JP2008195628A - Therapeutic agent for diseases caused by organ dysfunction or degeneration - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent for diseases induced by the dysfunction or degeneration of organs, more particularly a therapeutic agent for diseases containing a single-stranded hepatocyte growth factor (HGF) as an active component. <P>SOLUTION: Disclosed is a therapeutic agent for a renal disease, a therapeutic agent for a liver disease or a therapeutic agent for a heart disease, a growth stimulator for a renal tubular cell, an inhibitor for fibrosis of a glomerulus, and a therapeutic agent specific to a damaged organ, which comprises a single-stranded HGF as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for diseases caused by organ dysfunction or degeneration, and more particularly to a therapeutic agent for diseases containing a single-chain hepatocyte growth factor (hereinafter abbreviated as HGF) as an active ingredient.

HGF is synthesized as an inactive single-chain HGF in vivo and then activated by a protease to become an active double-chain HGF. Since single-chain HGF has no biological activity, conversion to double-chain HGF is essential for the expression of biological activity. Known proteases that activate HGF include HGF activator (HGFA), matriptase and the like contained in blood.
Single-chain HGF has no biological activity, but retains affinity for c-Met, which is a receptor (see Non-Patent Document 1). Therefore, when single-chain HGF is administered, it acts as an HGF antagonist and inhibits c-Met activation. Single-chain HGF also inhibits the activity of double-chain HGF (see Non-Patent Document 2). Thus, it has been considered desirable to use double-stranded HGF for treatment by administering HGF.
The EMBO Journal, 1992, Volume 11 (No. 7), p. 2503-2510 The Journal of Clinical Investigation, 2004, Volume 114 (No. 10), p. 1418-1432

  An object of the present invention is to develop a new use of single-chain HGF.

The present inventors have obtained the following findings as a result of various studies to solve the above problems.
(i) When single-chain HGF is administered to an animal that has induced acute renal failure and chronic renal failure, it has a physiologically or pathologically excellent therapeutic effect against any renal failure.
(ii) When single-chain HGF is administered to an animal in which hepatic diseases such as hepatitis and cirrhosis have been induced, an excellent therapeutic effect is exhibited for any liver disease.
(iii) When single-chain HGF is administered to an animal in which a heart disease has been induced, an excellent therapeutic effect is exhibited for any heart disease.
The present invention has been completed based on the above findings, and provides the following therapeutic agents.
Item 1. A therapeutic agent for diseases caused by organ dysfunction or degeneration, comprising single-chain HGF as an active ingredient.
Item 2. Item 2. The therapeutic agent according to Item 1, wherein the single chain HGF is HGF derived from human, dog, cat, mouse or rat.
Item 3. Item 3. The therapeutic agent according to Item 1 or 2, wherein the disease caused by organ dysfunction or degeneration is kidney disease.
Item 4. Item 4. The therapeutic agent according to Item 3, wherein the renal disease is acute renal failure or chronic renal failure.
Item 5. Item 4. The therapeutic agent according to Item 3, wherein the renal disease is acute renal failure caused by trauma, fever, infection, sepsis, hemolysis, ischemia or poisoning such as drugs.
Item 6. Item 4. The therapeutic agent according to Item 3, wherein the renal disease is glomerulonephritis, diabetic nephritis, tubular nephritis, nephrosclerosis, cystic kidney, chronic pyelonephritis, malignant hypertension, SLE, amyloidosis or gout. .
Item 7. Item 3. The therapeutic agent according to Item 1 or 2, wherein the disease caused by organ dysfunction or degeneration is liver disease.
Item 8. Item 3. The therapeutic agent according to Item 1 or 2, wherein the disease caused by organ dysfunction or degeneration is a heart disease.
Item 9. Item 9. The therapeutic agent according to any one of Items 1 to 8, which is specific to an injured organ.
Item 10. An agent for promoting the proliferation of tubular cells, comprising single-chain HGF as an active ingredient.
Item 11. A glomerular fibrosis inhibitor containing single-chain HGF as an active ingredient.
Item 12. An injured organ-specific therapeutic agent containing single-chain HGF as an active ingredient.

The therapeutic agent of the present invention has an excellent therapeutic effect on organ dysfunction or degeneration, particularly renal diseases such as renal failure, liver diseases, heart diseases and the like. In the treatment of renal diseases, proliferation of tubular cells can be promoted, and glomerular fibrosis can be suppressed.
In addition, the single-chain HGF used in the present invention can be active HGF in an injured organ, so that it can exert a therapeutic effect in the injured organ without affecting the healthy organ.

Single chain HGF
The single-chain HGF used in the present invention is a known substance, and any one prepared by various methods can be used as long as it is purified to the extent that it can be used as a medicine. As the single-chain HGF, for example, human-derived HGF represented by the amino acid sequence represented by registration number P14210 (SEQ ID NO: 1) or NP_001010932 (SEQ ID NO: 2) registered in the NCBI database or the like; registration number NP_001002964 (sequence) 3), a dog-derived HGF represented by an amino acid sequence represented by BAC57560 and the like; a cat-derived HGF represented by an amino acid sequence represented by registration number NP — 001009830 (SEQ ID NO: 4), BAC10545, BAB21499, and the like; (SEQ ID NO: 5), mouse-derived HGF represented by the amino acid sequence represented by BAA01065, BAA01064, etc .; or represented by the amino acid sequence represented by registration number NP — 058813 (SEQ ID NO: 6), etc. Tsu DOO derived HGF, etc. Preferably, but not limited thereto. The protein having the amino acid sequence represented by SEQ ID NOs: 1 to 6 is natural single-chain HGF, and when activated, has a migration activity as HGF, a morphogenic activity, and the like.

As long as single-chain HGF is activated and has substantially the same action as HGF, one or more (for example, about 2 to 20, preferably about 2 to 10, preferably about 2 to 10 in the amino acid sequence; The amino acid may be substituted, deleted or added, and the sugar chain may be similarly substituted, deleted or added. Such a single-chain HGF, for example, a single-chain HGF that is substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above, includes, for example, the 161st to 165th amino acid sequences represented by SEQ ID NO: 1. Accession No. 1 registered in the database of single-chain HGF (5 amino acid deletion type HGF) represented by SEQ ID NO: 2 in which 5 amino acid residues of SEQ ID NO: 2 are deleted or NCBI. Mention may be made of human-derived single-chain HGF such as BAA14348 or AAC71655, or the above-mentioned single-chain HGF of dogs, cats, rats or mice. Here, with respect to the amino acid sequence, “one or a plurality of amino acids are deleted, substituted or added” is generated by a well-known technical means such as a genetic engineering technique, site-directed mutagenesis, or the like, or occurs naturally. It means that a sufficient number (one to plural) is deleted, substituted or added. A single-chain HGF in which a sugar chain is substituted, deleted, or added is, for example, a single-chain HGF in which a sugar chain added to a natural single-chain HGF is treated with an enzyme or the like to delete the sugar chain, or a sugar Those in which the amino acid sequence at the glycosylation site is mutated so that the chain is not added, or those in which the sugar chain is mutated so that the sugar chain is added to a site different from the natural glycosylation site, etc. Say. Specifically, for example, with respect to human HGF with the registration number NP_001010932 registered in the NCBI database, the position 289 Asn of the glycosylation site is Gln, the position 397 Asn is Gln, the position 471 Thr is Gly, 561 Examples include HGF [Fukuta K et al., Biochemical Journal, 388, 555-562 (2005)] in which a sugar chain is prevented from being added by substituting position Asn with Gln and position 648 with Asn. it can.
Furthermore, it is a protein having at least about 80% homology with the amino acid sequence of single-chain HGF, preferably a protein having about 90% or more homology, more preferably a protein having about 95% or more homology. A protein having substantially the same activity as HGF when activated is also included in the single-chain HGF used in the present invention. “Homology” in the above amino acid sequences means the degree of coincidence of amino acid residues constituting each sequence by comparing the primary structures of proteins.

In the single-chain HGF used in the present invention, the C-terminus is either a carboxyl group (—COOH), a carboxylate [—COOM (M represents a metal)], an amide (—CONH ₂ ) or an ester (—COOR). There may be. Here, R in the ester is, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α -C6-12 aryl groups such as naphthyl, for example, C7-14 aralkyl groups such as phenyl-C1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl, C2-6 alkanoylmethyl groups such as acetyloxymethyl and pivaloyloxymethyl are used. When the single-chain HGF used in the present invention has a carboxyl group or a carboxylate other than the C-terminus, the single-chain HGF in the present invention is also a product in which the carboxyl group or carboxylate is amidated or esterified. include. As the ester in this case, for example, the above C-terminal ester or the like is used. Further, in the single-chain HGF used in the present invention, the amino group of the N-terminal methionine residue is a protecting group (for example, C1-6 such as a C2-6 alkanoyl group such as formyl group and acetyl) in the above-described protein. A group protected by an acyl group, a N-terminal side cleaved in vivo and a glutamyl group produced by pyroglutamine oxidation, a reactive group on the side chain of an amino acid in the molecule (for example, —OH, —SH) , An amino group, an imidazolyl group, an indolyl group, a guanidino group, etc.) protected with an appropriate protecting group (for example, a C1-6 acyl group such as a C2-6 alkanoyl group such as formyl group or acetyl), or Also included are complex proteins such as so-called glycoproteins to which sugar chains are bound.

  The single chain HGF used in the present invention may form a salt. Examples of the salt include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

  The single-chain HGF is cultured in an environment that does not contain an enzyme that activates HGF, such as primary cultured cells or cell lines that produce HGF without adding serum, and is separated and purified from the culture supernatant. Single-chain HGF can be obtained. Alternatively, a gene encoding single-stranded HGF is incorporated into an appropriate vector by genetic engineering techniques, and this is inserted into an appropriate host cell for transformation, and the transformant is cultured in a serum-free state, and the culture supernatant The desired recombinant single-stranded HGF can also be obtained from the above. (See, for example, JP-A No. 5-111383, Biochem. Biophys. Res. Commun. 1989, Vol. 163, p. 967). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as E. coli, yeast, bacilli, insect cells, plant cells or animal cells can be used. Alternatively, a method in which baculovirus is inoculated into larvae or pupae of silkworm or silkworm can be used (for example, the method described in WO01 / 092332). Alternatively, a method of incorporating an HGF gene into a genome gene of an organism that does not have an enzyme that activates HGF in silkworms, plants, etc. with an appropriate vector and expressing it in the body fluid of the resulting transgenic organism, etc. Can be used.

Renal disease In the present invention, “renal disease” refers to all conditions that cause a decrease in renal blood flow, and when undergoing tubular epithelial injury including interstitial nephritis, glomerulus including small vessel injury is damaged. including. Specific examples of the renal disease include acute renal failure and chronic renal failure.

  Acute renal failure is a condition in which renal function is suddenly reduced after several days or weeks due to damage to tubules including the stroma due to various causes. Examples include elevation of BUN and Cre, electrolyte abnormality, acidosis, oliguria, hematuria, elevation of urinary β2-microglobulin, elevation of urinary NAG (N-acetylglucosaminidase), and the like. Causes of acute renal failure include, for example, trauma, major injury and shock including fever, infection, sepsis, significant hemolysis, ischemia or drug addiction. Trauma includes crash syndrome and the like. Infectious diseases include pyelonephritis and infectious interstitial nephritis. Hemolysis includes paroxysmal nocturnal hemoglobinuria (PNH) characterized by early hemoglobinuria due to abnormally early destruction of red blood cells in the blood vessels, incompatible blood transfusion, or hemolysis when using cardiopulmonary bypass. Examples of the drug include β-lactams, aminoglycosides, thiazide diuretics, warfarin, non-steroidal anti-inflammatory analgesics and the like. The drug includes heavy metals such as mercury chloride; organic solvents such as methanol and carbon tetrachloride; agricultural chemicals such as paraquat, other uric acids, contrast agents, snake venom and the like.

Chronic renal failure refers to nephritis that has gradually progressed over several months to several years. The symptoms are the same as those of acute nephritis, but generally humans have a serum Cre value of 2 mg / dL or higher or a BUN of 20 mg / dL. Those that have sustained the above. In addition, dogs have a Cre value of 2 mg / dL or more, and cats have a Cre value of 3 mg / dL or more (international regional intellectual society; IRIS standards).
Causes of chronic renal failure include, for example, glomerulonephritis, diabetic nephritis, tubular nephritis, nephrosclerosis, cystic kidney, chronic pyelonephritis, malignant hypertension, SLE (systemic lupus erythematosus; systemic lupus erythematosus) and other immune diseases , Amyloidosis, gout and the like. Regarding dogs and cats, it is thought that chronic renal failure develops by a mechanism different from immune disease, but the cause has not been analyzed sufficiently.
Liver Disease In the present invention, the type of “liver disease” is not particularly limited. Symptoms include degenerative necrosis of the liver, inflammation of the liver, decreased liver synthesis function, decreased liver metabolic function, fibrosis, cholestasis, ischemia and the like. The names of diseases include chronic hepatitis, acute hepatitis, fulminant hepatitis, alcoholic hepatitis, cirrhosis, drug-induced (including toxic) liver damage, ischemic liver damage, lupoid hepatitis, fatty liver, and primary liver. Examples include cancer, metastatic liver cancer, hemolytic jaundice, and obstructive jaundice. Concerning dogs, congenital or acquired portal vein cava shunt is known (Comparative Hepatolory 2005 Dec 7; 4: 7), and single-chain HGF is effective for this treatment.
Heart Disease In the present invention, the type of “heart disease” is not particularly limited. For example, heart failure; bacterial or non-bacterial endocarditis; mitral valve stenosis, mitral regurgitation, aortic valve stenosis, aortic regurgitation; acute pericarditis, chronic Pericarditis such as contractile pericarditis; myocarditis; angina pectoris; myocardial infarction; cardiac asthma; pulmonary heart; sudden cardiomyopathy; For canine heart disease, single-chain HGF is effective in treating dilated cardiomyopathy with fibrosis. In addition, with regard to mitral regurgitation, which is common in dogs, single chain HGF is effective for treatment because it suppresses the accumulation of collagen fibers, which is considered to be a factor.

Treatment In the present invention, “treatment” refers to the amelioration or complete cure of symptoms of organ dysfunction and degeneration.
In the case of kidney disease, “treatment” includes, for example, an increase in blood urea nitrogen (hereinafter abbreviated as BUN) or creatinine (hereinafter abbreviated as Cre) in the kidney disease, electrolyte abnormality, It includes suppressing or preventing acidosis, oliguria or polyuria and normalizing them. The treatment also includes promoting the proliferation of tubule cells at sites where the tubule cells have degenerated or decreased. Therefore, single-chain HGF can also be used as an active ingredient of a proliferation promoting agent for tubular cells. The treatment also includes suppressing or preventing fibrosis of the glomerulus. As a result, the specific gravity of the urine is improved by improving the amount of glomerular filtration and improving the reabsorption capacity in the tubules. Effects such as improvements are also included. Therefore, single chain HGF can also be used as an active ingredient of a glomerular fibrosis inhibitor.
Remission or complete cure of liver disease includes one or more normalization or normalization tendency such as subjective symptoms and abnormal test values used for diagnosis of each liver disease. Remission or complete cure of a heart disease includes one or more normalization or normalization tendency such as subjective symptoms and abnormal test values used for diagnosis of each heart disease.
In addition, as described above, since single-chain HGF specifically treats dysfunction or degeneration of damaged organs without affecting normal organs, therapeutic agents containing single-chain HGF are specific for damaged organs. Can also be an agent. In this case, the treatment of the damaged organ, that is, the amelioration or complete cure of the organ damage, has one or more normalization or normalization tendency such as subjective symptoms and abnormal test values used for diagnosis of the organ damage. included.
Dosage form

  When the therapeutic agent of the present invention is administered to a patient, the preparation may take various preparation forms such as liquids, solids, etc., but generally only single-chain HGF, or it with a conventional carrier. It is preferably formulated into injections, sustained-release preparations (for example, depots) and the like. The injection may be either an aqueous injection or an oily injection. In the case of an aqueous injection, according to a known method, for example, an aqueous solvent (water for injection, purified water, etc.) is added to a pharmaceutically acceptable additive such as an isotonic agent (sodium chloride, potassium chloride, glycerin, mannitol, Sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon) Aminocaproic acid buffer, etc.), preservatives (methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boro Acid, borax, etc.), thickener (hydroxyethylcellulose) Hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.) or pH adjusters (hydrochloric acid, sodium hydroxide) , Phosphoric acid, acetic acid, etc.) can be prepared by dissolving single-stranded HGF in a solution appropriately added, and then sterilizing by filtration with a filter or the like, and then filling in an aseptic container. Further, a suitable solubilizing agent such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.) or nonionic surfactant (polysorbate 80, polyoxyethylene hydrogenated castor oil 50 etc.) may be further blended. Good. In the case of an oily injection, for example, sesame oil or soybean oil is used as the oily solvent, and benzyl benzoate or benzyl alcohol may be blended as a solubilizing agent. The prepared injection solution is usually filled in an appropriate ampoule or vial. The single-chain HGF content in the injection can be usually adjusted to about 0.0002 to 0.2 w / v%, preferably about 0.001 to 0.1 w / v%. It should be noted that liquid preparations such as injections are preferably stored after removing moisture by freeze storage or freeze drying. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.

  The single-chain HGF used in the present invention can be used as a sustained-release preparation (for example, a depot) together with a biodegradable polymer. By using single-chain HGF in particular as a depot, effects such as reduction in the number of administrations, sustained action and reduction of side effects can be expected. The sustained-release preparation can be produced according to a known method. The biodegradable polymer used in the sustained-release preparation can be appropriately selected from known biodegradable polymers. For example, polysaccharides such as starch, dextran or chitosan; proteins such as collagen or gelatin Polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine; polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer; polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, polyanhydride Or polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer; polyalkyl cyanoacrylic acid such as polyorthoester or polymethyl-α-cyanoacrylic acid; polycarbonate such as polyethylene carbonate or polypropylene carbonate, etc. It is. Polyester is preferable, and polylactic acid or lactic acid-glycolic acid copolymer is more preferable. When polylactic acid or lactic acid-glycolic acid copolymer is used, the composition ratio (polylactic acid or lactic acid / glycolic acid) (mol%) varies depending on the sustained release period. For example, the sustained release period is about 2 weeks to 3 months. In the case of about 2 weeks to 1 month, preferably about 100/0 to 50/50. The weight average molecular weight of the polylactic acid or lactic acid-glycolic acid copolymer is generally preferably about 5,000 to 20,000. Polylactic acid or lactic acid-glycolic acid copolymer can be produced according to a known production method, for example, the production method described in JP-A No. 61-28521. The blending ratio of the biodegradable polymer and the single-chain HGF is not particularly limited. For example, the single-chain HGF is preferably about 0.01 to 30 w / w% with respect to the biodegradable polymer.

Target of administration The therapeutic agent of the present invention is applied to mammals other than humans, such as dogs, cats, rats, mice, ferrets, monkeys, cows, horses, pigs, sheep, rabbits, hamsters, guinea pigs, chimpanzees, etc. Is also applicable. In particular, it can be suitably used for humans. When it is a therapeutic agent for heart disease, it is particularly effective for animals such as dogs.
In addition, when it is a therapeutic agent for kidney disease, it is preferably administered to a mammal suffering from each kidney disease. When it is a proliferation promoting agent for tubular cells, tubular cells are degenerated or decreased. It is preferably administered to a mammal, and when it is a glomerular fibrosis inhibitor, a mammal in which the glomerulus is becoming fibrotic or a mammal showing symptoms of proteinuria, which is the preceding stage, particularly In humans and animals, it is preferably administered to cats.
When it is a therapeutic agent for liver disease, it is preferably administered to a mammal suffering from each liver disease, and when it is a therapeutic agent for heart disease, it is administered to a mammal suffering from each heart disease. Administration is preferred.
Treatment Method Examples of the administration method of the therapeutic agent for diseases caused by organ dysfunction or degeneration of the present invention include subcutaneous, intramuscular and intravenous injection. In the treatment of renal diseases, a method of locally injecting the renal artery, injecting directly into a region close to the glomerulus of the kidney, or embedding a sustained-release preparation (depot agent) in a region close to the glomerulus of the kidney is also preferred. It is done. In the treatment of liver disease, a method of intravenous infusion is also preferable. In the treatment of heart disease, a method of local administration to the heart using a catheter is also preferred.
The dose is appropriately selected according to the type of animal to be administered, dosage form, degree or age of disease, body weight, etc., but is usually about 1 μg to 500 mg, preferably about 10 μg to 100 mg per dose. Preferably, it is about 50 μg to 15 mg. In addition, the number of administrations is appropriately selected depending on the dosage form, the degree of disease, age, etc., and can be administered once or continuously at a certain interval. In the case of continuous administration, the administration interval may be once a day to once every several months. For example, in the case of administration by a sustained-release preparation (depot), it may be once every several months.

Hereinafter, the present invention will be described using test examples, but the present invention is not limited thereto. In the following, the meaning of each abbreviation is as follows.
BUN: blood urea nitrogen Cre: creatinine Ccr: creatinine clearance PBS: phosphate buffered saline ELISA: enzyme immunoassay TGF-β1: Transforming growth factor-β1; tumor cell growth factor-β1
α-SMA: α-smooth muscle actin; α-smooth muscle actin BCA: bicinchoninic acid UUO: Unilateral ureteral obstruction; unilateral ureteral obstruction SDS-PAGE: SDS-Polyachrylamide gel electrophoresis; sodium dodecyl sulfate polyacrylamide gel electrophoresis PVDF: polyvinylidenedifluoride Polyvinylidene fluoride HRP: Horseradish Peroxidase; Horseradish peroxidase% in the examples indicates mass% unless otherwise specified.

[Test Example 1]
Effect of single-chain HGF on chronic kidney injury model cat
1. Method
(1) Test substance As a test substance, a recombinant feline single-chain HGF prepared by the method described in JP-A-2005-170835 (hereinafter abbreviated as “cat HGF”) is adjusted to a concentration of 80 μg / mL. A solution (referred to as cat HGF solution) dissolved in 10 mM citrate buffer (pH 6.0) containing 15 M sodium chloride and 0.01% Tween 80 was used.
As placebo, 10 mM citrate buffer (pH 6.0) containing 0.15 M sodium chloride and 0.01% Tween 80 was used.
(2) Test animals The test animals used were 12 hybrid male cats 10 months of age or older that were confirmed to have no clinical or hematological findings. The normal body weight before the start of the test (before model creation) was 4.38 ± 0.40 kg. The test animals were kept in a metabolic cage for each individual in a clean room where temperature and humidity were controlled. Feeding was limited to once a day, and 50 g of “Imus Chicken” (manufactured by AIMS Japan) was fed. For water supply, tap water was freely consumed.

(3) Production of kidney injury Production of kidney injury was performed by administering gentamicin (animal gentamine injection solution, Nippon Zenyaku Kogyo) subcutaneously to the back of the animal once a day for 5 days a week. . The single dose started from a 7.5-fold dose (30 mg / kg) of the normal dose (4 mg / kg) and gradually increased to a 11.25-fold dose. Administration of gentamicin was terminated for each individual when the BUN concentration was 50 mg / dL or more and the Cre concentration was 3 mg / dL or more in the blood test results conducted once a week in principle. The blood test was performed using the discrete rate clinical chemistry automatic analyzer (Synchron CX4 delta system; manufactured by Beckman Coulter), with BUN for the UV rate method and Cre for the rate Yaffe method.
Subsequently, the criteria at the start of test substance administration (hereinafter referred to as test criteria) were set as shown in Table 1.

1) Ccr was calculated by the following formula based on urine and serum Cre, urine volume, and body weight.
Ccr (mL / min / kg) = (Urine Cre x Urine volume) / (Serum Cre x 1440 x Body weight)
2) Urine specific gravity was measured using a digital clinical refractometer (SU-202, CIS).

After the end of administration of gentamicin, in principle, observation and examination for 3 weeks are performed to confirm whether the renal pathology in each test cat is stable in a state satisfying the test criteria, and the test substance administration start point (0d 6) of the index values of) were divided into the following two groups based on the Cre value.
HGF group (n = 6): group in which cat HGF is intravenously administered twice a day for 14 consecutive days Control group (n = 6): group in which placebo is administered intravenously twice a day for 14 consecutive days

(4) Test substance administration The HGF group had a cat HGF solution at a dose of 0.5 mL / kg (cat HGF 40 μg / kg), and the control group had a placebo at a dose of 0.5 mL / kg twice a day (at 9 o'clock). 16:00), it was intravenously administered for 14 consecutive days (0d-13d).

(5) During the clinical findings test period, the clinical findings (appetite) of each individual were observed and scored (score 0-4) according to the criteria in Table 2. In addition, the body weight of each individual was measured every day during the test period before drug administration. The average value ± standard deviation of each group was calculated from the measured values. The clinical findings score was examined using Mann-Whitney test for changes over time with reference to 0d.
For the intra-group variation in body weight, Dunnett's test was performed for differences from each time point with reference to 0d.

(6) Measurement of serum Cre As a rule, once a week during the gentamicin administration period, 0d (before the start of administration), 3d (administered on the fourth day of administration) during the administration period of the test substance and after the end of gentamicin administration Before, blood sampling was performed at 7d (before administration on day 8 of administration), 10d (before administration on day 11 of administration) and 15d (on day 2 of administration discontinuation).
For blood sampling, about 3 mL of blood was collected from the jugular vein of each test cat after the observation of each individual's findings and the like, and the serum was separated by centrifugation at 3000 rpm for 15 minutes to measure serum Cre. Serum Cre was measured by the rate-Jeffe method using a discrete clinical chemistry automatic analyzer (Synchron CX4 delta system; manufactured by Beckman Coulter).
The change in serum Cre was expressed as a relative value obtained by dividing the value at each time point by the reference value with 0d as the reference value, and the difference between the two groups at each time point was examined using the Tukey test.

(7) Measurement of urine specific gravity As a rule, once a week during the gentamicin administration period, 0d (before the start of administration), 3d (administration on the fourth day of administration) during the test substance administration period and after the end of gentamicin administration Urine sampling was performed before, 7d (before administration on day 8 of administration), 10d (before administration on day 11 of administration) and 15d (on day 2 of administration discontinuation).
Urine sampling is performed by storing the urine naturally excreted as 24-hour excretion urine of each test cat after the observation of each individual's findings, etc. in a glass Erlenmeyer flask placed under each metabolic gauge. Was measured using a graduated cylinder, and the urine specific gravity was measured using a digital clinical refractometer (SU-202 manufactured by CIS Co., Ltd.).
The change in urine specific gravity was expressed as a relative value obtained by dividing the value at each time point by the reference value with 0d as the reference value, and the difference between the two groups at each time point was examined using the Tukey test.

2. result
(1) Production of kidney injury and classification Test criteria for each group at 0d are BUN80.1 ± 21.6 mg / dL, Cre5.2 ± 1.5 mg / dL, endogenous Ccr0.60 ± 0 in the control group, respectively. .13 mL / min / kg and urine specific gravity 1.023 ± 0.005, BUN 92.0 ± 44.1 mg / dL, Cre5.3 ± 1.9 mg / dL, endogenous Ccr 0.69 ± 0.28 mL / in HGF group Min / kg and urine specific gravity were 1.021 ± 0.004. For these index values, both groups were equivalent populations (t-test p <0.05) showing equal variance (F-test p <0.05).

(2) Change in body weight No significant change in body weight over time was observed in both groups.
(3) Table 3 shows the results of the clinical findings appetite score. In the control group, there was no significant change in appetite over time, whereas in the HGF group, there was a significant increase in appetite over time.

(4) Serum Cre
The results are shown in FIG. The relative value of serum Cre was lower in the HGF group compared to the control group, and the Cre relative value at 15d was 0.82 ± 0.09 in the control group, compared to 0. 0 in the HGF group. A decrease of 71 ± 0.12 was observed (FIG. 1).

(5) The urine specific gravity results are shown in FIG. The relative urine specific value was unchanged from the decrease in the control group, while it was clearly increased in the HGF group. The relative urine specific value at 15d was 0.9997 ± 0.0033 in the control group. In contrast, the HGF group showed a significant increase of 1.0101 ± 0.0072 (FIG. 2).

[Test Example 2] Effect of single-chain HGF on renal tissue fibrosis
1. Method
(1) Test substance As a test substance, 0.15M sodium chloride and 0.01% Tween 80 are used so that a recombinant feline single chain HGF (hereinafter abbreviated as cat HGF) has a concentration of 80 μg / mL. A solution (referred to as a cat HGF solution) dissolved in 10 mM citrate buffer (pH 6.0) was used.
As placebo, 10 mM citrate buffer (pH 6.0) containing 0.15 M sodium chloride and 0.01% Tween 80 was used.
(2) UUO model cats A UUO model cat was created by ligating the left ureter of a mixed male cat 10 months of age or older, and a cat HGF solution administration group (hereinafter referred to as HGF group) and a placebo administration group (hereinafter referred to as control). Divided into groups).

(3) Measurement of fibrosis-related factors In the HGF group, the cat HGF solution 0.5 mL / kg (cat HGF 40 μg / kg) was administered intravenously twice a day for 6 days from the day after UUO model cat production (ureter ligation). Administered. In the control group, placebo was intravenously administered twice daily at a dose of 0.5 mL / kg for 6 days from the day after ureter ligation. After administration of cat HGF solution or placebo for 6 days, kidney tissue (left kidney presenting with hydronephrosis) was removed from each UUO model cat. The removed kidney tissue was frozen with liquid nitrogen and stored at −80 ° C. until the test. Stored renal tissue is obtained from Mizuno. According to the method of S et al. (Kidney Int. 2001 Apr; 59 (4): 1304-14), each was homogenized and centrifuged with PBS, and the resulting supernatant was used as a sample. For each sample, TGF-β1, TGF-β receptor 1, fibronectin and α-SMA were measured as fibrosis-related factors. Among the fibrosis-related factors, TGF-β1 was measured by ELISA, and TGF-β receptor 1, fibronectin, α-SMA, and marker actin were measured by Western blotting.

In the ELISA, first, the total protein concentration of a sample to be subjected to ELISA is measured by protein concentration measurement by BCA method (BCA kit; manufactured by PIERCE; 570 nm), and then TGF-β1 measurement ELISA kit (manufactured by BIOSOURCE) is used for TGF. -Β1 levels were measured.
The obtained data was calculated as the amount per 1 mg of protein, and statistical analysis (Tukey method, p <0.05) was performed on the difference between groups.

  In Western blotting, proteins in a sample were first separated by electrophoresis using 10% SDS polyacrylamide gel. Samples added during electrophoresis were unified at a protein concentration of 0.06 mg / lane, and a molecular weight marker was added in the first lane. After electrophoresis, it was transferred to a membrane (PVDF membrane). After completion of the transfer, the membrane was cut at the boundary between the molecular weight marker portion and the sample portion, and both were blocked with 5% skim milk / PBS for 1 hour. Subsequently, the membrane of the sample portion was subjected to antibody staining using a primary antibody and a secondary antibody. The reaction was performed for 3 hours for the primary antibody and 1 hour for the secondary antibody. When the antibody was changed, the reaction was performed 3 times with a washing solution (0.0005% Tween 20 added PBS). The membrane of the marker part was washed with washing solution three times after blocking, and then reacted with HRP (Precision Strep Tactin-HRP Conjugate (manufactured by IORAD)) All work was performed at room temperature. After washing with washing solution three times, a luminescent substrate (ECL Protein Biotinylation Module; manufactured by GE Healthcare Bioscience) was adsorbed, and each band was detected using a luminometer (Light-Capture; manufactured by ATTO). Mass analysis was performed on the detection band of CS using an image analysis software (CS Analyzer; manufactured by ATTO) Anti TGF-β1 SC-398 (manufactured by Santa Cruz) was used as the primary antibody of TGF-β1, and the primary of fibronectin was used. Anti fibronectin BD-610077 (Pharmingen (Astellas Pharma)) is used as the antibody, and Alpha-smooth Muscle antibody M0851 (DAKO) is used as the primary antibody of α-SMA. Was used. As the secondary antibody with HRP-labeled anti-mouse IgG antibody (manufactured by Cosmo Bio).

(4) Data analysis The TGF-β receptor 1, fibronectin and α-SMA levels of each sample were calculated as amounts obtained by relativizing each at the actin level (calculated by dividing each value by the actin level). For the obtained data, the mean value ± standard deviation in each group was calculated, and statistical analysis (Tukey method, p <0.05) was performed on the difference between groups.

2. Results The results in this test are shown in Table 4. In the HGF group in which feline HGF was systemically administered to UUO model cats that follow the renal fibrosis process after ureteral ligation, the TGF-β1 level in the kidney was lower than that in the control group. Similarly, the TGF-β receptor 1 level in the kidney tissue was lower in the HGF group administered with feline HGF than in the control group. This was considered that the administration of cat HGF suppressed the expression of TGF-β1 and the expression of TGF-β receptor present in the cell membrane. In the HGF group, fibronectin and α-SMA levels in the renal tissue were lower than in the control group, and administration of cat HGF suppressed the progression of renal fibrosis. From these results, it was found that feline HGF has an effect of suppressing renal fibrosis on damaged kidneys.

[Reference Example] Production of recombinant feline HGF in silkworm parasites PCR and cloning were carried out according to Example 1 (c) of WO01 / 092332 using feline leukocyte-derived cDNA as a template to obtain plasmid DNA. Recombinant baculovirus recombined with DNA encoding fHGF and dfHGF was obtained from the obtained plasmid DNA according to Superworm service of Katakura Industry Co., Ltd. according to Example 3 of WO01 / 092332. The obtained virus solution of the recombinant virus was administered to silkworm parasites, and after breeding for several days, silkworm body fluids were collected. The obtained silkworm body fluid was purified according to the example of Japanese Patent Application Laid-Open No. 2005-170835 and used in the above test as recombinant feline single chain HGF.
The obtained recombinant feline single-stranded HGF was subjected to SDS-polyacrylamide electrophoresis according to a conventional method, followed by Western blotting, and a recombinant fHGF protein was confirmed at a molecular weight of about 850,000. In addition, the obtained recombinant feline single chain HGF was cultured in MDCK cells in a DMEM medium supplemented with 9% serum according to Example 4 of WO01 / 092322, so that the single chain was produced by the action of the HGF activating enzyme in the serum. The biological activity of HGF converted from to active form was measured and confirmed to have HGF activity.

  The therapeutic agent or disease-suppressing agent of the present invention is useful as a drug for the treatment of renal diseases, particularly renal failure. It is also useful as a drug for the treatment of liver disease and heart disease. Furthermore, it is useful as a drug for specifically treating only damaged organs regardless of organs.

FIG. 1 is a view showing a change with time of a relative value of serum Cre in a chronic kidney injury model cat. In the figure, d indicates the day. FIG. 2 is a diagram showing the change over time of the relative value of urine specific gravity in a chronic kidney injury model cat. In the figure, d indicates the day.

Claims (12)

  A therapeutic agent for diseases caused by organ dysfunction or degeneration, comprising single-chain HGF as an active ingredient.   The therapeutic agent according to claim 1, wherein the single-chain HGF is HGF derived from human, dog, cat, mouse or rat.   The therapeutic agent according to claim 1 or 2, wherein the disease caused by organ dysfunction or degeneration is a renal disease.   The therapeutic agent according to claim 3, wherein the renal disease is acute renal failure or chronic renal failure.   The therapeutic agent according to claim 3, wherein the renal disease is acute renal failure caused by trauma, fever, infection, sepsis, hemolysis, ischemia or poisoning such as a drug.   The treatment according to claim 3, wherein the renal disease is glomerulonephritis, diabetic nephritis, tubular nephritis, nephrosclerosis, cystic kidney, chronic pyelonephritis, malignant hypertension, SLE, amyloidosis or gout. Agent.   The therapeutic agent according to claim 1 or 2, wherein the disease caused by organ dysfunction or degeneration is a liver disease.   The therapeutic agent according to claim 1 or 2, wherein the disease caused by organ dysfunction or degeneration is a heart disease.   The therapeutic agent according to any one of claims 1 to 8, which is specific to an injured organ.   An agent for promoting the proliferation of tubular cells, comprising single-chain HGF as an active ingredient.   A glomerular fibrosis inhibitor containing single-chain HGF as an active ingredient. An injured organ-specific therapeutic agent containing single-chain HGF as an active ingredient.