JP4566605B2 - Collagen synthesis inhibitor containing short peptide as active ingredient and method for producing the same - Google Patents

Description

  The present invention relates to a collagen synthesis inhibitor having a short peptide as an active ingredient and a method for producing the same.

Hypertrophic scars and keloids are diseases caused by prolonged healing of burns or wounds caused by burns, and are said to be caused by excessive production of scar tissue, mainly collagen fibers. As a result, scars bulge from the surroundings during the skin repair process, redness and hardening become prominent, often accompanied by itching and tenderness. In addition, the appearance of these pathological conditions is a serious mental burden on patients, especially for unmarried people.
Drugs currently used as treatments for hypertrophic scars and keloids include steroids and heparin analogs. However, steroid drugs have strong side effects as well as their strong anti-inflammatory effects, and hypertrophic scars, keloids, etc. have problems such as delayed wound healing, skin atrophy, and contact dermatitis-like rash. On the other hand, heparin-like substances have a weak effect on these diseases, and useful drugs with no side effects have been desired.
In addition, conventional collagen synthesis inhibitors have a problem in that they reduce the wound healing efficiency because they suppress not only cell collagen synthesis but also cell proliferation itself.

Prior art document information related to the invention of the present application is described below.
U.S. Pat. U.S. Pat. US Patent No. 4589881 US Patent No. 4589881 US Patent No. 4578079 US Patent No. 4578079 US Patent No. 4517686

  The present invention has been made in view of such circumstances, and an object thereof is to provide a highly safe collagen synthesis inhibitor that does not affect cell proliferation.

The present inventor has found that a short-chain peptide containing glycine and proline has an effect of inhibiting collagen synthesis, while conducting various studies to solve the above problems. When the influence of these peptides on cell proliferation was examined, these peptides did not affect cell proliferation, but specifically inhibited only collagen synthesis. Furthermore, in a rat cirrhosis model, these peptides improved serum GOT and GPT levels and suppressed liver collagen deposition. Therefore, the peptide identified by the present inventor can be used as a collagen synthesis inhibitor, and is considered to be particularly useful as a medicament with few side effects for the prevention and treatment of fibrotic diseases including cirrhosis.
That is, the present invention relates to a collagen synthesis inhibitor containing a short-chain peptide containing glycine and proline as an active ingredient, and a method for producing the same, and more specifically, provides the following invention.
(1) A collagen synthesis inhibitor comprising as an active ingredient a peptide comprising any one of the following amino acid sequences (a) to (c):
(A) Gly-Arg-Gly-Xn-Pro (SEQ ID NO: 1)
(Xn represents one or several amino acids)
(B) Gly-Pro-Ala (SEQ ID NO: 2)
(C) Gly-Pro (SEQ ID NO: 3)
(2) The collagen synthesis inhibitor according to (1), wherein Xn is a dipeptide consisting of two kinds of amino acids selected from the group consisting of Glu, Ser, Thr, Asp.
(3) The collagen synthesis inhibitor according to (1), wherein Xn is a dipeptide selected from the group consisting of Glu-Ser, Asp-Ser, and Asp-Thr.
(4) The collagen synthesis inhibitor according to any one of (1) to (3), which is a medicament for preventing or treating a disease caused by fibrosis.
(5) The disease caused by fibrosis is selected from the group consisting of hypertrophic scar, scar contracture, keloid disease, collagen disease, pulmonary fibrosis, diabetic nephropathy, fibrotic adhesions, and cirrhosis, (4) The collagen synthesis inhibitor described.
(6) A method for producing a collagen synthesis inhibitor, the method comprising a step of mixing a peptide comprising any one of the following amino acid sequences (a) to (c) with a pharmacologically acceptable carrier.
(A) Gly-Arg-Gly-Xn-Pro (SEQ ID NO: 1)
(Xn represents one or several amino acids)
(B) Gly-Pro-Ala (SEQ ID NO: 2)
(C) Gly-Pro (SEQ ID NO: 3)
(7) The production method according to (6), wherein Xn is a dipeptide consisting of two kinds of amino acids selected from Glu, Ser, Thr, and Asp.
(8) The production method according to (6), wherein Xn is a dipeptide selected from the group consisting of Glu-Ser, Asp-Ser, and Asp-Thr.

  According to the present invention, a collagen synthesis inhibitor comprising a short peptide containing glycine and proline as an active ingredient and a method for producing the same are provided. The collagen synthesis inhibitor of the present invention specifically suppresses only collagen synthesis without affecting cell growth, and thus is extremely safe and has diseases that exhibit collagen accumulation, particularly hypertrophic scars, keloidosis, It is useful for the prevention or treatment of fibrotic diseases such as collagen disease, pulmonary fibrosis, diabetic nephropathy, and cirrhosis.

  The present invention provides a collagen synthesis inhibitor having a short peptide containing glycine and proline as an active ingredient. The “collagen synthesis inhibitor” of the present invention may be a medicine or may be a reagent for research, but is preferably a medicine for the treatment or prevention of a disease exhibiting the above-mentioned collagen accumulation. .

In a preferred embodiment of the collagen synthesis inhibitor of the present invention, the short chain peptide containing glycine and proline is a peptide comprising an amino acid sequence having the following general formula.
Gly-Arg-Gly-Xn-Pro (SEQ ID NO: 1)
Here, Xn is one or several amino acids. For example, it is an amino acid having a chain length of 10 or less, 5 or less, or 3 or less, and most preferably an amino acid having a chain length of 2. The amino acid type of Xn is not particularly limited as long as it does not function inhibitory to the collagen synthesis inhibitory activity of the short chain peptide. Preferably, the amino acid is selected from Glu, Ser, Thr and Asp.
As a particularly preferred peptide as the collagen synthesis inhibitor of the present invention, for example, Gly-Arg-Gly-Glu-Ser-Pro (SEQ ID NO: 4) Gly-Arg-Gly-Asp-Ser-Pro (SEQ ID NO: 5) , Gly-Arg-Gly-Asp-Thr-Pro (SEQ ID NO: 6), Gly-Pro-Ala (SEQ ID NO: 2), Gly-Pro (SEQ ID NO: 3).
Thus, since the peptide of the present invention has a short chain and a low molecular weight, it has a great advantage that it can be absorbed percutaneously. Furthermore, it is less likely to cause allergies and is not harmful to the body or blood. It also has advantages.

  The above short peptide, which is an active ingredient of the collagen synthesis inhibitor of the present invention, can be used in the form of a general pharmaceutical preparation. The preparation is prepared using diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, lubricants and the like that are usually used.

As this pharmaceutical preparation, various forms can be selected according to the purpose of treatment, and representative examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, external preparations, poultices, capsules, suppositories, Injections (solutions, suspensions, etc.) and the like can be mentioned. In molding into a tablet form, various carriers well known in the art can be widely used as carriers.
Examples include lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and other excipients, water, ethanol, propanol, simple syrup, glucose solution, starch solution, Binders such as gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, lauryl sulfate Disintegrators such as sodium, stearic acid monoglyceride, starch, lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, quaternary ammonium base, absorption promoters such as sodium lauryl sulfate, Phosphorus, moisturizing agents such as starch, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, stearates, boric acid powder, a lubricant such as polyethylene glycol can be used. Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.
When forming into the form of a pill, those conventionally known in this field can be widely used as the carrier. Examples include excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin and talc, binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, and disintegrants such as laminaran and agar. Can be used.
In molding into a suppository form, conventionally known carriers can be widely used. Examples thereof include polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like. Capsules are usually prepared by mixing the active ingredient compound with the various carriers exemplified above and filling them into hard gelatin capsules, soft capsules and the like according to conventional methods.
When prepared as injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood, and are commonly used in this field as diluents when molded into these forms For example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, a sufficient amount of sodium chloride, glucose or glycerin may be contained in the pharmaceutical preparation to prepare an isotonic solution, and a normal solubilizing agent, buffering agent, soothing agent, etc. may be added. May be. Furthermore, a coloring agent, a preservative, a fragrance | flavor, a flavor agent, a sweetening agent, etc. and other pharmaceuticals can also be contained in a pharmaceutical formulation as needed.

  The amount of the active ingredient compound to be contained in the pharmaceutical preparation of the present invention is not particularly limited and is appropriately selected from a wide range, but is usually about 1 to 70% by weight, preferably about 1 to 30% in the pharmaceutical composition. It is good to set it as weight%.

  The administration method of the pharmaceutical preparation of the present invention is not particularly limited, and it is administered by a method according to various preparation forms, patient age, sex and other conditions, disease degree, and the like. Examples of dosage forms include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, etc., suspension injection, freeze-dried, etc., dissolution-type injection, suppository, nasal preparation, etc. Or oral preparations such as tablets, pills, capsules, granules, suspensions and the like. For formulation of these dosage forms, generally known methods are applied, and pharmaceutically acceptable preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting are used depending on the purpose. Contains agents, lubricants, colorants, fragrances, flavoring agents, skins, suspending agents, emulsifiers, solubilizers, buffers, tonicity agents, plasticizers, surfactants, soothing agents, etc. You may let them.

  The dosage of the pharmaceutical preparation of the present invention is appropriately selected depending on the usage, the patient's age, sex and other conditions, the degree of the disease, etc. Usually, the amount of the active ingredient is about 0.01 to 100 mg per kg body weight per day. It is preferable to set the range. In addition, the active ingredient compound is preferably contained in the dosage unit form in a range of about 1 to 200 mg.

  In the collagen synthesis inhibitor of the present invention, a component containing a drug that suppresses the synthesis of cytokines and leukotrienes and a drug effective in suppressing keloid formation such as fibrin glue can be used in combination.

Drugs that suppress the synthesis of cytokines and leukotrienes that can be used in combination with the present invention are selected from urinastatin, gabexate mesylate, nafamostat mesylate, camostat mesylate, salazosulfapyridine, metronidazole, 5ASA, prautotol, etc. Is available.
For example, urinastatin is commercially available from Mochida Pharmaceutical Co., Ltd. under the Miracled trademark. Gabexate mesylate is commercially available from Ono Pharmaceutical Co., Ltd. under the trademark FOW. Nafamostat mesylate is commercially available from Torii Pharmaceutical Co., Ltd. under the Fusan trademark. Camostat mesylate is commercially available from Ono Pharmaceutical Co., Ltd. under the FOUIPAN trademark. Salazosulfapyridine is commercially available from Midori Cross Co., Ltd. under the trademark of Sarazopyrine. Metronidazole is commercially available from Kayaku Co., Ltd. under the trademark Metronidazole. 5ASA is marketed by Nisshin Flour Milling Co., Ltd. under the trademark of mesalazine. Prounotol is commercially available from Sankyo Co., Ltd. under the Kernak trademark.

The fibrin glue that can be used in combination with the present invention comprises fibrinogen, fibrinogen solution (for example, aprotinin solution), thrombin, and thrombin solution (for example, calcium solution).
Fibrin glue is a two-component tissue adhesive that utilizes the above-described fibrinogen and thrombin-producing fibrin formation reaction. That is, fibrinogen becomes a fibrin monomer under the action of thrombin, gradually polymerizes to form a fibrin polymer, and is further cross-linked by the action of blood coagulation factor XIII to become a stabilized fibrin clot.
Fibrin glue is widely used for the purpose of adhering and closing tissues during surgery. For example, applied to the brain dura suture part, the sutured part after gastrectomy, the hepatectomy end part, the intestinal anastomosis part, the anastomosis part of the blood vessels and nerves, the tympanic membrane formation part, etc., generating fibrin on the application surface, By solidifying, it plays a role of preventing leakage of body fluids and blood. Fibrin glue is sold, for example, by the Chemical and Serum Therapy Laboratory under the Bolheel trademark.

The use of a drug effective for suppressing keloid formation that can be used in combination with the present invention may be applied or sprayed around the anastomotic region including the rectal mucosa remaining after ileal anal anastomosis for inflammatory bowel disease, for example. It is also useful in a similar manner of use for anastomotic stenosis due to keloid constitution, skin keloid formation, and the like. Furthermore, the effect of the drug of the present invention can be promoted by administering a cytokine synthesis inhibitor or a steroid together intravenously or intraarterially before and after surgery.
The present invention can also be used in combination with ketotifen or a salt thereof, lobenzarit salt, DADA, cimetidine or panthenol, which is already known to have a keloid inhibitory effect.

  The present invention also provides a method for producing the collagen synthesis inhibitor of the present invention. The production method of the present invention includes a step of mixing the short peptide with a pharmacologically acceptable carrier. The short peptide may be a synthetic peptide or a recombinant peptide. A recombinant peptide is prepared by preparing a transformant into which a DNA encoding a short peptide is introduced, culturing the transformant, and recovering the expressed peptide from the transformant or a culture medium thereof. can do.

  The DNA encoding the short chain peptide is not particularly limited, and includes a plurality of DNAs considering codon degeneracy. DNA encoding a short peptide can be prepared by synthesis using a DNA synthesizer. It can also be prepared from a biological sample according to the following method.

For example, RNA is first isolated from a human or rat tissue, preferably the liver, or a cultured cell line, preferably a cultured cell line derived from the liver, cDNA is synthesized using the RNA as a template, and the cDNA is incorporated into an appropriate plasmid vector. . Next, a host cell is transformed with the obtained recombinant vector to prepare a cDNA library, and a target clone is selected from the cDNA library to prepare DNA.
The steps of isolating RNA from a human or rat tissue, preferably the liver, or a cultured cell line, preferably a liver-derived cultured cell line, to prepare a cDNA library and the cloning step are carried out by biochemistry [Biochemistry, 18, 5294 ( 1977)], Japanese Patent Laid-Open No. 2-227075, or commercially available experimental documents such as gene manipulation experiment methods (Yasutaka Takagi, Kodansha), gene manual (Yasutaka Takagi, Kodansha), molecular cloning: a laboratory manual (No. 1) 2nd edition) and the like, or a commercially available kit can be used.
As a method for preparing total RNA from human or rat tissue, preferably liver, or cultured cell line, preferably cultured cell line derived from liver, guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], and the method for preparing poly (A) + RNA from total RNA is the oligo (dT) -immobilized cellulose column method [Molecular Cloning: A Laboratory Manual (2nd edition)]. can give. In addition, as a kit for preparing mRNA from human or rat tissue, preferably liver, or cultured cell line, preferably cultured cell line derived from liver, Fast Track mRNA Isolation Kit (Invitrogen) And a Quick Prep mRNA Purificatlon Kit (Pharmacia).
Methods for synthesizing cDNA from poly (A) + RNA or mRNA include Molecular Cloning: A Laboratory Manual (2nd edition), Current Protocols in Molecular Biology, Supplement 1-34, etc. The described method or a commercially available kit, for example, SuperScriptTM Preamplification System for First Strand cDNA Synthesis (Life Technologles) The method to use etc. are mention | raise | lifted.
cDNA library preparation methods include Molecular Cloning: A Laboratory Manual (2nd edition), Current Protocols in Molecular Biology, Supplement 1-34, etc., or commercially available kits. For example, SuperScriptTM Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) or Zap-cDNA Synthesis Kit [ZAP-cDNA Synthesis Kit , Manufactured by Stratagene, Inc.].
Cloning of a DNA encoding the short peptide of the present invention from a cDNA library can be performed by, for example, DNA encoding rat arginase reported by Otake et al. [J. Biol. 263, 2245, (1988)] and DNA encoding human arginase [Nucleic Acid Res., 16, 8789, (1988)] reported by Takiguchi et al., Using a colony hybridization method or a plaque hybridization method [ Molecular Cloning: A Laboratory Manual Second Edition].
Primers were prepared based on the base sequence of rat arginase reported by Otake et al. Or the base sequence of human arginase reported by Takiguchi et al., And a cDNA or cDNA library synthesized from poly (A) + RNA or mRNA was used as a template. As a result of the polymerase chain reaction (PCR) method [Molecular Cloning: A Laboratory Manual (1st edition), Current Protocols in Molecular Biology, Supplement 1-34], DNA encoding a chain peptide can also be prepared.
In order to prepare mutants of the DNA encoding the short peptide isolated in this way, Molecular Cloning: A Laboratory Manual (2nd edition) or Current Protocols in Molecular Bio The method described in the logy, supplement 1-34, etc. may be followed.

  The cDNA clone selected by the above method is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript KS (+) (Stratagene), and a commonly used nucleotide sequence analysis method such as dideoxy by Sanger et al. Method [Proc. Natl. Acad. Scl., U.S.A., 74, 5463 (1977)] and the like, the base sequence of the DNA can be determined. Base sequence analysis can be performed using an automatic base sequence analyzer, such as a 373A DNA sequencer (Applied Biosystems).

  A transformant expressing the short peptide of the present invention by constructing a recombinant vector in which the DNA encoding the short peptide of the present invention is inserted downstream of the promoter of an appropriate vector and introducing it into a host cell Can be obtained.

Any host can be used as long as it can express the target gene, such as bacteria, yeast, animal cells, and insect cells. Examples of bacteria include Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium flavum, Brevibacterium lactofermentum (Brevibacterium). Microorganisms such as Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, etc., such as lactofermentum), Corynebacterium glutamicum, Microbacterium ammoniaphilum Is exemplified. Examples of yeasts include Saccharomyces cerevisae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans Schneus, uv Is exemplified. Examples of animal cells include human cells such as Namalva cells, monkey cells, COS cells, Chinese hamster cells, CHO cells, and the like.
Insect cells are Spodoptera frugiperda oocytes Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, Oreilly, Miller, Lucor (Luckow), WHFreeman and Company, New York, 1992 edition (hereinafter abbreviated as “Baculoyl Squipression Vectors”), Examples include Tn5, which is a Trichoplusia ni egg cell and is commercially available as High5 from Pharmingen.
As the vector for introducing the DNA encoding the short peptide of the present invention, any vector can be used as long as it can incorporate the DNA encoding the short peptide of the present invention and can be expressed in a host cell.
When a bacterium such as Escherichia coli is used as a host, it is composed of a promoter, a ribosome binding sequence, a DNA encoding the short peptide of the present invention, a transcription termination sequence, and in some cases a promoter control sequence. It is preferable.
Examples of expression vectors include pKYP10 (Japanese Patent Laid-Open No. 58-110600), pLSA1 (Agricultural and Biological Chemistry (Agric. Biol. Chem.), 53,277, (1989)), pGEL1 (Proc. Natl. Acad. Sci., USA, 82, 4306, (1985)] pNS and the like are used.
Any promoter may be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli or phage, such as trp promoter (Ptrp), lac promoter (Plac), T7lac promoter, PL promoter, PR promoter, etc. are used. An artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrp × 2) or a tac promoter may be used.
As the ribosome-binding sequence, it is preferable to use it by adjusting an appropriate distance (for example, 6 to 18 bases) between the Shine-Dalgarno sequence (hereinafter abbreviated as SD sequence) and the start codon.
In the recombinant vector of the present invention, it is preferable to use the base sequence of the DNA encoding the short peptide of the present invention after substituting the base as necessary so as to be an optimal codon for expression in the host.
In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA encoding the short peptide of the present invention, but preferably a transcription termination sequence is preferably arranged immediately below the structural gene. .
As a method for introducing a recombinant vector into bacteria, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110-2114 (1972)], the protoplast method (Japanese Patent Laid-Open No. 63-2483942), and the like.
When yeast is used as a host, for example, Yep13 (ATCC37115), Yep24 (ATCC37051), YCp50 (ATCC37419) and the like are used as expression vectors.
Any promoter can be used as long as it can be expressed in yeast. For example, a promoter of a glycolytic gene such as hexose kinase, a gal 1 promoter, a gal 10 promoter, a heat shock protein promoter. MFα1 promoter, CUP 1 promoter and the like.
As a method for introducing a recombinant vector into yeast, any method can be used, for example, electroporation [Methods. Enzymol., 194, 182-287 (1990)], spheroplast method [Proc. Natl. Acad. Sci., USA, 84, 1929-1933 (1978)], lithium acetate method [J. Bacteriol., 153, 163-168 (1983)] or the like can be used.
When animal cells are used as hosts, examples of expression vectors include pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechnology, 3,133, (1990)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pAMoERC3Sc, pcDM8 (Nature, 329,840, (1987)), pcDNAI / Amp, pcDNAI (both manufactured by Funakoshi) and the like are used.
Any promoter can be used as long as it can be expressed in animal cells. For example, human cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 or metallothionein promoter, etc. can give. In addition, an IE gene enhancer of human CMV may be used together with a promoter.
As a method for introducing a recombinant vector into an animal cell, for example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) as long as DNA is introduced into animal cells. ), Lipofection method [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)] or the like can be used.
When using insect cells as a host, the protein is prepared by a method described in, for example, Current Protocols in Molecular Biology, Supplement 1-34, Baculovirus Expression Vectors, A Laboratory Manual, etc. Can be expressed. Specifically, the recombinant gene transfer vector and baculovirus described below are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into the insect cells, and the protein expressing insect Obtain cells.
Examples of the gene transfer vector include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, is used.
Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-27075), the lipofection method [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)].
In addition to direct expression, secretory production, fusion protein expression, and the like have been developed as gene expression methods, and any method can be used. For example, it can be performed according to the method described in Molecular Cloning: A Laboratory Manual (2nd edition).
When expressed in yeast, animal cells or insect cells, the short peptide of the present invention to which a sugar or sugar chain is added by exo or endoglycosidase can be obtained.

  As a plasmid vector for incorporating the DNA encoding the short chain peptide of the present invention, any plasmid vector can be used as long as it can incorporate the DNA encoding the short chain peptide of the present invention. PNS-hArg1, which is an expression plasmid vector for human arginase, which is one embodiment of the short-chain peptide, and pNS-rArg1, which is an expression plasmid vector for rat arginase, which is another embodiment. Escherichia coli BL21 / pNS-hArg1 which is Escherichia coli containing plasmid vector pNS-hArg1 and Escherichia coli BL21 / pNS-rArg1 which is Escherichia coli containing plasmid vector pNS-rArg1 Deposited as FERM BP-5605 and FERM BP-5606 respectively at the Industrial Technology Research Institute (1-3 East 1-3 Tsukuba, Ibaraki, Japan).

  The short-chain peptide of the present invention can be produced by culturing the transformant obtained as described above, generating and accumulating the short-chain peptide of the present invention in the culture, and recovering from the culture. .

  The method of culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host. The medium for cultivating transformants obtained using microorganisms such as E. coli or yeast as a host contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, so that transformants can be cultured efficiently. As long as it is a medium, either a natural medium or a synthetic medium may be used.

As the carbon source, carbohydrates such as glucose, fructose, sucrose, molasses, starch and starch hydrolysate, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.
Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic or organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein Hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented bacterial cells or digested products thereof, and the like are used.
Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
The culture is usually carried out at 15 to 40 ° C. for 16 to 96 hours under aerobic conditions such as shaking culture or deep aeration stirring culture. During the culture period, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, cultivate a microorganism transformed with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like using an expression vector using the trp promoter. Sometimes indoleacrylic acid (IAA) or the like may be added to the medium.
As a medium for culturing a transformant obtained by using animal cells as a host, a generally used RPMI1640 medium, Eagle's MEM medium, a medium obtained by adding fetal calf serum or the like to these mediums, or the like is used. The culture is usually carried out in the presence of 5% carbon dioxide at 35 to 37 ° C. for 3 to 7 days, and antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
As a medium for cultivating a transformant obtained using insect cells as a host, commonly used TNM-FH medium (Pharmingen), Sf900IISFM (Life Technologies), ExCell400, ExCell405 [both manufactured by JRH Biosciences] and the like are used. Culturing is performed at 25 to 30 ° C. for 1 to 4 days, and antibiotics such as gentamicin may be added to the medium as needed during the cultivation.
When the short peptide of the present invention is expressed in a dissolved state in the cell or insoluble in the cell, the cell is centrifuged after culturing, suspended in an aqueous buffer, The cells are disrupted by the method, the French press method, etc., and the collagen synthesis-inhibiting polypeptide of the present invention is recovered in the centrifuged supernatant.
Furthermore, when an insoluble substance is formed in the cell, the insoluble substance is solubilized with a protein denaturing agent, and then diluted in a dilute solution that does not contain the protein denaturing agent or the protein denaturing agent does not denature, Alternatively, it can be dialyzed to form a three-dimensional protein structure.
When the short peptide of the present invention or a derivative thereof such as a sugar or a sugar chain adduct thereof is secreted extracellularly, the short peptide of the present invention or their sugar or sugar chain adduct is added to the culture supernatant. The derivative can be recovered.

  For isolation and purification, solvent extraction, fractional precipitation with organic solvents, salting out, dialysis, centrifugation, ultrafiltration, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography , Separation operations such as crystallization and electrophoresis can be performed alone or in combination.

  The purified protein can be subjected to primary structure analysis according to the method described in Shinsei Kagaku Kenkyu Kogaku, Volume 1, Protein II, primary structure (Shinichi Chitani et al., Tokyo Kagaku Dojin). For example, determine the amino acid sequence of the N-terminal using an automated amino acid sequence analyzer, or determine the amino acid sequence of a peptide fragment obtained after hydrolysis of a protein with a protease such as trypsin using a mass spectrometer or automated amino acid sequence analyzer can do. Determined amino acid sequences are homologous with amino acid sequences in protein databases such as PIR-International Protein Sequence Database [Protein Identification Resource]. Can be attributed.

The short peptide of the present invention can be modified with a sugar, a sugar chain, a polyvalent sugar complex, a polymer substance, or the like. Methods for preparing modified forms of sugars, sugar chains or polyvalent sugar conjugates include methods of producing glycoproteins in animal cells, insect cells, yeast cells, etc., and sugars, sugar chains or polyvalent sugar conjugates in advance. And the like, and a method of adding it enzymatically or chemically.
Examples of methods for preparing sugars and sugar chains include chemical synthesis methods, polysaccharide decomposition methods, free sugar chains from glycoproteins, and purification of sugars or sugar chains having a specific structure as necessary. In addition, as a method for preparing a multivalent sugar complex, there is a method of binding a sugar or sugar chain to a multivalent carrier such as oligo or polylysine [Journal of Drug Targeting (hereinafter referred to as J. Drug Targetting). (Abbreviated), 3, 111, (1995)].
The obtained sugar, sugar chain, or polyvalent sugar complex can also be added using glycosyltransferase or the like. Moreover, it can also add using a diazo coupling method, an isothiocyanate coupling method, a guanidination method, an amidination method etc., for example. In the diazo coupling method and the isothiocyanate coupling method, it can bind to the tyrosine of protein, the benzene ring of phenylalanine or the imidazole group of histidine. In the guanidination method and amidination method, it can bind to the amino group of the lysine side chain or the amino group of the protein N-terminal [J. Drug Targetting, 3, 111, (1995)].
The polymer substance may be any substance as long as it has low antigenicity and toxicity. For example, polyethylene glycol, dextran, carboxymethyl cellulose, albumin, gamma globulin, polyamino acid, styrene maleic acid copolymer, antibody, etc. (Pharmaceutical Development Vol. 13, Drug Delivery, Hirokawa Shoten, 1989), (Inada Yuji, Protein Hybrid -Chemical Modification That Has Been So far, Kyoritsu Shuppan, 1987).
Modification methods for polymer substances or polyvalent sugar conjugates include acid amide bonding, thioamide bonding, ester bonding, disulfide bonding, diazo bonding, alkylation, triazine ring bonding, The urea bond method, imidocarboxylic acid bond method, carbamyl acid bond method, etc. have been developed (Yuji Inada, Protein Hybrid -Chemical Modifications So far, Kyoritsu Shuppan, 1987), Ichiro Chibata, Immobilized Biology (Catalyst, Kodansha, 1986), (Satoku Ono, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda, Chemical modification of proteins <top><bottom>, Shinzo Sugaya, Kensuke Shimura, Michinori Nakamura, Katsunori Funatsu, Biochemical experiment Method 12, Society of Science Publishing Center, 1981), any method can be used. In addition, the polymer substance and the collagen synthesis inhibiting polypeptide of the present invention can be bound by the above-mentioned binding method via a cross-linking agent (edited by Ichiro Chibata, Immobilized Biocatalyst, Kodansha, 1986), (Satoku Ono, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda, Chemical modification of proteins <top><bottom>, Shinzo Shibuya, Kensuke Shimura, Michinori Nakamura, Katsunori Funatsu, Biochemical Experimental Method 12, Society Press Center, 1981).
Furthermore, a sugar-modified polymer substance can be bound to the short peptide of the present invention. The short peptide of the present invention may be cross-linked by any method. For example, it can be performed according to the method described in Ichiro Chibata, Immobilized Biocatalyst, Kodansha (1986).
The short peptide of the present invention can be modified with a sugar, a sugar chain, a polyvalent sugar complex, a polymer substance, or the like. Methods for preparing modified forms of sugars, sugar chains or polyvalent sugar conjugates include methods of producing glycoproteins in animal cells, insect cells, yeast cells, etc., and sugars, sugar chains or polyvalent sugar conjugates in advance. And the like, and a method of adding it enzymatically or chemically.
Examples of methods for preparing sugars and sugar chains include chemical synthesis methods, polysaccharide decomposition methods, free sugar chains from glycoproteins, and purification of sugars or sugar chains having a specific structure as necessary. In addition, as a method for preparing a multivalent sugar complex, there is a method of binding a sugar or sugar chain to a multivalent carrier such as oligo or polylysine [Journal of Drug Targeting (hereinafter referred to as J. Drug Targetting). (Abbreviated), 3, 111, (1995)].
The obtained sugar, sugar chain, or polyvalent sugar complex can also be added using glycosyltransferase or the like. Moreover, it can also add using a diazo coupling method, an isothiocyanate coupling method, a guanidination method, an amidination method etc., for example. In the diazo coupling method and the isothiocyanate coupling method, it can bind to the tyrosine of protein, the benzene ring of phenylalanine or the imidazole group of histidine. In the guanidination and amination methods, it can bind to the amino group of the lysine side chain or the amino group of the protein N-terminal [J. Drug Targetting, 3, 111, (1995)].
The polymer substance may be any substance as long as it has low antigenicity and toxicity. For example, polyethylene glycol, dextran, carboxymethyl cellulose, albumin, gamma globulin, polyamino acid, styrene maleic acid copolymer, antibody, etc. (Pharmaceutical Development Vol. 13, Drug Delivery, Hirokawa Shoten, 1989), (Inada Yuji, Protein Hybrid -Chemical Modification That Has Been So far, Kyoritsu Shuppan, 1987).
Modification methods for polymer substances or polyvalent sugar conjugates include acid amide bonding, thioamide bonding, ester bonding, disulfide bonding, diazo bonding, alkylation, triazine ring bonding, The urea bond method, imidocarboxylic acid bond method, carbamyl acid bond method, etc. have been developed (Yuji Inada, Protein Hybrid -Chemical Modifications So far, Kyoritsu Shuppan, 1987), Ichiro Chibata, Immobilized Biology (Catalyst, Kodansha, 1986), (Satoku Ono, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda, Chemical modification of proteins <top><bottom>, Shinzo Sugaya, Kensuke Shimura, Michinori Nakamura, Katsunori Funatsu, Biochemical experiment Method 12, Society of Science Publishing Center, 1981), any method can be used. In addition, the polymer substance and the collagen synthesis inhibiting polypeptide of the present invention can be bound by the above-mentioned binding method via a cross-linking agent (edited by Ichiro Chibata, Immobilized Biocatalyst, Kodansha, 1986), (Satoku Ono, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda, Chemical modification of proteins <top><bottom>, Shinzo Shibuya, Kensuke Shimura, Michinori Nakamura, Katsunori Funatsu, Biochemical Experimental Method 12, Society Press Center, 1981).
Furthermore, a sugar-modified polymer substance can be bound to the short peptide of the present invention. The short peptide of the present invention may be cross-linked by any method. For example, it can be performed according to the method described in Ichiro Chibata, Immobilized Biocatalyst, Kodansha (1986).
The short peptides of the present invention and their modified products are effective for suppressing the fibrosis of living tissue. When the short peptide of the present invention or a modified product thereof is used as a medicine, as described above, it can be mixed with a pharmacologically acceptable carrier and used in various dosage forms depending on the administration form. it can.

Preferred embodiments of the invention

  EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Manufacture of a poultice preparation 32.9 g of sorbitol is used as a pre-formulation 1, and 2 g of gelatin and 1 g of PVA (polyvinyl alcohol) are added to 13 g of water and dissolved by heating to form a pre-formulation 2. Separately, 23 g of glycerin and 1.2 g of magnesium aluminate metasilicate, 1.0 g of CMC-Na (carboxymethylcellulose sodium), 1.1 g of polyacrylic acid starch / acrylic acid copolymer, and 3 g of sodium polyacrylate were sufficiently dispersed. Pre-formulation 3 was designated. Pre-formulation 4 was prepared by mixing 2 g of isopropyl myristate, 1 g of sorbic acid fatty acid ester and 0.3 g of l-menthol, and as pre-formulation 5 a solution of 0.4 g of methoxyethylene maleic anhydride copolymer in 5 g of water was further prepared. Preparation 6 was prepared by dissolving 0.6 g of polyethylene glycol monolaurate and 0.5 g of the peptide Gly-Arg-Gly-Glu-Ser-Pro of the present invention in 12 g of water. Add pre-formulation 2, pre-formulation 4, pre-formulation 5 and pre-formulation 6 to pre-formulation 1, then add pre-formulation 3 and stir and mix in a kneader for 30 minutes. This was spread and used as a poultice.

[Example 2] Manufacture of a gel agent To 30 g of citric acid 0.3 g and carboxyvinyl polymer 5 g added to 100 g of water and dissolved by heating, 30 g of ethanol, 3 g of propylene glycol, 31.2 g of water, the peptide Gly-Arg- of the present invention After adding 0.5 g of Gly-Glu-Ser-Pro, an appropriate amount of 10% sodium hydroxide solution was added to form a gel.

[Example 3] Production of cream preparation As formulation 1, sorbitan sesquioleate 3.0 g, polyoxyethylene hydrogenated castor oil 0.2 g, liquid paraffin 10.0 g, isopropyl palmitate 5.0 g, petrolatum 1.0 g, propyl parahydroxybenzoate 0.1 g Resinol 0.5 g and the peptide Gly-Arg-Gly-Glu-Ser-Pro 0.5 g of the present invention were dissolved by heating. As Preformulation 2, 0.1 g of methyl parahydroxybenzoate, 0.5 g of magnesium sulfate and 5.0 g of glycerin were dissolved by heating. Pre-formulation 2 was added to pre-formulation 1 to a total amount of 100 g with purified water, stirred for 10 minutes at 5000 rpm with a homomixer, and then stirred to 35 ° C. with normal stirring to obtain a cream.

[Example 4] Production of ointment As pre-formulation 1, sorbitan sesquioleate 5.0 g, polyoxycetyl ether 0.5 g, cetanol 6.0 g, beeswax 3.0 g, petrolatum 35.0 g, propyl parahydroxybenzoate 0.1 g, Peptide Gly-Arg-Gly-Glu-Ser-Pro 0.5g was dissolved by heating.
Further, as pre-formulation 2, 0.1 g of methyl parahydroxybenzoate and 0.2 g of EDTA-2Na (disodium ethylenediaminetetraacetate) were dissolved by heating with an appropriate amount of purified water. Pre-formulation 2 was added to pre-formulation 1 and stirred, and purified water was added to make the total amount 100 g, which was then left at 40 to 45 ° C. to make an ointment.

[Example 5] Production of lotion 2.1 g of stearyl alcohol was dissolved on a water bath, 5.5 g of span 20 and 40 g of liquid paraffin were added thereto, and the mixture was heated to 70 ° C. Dissolve 0.025 g of methyl parahydroxybenzoate and 0.015 g of propyl parahydroxybenzoate in boiling water, 5.5 g of Twin 20 and 1.0 g of sodium laurate sulfate, 0.5 g of peptide Gly-Arg-Gly-Glu-Ser-Pro of the present invention Add to dissolve and warm to 70 ° C. The aqueous phase was added to the oil phase, and the whole volume was adjusted to 100 ml with purified water, and stirred until cooling to 45 ° C. to obtain a lotion.

Example 6 Production of Tape Acrylic Copolymer Resin Adhesive 88.3g, Water 1.0g, Peptide Gly-Arg-Gly-Glu-Ser-Pro 0.5g of the Present Invention, Emazole 0.2g, Polyethylene Glycol Monolaurate 1.0 g, crotamiton 2.0 g, l-menthol 1.0 g, glycerin 6.0 g, and test drug 0.5 g were mixed and spreaded to obtain a tape preparation.

[Example 7] Manufacture of tablets The peptide Gly-Arg-Gly-Glu-Ser-Pro of the present invention was converted into 5 mg starch, 132 mg magnesium stearate, 18 mg lactose, 45 mg total, 200 mg according to a conventional method, Manufactured.

[Example 8] Production of injection 500 mg of the peptide Gly-Arg-Gly-Glu-Ser-Pro of the present invention, 0.3 g of polyethylene glycol (molecular weight: 4000), 0.9 g of sodium chloride, polyoxyethylene sorbitan monooleate 0.4 g Sodium metabisulfite 0.1g Methyl-paraben 0.18g, Propyl-paraben 0.02g, Distilled water for injection 100ml The above parabens, sodium metabisulfite and sodium chloride were stirred at 80 ° C in about half of the distilled water. Dissolve. The obtained solution is cooled to 40 ° C., the compound of the present invention, and then polyethylene glycol and polyoxyethylene sorbitan monooleate are dissolved in the above solution, and then distilled water for injection is added to the solution to obtain a final volume. The solution was sterilized by filtration using an appropriate filter paper and dispensed into ampoules by 1 ml to prepare an injection.

[Example 9] Measurement of collagen synthesis amount Collagen synthesis amount was measured by a non-protease collagenase assay.
Using human epidermal fibroblasts, the effect of the test drug on DNA synthesis of fibroblasts was examined. After 24 hours of human epidermis-derived 24th fibroblasts were cultured in Dulbecco's minimal essential medium (GIBCO) supplemented with the test drugs listed below and 10% calf serum, the cells were 10 μCi / mol ³ H- Treatment was carried out with proline (96 Ci / mmol, Amersham) or 25 μCi / mol ³⁵ S-methionine (1148 Ci / mmol, ICN Biomedical) for 23 hours. Thereafter, β-aminopropionitrile (50 μg / ml), ascorbic acid (50 μg / ml) and 2% calf serum were added and cultured. After culturing, the culture solution and the cell extract were separated, the cell extract was treated with 10 mM HCl at 37 ° C. for 100 minutes, and then the pH was neutralized with NaOH. Half of the sample was added with 1 ml of 50 mM Tris, 10 mM Caacetate, pH 7.2 and treated with non-protease collagenase (type III, 5 BTC units / ml, manufactured by Advanced Biofactures) at 37 ° C. for 3 hours. The other sample was not treated with this collagenase. Calf serum albumin (final concentration, 0.25 mg / ml) was added to each of the collagenase-treated solution and the collagenase-untreated solution. Both sample solutions were measured for total collagen. Total protein synthesis amount and total collagen synthesis amount (collagenase sensitive protein amount) were respectively measured as total radioactive activity.
The results are shown in Tables 1 and 2. From these results, it was shown that the test drug of the present invention specifically inhibits only collagen synthesis and does not affect the synthesis of other cell-derived proteins.

[Table 1]
Total collagen synthesis in culture (unit: 1 x 10 cpm)
Additive-free control 1935
Gly-Arg-Gly-Glu-Ser-Pro 547
Gly-Arg-Gly-Asp-Ser-Pro 508
Gly-Arg-Gly-Asp-Thr-Pro 492

[Table 2]
Total protein synthesis in culture (unit: 1 x 10 cpm)
Additive-free control 3901
Gly-Arg-Gly-Glu-Ser-Pro 3936
Gly-Arg-Gly-Asp-Ser-Pro 3824
Gly-Arg-Gly-Asp-Thr-Pro 3982

[Example 10] Fibroblast proliferation test Using human epidermal fibroblasts, the effect of the test drug on the proliferation of fibroblasts was examined. Passage 24 fibroblasts derived from human epidermis were cultured for 48 hours in Dulbecco's minimal essential medium (GIBCO) supplemented with the test drugs listed below and 10% calf serum. Next, the cells were cultured for 24 hours in a culture solution to which no test drug was added.
The cultured cells were treated with 100 mg NaCl and 10 mM Ca acetate with a type I, Sigma collagenase at a concentration of 3 mg / ml and treated for 100 minutes under conditions of pH 7.2 and temperature of 37 ° C. to separate the cells. The separated cells were treated with 0.05% trypsin (GIBCO) and 0.53 mM EDTA at 37 ° C. for 20 minutes. The treated cell sample was stained with trypan blue and the number of cells was measured.
The results are summarized in Table 3 below.
As is apparent from Table 3, there was no change in the control and fibroblast proliferation even when the test drug was administered, confirming the safety of the test drug of the present invention against fibroblasts.

[Table 3]
Number of cells (× 10 ³ / well)
Control 43.63
Gly-Arg-Gly-Glu-Ser-Pro 44.26
Gly-Arg-Gly-Asp-Ser-Pro 41.76
Gly-Arg-Gly-Asp-Thr-Pro 42.46
Mean n = 3

[Example 11] Measurement of DNA synthesis amount Using human epidermal fibroblasts, the influence of the test drug on DNA synthesis of fibroblasts was examined. Human epidermis-derived passage fibroblasts were cultured in Dulbecco's minimal essential medium (GIBCO) supplemented with the test drugs listed below and 10% calf serum for 21 hours, and the cells were 5 μCi / mol ³ H-thymidine ( 40 Ci / mmol, manufactured by ICN Biomedical Co., Ltd.) for 3 hours. After the treatment, the culture solution was removed and the cells were washed twice with PBS (−). The cultured cells were treated with 100 mg NaCl and 10 mM Ca acetate with a type I, Sigma collagenase at a concentration of 3 mg / ml and treated for 100 minutes under conditions of pH 7.2 and temperature of 37 ° C. to separate the cells. The separated cells were washed three times with 10% TCA at 4 ° C., and then the radioactivity was measured with a Beckman scintillation spectrometer in Budget-Solve (manufactured by Research Product International) to compare the number of cell divisions.
The results are summarized in Table 4 below. As is clear from the table, there was no significant difference in the amount of DNA even when the test drug was administered, and there was no change in the number of fibroblast divisions compared to the control.

[Table 4]
(Unit: 1x10cpm)
Additive-free control 19862
Gly-Arg-Gly-Glu-Ser-Pro 19923
Gly-Arg-Gly-Asp-Ser-Pro 19831
Gly-Arg-Gly-Asp-Thr-Pro 19870

[Example 12] Collagen gel atrophy suppression test A collagen gel atrophy test was conducted to examine the influence of the test drugs in Table 2 on the collagen gel atrophy. Dispense 2 ml of a 30% collagen solution, 10-fold concentration medium and buffer solution into a 6-well multi-well plate (1 hole 9.6 cm ² ) in a thermostat (37 ° C, 5% CO ₂ ). A collagen gel was prepared by heating for 1 hour. The concentration of each test drug was adjusted to 4 mM, 2 mM, 1 mM, and 0.5 mM, and 1 ml of this solution was dispensed into one well.
Next, 1 ml of fibroblast suspension (3 × 10 ⁴ cells / ml) is dispensed into each well (concentrations are 2 mM, 1 mM, 0.5 mM, and 0.25 mM, respectively). After 7 days, the size of the collagen gel was measured. The results are shown in Table 5 below. From Table 5, collagen gel atrophy was suppressed in the group to which the test drug was added.

[Table 5]
Atrophy area (cm ² )
Test drug concentration 0.25 mM 0.5 mM 1.0 mM 2.0 mM
Control 10.5
Gly-Arg-Gly-Glu-Ser-Pro 5.34 3.29 ± 0.65 1.75 ± 0.19 1.77 ± 0.85
Gly-Arg-Gly-Asp-Ser-Pro 3.92 ± 1.65 2.37 ± 1.62 0.52 ± 0.23 0.67 ± 0.29
Gly-Arg-Gly-Asp-Thr-Pro 2.52 ± 1.58 2.62 ± 0.49 2.78 ± 1.74 1.85 ± 0.47

[Example 13] Cut wound model The effect of the gel was examined using a cut skin model. As the gel, the gel of Example 2 containing each test drug in Table 3 at a concentration of 0.5% was used.
The back skin of an 8-week-old male male Wistar rat was shaved with an electric clipper, disinfected with alcohol cotton, and then a 3.4 cm incision was made along the midline with a scalpel. Sutures were applied at the center of the incision and at a total of three locations, 1 cm from the head and tail, and the sutures were removed after 3 days.
A gel preparation containing each test drug at a concentration of 0.5% was prepared, and 0.1 g each was applied twice in the morning and in the afternoon from the next day of skin cut preparation. Four days after skin cut creation, the skin including the center 2cm of the cut part is taken into a 3cm long strip, and one side is fixed and pulled. taking measurement.
The results are shown in Table 6. A significant decrease in wound strength was observed in the group to which the gel was applied, compared to the uncoated group.
[Table 6]
Test drug wound strength (g)
No application 152.6 ± 25.2
Gly-Arg-Gly-Glu-Ser-Pro 71.3 ± 12.3 *
Gly-Arg-Gly-Asp-Ser-Pro 84.4 ± 32.4 *
Gly-Arg-Gly-Asp-Thr-Pro 85.9 ± 26.9 *
Mean ± SE n = 6
*: P <0.05 Comparison with no application t test

[Example 14] Inhibitory effect on collagen synthesis of rat liver Ito cells Ito cells were collected from rat liver based on the method of Pinzani, M. et al. (J.clin. Invest. 84, 1786-1793 (1989)). That is, collagenase-added culture solution (HBSS-Ca, -Mg solution) was refluxed from the liver portal vein of SD rats, and the liver cells were separated after chopping the liver. After washing, the cells were overlaid with Ficol (specific gravity 1.053) solution and centrifuged at 20000 rpm, and Ito cells were separated from the upper layer.
The cells were subjected to the test after 10 days in the culture medium (Waymouth's MB752 + 10% horse serum + 10% bovine serum). Cells were seeded in 48-well plates at 1 × 10 ⁵ / ml. After 24 hours, the culture medium was replaced with D-MEM + 5.0% fetal bovine serum. Gly-Arg-Gly-Glu-Ser-Pro (hereinafter referred to as “the compound of the present invention”) containing glycine, arginine and proline (hereinafter referred to as “the compound of the present invention”) and ³ H-proline were added, followed by further incubation for 24 hours. did. After cultivation, the protein in the culture solution and cells was separated, and the protein was treated with collagenase. The amount of ³ H-proline of the protein degraded by collagenase was regarded as collagen, and the control of the compound as 100% was used to determine the collagen synthesis inhibitory effect of the compound of the present invention. As a result, the compound of the present invention showed 55.6% collagen synthesis inhibitory effect at 60 μM.

[Example 15] Effect of the compound of the present invention in a cirrhosis model caused by nitrosodimethylamine (NDMA) A cirrhosis model was prepared by the method of Baraona, E. et al. (Life sci. 52: 1249-1255 (1993)). That is, SD rats (male, 7 weeks old) were administered NDMA intraperitoneally at a dose of 10 mg / kg three times a week. The compound of the present invention was suspended in 1% carboxymethylcellulose and administered twice daily at a dose of 20 mg / kg. There were 13 rats in each group. Three weeks later, the rats were bled and the liver was removed. The extracted liver was fixed with formalin for tissue evaluation, and then subjected to Azan staining.
Serum GOT and GPT values were also determined. The results are shown in Table 7.
[Table 7]
GOT (U / ml) Inhibition rate (%) GOT (U / ml) Inhibition rate (%)
Normal rat 79 ± 4 35 ± 5
NDMA rat (control) 825 ± 45 210 ± 35
Rats administered the compound of the present invention 453 ± 34 45.1 121 ± 15 42.3
From Table 7, it can be seen that the compound of the present invention can suppress an increase in GOT value and GPT value, which are indicators of liver damage, at a dose of 20 mg / kg × 2.
In addition, the amount of collagen in the liver tissue was classified and evaluated according to the following criteria.
Score 0: Normal Score 1: Collagen fibers accumulate slightly around the hepatic lobule Score 2: Collagen fibers accumulate a little around the liver lobule Score 3: Collagen fibers accumulate slightly prominent around the liver lobule Score 4: Collagen fibers accumulate in the liver The results of accumulation around the leaflets are shown in Table 8 below.

[Table 8]
Liver fibrosis score inhibition rate (%)
NDMA rat (control) 1.81 ± 0.1
Rats administered with the compound of the present invention 1.29 ± 0.4 28.7
(Mean ± SE)
From Table 8, it can be seen that the compound of the present invention has an inhibitory effect on liver fibrosis even in tissue evaluation. From the above, the compound of the present invention showed an inhibitory effect on collagen synthesis in Ito cells, and also improved serum GOT and GPT values in a rat cirrhosis model and suppressed liver collagen deposition. It was considered effective for the prevention or treatment of fibrotic diseases.

[Example 16] Effect of short-chain peptide of the present invention in a patient who had undergone surgery A patient (46 years old, male) was injured with a laceration on the back of his left foot due to a traffic accident injury, and sutured in emergency surgery. After the healing, hypertrophic scar formation was observed in the wound. The scar containing 0.5 g of the short-chain peptide of the present invention was applied to the hypertrophic scar. The patient has been followed up, but after 3 months, the hypertrophic scars clearly show a tendency to disappear. Improved.

Claims (4)

A collagen synthesis inhibitor comprising as an active ingredient a peptide comprising the amino acid sequence of Gly-Arg-Gly-Glu-Ser-Pro . The collagen synthesis inhibitor according to claim 1 , which is a medicament for preventing or treating a disease caused by fibrosis. The collagen according to claim 2 , wherein the disease caused by fibrosis is selected from the group consisting of hypertrophic scar, scar contracture, keloidosis, collagen disease, pulmonary fibrosis, diabetic nephropathy, fibrous adhesions, and cirrhosis. Synthesis inhibitor. A method for producing a collagen synthesis inhibitor, comprising a step of mixing a peptide comprising an amino acid sequence of Gly-Arg-Gly-Glu-Ser-Pro with a pharmacologically acceptable carrier.