FR2972113A1 - Use of polysaccharide mixture comprising a hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparin, for forming a gel to protect and/or treat wound and skin lesions - Google Patents

Abstract

Use of polysaccharide mixture comprising a hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparin, for forming a gel, is claimed. ACTIVITY : Vulnerary. MECHANISM OF ACTION : None given.

Description

Description The present invention relates to the use of a mixture of different polysaccharide components individually present in the extracellular matrix and used for the manufacture of a medicament with healing activity, as well as a skin and dermal reconstruction biomaterial comprising this mixed. It is a mixture of polysaccharides derived from the extracellular matrix such as hyaluronic acid, chondroitin 6 and 4 sulfate, dermatan (chondroitin sulfate B) and high molecular weight heparin (and this depending on clinical cases) which makes it possible to obtain a complete reconstitution of cutaneous lesions whose size has been defined as critical.

The aim of this work is to evaluate the properties on cutaneous and dermal healing of an original composition of polysaccharides whose properties on bone healing have already been patented: FR2866571 (Al) Publication date: 2005-08-26, Inventor (s): ZANCHETTA PHILIPPE; Applicant (s): ZANCHETTA PHILIPPE [FR]; Classification: A61L27 / 26; A61L27 / 34; Application number: FR20040001700 20040220 To highlight the properties of this mixture on healing we used a model of healing in the rat. Problem The advantage of the present invention lies in the fact that the extended cutaneous skin losses are slow to heal and when the loss of surface material or depth exceeds a critical dimension, there can be no spontaneous healing and it is necessary to use external protective materials to prevent secondary infections and to guide healing. These protective materials, which lead or induce scarring, must then be used to protect the exposed deep tissue from loss of the dermis. There may be mentioned techniques derived from the technique of tulle gras or in the most critical cases the use of skin grafts. Autografts have the advantage of being perfectly immune. However, the graft surface that can be removed is limited in an individual and the additional surgical procedure that results from it carries risks of aesthetic and functional complications. It is possible to cultivate grafts from a donor sample on a synthetic or animal carrier with the problems of vascularization that may result. As for allografts or xenografts, they present among other risks of transmission of pathogens and may cause rejection phenomena due to reactions of the immune system.

Some materials have been proposed to solve these problems among which are the hyaluronic acid derivatives, which fix the growth factors present at the level of the lesion by protecting them from enzymatic degradations. But a loss of the dermal system requires complex healing to replace the different tissue layers disappeared first-line healing is impossible. Healing can only be done by the bottom by colonization of the clot and by the sides with significant cicatricial retraction phenomena. Contacting the wound margins with a suture would initiate first-line healing, which is not possible when there is extensive loss of substance, and healing is done according to the criteria for wound healing. second intention with budding and invasion from the bottom and the margins of the lesion. During the formation of the fleshy bud, the debridement phase occurs which aims to eliminate necrotic cells and tissues. Moreover, the protective or healing materials, as their names indicate, only lead to healing, without inducing skin or dermal tissue formation proper: They serve as supports for the biological phenomena of healing and are intended to be rejected outwardly in the form of an artificial crust after a certain time when the underlying scarring and the neoformed tissue have sufficiently completed their maturation.

As examples of healing materials used with more or less results, mention may be made of: -the fatty tulle, corticotullus-chemical sugar detergent (honey) -calginalginates of calcium and hydrocolloids-moisturizing dressings -the silicone derivatives-collagen and its derivatives - hyaluronic acid and its derivatives - cutaneous grafts (auto, allo or xenografts) Solutions In view of the literature, the different specialties proposed (and withdrawn) by the pharmaceutical industry and the shortcomings and disadvantages of this state of the art in the field of reconstruction of deep skin lesions, the inventors have therefore set themselves the goal of providing a biomaterial, usable in cutaneous and dermal reconstruction, which is adapted to the reconstruction of large losses of substances up to the muscular layer, that is defects whose dimension is called critical and whose dimensions lead to shrinkage and random or poor quality skin scarring, particularly in subjects suffering from certain pathologies such as diabetes. This biomaterial must be biocompatible, non-immunogenic and resorbable at the end or during cutaneous reconstruction, must avoid uncontrolled bud scarring and minimal retraction of the wound margins in case of loss of substance and allow reconstitution of the identical the loss of substance by accelerating the cicatricial phenomena without becoming anarchic or uncontrollable. The inventors have discovered that these goals were achieved by using a subtle mixture of particular polysaccharides, namely a physical mixture of different molecules such as hyaluronic acid, chondroitin sulfate (4 or 6) dermatan sulfate (chondroitin sulfate B) and sometimes heparin of high molecular weight. In the case where heparin is added to the mixture, it is at this time desirable to use this mixture on unopened wounds and to add in addition to the other ingredients of collagen or any natural haemostat, but necessarily bioabsorbable, although high molecular weight heparin has no anticoagulant activity. The subject of the invention is the use of a mixture, obtained by a simple pooling of the various components-without the need to first create chemical bonds between the different polysaccharide components encountered in the extracellular matrices and most of which have healing activities recognized in the literature. The advantage of the invention that we claim here lies in the fact that this original composition allows a healing activity in synergy of its various components and shows a reparative and detersive activity during the natural wound debridement phase on the skin tissues with destruction of the dermis that we have demonstrated by our experiments on animal models. The mixture used contains 49% of hyaluronic acid in the form of sodium salt, 49% of chondroitin 6 sulfate and 2% of chondroitin sulfate B (dermatan sulfate), all forming a homogeneous gel by the addition of 2.5 ml of sodium chloride. 9% sodium. It is also possible to use this mixture in freeze-dried form. The second mixture resulting from the first additionally contains 25,000 IU of heparin, respecting the same notion of purely physical mixing not seeking any chemical bond between the different components. Particularly advantageously, the polysaccharide mixtures defined above can be used for the preparation of a skin dressing intended to cover superficial and deep lesions of the dermis. The polysaccharides used in the invention may also be in a hydrated form, for example in the form of a physiological saline hydrogel or after lyophilization of the mixture which results in an object of the invention of fibrous appearance. . It is also possible to envisage using such a polysaccharide in the form of a solid three-dimensional cellular membrane or structure allowing a better volumetric maintenance of the loss of cutaneous substance and a better penetration by the clot or the cicatricial fluids. The subject of the invention is also a biomaterial for cutaneous and dermal healing, characterized in that it comprises a polysaccharide mixture as defined in the invention. In addition to the preceding provisions. This invention also includes other arrangements which will become apparent from the following description, which refers to examples of the use of the hyaluronic acid polysaccharide mixture of chondroitin sulfate, dermatan sulfate and sometimes in the presence of heparin in scarring and in the case of treatment of deep dermal lesions. It should be understood, however, that these examples are given solely by way of illustration of the subject matter of the invention, of which they in no way constitute a limitation in the composition of the polysaccharide mixture of which the percentages by weight are likely to vary from 1% to 99% of hyaluronic acid by modulating the masses of chondroitin sulfate (4 or 6) and dermatan sulfate and consequently as well as heparin.

EXAMPLE 1 Creation of a particular mixture of four polysaccharides One gram of hyaluronic acid in freeze-dried sodium form, 1 gram of chondroitin sulfate and 50 mg of dermatan sulfate (chondroitin sulfate B) are combined. In the first case, 2.5 ml of physiological saline are added to this mixture. The mixture is homogenized until a mixture without lumps and translucent. In the second case, it is added that 2.5 ml of physiological saline in which 25,000 IU of high molecular weight heparin (20,000 Da) have been dissolved.

EXAMPLE 2 In vivo use of the polysaccharide mixture defined by the invention as a skin healing biomaterial in the context of standard lesions with a diameter of 6 mm or very extensive lesions normally exceeding the cicatricial capacities of animals. a) Protocol Male Wistar rats of 275-299 grams are from January Laboratories. The animals are received one week before the date of the experiment and housed in individual cages with food and drink ad libitum. The object of the invention is used in two forms, either a maximum saturation gel of the polysaccharide mixture which is the subject of the invention in physiological saline, or in a lyophilized dry form. In this case, the subject of the invention is crushed before using a used biopsy punch as a punch with a diameter of 6 mm. This pellet of polysaccharides, object of the invention will be positioned directly in the defect created in the dermis of animals. Anesthesia is performed using Robinul® (Vétoquinol, Lure France) 0.1 ml / 100 g. and an analgesic supplement of Ketamine® 0.lml / 100g (Imalgene®, Mérial, Lyon, France) and Nesdonal® intraperitoneal (0.lml / 100g) (Specia, Rhône Poulenc Rorer, Montrouge France). The animals are shaved (with a mechanical mower and a bimetallic razor) on their dorsal part between the two shoulders on an approximate area of 80mm by 50mm. The area is not disinfected before perforating.

Standardized perforations are performed with a 6mm biopsy punch (Epitheasy®, Medax, Poggio Rusco, Italy). Because of the resistance of the epidermis of animals, each punch allows only two holes. A second series of animals is treated by performing - with scalpel and scissors - defects of 2 cm by 2 cm.

We realize three defects of 6mm of diameter on either side of the spine is 6 defects per animal.

The other animals treated receive the mixture of polysaccharides object of the invention in the defects created on the right side of the spine. The left side of the animal is considered as a specific control for each specimen. It will highlight a possible systemic effect. Lesions of 20 * 20mm are also filled with the material object of the invention. Animals do not receive any healing material, they are control animals. Digital photographs are taken daily to evaluate the scarring macroscopically. The animals are sacrificed at 2, 3, 5 and 7 days. Each series has 2 neutral witnesses and at least 3 animals treated with the object of the invention on the defects on the right side. The animals are sacrificed by intracardiac injection of penthobarbital after sedation with ether. Defects are taken with the underlying muscle part. They are fixed in formaldehyde before being treated for making serial histological sections stained with haemalun-eosin. b) Results. D + 2 Animals treated: We note the presence of the object of the invention (very disintegrated because not supporting the histological preparation treatments), a beginning of epithelialization (under the object of the invention), the presence of myofibroblasts that began to migrate, the presence of important cell infiltrate in the material (not in the control clot). The inflammatory infiltrate is important under the material and in the clot being organized and this inflammation is related to the increased presence of residues from the detersive phase amplified by the mixture of polysaccharides. Important angiogenesis is noted by the presence of numerous vessels in the granulation tissue The very strong angiogenesis in the fleshy bud is located under the material of the invention which does not serve as a matrix in its gel form but seems to guide healing. to his touch. There is an important lymphohistiocyte infiltrate associated with edema. There is more pronounced cell differentiation on the side that received the material than on the untreated side. Epithelialization has already begun under the crust whose formation is rapid. Control animals: There is no epithelialization, a slight fleshy bud, a small cell population and a macroscopic crust. Macroscopic and microscopic defect depression is important in the lesion. The differences are very marked with an animal treated with the object of the present invention.

D + 3 Here we find an intermediate ripening between what is observed at 2 days and 5 days. Healing is of much better quality on animals that have received healing material. Untreated animals show a very low level of epithelialization, significant edema and low cell differentiation in the lymphohistiocyte population.

For this series, the cell population is very marked as angiogenesis especially in contact with the subject of the invention. The fleshy bud is being organized with significant angiogenesis. The appearance of numerous myofibroblasts among the lymphohistiocyte population is significant. The presence of numerous bundles of collagen fibers is noted. The control has a crust that is not rich in cells but a marked angiogenesis on the surface. Wounds on both the control and the treated side show significant debridement with the expulsion of all cell residues into a fibrin clot, but the thickness of the residues from the debridement is much greater on the treated side. This mass is very important, seems non-fibrillar and gelled. It is present on both sides both on the side having received the object of the invention than on the qualified side of witness. Epithelialization is complete on both sides and keratinization is present above the defects. The treated side has a marked angiogenesis in contact with the object of the invention (visible). The epithelium seems to adhere to the object of the dry invention (torn off during the histological preparation and on 3 different sections). We can highlight a kind of double epithelium which corresponds to a rapid elimination of necrotic scar tissue on both sides of this "bubble" of waste disposal). This highlights the detergent properties of the invention when contacted with a large wound. It should be noted that the object of the invention no longer appears visible after 5 days (it can be guessed at 2 days in a fragmented form). Serum treated with the subject of the invention dry (lyophilized) Presence of a clot marked on the control side with marked cell cords and numerous myofibroblasts The crust is important and the epithelialization is in the process of formation. Note the characteristic presence of underlying cellular and muscular cords that go back into the fleshy bud. This depression characteristic of animals having received no object of the invention is not noted.

Witness Series Total epithelialization of the lesion at the surface is noted, but a weak underlying cellular organization, visible progression of healing from the bottom of the lesion (a sort of cellular elan). There is a large amount of lymphohistiocyte cells in a very oedematous tissue.

J + 7 The treated animals show a marked cell and tissue organization with significant angiogenesis. The presence of numerous collagen fibers of many capillaries associated with a cell population with many cells that come out by diapedesis. The cell population is much more varied. The fibroblasts are numerous and the superficial edema does not penetrate the deep layers. The collagen fibers are oriented parallel to the surface of the lesion. In some cases there is a reconstitution of appendices such as the hair system at the periphery of the lesions. The organization of the scar tissue is very marked, with an improvement in the quality on the sides that have been the object of the invention. Some animals still show this mass of elimination of cellular waste whose formation and volume are increased by the presence of the healing material object of the invention. Untreated controls show complete epithelialization (reconstruction of basal relief) with weak cellular organization underlying crustal remnants and muscle fibers. The subepithelial zone consists of a fleshy bud very weakly organized. It is more in the presence of an immature granulation tissue than that observed in animals treated with the polysaccharide mixture.

c) Comparative experiments. Few experiments of this kind are noted in the literature. Large lesions such as those we have practiced are undocumented. It thus appears that among the various polysaccharides tested, only the polysaccharide mixture, object of the present invention allows complete and rapid healing of the orifices formed in the dermal tissues of laboratory animals. It should be noted that the perioperative mortality rate is low and due to cardiorespiratory problems caused by anesthetic techniques.

Summary This invention relates to the use of a polysaccharide mixture composed of hyaluronic acid of chondroitin 6 sulfate, dermatan sulfate and sometimes high molecular weight heparin (depending on clinical situations) forming a gel for the manufacture a drug with healing and detersive activity during the first stages of healing, especially with activity on skin healing even in case of destruction of the dermis. The invention also relates to a skin healing biomaterial which comprises such a mixture of polysaccharides without constraints of modifications in the percentage of weight composition, as well as to a surgical device whose surface would be coated with such a polysaccharide mixture. .

d) Conclusion. The polysaccharide mixture developed by the inventors makes it possible, in a reproducible manner and within a few days, to heal cutaneous lesions of critical size (diameter of 6 mm) and of supercritical size in cutaneous tissues of rats up to muscles. Critical lesions are reconstituted ad-integrum with the hair system at an accelerated speed without uncontrolled effects observed without any noticeable side effects. This healing activity is manifested both on the treated side and the normally qualified side of control. The polysaccharide mixture used does not cause any inflammatory reaction, has detergent properties not explicitly described in the literature to date, and is completely resorbed by the surrounding tissues because they are constitutive elements of the extracellular matrix. The quality of the neoformed tissues is identical to that of the original skin without any hyper proliferative phenomenon and with reconstitution of the different histological structures, in particular the hair system. This mixture of polysaccharides thus constitutes a material for potentiating cutaneous cicatrization with a protective effect of the lesion even in case of destruction of the dermis.

Bibliographical references

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Claims (1)

CLAIMS 1 °) Use of a polysaccharide mixture composed of hyaluronic acid chondroitin sulfate dermatan sulfate and heparin (depending on clinical cases) forming a gel for the manufacture of a medicament with healing activity. 2) The use according to claim 1, characterized in that said medicament has an activity on cutaneous cicatrization. 3 °) Use according to claim 1 or claim 2, characterized in that said mixture of polysaccharides is used for the preparation of a repair material, protection or skin healing. 4 °) Use according to claim 1 or claim 2, characterized in that said mixture of polysaccharides is used for the preparation of a coating, dressing, cutaneous application gel or three-dimensional protective structure. 5 °) Use according to any one of the preceding claims, characterized in that said mixture of polysaccharides is likely to be obtained from a mixture of hyaluronic acid chondroitin sulfate dermatan sulfate and sometimes heparin forming a hydrogel or freeze-dried plastic structure without constraints relative to the respective weight percentages in the composition, this mixture being obtained by pooling the various components without seeking to create covalent chemical bonds. 6. Use according to claim 5, characterized in that said mixture of polysaccharides has a constant healing activity unrelated to a significant variation in its weight composition. 7 °) Use according to any one of the preceding claims, characterized in that said mixture of polysaccharides object of the polysaccharide invention is associated with another healing material and hemostatic. 8) Use according to any one of the preceding claims, characterized in that the mixture of polysaccharides based on hyaluronic acid, chondroitin sulfate, dermatan sulfate is used to obtain protection and / or healing of deep dermal wounds. 9 °) Use according to any one of the preceding claims, characterized in that this mixture of polysaccharides is used in all cases where protection, debridement and scarring of light skin lesions, extensive and / or deep will be necessary. 12