 11, 12). After 14 days, no changes were observed in all animals of the control and experimental groups.

In this regard, the conducted studies allow us to conclude that the drug for skin applications, according to the interstate standard GOST ISO 10993-10-2011, has a weak degree of response to irritation in guinea pigs. The application of a resolving dose of the drug to sensitized guinea pigs does not cause a response from the skin, which indicates the absence of allergenic properties in this composition.

When studying the therapeutic efficacy of the test samples for burns, wound suppuration was not observed. There were 4 cases of death in the control group and complicated course of the wound process. On the second day after the burn injury in all groups of animals, hyperemia of the skin is noted along the periphery of the burn. Necrosis is noted in the burn area, in some places only the surface layer of the skin itself is affected, in others the burn extends to its entire thickness, accompanied by complete necrosis of the papillary layer. A superficial dry whitish-gray scab is formed. On the second day after the burn injury, there was no outward difference between the animals of different groups. By the seventh day of the experiment in the first experimental group of animals, the formation of granulation tissue and the beginning of epithelialization from the side of healthy skin occur. On the 14th day of the experiment, there is a pronounced positive dynamics in the animals of the first experimental group, islands of epithelialization are noticeable on the granulations, and a noticeable rejection of the scab occurs. By the 20th day of the experiment, in the first experimental group of rats, the epithelial tissue is completely restored, the hairline grows from the side of healthy skin.

By the 20th day of the experiment, complete healing of the wound surface was noted in the first and second experimental groups of rats (Fig. 13, 14).

In the control group of animals, by the 20th day of the experiment, physiological processes of burn wound relief were noted. Intensive epithelialization of healthy skin. A noticeable decrease in the volume of the wound surface. 4 animals - lethal outcome.

Thus, based on the foregoing, it can be concluded that the obtained prototype of the drug "Sample No. 6" has pronounced wound healing properties. This is evidenced by the reduction in the time for complete recovery of the skin after burn injury in rats compared to control animals. The experimental sample of the cream "Sample No. 6" has a wound healing effect on a par with the generally accepted remedy "Panthenol", and also has a low local irritant effect, as evidenced by the results of intradermal injection of the sample and the application of skin applications.

Claims (1)

Wound-healing composition based on a lipid fraction isolated by pressing from the biomass of Hermetia illucens larvae, Aloe arborescens juice, allatoin and related components: dimethyl sulfoxide (DMSO), carboxymethylcellulose, glycerin, tween-80, talc, benzyl alcohol and water, containing: lipid fraction larvae of Hermetia illucens - 13%, allatoin - 10%, benzyl alcohol - 1%, DMSO - 10%, carboxymethylcellulose - 1%, glycerin - 5%, talc - 33%, Aloe arborescens juice - 10%, tween-80 - 5 % and water - the rest.

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