JP2004269542A - Wound-healing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a wound-healing agent containing a transglutaminase and a protein and/or particles, and, especially, to provide a wound-healing agent for preventing cicatricial contracture and easily performable of wound covering and hemostasis. <P>SOLUTION: The wound-healing agent, a cicatricial contracture inhibitor and a wound covering agent, which contain a calcium-independent transglutaminase and a protein containing an amino acid residue usable as a matrix for the transglutaminase, are provided. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to an agent for treating a wound, an agent for preventing scar contracture, an agent for covering a wound, and a hemostatic agent, which comprises a transglutaminase and a protein or / and a particle as active ingredients.

  The following facts have been known for medical-related uses of transglutaminase. First, Japanese Patent Application Laid-Open No. 62-74360 discloses an artificial skin containing, as a component, a membrane obtained by adding transglutaminase to a solution containing a protein and / or peptide. It is not only excellent in strength, elongation, flexibility and oxygen permeability, but also has excellent water vapor permeability and strong biocompatibility. Further, Japanese Patent Application Laid-Open No. 2-108631 discloses an immunosuppressant containing transglutaminase as an active ingredient, but the disclosure is limited to in vitro experiments and immunological considerations. In fact, actual wound treatment in vivo does not even suggest. Further, the use of such a transglutaminase is clearly different from that of the present invention, as will be understood from the description below.

  By the way, with regard to wound treatment, treatments such as primary suturing after disinfection are generally performed, but a surgical operation is indispensable for this. However, surgical suturing is difficult when the skin is severely damaged or burns.

  On the other hand, in the treatment of wounds, how to treat the wound without leaving behind is extremely important not only from an organic and functional viewpoint, but also from a cosmetic viewpoint.

  For such prevention of scar contracture, skin grafting, use of orthotic devices, drug therapy, radiation therapy, and the like are known, but all have drawbacks. In other words, in addition to the need for surgical treatment, skin grafting has a severe limitation on the pericarp area when the wound is extensive, such as severe burns, making ideal skin grafting difficult. It is pointed out that the wound contraction does not immediately stop, and the wound contraction progresses for a considerable period of time, such as at the adhesive fusion portion between the skin graft and the wound surface, as well as at the suture line at the edge of the skin graft. Regarding the use of the orthosis, there is a problem that, although it is an auxiliary means and long-term use is necessary, it is actually difficult to use it for a long time. Regarding drug therapy, a steroid ointment cannot be expected to have a clear effect, and tranilast, which has the effect of suppressing mast cell degranulation, can only be expected to have an auxiliary effect. Radiation therapy has the disadvantage that it suppresses the mechanism of wound contraction, but on the other hand, impairs wound healing.

  Some wound dressings have also been proposed and used, but all have drawbacks. That is, the freeze-dried pigskin needs to be dressed in order to thaw at the wound surface and needs to be regenerated with physiological saline before use. Collagen artificial skin sometimes causes mild pain and discomfort. Plasma membranes and fibrin membranes made from human blood do not have sufficient secretion outflow in the case of secretory wounds, and have a limited supply because human blood is used as a raw material. Pain and discomfort when worn are common to these wound dressings. In addition, these wound dressings do not have the necessary effects for drying, hemostasis, infection control and other courses of treatment of the affected area.

  As the hemostatic agent, gelatin, microfibrillar collagen, thrombin, and the like are known, and the development of an even more excellent hemostatic agent is desired.

  The present applicant has been focusing on a method for producing transglutaminase and developing a wide range of uses thereof, and has already filed many patent applications for the results thereof. Under the background of the above-mentioned conventional technology, it is an object of the present invention to provide a novel wound treatment agent containing transglutaminase as an active ingredient, which also adds a new use to transglutaminase.

  The present applicant has conducted intensive studies to achieve the object described in the preceding paragraph.As a result, transglutaminase has a wide-ranging wound healing action, this action includes a scar contracture prevention action, and furthermore, pain control, affected area dryness, and hemostasis. It has been found that the composition has a wound covering effect having a combined effect, and based on such findings, has completed the invention of a wound treatment agent comprising transglutaminase as an active ingredient, and has already filed a patent application for this. (International Publication No. WO93 / 20837), it has now been found that the action of such a wound healing agent is remarkably improved by blending proteins and / or particles, and the present invention has been completed. It should be noted that the hemostatic effect of the transglutaminase combined with the protein and / or the particles is not limited to the effect of wound covering, but also to the rescue treatment of wounds due to surgical areas and accidents. Of course it is useful.

  That is, the present invention relates to an agent for treating a wound, an agent for preventing scar contracture, an agent for covering a wound, and a hemostatic agent, comprising transglutaminase and a protein or / and a particle as active ingredients.

  Hereinafter, the present invention will be described in detail.

  Transglutaminase, which is one of the active ingredients of the agent for treating wounds of the present invention, is classified into a calcium-independent type and a calcium-independent type. Examples of the former include those derived from microorganisms (for example, see JP-A-64-27471), and examples of the latter include those derived from guinea pigs (for example, see Japanese Patent Publication No. 1-50382) and fish. (See, for example, Nobuo Seki et al., “Abstracts of Abstracts of the Meeting of the Fisheries Society of Japan 1988”, p. 167, and “Summary of the Abstracts of the Spring Meeting of the Fisheries Society of Japan, 1990” on page 219) and those derived from humans (eg, blood Factor XIII). Of these, calcium-independent ones are preferred, and their production can be carried out, for example, by the method of JP-A-64-27471.

  Examples of the protein as another active ingredient include a protein having an amino acid residue that can serve as a substrate for transglutaminase, that is, a protein having a glutamine residue and a lysine residue, such as collagen.

  Examples of the particles include particles having a property such that transglutaminase present in a solution does not partition, and any of solid particles and liquid particles may be used as long as the particles have such a property. Specific examples of the solid particles having such properties include cellulose particles. Further, specific examples of the liquid particles having the same properties include droplets (oil droplets) made of oil dispersed in an aqueous solution by adding a surfactant or the like. Oil droplets can be formulated, for example, in the form of an emulsion.

  Of course, as long as the effects of the present invention can be obtained, a plurality of types of proteins can be used in combination, and two or more types of particles can be used in combination. Further, the protein and the particles can be used alone or in combination. Can be simultaneously added to transglutaminase.

  The dosage form of the wound treatment agent of the present invention is not particularly limited as long as the effects of the present invention can be obtained. The active ingredient mixture itself, a solution containing the mixture, a semi-liquid excipient or an auxiliary agent is used. Examples of the dosage form suitable for topical application used include, for example, solutions, ointments, topical protective agents such as gauze containing a mixture of active ingredients, sprays, and emulsions. The solution can be prepared by dissolving transglutaminase and other active ingredients in a solvent such as sterile water or various buffers. There are no particular restrictions on the base of ointment, fats, fatty oils, lanolin, petrolatum, paraffin, wax, plasters, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents For example, the base can be appropriately adopted from ordinary raw materials.

  The content of transglutaminase in these solutions, ointments, topical protectants, sprays, emulsions and the like is not particularly limited, but is preferably about 0.1 to 1,000 U / g, preferably about 0.5 to 500 U / g. It is convenient. In addition, there is no particular limitation on the amounts of the protein and the particles as long as the reaction does not proceed before the application to the wound surface and the transglutaminase is not deactivated, and those skilled in the art can give a given amount. An appropriate amount can be easily determined depending on the case.

  The wound treatment agent of the present invention is administered to a wound by a method such as spraying, spraying, applying, compressing, and the like.

  When the wound treating agent of the present invention is used particularly as an agent for preventing scar contracture, the wound to be treated is, for example, a wound such as a burn or a wound such as a surgical operation.

  When used as a wound dressing, the target wound is, in addition to the above-mentioned wounds, wounds such as diabetic angiopathy, chronic skin ulcer, post-thrombotic ulcer, and pressure sore ulcer. The wound dressing of the present invention has a wound infection preventing action, a wound analgesic action, an affected area drying action, and the like. Incidentally, during the conventionally known infection prevention treatment, the cleaning of the wound using a disinfectant or the like is only a temporary treatment, and there is a concern that the use of an antibacterial agent may cause contamination with resistant bacteria. In addition, conventional wound dressing protection requires wound fixing, and has problems such as pain during use and detachment, discomfort, and outflow of secretion fluid.

  When used as a hemostatic agent, it is effective in stopping various bleedings including those in the surgical field.

  The wound treatment agent of the present invention exhibits a therapeutic effect widely on other wounds.

  ADVANTAGE OF THE INVENTION According to the wound treatment agent of this invention, scar contracture prevention, wound covering, and hemostasis can be performed very easily.

  The wound treatment agent of the present invention is used not only for treating wounds in humans, but also for treating wounds in animals other than humans.

  Hereinafter, the present invention will be further described with reference to examples.

  The transglutaminase used in the present example is a microorganism-derived transglutaminase produced according to the method of Example 1 described in JP-A-64-27471, and is abbreviated as TG below.

Example 1 (Preparation of therapeutic agent)
Therapeutic agents 3, 4, and 6 to 9 are of the present invention, and therapeutic agents 1, 2, and 5 are controls.

(A) Therapeutic agent 1 (negative control):
In the test of Example 2, the case where only the gauze is replaced is referred to as a therapeutic agent 1 for convenience.

(B) Therapeutic agent 2 (positive control for therapeutic agents 3 and 4):
With a TG3 unit / ml solution of 50 mM HEPES buffer (pH 7.0), 1 ml of this solution was added dropwise to one piece of gauze.

(C) Therapeutic agent 3 (containing solid particles):
A solution prepared by adding 15 ml of TG3 unit / ml solution to 3 g of white powdered cellulose “Avicel PH-M15” manufactured by Asahi Kasei Corporation with about 20 w / v% cellulose TG3 unit / ml solution. A value obtained by dividing the total volume by 15 (about 1.1 to 1.2 ml per animal) was dropped on one gauze.

(D) Therapeutic agent 4 (containing liquid particles):
A TG3 unit / ml emulsion solution prepared by collecting 0.1 ml of TG30 unit / ml solution and 1 ml of emulsion solution with a syringe and mixing. This 1.1 ml was dropped on one piece of gauze and then spread. The composition of this emulsion was 30 w / w% of corn oil, 70 w / w% of water and 0.6 w / w% of emulsifier (based on corn oil) (0.3 w / w% of “Sugar Ester S-1170” and xanthan gum) 0.3 w / w%), and the average emulsion diameter was 5 μm.

(E) Therapeutic agent 5 (positive control for therapeutic agents 6-9):
A TG30 unit / ml HEPES buffer solution prepared by collecting 1 ml of TG30 unit / ml solution and 1 ml of 50 mM HEPES buffer (pH 7.0) with a syringe and mixing. This was dropped on two pieces of gauze.

(F) Therapeutic agent 6 (protein combination (1)):
A TG30 unit / ml / collagen 1.5 mg / ml solution prepared by collecting 1 ml of the TG30 unit / ml solution and 1 ml of the collagen 1.5 mg / ml solution with a syringe and mixing. This was dropped on two pieces of gauze. The collagen solution was “Atelocollagen” manufactured by Koken, and was a 3% solution of 50 mM HEPES buffer (pH 7.0) with a protein purity of 99% or more.

(G) Therapeutic agent 7 (protein combination (part 2)):
A TG 30 unit / ml / collagen 3 mg / ml solution prepared by collecting a TG 30 unit / ml solution 1 ml and a collagen 3 mg / ml solution 1 ml with a syringe and mixing. This was dropped on two pieces of gauze. The collagen solution was the same as the therapeutic agent 6 except for the concentration.

(H) Therapeutic agent 8 (protein combination (part 3)):
A TG30 unit / ml / collagen 10 mg / ml solution prepared by collecting a TG30 unit / ml solution 1 ml and a collagen 10 mg / ml solution 1 ml with a syringe and mixing. This was dropped on two pieces of gauze. The collagen solution was the same as the therapeutic agent 6 except for the concentration.

(I) Therapeutic agent 9 (protein combination (part 4)):
A TG 30 unit / ml / collagen 30 mg / ml solution prepared by collecting a TG 30 unit / ml solution 1 ml and a collagen 30 mg / ml solution 1 ml with a syringe and mixing. This was dropped on two pieces of gauze. The collagen solution was the same as the therapeutic agent 6 except for the concentration.

(Example 2 (inspection of wound treatment effect))
(A) Test animals: 7-week-old Crj: CD (SD) SPF rats (108 males of Charles River Japan Co., Ltd. were divided into nine groups of the same number (12) in each group) Provided.

(B) Wound preparation method:
The wound site was anesthetized with pentobarbital sodium (40 mg / kg, ip), the back was shaved with a clipper (about 7 cm x 7 cm), disinfected with Hibiten, and sized to about 5 cm square using scissors. It was prepared by exfoliating the skin.

(C) Administration method and examination period:
Therapeutic agents 1-9 were tested using nine groups. Each group is referred to as groups 1 to 9 corresponding to the number of the test agent.

  The therapeutic agent was applied to one gauze (groups 2 to 4) or two (groups 5 to 9) having a size enough to completely cover the wound site, and about 1 ml (groups 2 to 4) or 2 ml (groups) per animal. 5-9) were applied and applied to the wound site. Thereafter, the sticking site was covered with an adhesive stretchable bandage (“High Latex Chu” manufactured by Iwatsuki Co., Ltd.), and further fixed thereon using surgical tape (manufactured by 3M Medical-Surgical Division) for about 24 hours.

  The dosing period was daily from 0 to 14 days after wound creation in groups 2 to 4, and on days 0, 4, 8, and 12 after wound creation in groups 5 to 9; Only gauze exchange was performed. In Group 1 (control group), only one piece of gauze was applied and exchanged every day.

  In addition, blood was collected and the wound site was collected from four animals in each group on days 2, 7, and 14 after the creation of the wound.

  As described above, 9 groups were provided by adding a non-administered control group to a total of 8 groups to which each therapeutic agent was administered every day or every 4 days as described above. As described above, the number of animals in one group was 12 males.

(D) Wound observation:
The wound site was observed every day during the observation period using the conditions listed in Tables 1 to 4 below as indices. The score for quantifying the observation result is also shown in each table.

(E) Observation results:
(1) The results of the formation of the film-like substance on the wound surface are shown in FIGS. 1A to 1I.

  The formation of a film-like layer on the wound surface was observed more extensively and thicker in the order of group 4> group 3> group 2> group 9 ≧ group 8 ≧ group 7 ≧ group 6> group 5> group 1 (control group). Was. In addition, in the coatings of Groups 2 and 3, yellowish animals were observed 6 to 9 days after the wound was made, and a part of the coating was peeled off when the gauze was replaced, and the peeled part was irregular in a bright pink to reddish tone. Was. Thereafter, a part of the film-like layer became crust-like, and, like the film-like layer, partly exfoliated when gauze was replaced, and the exfoliated part was whitish. The coating of Group 4 also showed a similar course, but the yellow color change of the coating and the time of partial peeling were extended to 7 to 12 days after the wound was created, and the film-like layer was crusted or partially peeled. The period of time was extended to 13 or 14 days after wound creation. On the other hand, in groups 5 to 9 in which the film-like layer was thin, only a small number of cases showed partial exfoliation of the coating, partial crust formation, and partial exfoliation of the crust.

  (2) The results on the state of exudate on the wound surface are shown in FIGS. 2A to 2I.

  The exudate on the wound surface was reduced 7 or 8 days after wound creation in Group 4 where the coat-like layer was extensively thickened, 4 to 8 days after wound creation in Group 3 and 7 days after wound creation in Group 2 However, exudates were always observed in the exudation state of the other administration groups including the control group, and it was considered that there was no difference.

  (3) The results regarding the state of pain are shown in FIGS. 3A to 3I.

  The number of animals that hurt when the adhesive gauze was taken was smallest in Group 4 in which the wound-like layer on the wound surface was extensively thick, and in the following Group 9 ≦ Group 8 = Group 7 = Group 6 = Group 5 = Group 3 = Group 2 <Group 1 (control group).

  (4) The results of the bleeding state on the wound surface are shown in FIGS. 4A to 4I.

  Hemorrhage on the wound surface after removing the adhesive gauze is reduced as the wound-like layer on the wound surface becomes thicker over a wide area, and the average score is group 4 <group 3 <group 2 = group 9 = group 8 = group 7 = group 6 = group 5 <group 1 (control group), with group 4 having the least bleeding. Bleeding in each group decreased after 11 days after wound creation.

(F) Discussion and Summary Therapeutic agents 2 to 4 were repeatedly applied to back wounds of 7-week-old Crj: CD (SD) SPF rats (12 males per group) for 14 days, and therapeutic agents 5 to 9 Repeatedly applied 24 times for 24 hours each at daily intervals, and on days 2, 7, and 14 after wound creation, blood samples and wound sites were collected for each group of 4 animals, and the therapeutic agent that would yield the most effective therapeutic effect was examined. did. Incidentally, it is as described above that the therapeutic agents 3 to 4 and 6 to 9 are related to the present invention.

  Formation of the film-like layer on the wound surface was faster, wider, and thicker in the order of group 4> group 3> group 2> group 9 ≧ group 8 ≧ group 7 ≧ group 6> group 5> group 1. .

  In group 4 where the formation of this coating-like layer was the thickest, the animals did not hurt (do not sound) when the applied gauze was taken, and there was less bleeding on the wound surface after removing the applied gauze. However, there was a weak point that the exudate decreased on the 7th or 8th day after the creation of the wound and the reduction of the wound area on the 7th day after the creation of the wound was delayed.

  In groups 3 and 2 in which the formation of the coat-like layer was thicker next to group 4, less bleeding was observed on the wound surface after removing the applied gauze, but the effect was weaker than in group 4. In addition, pain when taking the applied gauze was also suppressed, but the effect was weaker than in groups 4 and 9, and a weak point was observed in which exudate was temporarily reduced during 4 to 8 days after wound preparation.

  In groups 5 to 9, the pain when taking the applied gauze was suppressed in group 9 having the highest collagen concentration, but the effect was weaker than in group 4, and the bleeding on the wound surface after taking the applied gauze was also suppressed. However, the effect was weaker than groups 4 and 3. However, exudate on the wound surface was always observed and did not affect the reduction of the wound area. In groups 8, 7, 6 and 5, the effects of suppressing pain and bleeding were comparable or slightly weaker than in group 9.

(The invention's effect)
According to the present invention, a new therapeutic agent for wounds, particularly an excellent therapeutic agent for wounds, which can easily prevent scar contracture, cover wounds and stop bleeding has been newly provided.

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Claims (4)

  An agent for treating wounds, comprising as active ingredients calcium-independent transglutaminase and a protein having an amino acid residue that can serve as a substrate for the transglutaminase.   An agent for preventing scar contracture, comprising as active ingredients a calcium-independent transglutaminase and a protein having an amino acid residue that can serve as a substrate for the transglutaminase.   A wound dressing comprising, as active ingredients, calcium-independent transglutaminase and a protein having an amino acid residue that can serve as a substrate for the transglutaminase.   A hemostatic agent comprising, as active ingredients, calcium-independent transglutaminase and a protein having an amino acid residue that can serve as a substrate for the transglutaminase.

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