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WO1996033733A1 - Nouveau remede pour affections cutanees - Google Patents

Nouveau remede pour affections cutanees Download PDF

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Publication number
WO1996033733A1
WO1996033733A1 PCT/JP1996/001108 JP9601108W WO9633733A1 WO 1996033733 A1 WO1996033733 A1 WO 1996033733A1 JP 9601108 W JP9601108 W JP 9601108W WO 9633733 A1 WO9633733 A1 WO 9633733A1
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WIPO (PCT)
Prior art keywords
therapeutic agent
skin
agent according
tissue inhibitor
wound
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PCT/JP1996/001108
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English (en)
Japanese (ja)
Inventor
Tamotsu Kanzaki
Akira Shinagawa
Hidekuni Shima
Takanori Aoki
Shin-Ichi Yoshida
Takashi Shinya
Original Assignee
Fuji Yakuhin Kogyo Kabushiki Kaisha
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Publication of WO1996033733A1 publication Critical patent/WO1996033733A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans

Definitions

  • 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.
  • TIMPs tissue inhibitors metabolic proteases
  • 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.
  • 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.
  • TIs tissue inhibitor 1
  • TIMP-1 tissue inhibitor 1
  • TIMP-2 tissue inhibitor 1
  • issue 2 nh ibitorofmata 1 l prorotei na ses—2 A preparation containing TIMP-2
  • 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.
  • rTIMPs recombinant tissue inhibitors of metallo-oral proteases
  • 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.
  • 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.
  • macrophages and lymphocytes serve as a source of necrotic tissue removal, growth factors, and cytodynamics at the wound site.
  • the intense factor colony sti mu factor factor: CSF
  • interleukin-8 IL-8
  • 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.
  • fibronectin, collagen, hyaluronic acid, etc. make up the extracellular matrix of the new dermis.
  • PDGF, bFGF promotes cell division of skin cells
  • KGF keratinocyte growth factor
  • cytokines such as growth factors, growth factors, etc.
  • cytokine is key to the healing process. Absent.
  • 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.
  • tissue degeneration and necrosis 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.
  • tissue degeneration and necrosis 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.
  • MM P matrix metallobu orase
  • Matrix meta-oral proteases 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.
  • 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.
  • An object of the present invention is to provide a pharmaceutical composition containing ⁇ 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 ⁇ trauma scars.
  • 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.
  • the recombinant TIMPs rTIMPs
  • the present invention has been completed.
  • the present invention is a.
  • 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) ),
  • 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.
  • 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.
  • 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.
  • tissue inhibitor metabolic protease 1 and tissue inhibitor metabolic protease 2 as the tissue inhibitor metabolic proteases of the above-mentioned (18).
  • the therapeutic agent as described in and
  • the dosage form of the therapeutic agent is ointment, plaster, solution, solution, oil, cream, paste, cataplasm, liniment, lotion, spray, suspension, emulsion
  • 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
  • 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.
  • 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.
  • PCR primer primer T2F7: 5'-AAAGTCGACCATGGGCGCCGC GGCCCGCACCCT—3 'and primer T2R5; 5'-TT
  • the resulting TIMP-2 gene was subcloned into an appropriate cloning vector, for example, pUC13, and then an appropriate expression vector was added.
  • an appropriate cloning vector for example, pUC13
  • an appropriate expression vector was added.
  • 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).
  • the therapeutic agent according to (1) which comprises rT IMP-2 prepared as described in Example 1 as an active ingredient.
  • 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.
  • FIG. 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.
  • 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.
  • TIMP-2 has many excellent properties as a therapeutic agent for skin deficiencies, particularly as a therapeutic agent for skin fulminants and a wound.
  • 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.
  • ⁇ Culture of pulp-derived cells ⁇ 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.
  • 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).
  • 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.
  • TIMPs in quantity and quality that can be used in animal experiments and clinical tests required for approval of the production of pharmaceuticals.
  • 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.
  • 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.
  • eukaryotic hosts such as yeast and Chinese hamster ovary cells (CHO cells), prokaryotic hosts such as Escherichia coli, and insect cells such as Sf21
  • eukaryotic hosts such as yeast and Chinese hamster ovary cells (CHO cells)
  • prokaryotic hosts such as Escherichia coli
  • insect cells such as Sf21
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • rT IMPs does not contain contaminants that are normally involved in non-recombinant cells (non-transformants).
  • 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.
  • 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.
  • it can be used in combination with other drugs such as hamagi and wound treatments.
  • 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.
  • 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.
  • compositions of the present invention 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.
  • 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.
  • any method and means known in the art can be used.
  • any TIMPs that inhibit MMPs activity can be used.
  • a large amount of homogeneous TIMPs can be obtained.
  • artificially prepared r TIMPs can be used.
  • 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.
  • strict requirements are required for the stability of the preparation. TIMP-2 also has excellent properties in this respect.
  • 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.
  • 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.
  • T IMPs-containing solution vehicle 0.005% benzonalconium chloride-saline: 0.005% benzalkonium chloride 0.15M NaCl solution, pH 7.2
  • 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
  • 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.
  • 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 ⁇ hypertrophic scars and prevent traumatic scars in skin * defective wounds after surgery.
  • 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.
  • 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 4 ⁇ 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.
  • 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.
  • TIMP-1 gene After subcloning the obtained TIMP-1 gene into an appropriate cloning vector, for example, pUC13, an appropriate expression vector, for example, pME
  • an appropriate expression vector for example, pME
  • Mneo Leeeta l., Nature, 294, 228-2232, 19981
  • 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.
  • 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
  • ointment preparations various publicly known ointment bases can be used.
  • rTIMP-1 can be added to macrogol ointment and formulated as follows.
  • Example 3 Preparation of ointment containing rTIMP-2
  • Example 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
  • 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.
  • 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.
  • the rT IMP-2 containing solution prepared in Example 5 was used as the rT IMP-2 containing test drug.
  • a physiological saline solution containing 0.005% benzalkonium chloride sterilized with a membrane filter having a pore size of 0.1 / m was used.
  • 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.
  • 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.
  • the change in the area of the skin defect was determined from the daily change in the calculated area ratio.
  • 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.
  • mice 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.
  • 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).
  • 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.
  • 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
  • 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.
  • mice 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
  • 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).
  • rTIMP-2 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.
  • 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.
  • FIG. 8 and FIG. 9 which is an enlarged photograph of a part of the histological image shown in FIG. 8).
  • 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).
  • the histological findings of the wound were classified, quantified and evaluated according to the degree as follows.
  • 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.
  • Example 10 Effect of a solution containing 10 rT IMP-2 on proliferation of human normal skin fibroblasts
  • 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 ⁇ m and stored refrigerated (4 ° C) until use.
  • 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.
  • 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.
  • 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.
  • a physiological saline solution containing 0.005% benzaluconium chloride sterilized with a membrane filter having a pore size of 0.1 m was used.
  • mice 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.
  • 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 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 * ⁇ 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.
  • vasculopathy hamsters represented by arterial occlusive lower limbs * ⁇ 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.
  • 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.
  • 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.
  • TIMPs are useful as a therapeutic agent for chronic (refractory) Hama fistula.
  • 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.
  • 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.

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Abstract

L'invention porte sur des médicaments jouant par eux-mêmes un rôle efficace dans le difficile processus de guérison des traumatismes et affections cutanés tels que les plaies et les ulcères, y compris les ulcères chroniques incurables, et sur des médicaments agissant sur les plaies et traumatismes opératoires pour en favoriser la guérison et en réduire les cicatrices. L'invention porte également sur des médicaments comportant au moins une fraction d'inhibiteurs tissulaires de métalloprotéases (TIMP) agissant sur les méalloprotéases matrices (MMP) et s'avérant très efficaces dans le difficile processus de guérison des traumatismes et affections cutanés tels que les plaies et les ulcères. Le TIMP-2 en particulier s'avère être un excellent médicament, et le rTIMP-2, un remède pratique pour le traitement des affections cutanées tels que les plaies et les ulcères et comme médicament agissant sur les plaies et traumatismes opératoires pour en favoriser la guérison et en réduire les cicatrices.
PCT/JP1996/001108 1995-04-25 1996-04-24 Nouveau remede pour affections cutanees WO1996033733A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002056901A3 (fr) * 2001-01-22 2002-11-21 Henkel Kgaa Preparations cosmetiques ou pharmaceutiques pour le traitement du tissu epithelial externe
WO2004043481A3 (fr) * 2002-11-07 2004-10-28 Procyte Corp Compositions contenant des complexes de cuivre-peptide et des inhibiteurs de metalloproteinase, et methodes s'y rapportant
JP2011520902A (ja) * 2008-05-16 2011-07-21 コーセラ，インコーポレーテッド 創傷の治癒を促進する方法

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WO1992021360A1 (fr) * 1991-05-28 1992-12-10 Merck & Co., Inc. Derives substitues n-carboxyalkylpeptidyle en tant qu'agents antidegeneratifs
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JPH0625284A (ja) * 1991-06-27 1994-02-01 Glaxo Inc 環状イミド誘導体
JPH0625183A (ja) * 1991-12-04 1994-02-01 Banyu Pharmaceut Co Ltd 生理活性物質ｂｅ−１９０９３及びその製造法
JPH0687813A (ja) * 1992-06-25 1994-03-29 F Hoffmann La Roche Ag ヒドロキサム酸誘導体
JPH06256209A (ja) * 1993-03-03 1994-09-13 Fuji Yakuhin Kogyo Kk 眼球及びその周辺部組織の潰瘍に対する予防・治療剤
JPH06300757A (ja) * 1993-04-12 1994-10-28 Fuji Yakuhin Kogyo Kk ヒト間質型コラゲナーゼと阻害剤との複合体の免疫学的定量法および臨床診断への応用

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JPS6245529A (ja) * 1985-08-22 1987-02-27 Asahi Chem Ind Co Ltd 新たなコラゲナ−ゼインヒビタ−
JPH01146896A (ja) * 1987-12-04 1989-06-08 Fuji Yakuhin Kogyo Kk 新規なペプチジルヒドロキサム酸誘導体
JPH01160997A (ja) * 1987-12-17 1989-06-23 Fuji Yakuhin Kogyo Kk 新規なヒドロキサム酸誘導体
JPH01222782A (ja) * 1988-02-29 1989-09-06 Toray Ind Inc コラゲナーゼインヒビターの精製法
WO1990011287A1 (fr) * 1989-03-21 1990-10-04 The United States Of America, Represented By The Secretary, United States Department Of Commerce Peptides inhibiteurs de metalloproteinases matricielles
WO1992021360A1 (fr) * 1991-05-28 1992-12-10 Merck & Co., Inc. Derives substitues n-carboxyalkylpeptidyle en tant qu'agents antidegeneratifs
JPH0625284A (ja) * 1991-06-27 1994-02-01 Glaxo Inc 環状イミド誘導体
JPH05199868A (ja) * 1991-07-18 1993-08-10 Fuji Yakuhin Kogyo Kk ティシュ・インヒビター・オブ・メタロプロテイナーゼ含有組織培養用無血清培地と細胞増殖方法
JPH0625183A (ja) * 1991-12-04 1994-02-01 Banyu Pharmaceut Co Ltd 生理活性物質ｂｅ−１９０９３及びその製造法
JPH0687813A (ja) * 1992-06-25 1994-03-29 F Hoffmann La Roche Ag ヒドロキサム酸誘導体
JPH06256209A (ja) * 1993-03-03 1994-09-13 Fuji Yakuhin Kogyo Kk 眼球及びその周辺部組織の潰瘍に対する予防・治療剤
JPH06300757A (ja) * 1993-04-12 1994-10-28 Fuji Yakuhin Kogyo Kk ヒト間質型コラゲナーゼと阻害剤との複合体の免疫学的定量法および臨床診断への応用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002056901A3 (fr) * 2001-01-22 2002-11-21 Henkel Kgaa Preparations cosmetiques ou pharmaceutiques pour le traitement du tissu epithelial externe
WO2004043481A3 (fr) * 2002-11-07 2004-10-28 Procyte Corp Compositions contenant des complexes de cuivre-peptide et des inhibiteurs de metalloproteinase, et methodes s'y rapportant
JP2011520902A (ja) * 2008-05-16 2011-07-21 コーセラ，インコーポレーテッド 創傷の治癒を促進する方法

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