WO2020032902A2

WO2020032902A2 - Aksolotl blastema for use in the treatment of wounds and burns

Info

Publication number: WO2020032902A2
Application number: PCT/TR2019/050666
Authority: WO
Inventors: Turan DEMIRCAN; Gürkan Öztürk; İlknur KESKİN; Mustafa KESKİN; Hasan ÇELİK
Original assignee: T.C. Istanbul Medipol Universitesi
Priority date: 2018-08-10
Filing date: 2019-08-08
Publication date: 2020-02-13
Also published as: WO2020032902A3

Abstract

The present invention relates to the use of axolotl blastema in the treatment of wounds and burns.

Description

AKSOLOTL BLASTEMA FOR USE IN THE TREATMENT OF WOUNDS AND

BURNS

Technical Field

State of the Art

The skin is a bilayered organ that is necessary for the continuity of the life. The skin is the heaviest organ of the body and constitutes 16% of the total body. It is the largest organ and also it is the organ that is the most exposed to trauma in the body. It is important to maintain continuity and integrity of the skin as it has very important functions for survival. Skin consists of two layers, namely epidermis and dermis .The main task of the epidermis is to protect the body from external factors with a hard outer layer. Its thickness varies with respect to anatomical regions. It is composed of 4 different cells: melanocyte, keratinocyte, merkel and langerhans cells. Dermis is the part of the skin that provides strength. It has two parts, papillary and reticular. Hair follicles, sweat and sebaceous glands, vessels and nerves are located in the dermis. 95% of the epidermis cells are keratinocytes. They form well-bounded layers from the basal layer to the skin surface. The part under the dermis is called hypodermis. It binds the skin to neighboring and surrounding tissues. The skin, which acts as a receptor that provides communication with the environment, protects the organism against friction and impact.

The condition that occurs as a result of disruption of functional and anatomical continuity of the skin or mucosa due to illness, trauma or surgical intervention is called a wound. Tissue damage caused by body contact with fire, steam or hot objects is called burn .It is vital to have a product that can be safely used in acute wounds and in chronic wounds where the inflammatory period is and for burns with limited therapeutic options. It is a long-standing research topic for researchers in the health and pharmaceutical sector to treat burns and injuries caused by accidents, wars, natural disasters, to achieve functional recovery without scarring, to reduce treatment costs and to shorten treatment time. The development of a treatment option that minimizes economic, aesthetic and functional losses has been the mainstay of the present invention. Existing burn and wound treatment methods have technical limitations in terms of prolonged treatment period, limited benefit in severe injuries, and inability to reproduce certain cells that would allow functional recovery. In addition, mortality and morbidity are high in some severe injuries and burns.

Currently, it is aimed to reduce the mortality and morbidity rates by minimizing wound sepsis in patients with large burns and injuries by using topical chemotherapy in burns and injuries. In such cases, many ointments are used to temporarily cover wounds. The simplest of these is bacitracin, a petroleum-based antibiotic .The most commonly used is silver sulfodiazine, which has moderate penetration to the wound. The use of silver sulfadiazine as a topical antibacterial in burns and wounds dates back to the l970s. Today, silver containing therapeutics and silver release garments have been used to prevent infections in burns, open wounds and chronic ulcers.

Although silver is widely used, the concentrations required to benefit from the antibacterial effect of silver therapeutics can have a lethal effect on both keratinocytes and fibroblasts. Silver-containing products damage pathogenic bacteria and damage healthy cells for wound healing, and cannot distinguish between these two.

In addition, silver sulfadiazine or other chemotherapeutic-phytotherapeutic agents have a positive effect on wound closure, but cannot shorten the healing time and improve the quality of healing. In addition to these reasons, their major disadvantages are the lack of a significant contribution to epithelialization.

The ultimate goal of burn and wound treatment is to achieve functional healing and epithelialization of the wound as quickly as possible by minimizing functional and aesthetic losses and preventing infection. Any technical development that will allow these criteria to be fulfilled will lead to new alternatives for the treatment of wounds and burns.

The main rationale underlying the invention is therefore to develop an approach that will shorten the treatment time compared to the options used in the prior art, thereby providing early healing potential and, in addition to faster wound healing, triggering a better healing process in terms of functionality and aesthetic appearance.

Detailed Description of the Invention

In other words, the invention relates to axolotl blastema for use in the treatment of wounds and burns.

As used in the context of the invention, the term "axolotl blastema" refers to tissue that can proliferate and regenerate after injury or amputation in axolotl organisms.

After injury or amputations that do not kill the axolotls a tissue called blastema forms and regeneration occurs without scarring and normal skin is formed. A few hours after injury or amputation in the axolotls, an epithelial layer that surrounds the open part due to amputation or injury is formed. The structure known as apical epithelial cap (AEC) formed by the migration of keratinocytes will become a special signal center in the following stages. With the help of the signals originating from this center a regeneration of the cells present in the amputation area takes place and these cells gain stem cell property and undergo a fast proliferation period thus forming the tissue named as blastema. The molecular content of the cells in this tissue makes them cells that are allowed to immuno-modulation, division and differentiation, thereby performing functional restoration.

It was found that when the blastema obtained from the axolotls are used directly as tissue or when the tissue is dissociated into its cells and applied as blastema cells or when the blastema cells are applied after homogenization, it shortens the healing time in wound and burn tissues, minimizes the scar formation and that it is non-toxic and hypoallergic and it provides generation of many types of cells that are lost due to the wound formation and also it has a positive effect on indicators of functional healing such as immunomodulation, angiogenesis, granulation and connective tissue development and epitalization stages when compared to other known methods. As is known, the axolotl blastema does not consist of a single substance and contains tens of thousands of different proteins and/or enzymes. The inventors have found that the favorable effect of applying axolotl blastema to the wound and burn sites is not due to a single enzyme present in the blastema, but to the synergistic effect of the enzyme composition present in the blastema.

The inventors have also surprisingly found that axolotl blastemas have a beneficial effect not only in wound healing but also in burn healing.

In another aspect, the present invention relates to pharmaceutical compositions comprising Axolotl blastema.

In a preferred embodiment of the invention, in the pharmaceutical composition, Axolotl blastema can be used as obtained from axolotl, i.e., without any processing, or after being obtained from axolotl, by separating into its cells or by homogenizing.

The pharmaceutical compositions comprising Axolotl blastema according to the invention may contain at least one excipient in addition to Axolotl blastema.

The excipient can be selected from the group comprising solvents, lubricants, glidants, fillers, density enhancers, oils, emollients, penetration enhancers, binders, dispersants, preservatives.

Pharmaceutical compositions comprising axolotl blastema according to the invention can be in a dosage form selected from a group comprising tablets, sublingual tablets, liquid solutions, suspensions, pastes, drops, ointments, aerosols, vaporizers, as solutions for intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous administration, creams, gels, dressings or skin patches.

The pharmaceutical compositions comprising Axolotl blastema according to the invention can be used in the treatment of wounds and / or burns.

As used in the context of the invention, the term "wound" refers to acute wounds, chronic wounds, infected wounds, abrasion wounds (rupture of the outer skin), tearing wounds, bruise wounds (wounds of swollen bruises resulting from collection of dead cells and blood under the skin), concussion wounds (damage to organs and tissues under the skin or skull despite there is no visible wound), knife wounds, skin cuts, surgical wounds (intentional wounds to the skin to perform a surgical procedure), firearm wounds, thermal wounds (wounds due to extreme heat, extreme cold, cold burn, sunburn and the like), chemical wounds (skin wounds caused by contact with chemicals or wound in the lungs due to inhalation of these substances), bites and stings (wounds due to bites of human, bee, dog, cat, bird, bat, rodent, snake, scorpion, spider and lice and the like), electricity originated wounds.

The term "burn" as used in the invention includes first degree burns (red, non-blistered skin), second degree burns (blistered skin and skin thickening), third degree burns (widespread skin thickening with white leathery appearance) and fourth degree burns (burns extending to the tendons and/or bones).

In another aspect, the present invention relates to a method for use in the preparation of Aksolotl blastema; said method comprises the steps of

a. Amputation of right or left, anterior or posterior limb, or tail region of the Axolotl under anesthesia,

b. Allowing the amputated limb or extremities to regenerate,

c. Isolation of blastema tissue from the organism under anesthesia during blastema stage,

d. Homogenizing the isolated blastema tissue in physiological saline solution.

Accordingly, in a preferred embodiment of the invention, the blastema stage is the duration between 24 hours to 4 weeks after amputation. In a particularly preferred embodiment of the invention, blastema isolation is carried out in the early stages of the blastema stage, for example on day 7 or on day 10 or on day 15 after the amputation.

In a preferred embodiment of the invention, the homogenization mentioned in step d) is carried out by mechanical, chemical or enzymatic means.

In another aspect, the invention relates to an axolotl blastema for use in treatment of wounds or burns obtainable by a method comprising the steps of

b. Allowing the amputated limb or extremities to regenerate, c. Isolation of blastema tissue from the organism under anesthesia during blastema stage,

d. Homogenizing the isolated blastema tissue in physiological saline solution.

Around these basic concepts, it is possible to develop a wide variety of applications relating to the subject matter of the invention, the invention is not limited to the examples described herein, but is essentially as set forth in the claims.

It is evident that a person skilled in the art can demonstrate the novelty disclosed in the invention using similar embodiments and / or may apply this embodiment to other fields of similar purpose as are used in the art. It is therefore evident that such embodiments will lack novelty and in particular inventive step.

Claims

1. Axolotl blastema for use in the treatment of wounds and burns.

2. Use according to claim 1, wherein the wound is acute wounds, chronic wounds, infected wounds, abrasion wounds, tear wounds, bruise wounds, concussion wounds, knife wounds, skin cuts, surgical wounds, gunshot wounds, thermal wounds, chemical wounds, bites and stings, electric-induced wounds.

3. Use according to claim 1, wherein the burn may be first degree burn, second degree burn, third degree burn and fourth degree burn.

4. Pharmaceutical compositions comprising axolotl blastema for use in the treatment of wounds and burns.

5. The pharmaceutical composition according to claim 4, which comprises at least one excipient in addition to axolotl blastema.

6. A pharmaceutical composition according to claim 5, wherein the excipient is selected from the group comprising solvents, lubricants, glidants, fillers, density enhancers, oils, emollients, penetration enhancers, binders, disintegrants, preservatives.

7. A pharmaceutical composition according to claims 4-6 wherein pharmaceutical composition is in a dosage form selected from the group comprising tablet, sublingual tablet, liquid solution, suspension, paste, drop, ointment, aerosol, vaporizer, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, cream, gel, wound dressing, skin plaster.

8. A pharmaceutical composition according to claims 4-7 for use in the treatment of wounds and burns.

9. A method for the preparation of axolotl blastema comprising the steps of;

a. Amputation of right or left, anterior or posterior limb, or tail region under anesthesia,

d. Homogenizing the isolated blastema tissue in physiological saline solution.

10. A method according to claim 9, wherein the blastema stage is the period between 24 hours to 4 weeks after the amputation.

11. The method according to claim 10, wherein the blastema stage is for example on day 7 or day 10 or day 15 after amputation.

12. A method according to claims 9-11, wherein said homogenization in step d) is carried out by mechanical, chemical or enzymatic means.