WO1996033733A1 - Novel remedy for skin deficiencies - Google Patents

Abstract

Medicines which exhibit an efficacious activity by themselves by acting in the complicated processes of healing skin deficiencies, particularly wounds and skin ulcers, and also chronic (intractable) skin ulcers; and medicines which act on surgical wounds and traumas to promote healing thereof and reduce surgical and traumatic cicatrices. Medicines containing at least one member of tissue inhibitors of metalloproteases (TIMPs) as inhibitors against matrix metalloproteases (MMPs) (exhibit excellent effects in the complicated processes of healing skin deficiencies such as wounds and skin ulcer). Particularly TIMP-2 has excellent characteristics as the medicine, and rTIMP-2 is useful as a practical remedy for skin deficiencies such as wounds and skin ulcers and as a medicine which acts on surgical wounds and traumas to promote healing thereof and reduce surgical and traumatic cicatrices.

Description

 Injury New therapeutic agent for skin deficiency Industrial application

The present invention is characterized in that at least one tissue inhibitor selected from the group consisting of tissue inhibitors metabolic proteases (tissue inh ibitorof metalallproteases: TIMPs) is used as an active ingredient. A therapeutic agent for skin deficiency, particularly a therapeutic agent for skin fulminant applied to the affected area of a patient with skin bruises and a therapeutic agent for a wound applied to the affected area of a wound patient. The present invention relates to a therapeutic agent that is effective for reducing surgical scars and trauma scars by preventive treatment of wounds and wounds. More specifically, the present invention relates to a tissue inhibitor 1 (TIs) or tissue inhibitor 1 (TIMP-1) or issue 2 nh ibitorofmata 1 l prorotei na ses—2: A preparation containing TIMP-2) as the main active ingredient, effective for the treatment of skin * ulcers and wounds, and various surgical operations. In another aspect, the present invention relates to a group of recombinant tissue inhibitors of metallo-oral proteases (rTIMPs), which are characterized by reduction of surgical scars and trauma scars by preventive treatment of wounds and wounds. Therapeutic agent for skin deficiency, comprising at least one tissue inhibitor selected from the group consisting of an active ingredient, particularly a recombinant tissue inhibitor Meta-oral protease 1 (r TIMP-1) and recombinant tissue A therapeutic agent for skin deficiency, which further comprises at least an active ingredient selected from the group consisting of inhibitors of meta-oral protease _2 (rTIMP-2); The present invention relates to a medicament for the prevention and treatment of cutaneous slides such as ulcers such as decubitus ulcers, inflammatory fistulas and simple hammers. Background art

The healing process of wounds and waves goes through (1) inflammation, (2) cell proliferation, and (3) reconstitution of the epidermal and dermal components, with characteristic cells at each stage. The first reaction to the wound is the synthesis of extracellular matrix components such as fibronectin, fibrinogen, vitronectin, etc., providing a matrix that serves as a scaffold for cells to migrate to the upper part. Activated platelet strength, platelet-derived growth factor (PDGF), transforming growth factor-a (transformation factor, TGF-), transforming growth Factor-1 (TGF- ^), insulin-like growth factor-I (I: IGF-I), etc. are released, and inflammatory cells migrate to the wound. Two days later, macrophages and lymphocytes appear and hold the key to wound healing. That is, macrophages and lymphocytes serve as a source of necrotic tissue removal, growth factors, and cytodynamics at the wound site. The TGF secreted by the macula phage TGF-) S, I GF-I, colony! The intense factor (colony sti mu factor factor: CSF), interleukin-8 (IL-8), promotes tissue regeneration from the inflammatory process by migrating cells to the wound. Move to the process. Heparin-binding epidermal growth factor secreted by lymphocytes (he arinbi d inng epi de rma lgro wt hf ac tor: .HB-EGF), basic fibroblast growth factor (basic fibl ob lastg rowt hfactor) : B FGF) induces proliferation of fibroblasts, vascular endothelial cells, and vascular smooth muscle cells, and promotes angiogenesis.

Subsequently, fibronectin, collagen, hyaluronic acid, etc. make up the extracellular matrix of the new dermis. PDGF,

bFGF promotes cell division of skin cells, and keratinocyte growth factor (KGF) acts only on epithelial cells and promotes proliferation. It is believed that keratinocytes produce cytokines that promote fibroblast proliferation and collagenogenesis, and determine the ultimate quality of skin repair.

 Expression of cell surface receptors for these various growth factors is thought to play an important part in the healing process of cell migration and proliferation.

 The effects of cytokines such as growth factors, growth factors, etc., are based on complex crosstalk, and it is not necessarily the case that a type of cytokine is key to the healing process. Absent. In addition, the expression of the cell adhesion factor receptor on the cell surface plays a role in adhesion between cells, adhesion between cells and extracellular matrix, contact of wounds and imparting contractility.

In the process of tissue destruction and reconstruction during the healing process of wounds and ulcers, tissue degeneration and necrosis, leaching based on circulatory disorders, and inflammation reactions involving proliferation of tissue cells and free cells, inflammatory cells and proteases derived from tissue cells Is known to play an important role. In fact, in skin sores, corneal hamsters, burn hammers, skin bullous diseases, etc., certain types of matrix metallobu orase (MM P) activity Has been observed.

Matrix meta-oral proteases (MM P s) are a group of neutral proteases that form famili, and have Zn ² — at the active center. It is well known that MMPs plays an important role in the degradation of extracellular matrix, and more than 10 types of MMPs are known to date. Destruction of the extracellular matrix by MMPs is one of the major causes of delay in the cure of intractable diseases involving tissue destruction, and this increase in MMP activity is associated with endogenous MMPs inhibitors. It is thought to be the result of a quantitative imbalance with TIMPs.

 Among inflammatory cells derived from blood cells, neutrophils and macrophages have the ability to secrete various types of MMPs, but their expression is increased during inflammation. Induction of MMPs production is regulated in a complex manner.

 Skin * In addition to vasculopathy beach pain, such as arterial obstructive cranial fistula and venous stagnation cranial fistula, ulcers caused by persistent pressure irritation as persistent scars are difficult to treat. In addition to diabetic beaches, there are burned beaches, traumatic carcinogenesis, radiation beaches, drug beaches, postoperative beaches, inflammatory beaches, and simple beaches, depending on the cause. Chronic (refractory) skin carcinoma involves a variety of factors, such as aging, physical factors, or the underlying underlying illness, and it is not uncommon to have wound infections Therefore, many require long-term treatment. For the treatment of these difficult-to-treat skin ulcers, especially chronic (refractory) clarifiers, purified sucrose “popidone syrup, tocoletinate, bucladecine sodium, dexdexomer / iodine, etc. are currently used.

Most of these drugs are drugs for the sterilization and cleansing of the affected area, and wound poisoning agents.They have been developed with the aim of actively promoting cell proliferation and tissue regeneration in the affected area. There are few things. Chronic (refractory) Because the site, size, pathological condition, and other factors are different and the factors that prevent healing are still unclear, no drug that has a strong effect alone has appeared so far. In the future, it is expected that the proportion of elderly people over the age of 65 to the population will increase and the number of ill elderly people will also increase, including chronic (refractory) ulcers such as decubitus humor and lower limb pain. Since the number of patients is expected to increase, it is desired to develop a novel (intractable) dermatological skin fistula alone and a novel drug that acts widely on wounds.

 It is said that millions of operations are performed annually in Japan, but at least if the healing of surgical scars in simple surgery can be shortened, the hospitalization period can be shortened. This in turn will reduce the economic burden on healthcare financing, such as health insurance. Furthermore, the prevention of keloid II hypertrophic scars and the prevention and treatment of trauma scars in postoperative skin defect wounds are extremely useful from the aesthetic point of view, which is an important element of qualityofi1fe. However, wound healing agents with direct medicinal effects that are extremely effective for such purposes, healing promoters for surgical wounds by surgery, and agents for preventing the development of keloids and hypertrophic scars in postoperative skin defect wounds have been developed to date. There is no actual situation. For clinical considerations and explanations related to skin deficiencies, see, for example, “Christopher Surgery (Second Edition)” (translated by Koichi Ishikawa and one other, published by The Medical College) Truss, Diagnosis and Treatment "(Jun Kukida et al. Supervised and edited by Clinical Pharmaceutical Research Group, Chugai Medical)

"Recent Advances in Wound Healing" (edited by Kiyonori Harii, edited by Takahiko Moriro, published by Katsu Seido Shuppan Co., Ltd., 1993), "Dressing New Wound Management" (edited by Sadao Anazawa, Aki Kuramoto et al. 3) (Edited name, published, published in 1995), etc., and these descriptions are referred to as appropriate and used to understand problems. Disclosure of the invention

 An object of the present invention is to provide a pharmaceutical composition containing τ IMPs as a main active ingredient to treat or prevent chronic (refractory) beach pain, treat a wound, and furthermore, to remove surgical scars caused by various surgical operations. An object of the present invention is to provide a treatment means characterized by prevention and treatment of scars and reduction of surgical scars ゃ trauma scars.

 The present inventors have determined that TIMPs is an endogenous inhibitor present in tissues and body fluids and specifically inhibits the activity of MMPs, a growth factor-like activity on cultured epidermal keratinocytes and cultured fibroblasts. Focusing on the fact that TMPs inhibit the activity of MMPs, it is possible to provide an effective treatment for chronic (refractory) actin, and we conducted various studies of its clinical usefulness. Thus, the present invention succeeded in providing a therapeutic agent for skin deficiency deficiency comprising T IMPs as an active ingredient, in particular, a therapeutic agent for treating skin hamlet and a therapeutic agent for wound.

 The present inventors set up TIMPs, which were conventionally difficult to obtain in large quantities from tissues or cultured cells or blood derived from animals including humans, and it was difficult to apply or use them strictly in actual practice. In addition to obtaining large quantities using the recombinant DNA technology, the recombinant TIMPs (rTIMPs) thus obtained can be used to confirm what kind of pharmacological action they actually have, and that they can be used clinically. Thus, the present invention has been completed.

 The present invention

 (1) An agent for treating skin deficiency, which comprises at least one tissue inhibitor selected from the group consisting of meta-proteases as an active ingredient (a pharmaceutical composition for treating skin deficiency, hereinafter the same) ),

(2) The therapeutic agent is used in the treatment of skin deficiency, including the prevention of scarring due to skin deficiency and the preventive treatment for reducing the scar. The therapeutic agent according to the above (1), wherein

 (3) The therapeutic agent according to the above (1), wherein the skin deficiency is a wound,

 (4) The therapeutic agent according to (3), wherein the wound is a surgical wound, and the surgical wound or a surgical scar related thereto is reduced by preventive treatment.

 (5) The therapeutic agent according to the above (3), wherein the wound is a wound wound, and the wound wound or a wound scar related thereto is reduced by preventive treatment.

 (6) The therapeutic agent according to the above (1), wherein the skin deficiency is a cutaneous gallbladder.

 (7) The method according to the above (6), wherein the skin bleeding is postoperative fresh sputum, and the postoperative phlegm or related postoperative scar is reduced by preventive treatment. Therapeutic agents,

 (8) The above-mentioned (6), characterized in that the cutaneous pain is a traumatic beach, and the traumatic tumor or related scars associated therewith is reduced by preventive treatment. Described therapeutic agent,

 (9) The therapeutic agent according to the above (6), wherein the cutaneous fistula is renal dysfunction.

 (10) The therapeutic agent according to (6), wherein the skin slide is decubitus fulminans.

 (1 1) The therapeutic agent according to (6), wherein the skin acne is a vasopathic fistula,

 (1 2) The therapeutic agent according to the above (6), wherein the skin phlegm is inflammatory phlegm,

(13) The description of (6) above, wherein the skin rash is a simplicity exercise. Therapeutic agents,

 (14) The therapeutic agent according to any one of the above (1) to (13), which comprises a tissue inhibitor of meta-oral proteinase 11 as an active ingredient.

 (15) The therapeutic agent according to the above-mentioned (14), which comprises recombinant tissue inhibitor metabolic protease 1 as tissue inhibitor metabolic protease-11.

 (16) Recombinant Tissue Inhibitor Metallob Tissue 1 is expressed by transformants obtained using Escherichia coli, CH0 cells or C0S-1 cells as host cells, and the obtained gene product is purified. The therapeutic agent according to the above (15), wherein

(17) The therapeutic agent according to any one of the above (1) to (13), wherein the therapeutic agent comprises a tissue inhibitor metabolic proteinase 12 as an active ingredient.

 (18) The therapeutic agent according to the above (17), which comprises a recombinant tissue inhibitor metabolic proteinase 2 as tissue inhibitor metalloproteases 2.

(19) Recombinant tissue inhibitor meta-proteinase 2 is expressed by transformants obtained using Escherichia coli, CHO cells or COS-1 cells as host cells, and the resulting gene product is purified. (20) a mixture of tissue inhibitor metabolic protease 1 and tissue inhibitor metabolic protease 2 as the tissue inhibitor metabolic proteases of the above-mentioned (18). A therapeutic agent according to any one of the above (1) to (13), wherein (21) tissue inhibitor metabolic protease 1 (20) characterized in that it contains recombinant tissue inhibitor metabolic protease 1 and recombinant tissue inhibitor metabolic protease 2 as tissue inhibitor metabolic protease 12 respectively. The therapeutic agent as described in and

 (22) Transformation obtained by using recombinant tissue inhibitor meta-oral protease 1 and recombinant tissue inhibitor metabolic protease 2 using Escherichia coli, CHO cells or COS-1 cells as host cells, respectively. The therapeutic agent according to the above (21), which is obtained by purifying the obtained gene product expressed by somatic cells, is provided.

 Further, in the present invention,

 (23) The therapeutic agent according to any one of the above (1) to (22), wherein the therapeutic agent is in a dosage form suitable for local administration to an affected part.

 (24) The dosage form of the therapeutic agent is ointment, plaster, solution, solution, oil, cream, paste, cataplasm, liniment, lotion, spray, suspension, emulsion The therapeutic agent according to any one of the above (1) to (23), which is one selected from the group consisting of a tincture for external use, a skin solution, an irrigation agent and a patch. ,

(25) The above-mentioned (1), wherein the dosage form of the therapeutic agent is one selected from the group consisting of a dressing agent, a skin dressing or a descendant, a skin adhesive, and a bandage. ) To (23), (26) a pharmaceutically acceptable carrier, excipient, or diluent. (27) a tissue inhibitor, wherein the effective amount of at least one tissue inhibitor selected from the group consisting of metallobutal proteases is A method of treating a skin deficiency characterized by administering to a patient,

 (28) The method according to the above (27), wherein the method is intended to prevent scarring of skin deficiency and to reduce the scarring.

 (29) Wounds, cutaneous fistulas, surgical wounds due to surgery, trauma wounds, vasopathic phallic ulcers, including arterial occlusive lower ulcer fistulas and venous stasis lower limb fistulas, pressure sores due to continuous compression Any one of the diseases selected from the group consisting of diabetic sore, burn sore, traumatic lysis, radiation beach fistula, drug fistula, postoperative fistula, inflammatory acne and simple fistula. (27) or (28), which comprises treating

 (30) «Local administration to fistulas and wounds

7) The method according to any one of (29) to (29),

 (31) Tissue inhibitor characterized by using an effective amount of at least one tissue inhibitor selected from the group consisting of meta-oral proteases in artificially cultured skin) How to treat, and

 (32) To provide a use of a tissue inhibitor selected from the group consisting of tissue inhibitors of meta-oral proteases for producing a pharmaceutical composition for treating or preventing dermatosis deficiency.

 According to the present invention,

(33) Kodama eta l., Collagen Re l.Res., 7, 34 1-350, 1 987 or Kishiand Hayayawa, J. Bioch em., 96, 395-404, 1 According to the method described in 984, 培養 culture medium of cells derived from pulp pulp 軟 cartilage, pulp, gingiva, uterus, placenta, placenta, skin, and articular cartilage derived from humans, psoriasis, and other animals containing human serum Cells, osteoblasts, fibroblasts from various tissues, or epithelial cells, amniotic fluid, serum, plasma, platelets, monocytes, macrophages, Or the therapeutic agent according to the above (1), which comprises T IMP-1 obtained from the culture solution as an active ingredient.

 (34) Kodama eta 1., J. Immunol 1. Contains TIMP-1 obtained from human placenta as an active ingredient according to the method described in Methods, 127, 103-108, 1990. The therapeutic agent according to the above (1),

 (35) Fu jimo toeta l., CI in. Chim. Acta, 220, 31-45, 1993 or according to the method described in JP-A-6-300757. Other animal-derived cartilage, dental pulp, gingiva, uterus, skin, etc., articular chondrocytes, osteoblasts, fibroblasts from various tissues, or epithelial cells, amniotic fluid, serum, plasma, platelets, monocytes, macrophages Or the therapeutic agent according to the above (1), comprising T IMP-2 obtained from a culture thereof as an active ingredient,

(36) W i 11 i ams on, eta l., Biochem. J., 268, 267-274, 1990, Boone, eta 1., Proc. Natl. Ac ad. USA., 87, 2800-28004, 1990. The therapeutic agent according to the above (1), which contains rTIMP-1 or NO and rTIMP-2 obtained as active ingredients, according to the method described in (1), (37) Synthesis of 1stst rand cDNA by reverse transcription reaction using poly (A) mRNA prepared from human normal gingival fibroblasts (Gin-1 cells) as type I and oligo dT (15-18) as primer Then, the PCR primer was used to amplify the TIMP-1 gene using the PCR primer: primer Primer T1 F1; 5'-ATGGCCCCCT TTGAGCCCCTG-3 'and primer T1RI; After subcloning the obtained TIMP-1 gene into a suitable cloning vector, for example, pUC13, Cloning into a current vector, for example, pMEMneo (Le eeta 1., Nature, 294, 228-232, 19981), introducing it into a host cell, and obtaining the TIMP-1 transgenic yeast and CHO cell thus obtained. The therapeutic agent according to the above (1), which comprises r TIMP-1 prepared as an active ingredient by culturing the transformant containing

 (38) Synthesis of 1ststr and cDNA by reverse transcription reaction using poly-A mRNA prepared from human G in-1 cells as type I and oligo dT (15 to 18) as primers. PCR primer: primer T2F7: 5'-AAAGTCGACCATGGGCGCCGC GGCCCGCACCCT—3 'and primer T2R5; 5'-TT

After amplification of the TIMP-2 gene by PCR reaction using AGGAATTCTTGC-3 ', the resulting TIMP-2 gene was subcloned into an appropriate cloning vector, for example, pUC13, and then an appropriate expression vector was added. As an active ingredient, rTIMP-2 prepared by cloning into pKG, introducing into host cells, and culturing the thus obtained transformant cells including the transfected CHO cells with TIMP-2 as an active ingredient (see above). 1) The indicated therapeutic agent,

 (39) The therapeutic agent according to the above (1), which contains T IMP-1 prepared as described in Example 1 as an active ingredient.

 (40) The therapeutic agent according to the above (1), which contains T IMP-2 prepared as described in Example 1 as an active ingredient.

 (41) The therapeutic agent according to the above (1), which comprises rT IMP-1 prepared as described in Example 1 as an active ingredient.

(42) The therapeutic agent according to (1), which comprises rT IMP-2 prepared as described in Example 1 as an active ingredient. (43) The therapeutic agent according to the above (1), which contains rTIMP-1 as an active ingredient, prepared according to the method described in JP-A-5-199868, and

 (44) The therapeutic agent according to (1) above comprising rT IMP-2 as an active ingredient, which is prepared according to the method of A0ki et al. (Connecti ve Tissue, 1995; 26: 281-290). Is done. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the results of a stability test of a liquid formulation containing rTIMP-2.

 Figure 2 shows the change in the area of the skin defect due to the administration of rTIMP-2 in the SD rat experimental skin loss model.

 FIG. 3 shows the change in the area of the skin defect due to the administration of rTIMP-2 in an experimental model of skin deficiency in aged ICR mice.

 FIG. 4 shows the change in the area of the skin defect due to administration of rTIMP-2 in an experimental diabetic mouse skin deficiency model.

 FIG. 5 shows the results of a dose-dependent test of the therapeutic effect on skin deficiency of a test drug containing rTIMP-2.

 FIG. 6 is a photograph of a typical histopathological finding of the group treated with a test drug containing rTIMP-2 on day 15 of the test in a diabetic mouse experimental skin defect model, showing a photograph of the form of the organism.

 Fig. 7 shows typical histopathological findings of the group treated with the test drug containing rTIMP-2 on day 5 in the diabetic mouse experimental skin defect model. Fig. 6 is an enlarged view of the boxed part in Fig. 6. The photo shows a picture of the creature's form.

Figure 8 is a photograph of typical histopathological findings of the control group versus the test drug treatment group containing rTIMP-2 in the diabetic mouse experimental skin deficiency model. A photograph is shown. FIG. 9 is a magnified photograph of a boxed part of FIG. 8 showing a typical histopathological finding of a control group in a test with an experimental skin defect model in diabetic mice, and shows a photograph of the form of an organism.

 FIG. 10 shows the growth promoting effect of rT IMP-2 on human normal dermal fibroblasts.

 FIG. 11 shows the growth promoting effect of rT IMP-2 on normal skin fibroblasts in percentage relative to control.

 FIG. 12 shows the results of comparing the therapeutic effects of the test drug containing rT IMP-1 and the test drug containing rT IMP-2 on skin defects. BEST MODE FOR CARRYING OUT THE INVENTION

 The TIMPs used in the present invention may include inhibitors for the MMP family, and for example, inhibitors for MMPs such as TIMP-1 and TIMP-2 can be used. In particular, TIMP-2 has many excellent properties as a therapeutic agent for skin deficiencies, particularly as a therapeutic agent for skin fulminants and a wound. In particular, rTIMP-2 shows excellent properties.

TI MP-1 is a glycoprotein, and is used in tissues such as cartilage, dental pulp, gingiva, uterus, placenta, and skin from humans, pests and other animals, as well as articular osteocytes, osteoblasts, and fibroblasts from various tissues It is produced from cultured cells such as epithelial cells, amniotic fluid, serum, plasma, platelets, monocytes, macrophages, etc., and can be prepared from a condition culture of these tissues and cultured cells. For example, Kodama et al., Collagen Re 1. Res., 7, 341-350, 1987, and Kishi and Hayakawa, J. Biochem., 96, 395-404, 1984. According to the method described in, 培養 Culture of pulp-derived cells 細胞 Monochrome from human serum It can be isolated by affinity chromatography using a null antibody. Also, it can be obtained from human placenta or the like according to the method described in Kodama eta 1., J. Immuno 1. Methods, 127, 103-108, 1990.

 TIMP-2 was found relatively recently and has about 40% homology with TIMP-1 in amino acid sequence, but differs from TIMP-1 — binds conjugated oligosaccharide chains I haven't. TIMP-2 can also be obtained from various tissues, culture solutions of tissue-derived cultured cells, and the like as in the case of TIMP-1. For example, according to the method described in Fu jimo toeta]., Clin. Chim. Acta, 220, 31-45, 19993, JP-A-6-300757, etc., monoclonal antibodies can be obtained from human placenta or the like. It can be obtained by affinity chromatography or the like. Although TIMP-2 has a different property from TIMP-1 and a different molecular structure without N-linked oligosaccharide chains, TIMP-2 has a direct therapeutic effect on wounds, It is very useful as an agent for promoting healing, and as an agent for preventing the development of keloid II hypertrophic scars in skin defect wounds after surgery.

TI MP-1 and TI MP-2 are based on recombinant DNA technology (Maniatis, T. eta l., Molecular Cloning, Co Id Sprin Harbo r Labo ratory, Co) d Sp ri ng Harbor, ew York, 1982). There are certain limitations inherent in the use of conventional cultures of cell cultures or serum, or even from the placenta.However, the production method using recombinant DNA technology imposes such restrictions. Without receiving it, it is possible to obtain TIMPs in quantity and quality that can be used in animal experiments and clinical tests required for approval of the production of pharmaceuticals. In particular, the healing process of wounds and skin Ithamaki involves a complex multi-step process as described above, and each stage involves various in vivo cells and bioactive factors that are different from each other. Because they work in a variety of balances, it is possible to determine what role TIMPs actually plays under pathological conditions, what kind of pharmacological activity and biological activity, and what kind of disease is effective. It should be understood that is difficult. From this point of view, recombinant DNA technology can be used to produce the product in direct form and in sufficient quantity and quality to confirm its role in the healing process of complex wounds and skin cancer. In addition, rT IMPs obtained in a quantity and quality that can be used in animal experiments and clinical trials required for the approval of the manufacture of pharmaceuticals can be used for the treatment of skin deficiency, especially for the treatment or prevention of skin blemishes and wounds. It is noteworthy to find therapeutic and healing promoting activities.

 For example, using nucleic acid sequences encoding TIMP-1 and TIMP-2, eukaryotic hosts such as yeast and Chinese hamster ovary cells (CHO cells), prokaryotic hosts such as Escherichia coli, and insect cells such as Sf21 For example, it can be obtained by using all hosts commonly used for recombinant DNA technology and expression vectors corresponding to the hosts. In the case of TIMP-1 which is a glycoprotein, a host vector system using eukaryotic cells as a host is preferably used. Acquisition of rTIMP-1 and rTIMP-2 by the recombinant DNA method is performed, for example, according to Williams on, eta 1., Biochem. J., 268, 267-274, 1990, Boone, USA., 87, 2800-2804, 1990. The method described in eta 1., Proc. Natl.

For example, rTIMP-1 is 1ststrand by reverse transcription reaction using poly-A mRNA prepared from human normal gingival fibroblasts (Gin-1 cells) as type I and oligo dT (15-18) as a primer. c DNA Then, using a PCR primer, for example, primer TIF1; 5'-ATGGCCCCCTTTGAGCCCCTG-3 'and primer T1R1; 5'-CAGGATTCAGGCTATCTG-3', amplify TIMP-1 gene appropriately by PCR reaction. After subcloning the obtained TI MP-1 gene into an appropriate cloning vector, for example, pUC13, an appropriate expression vector, for example, pMEMne0 (Leeeta 1., Nature, 294, 228-232, 198 1), and introduced into CH 0 cells and the like by a common calcium phosphate coprecipitation method. Transformants, such as TIMP-1 transgenic yeast and CHO cells, are cultured by a suitable method such as microcarrier culture or high-density continuous culture, and rTIMP-1 is prepared from the resulting condition medium. I do. It should be understood that, depending on the host cell into which the expression vector has been introduced, the sugar structure of the gene product may be different from that of the natural product.

RT IMP-2 can be obtained by reverse transcription using, for example, poly A "mRNA prepared from human G in-1 cells as a type I and oligo dT (15 to 18) as a primer. d Perform cDNA synthesis and then use PCR primers, for example, primer T2F7; 5'-AAAAGTC GACCATGGGCGCCGCGGCCCGCACCCT-3 'and primer T2R5; The gene is amplified, and the obtained TIMP-2 gene is subcloned into an appropriate cloning vector, for example, pUC13, and then cloned into an appropriate expression vector, for example, pKG. Transfected cells such as CHO cells were cultured by an appropriate method such as microcarrier culture or high-density continuous culture. Prepare rT IMP-2 from the medium.

 RT IMPs obtained by using recombinant DNA technology may have one or more mutations in its constituent amino acids, such as deletions, substitutions, insertions, transpositions, etc., so long as they are compatible with the purpose of the present invention. The amino acid sequence can be different due to addition. Using recombinant DNA technology, for example, one or more amino acids can be deleted, substituted, or inserted by mutagenizing a specific site of the basic DNA sequence. A representative method is the position-directed mutagenesis method (edited by The Biochemical Society of Japan, "New Biochemistry Experiment Course 2, Nucleic Acid 111, Recombinant DNA Technology", Tokyo Kagaku Dojin, pages 233 to 251, October 5, 1992) ).

 TIMPs derived from these various sources can be obtained by conventional methods used for the purification of normal proteins, such as salting out using ammonium sulfate and sodium sulfate, gel filtration carriers and ion exchange carriers. Chromatography, electrophoresis, monoclonal antibodies against each TIMPs, affinity chromatography using ConA, etc., high performance liquid chromatography, etc. can be used alone or in any combination according to the properties of each TIMP. It can be used and purified as appropriate. By the way, there is a limit in obtaining a natural product as a homogeneous product from a microscopic point of view.For example, TIMP-1 which is a natural substance is contained in a molecule due to the effect of processing during the process of isolation or isolation. In fact, it is a product that has various forms of sugar chains and is therefore composed of substances with various molecular weights and the like.

Since it has a small amount of biological activity and is difficult to completely separate and purify, it is considered that rT IMPs does not contain contaminants that are normally involved in non-recombinant cells (non-transformants). In particular, rTIMP-2 can be obtained as a final product containing little denatured protein and the like after purification and isolation. rT IMP-2 is for the treatment of complex wounds and skin sores It is said to be obtained in a sufficient amount and purity to confirm its function in the healing process and to be sufficient for use in animal tests and clinical tests required for the approval of the production of pharmaceuticals. Its properties are recognized and it is excellent as an active ingredient of medicine.

 The present invention provides a pharmaceutical composition containing TIMPs as an active ingredient and containing a pharmaceutically and pharmaceutically acceptable pharmaceutical additive. The active ingredient selected from the group consisting of TIMPs can be administered in the range of about 0.000 lng to about 100 mg per day, but of course the symptoms, age, degree of disease, Depending on the concomitant drug, etc., the dose can be selected so as not to cause any side effects, and the number of administrations can be appropriately selected, such as once to multiple times per day. The active ingredient selected from the group consisting of TIMPs can be incorporated into the drug product in the range of 0.001% to 50%, but the amount can be appropriately selected as necessary. It is possible.

The pharmaceutical composition of the present invention is prepared by mixing and the like, and if necessary, stabilizers, PH regulators, surfactants, mouthwashes, fragrances, preservatives, bases, solvents, diluents, fillers Agent, extender, solubilizer, solubilizer, isotonic agent, emulsifier, suspending agent, dispersant, thickener, gelling agent, curing agent, absorbent, adhesive, elasticizer, plastic Agents, binders, disintegrants, radiation agents, preservatives, antioxidants, sunscreens, humectants, emollients, antistatic agents, soothing agents, etc., alone or in combination. Stabilizers include amino acids such as glycine or salts thereof, sugars such as glucose and sucrose, sugar alcohols such as mannitol and sorbitol, oligosaccharides, polysaccharides, albumin, gelatin, proteins such as globulin and protamine, and peptides And the like. Examples of the pH regulator include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and carbonic acid, organic acids such as citric acid, and salts thereof. Other, distributed to pharmaceuticals It can be appropriately selected as necessary from those known to be used in combination.

 The pharmaceutical composition of the present invention can be adapted for oral, topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraarticular administration, but is preferably applied locally to a fistula or a wound. Of course, it can be used in combination with other drugs such as hamagi and wound treatments. In addition, cytokines such as PDGF, TGF-, growth factors such as TGF- / 3, IGF-I, CSF, IL-8, EGF, bFGF, KGF, and other physiologically active substances are also available. Mixing is also possible if it is effective for the treatment of skin deficiency of the present invention. In some cases, it can be used together with artificially cultured skin, and it can also be used in combination with medical materials for treatment or medicines that are considered to be effective for treating skin deficiency symptoms.

 When the pharmaceutical composition of the present invention is to be administered locally to the affected part, ointments, descendants, solutions, solutions, oils, creams, pastes, cataplasms, remedies, lotions, sprays Preparations, suspensions, emulsions, tinctures for external use, skin solutions, irrigation agents, patches and the like can be arbitrarily selected. Further, a liquid agent or the like can be used by impregnating gauze, absorbent cotton, a wound dressing material, an adhesive blaster, or the like.

 In the manufacture of pharmaceuticals, the additives and preparation methods are, for example, supervised by the Japan Public Health Association, 11th revised edition of the Japanese Pharmacopoeia, published on July 18, 1986, published by Hirokawa Shoten Co., Ltd. Pharmaceutical Development Vol. 12 (Pharmaceutical Ingredient [I]), published on October 15, 1990, Hirokawa Shoten Co., Ltd .; Pharmaceutical Development Vol. 12 (Pharmaceutical Materials [11 )) Published on October 28, 1990, referring to the descriptions of Hirokawa Shoten Co., Ltd., etc., and selecting from them as needed and applying them.

When used for the treatment of skin deficiencies, absorption of active ingredients through the skin, etc. Although considered important, moistening may generally improve drug absorption. As long as it is effective for the treatment of skin ia deficiency of the present invention, any method and means known in the art can be used. As the TIMPs contained in the pharmaceutical composition of the present invention, any TIMPs that inhibit MMPs activity can be used. Preferably, a large amount of homogeneous TIMPs can be obtained. In this respect, artificially prepared r TIMPs can be used. In particular, rTIMP-2 can be obtained in an amount and purity that is homogeneous and effective in the healing process of wounds and skin ulcers, and has excellent properties in handling. When used as a medicine, strict requirements are required for the stability of the preparation. TIMP-2 also has excellent properties in this respect.

 The quantification of TIMPs required for preparing the pharmaceutical composition according to the present invention was performed according to the methods described in JP-A-63-210665 and JP-A-5-244895, TIMP-1 or TIMP-2 can be performed by enzyme immunochemistry using a sandwich assay using two types of monoclonal antibodies that specifically recognize different epitopes.

 The confirmation of the effect of the pharmaceutical composition according to the present invention is typified by various wounds, skin ulcers, surgical wounds by various surgical operations, trauma wounds, arterial occlusive lower limb veins, and vein g stagnating lower limbs * ulcers Vascular disorder fistula, pressure ulcer due to sustained pressure stimulus, diabetic ulcer, burn injury, traumatic fistula, radiation bleb, drug phlebotomy, postoperative phlebology, inflammatory fistula, simple It can be confirmed by using a model suitable for evaluating the activity of treating skin deficiency, in particular, the activity of treating or preventing skin ulcers and the treatment of wounds, and the activity of promoting healing, using a model such as rosacea.

In particular, in the test for confirming the effect of the pharmaceutical composition according to the present invention, various methods besides the power that can be performed using the experimental skin defect (¾ pain and wound) model and the experimental surgical wound model described below are used. Understand that you can do Should.

 The effect confirmation test using an experimental skin defect model can be evaluated as follows. Skin defects are made in two places with a circular punch (8 mm ID) on the back skin of rats or mice that have been shaved in advance under ether anesthesia. T IMPs-containing solution (vehicle 0.005% benzonalconium chloride-saline: 0.005% benzalkonium chloride 0.15M NaCl solution, pH 7.2) was used as a test drug daily for the skin defect. Twenty-two times a day. -On the other hand, as a control, only the physiological saline containing 0.005% benzaluconium chloride without TIMPs is administered to the affected area every day. As a result, compared with the control group, in the group to which the pharmaceutical composition of the present invention was administered, reduction of the defect area of the skin * -deficient lesion was promoted, and the number of days to complete healing was shortened. The improvement in blood color was also remarkable. More notably, the re-epithelialization of the affected area of the skin defect, the formation of granulation tissue, and angiogenesis were promoted, the repair of the affected area was promoted, and the treated scar was small and less noticeable. This effect is not only useful as a therapeutic agent for chronic (refractory) nausea, but also can be used to reduce general surgical scars and trauma scars. It was judged to be very useful from the perspective of aesthetics occupying the elements.

 The effect confirmation test using the experimental surgical wound model can be evaluated as follows. For example, diabetic mice (KK—Ay ZT a Jc 1), 6 weeks old, weighing about 30 g, were subjected to Nembutal anesthesia (3 Omg / kg, ip) and pre-shaved and disinfected from the back to the abdomen. Then, make a flank incision about 10 mm in length perpendicular to the midline with a scalpel. Immediately on one incision, a solution containing TIMPs as a test drug (vehicle 005% saline containing benzalkonium chloride; 0.005

0.1% 5M NaCl solution, pH 7.2 containing 5% benzalkonium chloride) Two drops (approximately 22 1) of a control saline solution containing 0.05% benzalkonium chloride as a control were added to the other cut using a syringe with a 26 G needle. Suture using silk thread. After that, the test drug or control solution is continuously applied topically (drip) once a day for 7 days. The wound site should be hermetically sealed with a coating (bi0c1 usi Ve) and an elastic bandage (Silkitex) to prevent contamination. The thread is removed on the 7th day of the test, and the healing status of the affected area and the degree of scar formation are determined with the passage of time. The surface tissue of the affected area is collected and histopathologically examined to evaluate the healing effect. The gross findings are evaluated based on the size of the suture wound compared to the control group and the blood color of the suture wound and the surrounding area. Histopathological findings are evaluated based on the balance between degeneration, necrosis and reconstructed images, fibrosis and infiltration of inflammatory cells and edema compared to the control group.

 In addition, no side effect judged visually was found at the site to which the pharmaceutical composition according to the present invention was applied.

Drugs containing at least one tissue inhibitor selected from the group consisting of TIMPs as an active ingredient in this manner include, in addition to vasculopathy beach fistulas represented by arterial occlusive lower limb fistula and venous g lower limb fistula, Pressure ulcers and diabetic beach pain due to persistent pressure irritation, which are difficult to treat, fever burn sores, traumatic necrosis, radiation fistula, drug ulcer, postoperative beach fistula, inflammatory It is considered to have a preventive or curative effect on the nodules, simple nodules, etc., and a symptom improving effect. Drugs containing at least one tissue inhibitor as an active ingredient selected from the group consisting of TIMPs include chronic (refractory) skin ulcers and other factors such as aging, physical factors, or serious underlying diseases. Is involved in various factors, such as the presence of a background in some cases, and is considered to have significant activity against symptoms and diseases that are not rarely associated with wound infections. At least selected from the group consisting of TIMPs A drug containing one tissue inhibitor as an active ingredient is useful alone as a drug having a strong effect. In addition, drugs containing at least one tissue inhibitor selected from the group consisting of TIMPs as an active ingredient have been shown to prevent the development of keloid ゃ hypertrophic scars and prevent traumatic scars in skin * defective wounds after surgery. It is useful as a therapeutic agent for wounds having a therapeutic effect, an agent for promoting healing of surgical wounds by surgery, and an agent for preventing the occurrence of keloid-thickening scars in skin defect wounds after surgery. In addition, TIMPs are useful as therapeutic agents for chronic (refractory) ulcers.

 Drugs containing at least one tissue inhibitor selected from the group consisting of rTIMPs, in particular, rTIMP-2 as an active ingredient, have the advantages described above, in addition to their advantages, including their processing as pharmaceuticals. It has excellent activity on various kinds of wounds, wounds caused by surgery, skin ulcers and the like. The therapeutic agent for skin deficiency containing rT IMP-2 can be expected to have stable biological activity even after long-term storage, and can be expected to have excellent biological activity even after being applied to the affected area. Example

 Next, the present invention will be described more specifically with reference to examples. However, it should be understood that the present invention is not limited to these specific examples and can be implemented in various forms as long as the idea is followed. It is.

Example 1 Preparation of TIMPs

TIMP-1 can be obtained from human normal gingival fibroblasts (Gin-1 cells) according to the method described in, for example, Kodamaeta 1., Co 11 age Re 1. Res., 7, 341-350, 1987. U 1 troge 1 Ac A-44 (LKB), Con A Sepharose, anti-TIMP-1 monoclonal antibody (for example, clone No. 7 disclosed in JP-A-63-219392). — 2 IB 12, etc.) Join Purify using a Sepharose 4Β affinity column. Finally, treat at 35 ° C for 30 minutes in 4M guanidine monohydrochloric acid, and then test on a Bio-gel P60 column. Protein factors that may have bound to 1 were reconstituted and removed by fractionation to prepare.

 T IMP—2 is, for example, F u j i mo t o e t a 1., C l i n.

According to the method described in Chim. Acta, 220, 31-45, 19993 and Japanese Patent Application Laid-Open No. Hei 6-300757, the placenta was shredded, and the mixture was stirred in a loose mouth liquid to obtain a mixture. The supernatant was subjected to an anti-TIMP-2 monoclonal antibody (for example, clone No. 67-4H11 disclosed in Japanese Patent Application Laid-Open No. Hei 5-244985) and bound to a Sepharose 4B affinity column. Thereafter, it was prepared using U1troge1AcA-44 (LKB).

 rTIMP-1 was prepared, for example, according to the method described in JP-A-5-199868. From a total RNA fraction prepared from human normal gingival fibroblasts (Gin-1 cells), poly (A) -mRNA was purified and concentrated using an oligo (dT) -cellulose column. The reverse transcription reaction using polyA mRNA as type I and oligo dT (15 to 18) as primers resulted in the synthesis of 1ststrandcDNA. Refer to the cDNA sequence of TIMP-11 described in Dochertyeta 1., Nature, 318, 66-69, 1985, PCR primer, primer TIF1; 5'-ATGGCCCCCTTTGAGCCCCTG-3 ' And primer T 1 R 1; 5′-one CAGGATTCAGGCTATCTG-3 ′ were prepared, and the TI MP-1 gene was amplified by PCR using these PCR primers with the previously prepared 1 ststrand cDNA as type III. .

After subcloning the obtained TIMP-1 gene into an appropriate cloning vector, for example, pUC13, an appropriate expression vector, for example, pME The clone was cloned into Mneo (Leeeta l., Nature, 294, 228-2232, 19981) and introduced into CH0 cells by a common calcium phosphate coprecipitation method. The TIMP-1 gene-introduced CH0 cells were cultured by an appropriate method such as micromouth carrier culture or high-density continuous culture, and rTIMP-1 was prepared from the obtained condition medium.

The prepared rTIMP-1 was observed as a single band of about 30 kDa on sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE, 12% homogeneous gel, under reduced conditions). Western blotting using a labeled mouse anti-human TIMP-1 antibody also revealed a single band of about 30 kDa. rT I MP- 1 of human fibroblasts (CCD-4 1 SK cells) inhibition activity against derived MMP- 1 is, IC _5. There was approximately 1 X 1 0- ⁹ M.

r TIMP-2 was prepared, for example, according to the method of Aoki et al. (Continuous T issue, 19995: 26: 281-290). Poly-A mRNA was purified and concentrated using an oligo (dT) -cellulose column from the total RNA fraction prepared from human G in-1 cells. 1st strand cDNA was synthesized by a reverse transcription reaction using poly-A mRNA as type I and oligo dT (15 to 18) as primers. Sci. USA., 87, 2800-2804, 1990. PCR primers based on the cDNA A sequence of TIMP-2 as described in Bone, eta 1., Proc. Natl. Ac. , Primer T2 F 7; 5′-AAA and Primer T2 R 5; 5 ′ — TTAAGATCTGTCGACTT AAGGATCCTCGATATCGAGGAATTCTTGC-3 ′ TIMP—2 遗 Gene amplification was performed. The resulting TIMP-2 gene is subcloned into an appropriate cloning vector, for example, pUC13, and then cloned into an appropriate expression vector, for example, pKG, and introduced into CHO cells by a common calcium phosphate coprecipitation method. did. TIMP-2 transfected CHO cells were cultured by an appropriate method such as microcarrier culture or high-density continuous culture, and rTIMP-2 was prepared from the obtained condition medium. The prepared TIMP-2 was observed as a single band of about 24 kDa on SDS-PAGE (12% homogeneous gel, normal conditions), and was subjected to Western blotting using mouse anti-human TIMP-2 monoclonal antibody. In this case, it was recognized as a single band of about 24 kDa. The inhibitory activity against MMP-1 derived from CCD-41 SK cells was 1; There was about 1. 1 X 1 0- ⁹ M. The pharmaceutical composition provided by the present invention has the ability to use any TIMPs that inhibit MMPs activity. Preferably, a large amount of homogeneous TIMPs can be obtained. R TIMP s artificially prepared can be used. Example 2 Preparation of ointment containing rT IMP-1

 As ointment preparations, various publicly known ointment bases can be used. For example, rTIMP-1 can be added to macrogol ointment and formulated as follows.

 To 5 g each of Macrogol 400 and Macrogol 4000, 5 mg of paraoxyethyl benzoate and 11 mg of TIMP-11 obtained in Example 1 were added, and the mixture was uniformly mixed by a conventional method to prepare an ointment. Example 3 Preparation of ointment containing rTIMP-2

r The ointment containing TIMP-2 was applied in the same manner as described in Example 2. The rTIMP-2 obtained in Example 1 can be formulated by adding it to Macrogol ointment. Example i Preparation of rT IMP-1 containing solution

 R TIMP-1 obtained in Example 1 was added to a final concentration of 0.15% in a physiological saline solution containing 0.005% benzalkonium chloride (0.15 M NaCl solution containing 0.055% benzalkonium chloride, pH 7.2). %. The prepared solution was sterilized with a membrane filter with a pore size of 0.1 t / m, and stored refrigerated (4 ° C) until use. Example 5 Preparation of rTIMP-2 containing liquid

 The rTIMP-2 containing liquid preparation can be formulated using rTIMP-2 obtained in Example 1 in the same manner as in the method described in Example 4. Example 6 Daily stability of rT IMP-2 containing liquid preparation

 The stability of the rTIMP-2 containing solution stored at 4 ° C was evaluated by its inhibitory activity against purified human MMP-1 in invitro. The preparation of rTIMP-2 containing solution was carried out according to the method described in Example 5. Human latent MMP-1 can be obtained, for example, according to the method described in Fujimo toeta 1., C1 inn. Chim. Acta, 219, 1-14-1993, and human neonatal fibroblasts. (NB 1 RGB) can be purified from the culture supernatant.

 (1) Inhibition of inv itro inhibition activity of rTIMP-2 containing solution

 Measuring method

The measurement of the inhibitory activity was carried out as described in Nagaieta 1., Inflammation, 4, 123-130, 1984 and Nagaieta, Inflammation, 4, 24- 254, 1984.

 0.02% BSA, 0.2 M NaCl, 5 mM CaC solution containing 50 mM Tris-HC1 (pH 7.5) Add an equal volume of 2 mM 4-Aminophenyl mercuricacetate, 35. Incubate at C for 2 hours. To this was added 60 μl of r TIMP-2 containing solution diluted stepwise with 50 mM Tris-HC 1 (pH 7.5) buffer containing 0.2 M Na C 1 and 5 mM Ca C 1. (At this time, add only 60/1 of the mouth liquid as a control.) Incubate at 35 ° C for another 15 minutes. In addition, 0.1M Tris-HC1 (pH 7.5) buffer containing 0.4M NaC1.10mM CaC is used. Add 1 and incubate at 35 ° C for 2 hours (boil the control reaction at 100 ° C for 5 minutes). Stop the reaction by adding 10 l of o-L-n-anhydrous dissolved in 50% ethanol. Add 0,02% BSA, 0.2M NaC1, 5mM CaC and add 50mM Tris-HC1 (pH 7.5), diluted appropriately with Elastase 2001, and add 10 to 35. Incubate for minutes. To this is added a buffer of Tris-HC1 (pH 9.5) containing 400% of 70% ethanol and 0.67 M NaC1 and stirred with a mixer for 30 seconds. The fluorescence of the obtained supernatant is measured at a wavelength of Ex; 495 nm, Em; 520 nm.

 Assuming that the fluorescence intensity of the control was 100% of the substrate degraded, the difference in the fluorescence intensity of the sample was calculated, and the 50% inhibitory concentration (IC: ,, value) was determined.

 (2) Storage stability

The stability of r TIMP-2 containing liquid stored at 4 ° C was determined by IC. Evaluation was based on the values. As a result, this solution has at least 12 weeks of inhibitory activity. It was judged to be stable, and stable bioactivity could be expected even after storage for a relatively long time in refrigeration. The results of weekly inhibitory activity measurements are shown in FIG. Actual | Example 7_ Therapeutic effect of skin defects (wounds and ulcers) of agents containing rT IMP-2 (1) Preparation of test drugs containing rT IMP-2

 The rT IMP-2 containing solution prepared in Example 5 was used as the rT IMP-2 containing test drug. As a control, a physiological saline solution containing 0.005% benzalkonium chloride sterilized with a membrane filter having a pore size of 0.1 / m was used.

 (2) Test animals and experimental skin

 As test animals, 8 SD rats (12 weeks old, body weight: 254 to 385 g), 9 retired ICR mice (early 30 weeks old, body weight: 20 to 56 g) and 9 diabetic mice (KK-AyZTa J cl) ) (Early 6 weeks old, body weight 26-34 g) Using 6 animals, shaved and disinfected beforehand under ether anesthesia Use a circular punch (8 mm ID) on the back skin of these rats or mice. Two experimental skin defect sites were created symmetrically at the center. One site of skin frost deficiency was the site of administration of the test drug containing rTIMP-2, and the other site was the site of administration of the control solvent.

 (3) Administration of test drug containing rT IMP-2 and observation of affected area

 The test drug containing rTIMP-2 and the solvent for the control were continuously administered by injecting two drops (approximately 221) directly into the affected area using a syringe with a 26G needle once per test. For aged (retire) ICR mice and diabetic mice, the affected area was hermetically sealed with a covering agent (bioc1usiVe) and an elastic bandage (Silky Tex) so as not to contaminate the affected area of the skin defect.

Every day, measure the major axis and minor axis of the skin defect area with a caliper and use the following method. The change in wound area during the healing process was determined. Assuming that the product of the major axis and minor axis of the skin defect creation day (Day O) was 100%, the ratio to this was calculated by the following formula.

The major axis X minor axis of the skin defect (observed)

 Area ratio (%) = X100

 Large diameter X short diameter (Day O)

The change in the area of the skin defect was determined from the daily change in the calculated area ratio.

① SD rat test results

 Compared with the control group, the group administered the rTIMP-2 containing test drug significantly promoted the reduction of the skin defect area from day 4 onward, and showed a slight, but shorter, cure period. Typical results of the daily healing process are shown in FIG.

 ② Test results of aged ICR mice

 Compared with the control group, the group treated with the rTIMP-2 containing test drug significantly accelerated the reduction of the skin defect area from day 3 onward, and also showed a reduction in the number of days to complete healing. Figure 3 shows the average of the test results and the healing process.

 (3) Test results of diabetic mice

 The group treated with the rTIMP-2 containing test drug significantly promoted the reduction of the skin defect area from day 3 onwards as compared with the control group, and also showed a reduction in the number of days to complete healing. Figure 4 shows the average of the test results and the healing process.

The gross findings in all three of the test animals clearly showed that the test compound-administered group had good blood color at the skin defect site and a high degree of drying on the third day of the test as compared with the control group. On the 9th and 10th day of the test, the area of the skin defect site in the control group was significantly reduced and the blood color was also improved. I was approaching. Comparison of treatment site between control group and test drug administration group when skin defect is cured The treatment scar was smaller and less noticeable in the study drug administration group. In other words, what is noteworthy in the results of this study is that the treatment scar is small and less conspicuous as well as accelerates the healing of the skin defect, and when applied to humans, the occurrence of keloid ゃ hypertrophic scar in the skin defect wound And to prevent trauma. In these studies, no adverse reactions were observed at all. Example 8 Dose Dependence of Treatment Effect for Skin Defects of r TI MP-2 Containing Agent

(1) Preparation of test drug containing rT IMP-2

 The rTIMP-2 containing liquid prepared in Example 5 was used as the rTIMP-2 containing test drug. However, the content of rTIMP-2 was set to four types of 0.05%, 0.01% and 0.001% in addition to 0.1%. As a control, a physiological saline containing 0.005% benzalkonium chloride was used.

 (2) Preparation of test animal and experimental skin defect model

 Using 30 diabetic mice (KK-AyZTaJc1) (13 weeks old, body weight 39-55 g) as test animals, the site of an experimental skin defect on the back skin of the mouse by the method described in Example 7 Was made in two places. Each wound was used as a site for administration of a test drug or control solvent containing rTIMP-2 at different concentrations.

 (3) Administration of rTIMP-2 containing test drug and observation of affected area

 The test drug containing rTIMP-2 and the control were directly instilled once daily into the affected area as described in Example 7 and administered daily until wound closure. The affected area of the skin defect was sealed and bandaged with a covering agent (bi0c1usiVe) and an elastic bandage (Silky Tex).

Every day, the major axis and minor axis of the skin defect area were measured with a caliper, and the change in the area of the affected area was calculated by the method described in Example 7. The combination of different concentrations of rTIMP-2 test drug administered to the two wound sites

 (B) Control (0%) and 0.001%,

 (Mouth) 0.001% and 0.01%,

 (C) 0.01% and 0.05%,,

 (Ii) 0.05% and 0.1%,

 (E) 0.0 1% and 0.1%

 And 6 mice were used for each combination.

 Fig. 5 shows the change over time in the area of the wound after treatment with various concentrations of the test drug containing rTIMP-2. Compared with the control, the test drug containing rTIMP-2 at a concentration of at least 0.001% or more showed a clear wound healing promoting effect. There is a dose-dependent trend in the strength of the action, but the effects of 0.001% drug and 0.01% drug are almost the same, and 0.05% drug and 0.1% drug Showed almost the same effect. Example 9 Histological evaluation of the therapeutic effect of rT IMP-2 containing agent on skin defect

(1) Preparation of test drug containing rT IMP-2

 As the rTIMP-2 containing test drug, the rTIMP-2 containing liquid preparation prepared in Example 5 was used, and as a control, 0.005% benzalkonium chloride-containing physiological saline was used.

 (2) Preparation of test animal and experimental skin loss model

Using 30 diabetic mice (KK-AyZTa J cl) (8-13 weeks old, body weight 36-53 g) as test kills, an experimental skin defect site was formed on the back skin of the mice by the method described in Example 7. We made two places. One skin deficiency site was the site where the rTIMP-2 containing test drug was administered, and the other site was the site where the control solvent was administered. (3) Administration of r TIMP-2 containing test drug and histological evaluation of affected area

 The test drug containing rTIMP-2 and the control were directly instilled once a day at the dose described in Example 7 into the wound site and administered daily until wound closure. The affected area of the skin defect was hermetically sealed and bandaged with a coating agent (bioc1usiVe) and an elastic bandage (Silky Tex).

 On days 0, 5, 10, 15, 15 and 20, six animals were sacrificed by intraperitoneal administration of pentobarbital. Skin * was cut into a square at the depth above the fascia so as to include the entire wound, fixed with 10% formalin, and then a paraffin-embedded block was prepared. From each block, 5 m sectioned slides were prepared, and each slide was stained with hematin-lin-eojin.

 (4) Histological evaluation

 As the number of treatment days elapsed, a clear reconstructed image of the epidermis was observed in the group treated with the rTIMP-2-containing test drug as compared with the control group, and granulation tissue formation, dense matrix formation, and Angiogenesis was clearly promoted. In particular, re-epithelialization was remarkable, which was considered to be the result of histologically promoted migration of keratinocytes. In addition, it was considered that rTIMP-2 also has an action of promoting granulation.

Histological images of the control group and the test drug-treated group containing rTIMP-2 on the 15th day of the test are shown in FIGS. As can be seen from Fig. 6 and Fig. 7, which is an enlarged photograph of a part of the histological image shown in the figure, r TIMP-2 treated mice are completely healed (the wound is small) ( Figure 7 shows that the thickness of the epidermis at the healed site is the same as the thickness of the epidermis outside the wound, indicating that healing is more complete.) This is more apparent when comparing control mice (FIG. 8 and FIG. 9 which is an enlarged photograph of a part of the histological image shown in FIG. 8). Is insufficient, and the epithelial site is also healed in the group treated with the test drug containing TIMP-2. It can be seen that the thickness of the epidermis is thinner than the position). In addition, the histological findings of the wound were classified, quantified and evaluated according to the degree as follows.

 (a) Re-epithelialization: The image immediately after wound creation was set to 0, the time of complete healing was set to 10 and the degree of re-epithelialization was evaluated on a 10-point scale.

 (b) Granulation: Evaluated in the following four stages.

 1: light granulation 2; medium granulation 3; thick granulation 4; very thick granulation

(c) Matrix density: Evaluated in the following four steps.

 1; edema 2; small amount of coarse matrix 3; medium matrix 4; dense matrix

 (d) Number of cells infiltrated (fibroblasts, macrophages): evaluated in the following four steps.

 1; almost none 2; medium 3; many 4; very many

 (e) Number of capillaries per wound: evaluated in the following steps.

 0; 0 to 4 1; 5 to 14 2: 15 to 24 3: 25 to 3 4

 10; 95 to 104 e t c.

 For example, the results of quantifying and evaluating each histological finding of Study 10 are shown in the table.

Shown in 1. Numerical evaluation also confirmed that re-epithelialization, granulation, increased matrix density, and an increase in the number of capillary tubes were observed in the r TIMP-2 containing test drug-treated group compared to the control group. It was remarkable.

Table 1. Wound healing promoting effect of rTIIIIP-2 by histological evaluation. Treatment «1 ^ Re ± J¾ ^ ΙΒ» Matrix density a

^{Χ ^^ Ο ΒΡΒΙ 'RA P "} 2 3 4 .0 Sat ^{0 2.3 ± 0.6 2.3 ± 0.6 2.3} ± 0.6 13 ± 0.6

² X ^MQ ] ^MP " ^{2 3} 7.0 ± 1.7 3 ^Τ 3.0 ± 0 3.0 ± 0 2.3 ± 0.6 9.7 ± 1.

ρ <0.05

Example 10 Effect of a solution containing 10 rT IMP-2 on proliferation of human normal skin fibroblasts

 (1) Preparation of rT IMP-2 test drug

 The rTIMP-2 obtained in Example 1 is added to a physiological saline (0.15 M NaCl solution, pH 7.2), and the final concentration is adjusted to, for example, 1 Omg / m1. The prepared solvent was sterilized with a membrane filter having a pore size of 0.1 μm and stored refrigerated (4 ° C) until use.

 (2) Culture of human normal skin fibroblasts and determination of the effect of promoting proliferation Human normal skin fibroblasts are obtained by primary culture of human skin, or are commercially available, for example, commercially available from Conetics. Infant-derived normal skin fibroblasts can be used.

 Cell proliferation was measured by a dye extraction method.

Cells cultured in a single MEM medium containing 10% fetal bovine serum were inoculated in a 96-well microplate at 10 ³ ce 11 sZO. After overnight culture in a 5% CO-incubator at 37 ° C, the cells were washed three times with PBS to remove the medium. Eagle MEM medium containing fetal bovine serum supplemented with various concentrations of rTIMP-2 was added to the washed cells, and cultured at 37 ° C, 5% CO 'for 5 days. At this time, an Eagle MEM medium containing 0%, 1.25%, 2.5%, 5%, and 10% of fetal calf serum was used as a basal medium to which rTIMP-12 was added.

After completion of the culture, the medium was removed by suction, and 0.5% crystal violet dissolved in 20% methanol was added, followed by staining for 30 minutes. After gently washing the cells with distilled water and drying, the crystal violet taken up by the cells was eluted with 30% acetic acid. The eluted crystal violet was measured at an absorbance of 570 nm using a microplate reader (manufactured by Tosoichi, MPRA4). r The degree of cell proliferation was determined from the difference from the absorbance of the control cultured without TIMP-2. FIG. 10 shows the degree of proliferation of cells cultured at various rT IMP-2 ′ concentrations by absorbance. FIG. 11 shows the degree of proliferation relative to the control as a percentage.

 As a result, the proliferation of human normal skin fibroblasts was promoted more dose-dependently than the rTIMP-2 concentration SzgZml. In addition, when the rT IMP-2 concentration was 10 OjUgZml, the proliferation was enhanced by 40 to 50% compared to the control. As is evident from FIG. 11, regardless of the amount of fetal fetal serum contained in the medium, the growth promoting effect was exhibited uniformly depending on the rTIMP-2 concentration in the medium. That is, this effect does not depend on the amount of fetal calf serum in the medium, and can be estimated to be the effect of rTIMP-2 itself. Example 11 1 Wound healing promoting effect of rT IMP-1 and rT IMP-2

 comparison

 (1) Preparation of test drug containing rTIMP-1 and test drug containing rTIMP-2

 The rTIMP-1 containing liquid prepared in Example 4 was used as the rTIMP-1 containing test drug, and the rTIMP-2 containing liquid prepared in Example 5 was used as the rTIMP-2 containing test drug. As a control, a physiological saline solution containing 0.005% benzaluconium chloride sterilized with a membrane filter having a pore size of 0.1 m was used.

 (2) Test animals and experimental skin

Using 10 diabetic mice (KK-AyZTa J cl) (early 8 weeks old, body weight 31-36 g) as test animals, two experimental skin defect sites were created on the back skin of the mice by the method described in Example 7. did. The five mice were used as rTIMP-1 administration group and rTIMP-2 administration group, respectively. 1 place The site of skin deficiency was the site of administration of the test drug containing rTIMP-1 or rTIMP-2, and the other was the site of administration of the control solvent.

 (3) Administration of test drug and observation of affected area

 The test drug and control were applied once a day, and the dose described in Example 7 was directly dropped into the affected area and administered daily until wound closure. The protection of the skin It deficient part and the change in the area of the affected part were calculated by the method described in Example 7.

 Fig. 12 shows the daily changes in the area of the part when treated with rTIMP-1 and rTIMP-2. Both groups showed almost the same wound healing process, with no significant difference. There was no significant difference between the two groups when comparing the number of days required for 50% healing. That is, the concentration used in this test was 0.

At 1%, rT IMP-1 and rT IMP-2 were shown to have nearly equivalent wound healing effects.

At least one tissue inhibitor selected from the group consisting of rTIMPs, in particular, a drug containing rTIMP-2 as an active ingredient has the advantages described above, in addition to its advantages, including its processing as a formulation. It has excellent activity on various kinds of wounds, wounds caused by surgery, cutaneous fistula, etc. The therapeutic agent for rT IMP-2 containing skin deficiency can be expected to have stable biological activity even after long-term storage, and can be expected to have excellent biological activity even after being applied to the affected area. Drugs containing at least one tissue inhibitor as an active ingredient are selected from the group consisting of vasculopathy hamsters represented by arterial occlusive lower limbs * 瘼 and venous duct Pressure ulcers or diabetic hamaki due to a certain sustained pressure stimulus, burns * ulcers, traumatic ham fistulas, radiation ulcers, drug hammers, postoperative hammers, inflammatory nodules, simplicity * It is judged to have a preventive or therapeutic effect on fistulas, etc., and an effect of improving symptoms. Selected from the group consisting of TI MPs Drugs containing at least one tissue inhibitor as an active ingredient include chronic (refractory) dermal fistulas, which may be due to aging, physical factors, or serious underlying diseases. These factors are considered to have significant activity against symptoms and diseases that are not rarely associated with wound infections. Drugs containing at least one tissue inhibitor as an active ingredient, selected from the group consisting of TIMPs, are useful alone as potent drugs. In addition, a drug containing at least one tissue inhibitor selected from the group consisting of TIMPs as an active ingredient has been shown to have the activity of preventing the development of keloid hypertrophic scars and the prevention of trauma scars in skin defect wounds after surgery. It is useful as a therapeutic agent for wounds with therapeutic efficacy, an agent for promoting the healing of surgical wounds by surgery, and as an agent for preventing the occurrence of keloids and hypertrophic scars in skin defect wounds after surgery. Furthermore, TIMPs are useful as a therapeutic agent for chronic (refractory) Hama fistula. Industrial applicability

 The present invention provides a pharmaceutical composition containing TIMPs as a main active ingredient. The drug composition containing TIMPs as the main active ingredient according to the present invention can promote the healing of a difficult-to-treat, difficult-to-treat (refractory) sarcoma fulminosa. It is small and less noticeable. This effect is very useful from the aesthetic point of view, which is an important factor in qua1ityof1ife. Furthermore, the treatment using the pharmaceutical composition according to the present invention makes it possible to at least accelerate the healing of the surgical wound in simple surgery, thereby shortening the length of hospital stay. This in turn contributes to reducing the financial burden on healthcare financing such as health insurance.

Claims

The scope of the claims

1. An agent for treating skin deficiency, comprising as an active ingredient at least one tissue inhibitor selected from the group consisting of tissue inhibitors and metallobule-oral proteases.

 2. The therapeutic agent is used in the treatment of skin deficiency, including the prevention of scar * from skin * deficiency and the preventive treatment for reducing the scar. Item 7. The therapeutic agent according to Item 1.

 3. The therapeutic agent according to claim 1, wherein the deficiency is a wound.

 4. The therapeutic agent according to claim 3, wherein the wound is a surgical wound, and the surgical wound or a surgical scar related thereto is reduced by preventive treatment.

 5. The therapeutic agent according to claim 3, wherein the wound is a wound wound, and the wound wound or a wound scar related thereto is reduced by preventive treatment.

 6. The therapeutic agent according to claim 1, wherein the skin frost deficiency is dermatitis.

 7. The therapeutic agent according to claim 6, wherein the cutaneous fistula is a postoperative fistula, and the postoperative fistula or postoperative scar associated therewith is reduced by preventive treatment.

8. The therapeutic agent according to claim 6, characterized in that the dermatological clearing is a traumatic fulminant, and the traumatic ham fistula or the related traumatic scar is reduced by prophylactic treatment. .

9. The therapeutic agent according to claim 6, wherein the cutaneous beach fistula is a chronic beach fistula.

10. The therapeutic agent according to claim 6, wherein the skin I »ulcer is a decubitus ulcer.

 1 1. The therapeutic agent according to claim 6, wherein the cutaneous fistula is a vasculopathy gall ulcer.

 1 2. The therapeutic agent according to claim 6, wherein the skin ulcer is an inflammatory ulcer.

 1 3. The therapeutic agent according to claim 6, wherein the skin ulcer is a simple ulcer.

 1 4. The therapeutic agent according to any one of claims 1 to 13, further comprising tissue inhibitor metallobrothase-1 as an active ingredient.

 15. The therapeutic agent according to claim 14, characterized in that it contains recombinant tissue inhibitor metabolic protease 1 as tissue inhibitor metabolic protease 1.

 1 6. Recombinant tissue inhibitor meta-oral protease-1 is expressed by transformed cells obtained using E. coli, CHO cells or COS-1 cells as host cells, and the resulting gene product is purified. 16. The therapeutic agent according to claim 15, wherein the therapeutic agent is used.

 1 7. The therapeutic agent according to any one of claims 1 to 13, wherein the therapeutic agent comprises a tissue inhibitor metabolic proteinase-2 as an active ingredient.

 18. The therapeutic agent according to claim 17, wherein the agent comprises recombinant tissue inhibitor metabolic protease 2 as tissue inhibitor metabolic protease 2.

1 9. Recombinant tissue inhibitor meta-oral protease-2 is used as a host cell for Escherichia coli, CHO cells or COS-1 cells 19. The therapeutic agent according to claim 18, wherein the therapeutic agent is expressed by a transformant cell obtained using the above, and the obtained gene product is purified.

 20. The method according to any one of claims 1 to 3, wherein a mixture of tissue inhibitor metabolic protease 1 and tissue inhibitor metabolic protease 12 is used as an active ingredient as tissue inhibitor metabolic proteases. 13. The therapeutic agent according to any one of 13.

 2 1. Recombinant tissue inhibitor metabolic protease 1 as tissue inhibitor 1 metabolic protease 1 and recombinant tissue inhibitor metabolic protease 1 as tissue inhibitor metabolic protease 1 2. 21. The therapeutic agent according to claim 20, which comprises:

 2 2. Recombinant tissue inhibitor metab-proteinase 1 and recombinant tissue inhibitor metabolic protease-2 obtained using Escherichia coli, CHO cells or COS-1 cells as host cells, respectively. 22. The therapeutic agent according to claim 21, wherein the gene product is expressed and purified.