JP2009120607A - New prophylaxis or therapeutic agent for hepatic disease, and insulin resistance ameliorating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a prophylaxis or therapeutic agent for hepatic disease, and to provide an insulin resistance ameliorating agent. <P>SOLUTION: The prophylaxis or therapeutic agent for hepatic disease contains N-(3,4-dimethoxycinnamoyl)anthranilic acid or its pharmacologically permissible salts as an active ingredient, and the insulin resistance ameliorating agent contains N-(3,4-dimethoxycinnamoyl)anthranilic acid or its pharmacologically permissible salts as an active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a preventive or therapeutic agent for liver diseases, particularly nonalcoholic steatohepatitis. The present invention further relates to an insulin resistance improving agent.

  Nonalcoholic fatty liver (NAFLD) is the most common liver disease in developed countries (see Non-Patent Documents 1 and 2). NAFLD includes a wide range of liver diseases, ranging from simple fatty liver to steatohepatitis and cirrhosis. Nonalcoholic steatohepatitis (NASH) is a severe form of NAFLD, characterized by steatosis, inflammation, and fibrosis. Recently, NAFLD has been recognized as an element of metabolic syndrome (see Non-Patent Documents 3 to 5). Insulin resistance is a pathological condition underlying metabolic syndrome, and plays an important role in the pathogenesis of NASH. The present inventors recently created a rat model of NASH with insulin resistance, and pioglitazone, an agonist of PPARγ (peroxisomal proliferator activated receptor-γ), prevents the progression of NASH through improvement of insulin resistance. (See Non-Patent Document 6). However, it is not clear whether all NASH patients are responders of insulin sensitizers such as PPARγ agonists, and the optimal treatment for NASH has not been established at present.

  TGF-β (Transforming growth factor-β) is a multifunctional peptide and has an important role in fibril formation (see Non-Patent Documents 7 to 10). Expression of TGF-β is enhanced in liver fiber tissue, and TGF-β is known to function as a fibrosis mediator in many liver diseases (see Non-Patent Document 11). It has been reported that TGF-β stimulates collagen production in cultured stellate cells, and overexpression of TGF-β in the liver causes liver fibrosis (see Non-Patent Documents 12 and 13). Inhibition of TGF-β synthesis and TGF-β signal has been reported using different experimental systems to prevent liver fibrosis (see Non-Patent Documents 14 to 17). The TGF-β signal may be recognized as an anti-fibrotic target for fibrosis-related liver disease.

  Tranilast (N- (3 ', 4'-dimethoxyciannamoyl) -anthranilic acid) is a drug originally developed as an antiallergic agent and is used for bronchial asthma, atopic dermatitis and the like (see Non-Patent Document 7). . Recently, it was reported that tranilast exerts an anti-fibrotic effect by inhibiting the action of TGF-β. In fact, tranilast is used for fibrosis-related skin diseases such as skin scars and sclerosis (see Non-Patent Documents 18 and 19). However, there are no reports to date that have proved usefulness of tranilast for liver disease in animal models.

Reid AE. Et al. Gastroenterology 2001; 121: 710-723. Angulo P. et al., N Engl J Med 2002; 346: 1221-1231. Marchesini G. et al, Hepatology 2003; 37: 917-923. Marchesini G. et al., Diabetes 2001; 50: 1844-1850. Marceau P. et al., J Clin Endocrinol Metab 1999; 84: 1513-1517. Ota T. et al, Gastroenterology 2007; 132: 282-293. Suzawa H. et al., Jpn J Pharmacol 1992; 60: 85-90. Bonnet F. et al., Diabetes Metab 2003; 29: 386-392. Ikeda H. et al., Biochem Biophys Res Commun 1996; 227: 322-327. Miyazawa K. et al., Atherosclerosis 1995; 118: 213-221. Liu X. et al., Liver Int 2006; 26: 8-22. Chapman HA. Et al., J Clin Invest 2004; 113: 148-157. Casini A. et al., Gastroenterology 1993; 105: 245-253. George J. et al., Proc Natl Acad Sci U S A 1999; 96: 12719-12724. Arias M. et al., BMC Gastroenterol 2003; 3:29. Qi Z. et al., Proc Natl Acad Sci U S A 1999; 96: 2345-2349. Nakamura T. et al., Hepatology 2000; 32: 247-255. Shigeki S. et al, Scand J Plast Reconstr Surg Hand Surg 1997; 31: 151-158. Yamada H. et al., J Biochem (Tokyo) 1994; 116: 892-897.

  An object of the present invention is to provide a preventive or therapeutic agent for liver diseases, particularly non-alcoholic steatohepatitis. Another object of the present invention is to provide an insulin resistance improving agent.

  In previous reports, we found in animal models that tranilast prevents renal fibrosis in diabetic nephropathy by inhibiting TGF-β (Akahori H. et al., J Pharmacol Exp Ther 2005; 314: 514-521.). TGF-β is known to play a central role in the development of NASH (Williams EJ et al., J Hepatol 2000; 32: 754-761.). Therefore, the present inventors hypothesized that administration of tranilast improves the pathological condition of NASH by suppressing TGF-β action and liver fibrosis. To test this hypothesis, the present inventors examined the effects of tranilast using an established animal NASH model of obese diabetic rats fed a methionine choline deficient diet.

  As a result, it was found that tranilast is effective in the treatment of liver diseases, in particular, suppression of liver fibrosis and liver fatification, and improvement of liver inflammation, and the present invention has been completed.

Furthermore, the present invention examined the effect of tranilast on macrophage activation. As a result, it was found that tranilast can suppress macrophage activation, thereby improving insulin resistance, improving metabolic syndrome, and preventing or treating diabetes and improving obesity, thereby completing the present invention. It was.
That is, the present invention is as follows.

[1] A preventive or therapeutic agent for liver disease comprising N- (3,4-dimethoxycinnamoyl) anthranilic acid or a pharmacologically acceptable salt thereof as an active ingredient.
[2] The preventive or therapeutic agent for liver disease according to [1], wherein the liver disease is non-alcoholic fatty liver injury (NAFLD).
[3] The preventive or therapeutic agent for liver disease according to [1], wherein the liver disease is nonalcoholic steatohepatitis (NASH).
[4] The preventive or therapeutic agent for liver disease according to [1], wherein the liver disease is liver fibrosis associated with nonalcoholic steatohepatitis (NASH).
[5] The preventive or therapeutic agent for liver disease according to [1], wherein the liver disease is hepatic steatosis associated with nonalcoholic steatohepatitis (NASH).
[6] The preventive or therapeutic agent for liver disease according to [1], wherein the liver disease is liver inflammation associated with nonalcoholic steatohepatitis (NASH).
[7] An insulin resistance improving agent comprising N- (3,4-dimethoxycinnamoyl) anthranilic acid or a pharmacologically acceptable salt thereof as an active ingredient.
[8] A metabolic syndrome improving agent comprising the insulin resistance improving agent of [7].

  The pharmaceutical composition comprising tranilast (N- (3,4-dimethoxycinnamoyl) anthranilic acid) of the present invention as an active ingredient suppresses liver disease, particularly liver fibrosis and hepatic steatosis associated with nonalcoholic steatohepatitis. Improves liver inflammation associated with non-alcoholic steatohepatitis. Furthermore, the pharmaceutical composition containing tranilast of the present invention as an active ingredient can improve insulin resistance, improve metabolic syndrome, and can be used for the prevention and treatment of diabetes and the improvement of obesity.

Hereinafter, the present invention will be described in detail.
The present invention relates to treatment of liver diseases comprising tranilast (N- (3,4-dimethoxyciannamoyl) -anthranilic acid; N- (3,4-dimethoxycinnamoyl) anthranilic acid) or a pharmacologically acceptable salt thereof as an active ingredient. Or it is a pharmaceutical composition used for prevention. Liver diseases include, but are not limited to, liver disease with liver fattening, fibrosis, and inflammation. Non-alcoholic fatty liver disorder (NAFLD) is preferable. Nonalcoholic fatty liver disease (NAFLD) ranges from simple fatty liver to steatohepatitis and cirrhosis, and the pharmaceutical composition of the present invention is useful for their prevention or treatment. When non-alcoholic fatty liver disorder becomes severe, non-alcoholic steatohepatitis (NASH) occurs, and liver steatosis, inflammation, and fibrosis progress. The pharmaceutical composition of the present invention contains a prophylactic or therapeutic agent for nonalcoholic steatohepatitis (NASH), and further contains a prophylactic or therapeutic agent for fattening, inflammation, or fibrosis associated with nonalcoholic steatohepatitis. The pharmaceutical composition of the present invention is a liver fibrosis inhibitor and a hepatic steatosis inhibitor that suppress such liver fibrosis and hepatic steatosis, and further a liver inflammation improving agent that improves liver inflammation.

  Tranilast suppresses the expression of TGF-β and improves various liver disorders.

  Furthermore, the present invention relates to insulin containing tranilast (N- (3,4-dimethoxyciannamoyl) -anthranilic acid; N- (3,4-dimethoxycinnamoyl) anthranilic acid) or a pharmaceutically acceptable salt thereof as an active ingredient. It is a resistance improving agent and a glucose metabolism improving agent, a metabolic syndrome improving agent, an anti-obesity agent, or a prophylactic or therapeutic agent for diabetes containing the insulin resistance improving agent.

  Tranilast suppresses macrophage activation, suppresses / improves insulin resistance, improves glucose metabolism, and can also be used for the prevention or treatment of diabetes. Furthermore, tranilast suppresses macrophage activation, improves metabolic syndrome, and can also be used as an anti-obesity agent.

  Tranilast or a pharmacologically acceptable salt thereof can be produced by chemical synthesis, or a commercially available product may be used. Tranilast or a salt thereof is, for example, JP-B-56-40710, JP-B-57-36905, JP-B-58-17186, JP-B-58-48545, JP-B-58-55138, JP-B-58. -55139, JP-B-1-28013, JP-B-1-50219, JP-B-3-37539 and the like.

  Examples of pharmacologically acceptable salts of tranilast include salts with inorganic bases such as sodium salts and potassium salts, salts with organic amines such as morpholine, piperidine and pyrrolidine, and salts with amino acids. .

  The pharmaceutical composition containing the tranilast of the present invention or a pharmacologically acceptable salt thereof as an active ingredient may contain a pharmaceutically acceptable carrier or diluent. Carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, buffered saline, and the like.

  The administration route includes oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous. Desirable is oral administration. As the dosage form, it can be administered in various forms such as capsules, tablets, granules, syrups, fine granules, sprays, emulsions, suppositories, injections, ointments, tapes. Agents and the like.

  Liquid preparations such as emulsions and syrups are composed of saccharides such as water, sucrose, sorbitol and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; p-hydroxybenzoic acid Preservatives such as esters; flavors such as strawberry flavor and peppermint can be used as additives.

  For capsules, tablets, powders, granules, fine granules, etc., excipients such as lactose, glucose, sucrose and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; It can be produced using a binder such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin; a surfactant such as a fatty acid ester; a plasticizer such as glycerin as an additive.

  Injections include sugars such as water, sucrose, sorbitol, xylose, trehalose, and fructose; sugar alcohols such as mannitol, xylitol, and sorbitol; buffers such as phosphate buffer, citrate buffer, and glutamate buffer; fatty acid esters A surfactant such as can be used as an additive.

  The pharmaceutical composition of the present invention may further contain a pH adjuster, an antioxidant, a stabilizer, a preservative, a thickener, a chelating agent, a humectant, a colorant, an isotonic agent and the like.

  The dosage of the pharmaceutical composition containing the tranilast of the present invention or a pharmacologically acceptable salt thereof as an active ingredient varies depending on symptoms, age, body weight, etc. The dose is about 0.01 mg to 50 g, and these can be administered once or divided into several times. Per kilogram of body weight is about 0.002 mg to 1 g per day. In parenteral administration, about 0.01 mg to 50 g (about 0.002 mg to 1 g per day per kg of body weight) can be administered by subcutaneous injection, intramuscular injection or intravenous injection.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Effect of tranilast on non-alcoholic steatohepatitis (NASH) animal model Examples of the present invention were carried out by the following method.
Animal Model and Experimental Design Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (Otsuka Pharmaceutical, Tokushima, Japan), established as an animal model for obese type 2 diabetes, were used. Spontaneous type 2 diabetic OLETF rats developed obesity and hyperinsulinemia from the 8th week of life. Since these animals show increased fat accumulation in the liver depending on age, NAFLD is studied using OLETF rats. All rats in the OLETF group developed diabetes at 24 weeks of age.

  As control animals, 4-week-old OLETF rats and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats of the same colony origin as OLETF were obtained, controlled temperature (25 ° C), humidity and lighting (12 hours) Cultivated under artificial light-dark cycle). Animals were allowed free access to a standard rat diet and tap water. At 24 weeks of age, OLETF rats were divided into two experimental groups and raised for 8 weeks as follows: MCD diet (Oriental Yeast Industry, Tokyo, Japan; OLETF-MCD, 21% fat, n = 11) and MCD-tranilast diet (OLETF-MCD-trani; tranilast concentrations are 1%, n = 3; 1.4%, n = 3; 2%, n = 10). In addition, at 24 weeks of age, male LETO rats were also divided into two groups: MCD diet (LETO-MCD, n = 9) and MCD-tranilast diet (LETO-MCD-trani; tranilast 2 % (N = 5)). Rats were bred under free drinking and free feeding. The body weight and food intake of each group of rats was recorded weekly.

Blood sample collection and analysis
After 8 weeks, the rats were sacrificed after 12 hours of fasting and blood samples were taken from the heart. Plasma glucose was measured with a Freestyle Kissei monitor (Kissei Pharmaceutical, Matsumoto, Japan). Blood samples were centrifuged and serum was frozen at 70 ° C. for determination of serum alanine aminotransferase (ALT), triglycerides, free fatty acids (FFA), cholesterol (TC) and HDL cholesterol (HDLC) concentrations. Serum ALT was measured spectrophotometrically (Sigma, St. Louis, MO, USA). Serum FFA levels were measured by non-ester fatty acid (NEFA) C-test (Wako, Osaka, Japan). TC and HDLC concentrations were quantified by colorimetric analysis. Serum insulin levels were measured by ELISA (Mercodia, Uppsala, Sweden).

Plasma glucose level measurement
After 8 weeks, all rats received an oral glucose tolerance test after 12 hours of fasting. 2 g of glucose per kg body weight was administered orally. Blood was collected from the tail vein after 0, 60, 120 minutes for measurement of serum glucose concentration.

Serum and hepatic neutral fat (hereafter unified with either triglyceride or neutral fat) level measurement
After 8 weeks, the rats were sacrificed and liver weight and liver triglyceride content were measured. For quantification of liver triglyceride content, the liver was lysed with a buffer from a commercial kit (TG E test; Wako).

Morphological analysis and immunohistochemistry
After 8 weeks, the rats were sacrificed and the liver was fixed with 10% formalin buffer and embedded in paraffin. The severity of liver tissue changes was assessed by pathologists with H & E staining and Azan staining. Fatty, fibrotic and disease activity were assessed semi-quantitatively according to standard NASH grading criteria. The degree of fatification was recorded as a percentage of hepatocytes containing lipid droplets. Fibrosis scores were as follows: 1, pericellular and perivenous fibrosis; 2, localized bridging fibrosis; 3, bridging fibrosis due to lobular deformation; 4, cirrhosis. Disease activity was assessed as the sum of scores of acinar inflammation (score 0-3) and portal vein inflammation (score 0-3) (score 0-6).

  About the slide which carried out Azan dyeing | staining, the fibrosis area | region (area | region dye | stained blue by Azan dyeing | staining) was measured under the microscope using the image analysis system.

  In addition, slides were immunostained with monoclonal mouse anti-human α-smooth muscle actin (α-SMA) (Dako, Kyoto, Japan) and anti-rabbit TGF-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA). . This was followed by application of immunoperoxidase using the Envision kit (Dako). Peroxidase activity was confirmed by the reaction of 3 ', 3'-diaminobenzidine (Sigma). Two independent observers with no prior knowledge of experimental design evaluated each section. Staining with anti-α-SMA or anti-TGF-β was expressed using WinROOF version 5.71 (Mitani Corporation, Fukui, Japan) as a percentage of the observed background area of the positive area.

Real-time PCR
Total RNA was extracted from each liver using the RNeasy Mini Kit (Qiagen, Tokyo, Japan). Real-time PCR for TGF-β, α1 (I) procollagen (collagen I), plasminogen activator inhibitor-1 (PAI-1), carnitine O-palmitoyltransferase-1 (CPT-1), and PPAR-α mRNA Performed using ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The set of primers for collagen I and the TaqMan probe were owned by Applied Biosystems (TaqMan Gene Expression Assays product). In order to control changes in the amount of DNA obtained by PCR of different samples, gene expression of the target sequence was normalized relative to the expression of the endogenous control 18SrRNA (TaqMan Control Reagent Kit; Applied Biosystem). PCR conditions were 50 ° C for 2 minutes and 95 ° C for 10 minutes for one cycle, and 50 cycles were performed.

Statistical analysis All results are expressed as mean or mean ± SEM. Data were analyzed by one-way analysis of variance and P <0.05 appeared to be statistically significant. All calculations were performed with the Windows version of SPSS version 11.0 (SPSS, Chicago, IL, USA).

  The following results were obtained by this example.

  Tranilast was well tolerated in rats fed the MCD diet.

Spontaneous type 2 diabetic OLETF rats were given an MCD diet for 8 weeks. MCD diet caused severe lobular inflammation in zone 3 and peri-venous and peri-cellular fibrosis (FIGS. 1A and B). To ascertain the most effective amount of tranilast, OLETF-MCD rats were treated with tranilast at three concentrations, 1%, 1.4% and 2%, respectively, averaging 160, 190 and 320 mg kg ^-1 day ^-1 . These are reported to be reasonable daily doses for animal experiments. Tranilast did not affect physical findings, stool composition, food or water intake, indicating that tranilast was well tolerated. Treatment with tranilast improved hepatic pathology of fatring, inflammation and fibrosis in a dose-dependent manner in MCD-OLETF rats (FIGS. 2A-C). The effect was more prominent in the 2% tranilast treatment group, so the following study was conducted in this group:

Metabolic parameters
Table 8 shows the metabolic parameters at 8 weeks of OLTF rats fed MCD diet, 2% tranilast-containing diet, and non-diabetic control LETO rats. Tranilast significantly reduced serum ALT and increased liver weight in both OLETF-MCD and LETO-MCD rats. Blood glucose and triglycerides were not affected by tranilast. Furthermore, an insulin tolerance test suggested that rats receiving tranilast treatment did not improve insulin sensitivity (data not shown).

Tranilast was an animal model of dietary NASH that prevented stellate cell activation and progression of liver fibrosis.
Histological analysis with Azan staining showed that treatment with 2% tranilast dramatically prevented the development of liver fibrosis in LETO-MCD and OLETF-MCD rats (FIGS. 3 and 4). FIG. 3 shows a stained image of Azan staining, and FIG. 4 shows the ratio of the region stained by Azan staining. It is known that activation of hepatic stellate cells plays a central role in the progression of liver fibrosis. To investigate the effect of tranilast on stellate cell activation, we performed an immunohistochemical analysis of α-SMA, an indicator of stellate cell activation. α-SMA positive cells were significantly increased in the livers of OLETF-MCD rats compared to LETO-MCD rats. Tranilast reduced the number of these cells in both LETO-MCD and OLETF-MCD rats (FIGS. 5 and 6). FIG. 5 shows a stained image using monoclonal mouse anti-human α-smooth muscle actin (α-SMA), and FIG. 6 shows the ratio of the stained region.

Tranilast prevented the progression of hepatic steatosis.
To date, little has been reported on the effects of tranilast on intracellular lipid accumulation. However, histological analysis unexpectedly showed that tranilast treatment clearly improved hepatic steatosis in LETO-MCD and OLETF-MCD rats (FIG. 7). Consistent with histological findings, tranilast significantly reduced hepatic triglyceride content in these two groups of rats (FIG. 8).

Tranilast was a dietary NASH animal model that suppressed TGF-β expression in the liver.
To clarify the mechanism by which tranilast prevents liver fibrosis in rats, the expression of TGF-β, the main regulator of liver fibrosis progression, was evaluated by real-time PCR (FIG. 9). Treatment with tranilast tended to suppress the expression of TGF-β mRNA in the liver of OLETF-MCD rats and to suppress the expression in the liver of LETO-MCD rats. Furthermore, tranilast suppressed the expression of TGF-β target genes, collagen I and PAI-1, which are related to fibrosis (FIGS. 10 and 11). Consistent with the mRNA findings, immunohistochemical analysis showed suppression of TGF-β protein expression in the liver of rats receiving tranilast treatment (FIG. 12).

Tranilast increased mRNA expression of genes related to fatty acid β-oxidation.
To clarify the mechanism by which tranilast improves hepatic steatosis in rats, the expression of genes involved in fatty acid β-oxidation was measured by real-time PCR (FIGS. 13A and B). The mRNA expression of PPAR-α, a transcription factor that plays a central role in the control of β-oxidation, was significantly enhanced by tranilast in the livers of OLETF-MCD and LETO-MCD rats. In addition, tranilast also induced mRNA expression of CPT-1, the rate-limiting enzyme of fatty acid β-oxidation to mitochondria.

  From the above results, the following was found.

  Fibrosis is well known as a pathological biochemical feature of NASH. The progression of fibrosis causes liver failure and portal hypertension and is a risk factor for liver cancer. Therefore, hepatic fibrosis is an important therapeutic target for NASH, and various fibrosis inhibitors have been investigated as candidates for NASH therapy (Gressner AM et al., J Cell Mol Med 2006; 10: 76-99 .).

  This example demonstrates for the first time that tranilast, an antifibrotic agent, improves liver fibrosis in an animal model of NASH. Tranilast administration inhibits hepatic stellate cell activation and attenuates the expression of TGF-β and its target molecule, which probably prevented fibrosis. This study provided evidence that anti-fibrotic treatment with tranilast is a beneficial and promising approach to NASH.

  Among many mediators having fibrosis-inducing ability, TGF-β is considered to have the most potent effect (Gressner AM et al., Front Biosci 2002; 7: d793-d807.). Inhibition of TGF-β synthesis and TGF-β signaling has been reported using different experimental systems to prevent liver fibrosis (George J. et al., Proc Natl Acad Sci USA 1999; 96: 12719- 12724 .; Arias M. et al., BMC Gastroenterol 2003; 3:29 .; Qi Z. et al., Proc Natl Acad Sci USA 1999; 96: 2345-2349 .; Nakamura T. et al., Hepatology 2000; 32: 247-255.). In this example, it has been demonstrated that administration of tranilast attenuates both TGF-β gene expression and α-SMA positive cells in the liver, and tranilast directly suppresses TGF-β expression, resulting in liver fibrosis. It is estimated that hepatic disorder was improved. This finding is consistent with previous reports that have demonstrated in vitro that administration of tranilast suppresses activation of hepatic stellate cells by reducing TGF-β gene expression (Ikeda H. et al., Biochem Biophys Res Commun. 1996; 227: 322-327.). In addition, tranilast also attenuated the expression of PAI-1, one of the TGF-β target genes. PAI-1 has been reported to be a therapeutic target for metformin for ethanol-induced liver injury (Bergheim I et al., Gastroenterology 2006; 130: 2099-2112.). Therefore, tranilast may be useful for alcoholic liver disease.

  Obesity, type 2 diabetes, and dyslipidemia frequently accompany NASH. Insulin resistance is an element that forms the basis of these metabolic disorders and is considered to play an important role in the pathogenesis of NAFLD. It has been shown that insulin sensitizers improve the pathological findings of NASH, a severe form of NAFLD, in both animal models and humans (Ota T. et al, Gastroenterology 2007; 132: 282-293 .; Belfort R et al., N Engl J Med 2006; 355: 2297-2307.). In the study in this example, tranilast administration did not affect insulin sensitivity in rats. Furthermore, unlike the action of pioglitazone (Ota T. et al, Gastroenterology 2007; 132: 282-293 .; Belfort R et al., N Engl J Med 2006; 355: 2297-2307.), Tranilast is insulin resistant It was also effective for LETO rats, a model of NASH that does not have. The effect of tranilast on NASH may be via a route that does not depend on insulin resistance, and administration of tranilast may also be effective for a non-responder of an insulin sensitizer.

  In this example, it was surprisingly found that tranilast suppresses the development of hepatic steatosis as well as liver fibrosis. There were no reports demonstrating the effects of tranilast on intracellular fat accumulation, and this finding was unexpected. Realtime-PCR analysis revealed that expression of fatty acid β-oxidation-related genes such as CPT-I and PPAR-α was enhanced by tranilast administration. This suggests that the enhancement of β-oxidation induced by tranilast attenuated hepatic steatosis in rats.

  As noted above, this example demonstrates that tranilast prevents the development of nonalcoholic steatohepatitis in a rat model, and that this effect is likely by a pathway independent of insulin resistance. As a result, it was suggested that targeting to TGF-β by tranilast leads to a novel treatment for NASH.

Example 2 Effect of tranilast on nonalcoholic steatohepatitis (NASH) human patients A 32-year-old woman being treated for hypertension and obesity diagnosed with nonalcoholic steatohepatitis (NASH) from liver biopsy and clinical findings The patient was treated with tranilast. Since there was a complication of perennial nasal allergy, tranilast was started at 300 mg / day. The dosing interval was 3 times a day after each meal. A liver biopsy was performed 22 months after the start of administration. Compared with the start of administration, body weight (83.4 → 82.0 kg), blood pressure and glucose tolerance were not significantly changed (FIG. 14), and liver function (AST63 → 24, ALT89 → 30, γGTP41 → 18) has been greatly improved (Fig. 15), and liver tissue is also changed from NASH to fatty liver (NAFLD class 3 → 2/4, Grade 2 → 2/3, Stage 2 → 1/4) It was improved (FIG. 16). In addition, improvements were observed in fat formation, Mallory body, acinar inflammation, portal vein inflammation and balloon-like degeneration (FIG. 17). That is, tranilast acted directly on the liver to improve liver function and markedly suppressed NASH pathology, especially liver inflammation and fibrosis (FIG. 18).

  Therefore, tranilast was effective in human NASH patients.

Example 3 Effect of tranilast on fatty liver, diet-induced obesity, metabolic disorder animal model Chronic over-nutrition causes a macrophage infiltration in adipose tissue, which may lead to local inflammation and enhance insulin resistance Has been speculated.

  Therefore, we investigated whether tranilast could suppress macrophage activation and improve insulin resistance in the livers of hypertrophic obese diabetic model rats.

(1) Inhibition of macrophage activation in vitro Mouse macrophage cell lines (RAW cells) were stimulated with Lipopolysaccharide (LPS), and tranilast was activated by macrophages using Real-Time RT-PCR and ELISA. We examined whether to suppress the conversion.

  RAW cells were cultured in the presence of 1 μg / mL LPS. At this time, tranilast was added at a concentration of 30 or 300 μmol / L. After culture, mRNA expression of TNF-α, IL-6, and iNOS was measured by Real-Time RT-PCR.

  The results are shown in FIG. As shown in FIG. 19, in RAW cells, tranilast suppressed the expression of TNF-α, IL-6 and iNOS mRNA, which are indicators of macrophage activation increased by LPS.

  Further, TNF-α and IL-6 concentrations in the culture medium of cultured RAW cells were measured by ELISA.

  The results are shown in FIG. As shown in FIG. 20, in RAW cells, tranilast decreased the concentration of TNF-α and IL-6, which are indicators of macrophage activation, in the supernatant.

(2) Inhibition of macrophage activation in vivo As follows, each rat was given a diet containing or not containing tranilast (Tra) for 8 weeks, and macrophage activation and insulin resistance in the liver were examined.
1. Control rat (LETO rat; 25 weeks old) + normal diet (CON group)
2. Obese diabetic model rat (OLETF rat; 25 weeks old) + high fat diet (DM group)
3. OLETF rat + Tra (1.6%) mixed high fat diet (DM + Tra group)

  FIG. 21 shows photographs of 25-week-old LETO rats and OLETF rats. FIG. 22 shows the appearance of pathological conditions according to the age of LETO rats and OLETF rats.

  FIG. 23 shows the body weight and the like after 8 weeks of the above-mentioned CON group, DM group and DM + Tra group. As shown in FIG. 23, tranilast did not change the amount of food intake, but decreased the body weight from 687 ± 15 to 605 ± 27 g and the visceral fat amount from 72 ± 6 to 53 ± 5 g. It also decreased fasting blood glucose levels.

  The used rat liver was collected and subjected to H & E staining. FIG. 24 shows the result of HE staining in the liver. As shown in FIG. 24, tranilast histologically suppressed hepatic steatosis and decreased the neutral fat content of the liver.

  In addition, immunostaining was performed using ED-1 (macrophage marker). The results are shown in FIG. As shown in FIG. 25, tranilast reduced the number of ED-1 positive cells.

  Further, using the collected liver, mRNA expression levels of ICAM and VCAM, which are cell adhesion factors in the liver, were measured by Realtime PCR. The results are shown in FIG. As shown in FIG. 26, tranilast decreased the expression level of ICAM mRNA, which is a cell adhesion factor, in the liver.

  Furthermore, a glucose tolerance test and an insulin tolerance test were performed. The respective results are shown in FIGS. FIG. 29 shows blood glucose level (A), serum insulin level (B), and blood glucose level × insulin level (C) as needed. Tranilast decreased blood glucose levels at 0 and 120 minutes after glucose load from 141 ± 6 to 102 ± 3 mg / dl and from 444 ± 33 to 231 ± 53 mg / dl, respectively. In addition, the fasting blood insulin level was reduced from 107 ± 12 to 38 ± 14 μU / ml. From these results, it was found that tranilast improved insulin resistance.

  Furthermore, mRNA expression levels of G6PC (Glucose-6-phosphatase, catalytic subunit) and IRS1 (Insulin receptor substrate-1) in the liver were measured by Realtime PCR. Each result is shown in FIGS. 30A and 30B. This result indicates that the improvement in insulin resistance by tranilast is not via a direct effect on G6PC and IRS1 expression.

  From the above in vitro and in vivo studies, it was found that tranilast suppresses macrophage activation in vitro. Furthermore, tranilast was found to suppress body weight, visceral fat, hepatic steatosis, hepatic macrophage infiltration and insulin resistance in obese diabetic rats. These results suggest that tranilast can improve metabolic syndrome and insulin resistance through the macrophage activation inhibitory action.

The figure which shows the H & E (FIG. 1A) and Azan staining (FIG. 1B) image which show the dose-dependent effect of tranilast in the liver pathology (adipose, inflammation, and fibrosis) of a diabetic rat (OLETF) and a non-diabetic rat (LETO) The magnification is x100). FIG. 2 shows the dose-dependent effect of tranilast in liver pathology (fatting, inflammation and fibrosis) in rats. It is a figure (magnification is x100) showing an Azan-stained image of the liver in diabetic rats (OLETF) and non-diabetic rats (LETO) administered or not administered with tranilast. It is a figure which shows the ratio of the Azan dyeing | staining area | region of a liver in the diabetic rat (OLETF) and non-diabetic rat (LETO) which administered or not administered tranilast. The figure which shows the dyeing | staining image using the monoclonal mouse anti-human alpha-smooth muscle actin antibody of the liver in diabetic rats (OLETF) and non-diabetic rats (LETO) to which tranilast was administered or not (magnification is × 100) is there. It is a figure which shows the ratio of the dyeing | staining area | region by the monoclonal mouse | mouth anti-human alpha-smooth muscle actin antibody of the liver in the diabetic rat (OLETF) and the non-diabetic rat (LETO) which administered or not administered tranilast. It is a figure (magnification is x100) showing an H & E stained image of the liver in diabetic rats (OLETF) and non-diabetic rats (LETO) administered or not administered with tranilast. It is a figure which shows the liver triglyceride content in the diabetic rat (OLETF) and non-diabetic rat (LETO) to which tranilast was administered or not. It is a figure which shows the mRNA expression level of TGF- (beta) in the liver in the diabetic rat (OLETF) and non-diabetic rat (LETO) to which tranilast was administered or not. It is a figure which shows the mRNA expression level of the collagen I in the liver in the diabetic rat (OLETF) and non-diabetic rat (LETO) to which tranilast was administered or not. It is a figure which shows the mRNA expression level of PAI-1 in the liver in the diabetic rat (OLETF) and non-diabetic rat (LETO) to which tranilast was administered or not. It is a figure (magnification magnification is x100) which shows the immuno-staining image which shows the expression level of TGF- (beta) in the liver in a diabetic rat (OLETF) and non-diabetic rat (LETO) to which tranilast was administered or not. It is a figure which shows the expression of the gene relevant to fatty-acid (beta) -oxidation in the diabetic rat (OLETF) and non-diabetic rat (LETO) which administered tranilast or not. FIG. 13A shows PPARα mRNA expression, and FIG. 13B shows CPT1 mRNA expression. It is a figure which shows the change of the body weight in a human NASH patient who administered tranilast, and glucose tolerance. It is a figure which shows the change of the liver function in the human NASH patient who administered tranilast. It is a figure which shows the improvement effect of NASH in the human NASH patient who administered tranilast. It is a figure which shows the change of fattening of a liver, Mallory body, acinar inflammation, portal region inflammation, and balloon-like degeneration in the human NASH patient who administered tranilast. It is a figure which shows the summary of the effect in the human NASH patient who administered tranilast. It is a figure which shows the expression of mRNA of TNF- (alpha) (A), IL-6 (B), and iNOS (C) by tranilast in a cultured cell. It is a figure which shows the TNF- (alpha) (A) and IL-6 (B) density | concentration in the culture medium by tranilast in a cultured cell. It is a photograph of LETO rat and OLETF rat. It is a figure which shows the mode of the onset of the disease state by the age of a LETO rat and an OLETF rat. It is a figure which shows the body weight etc. of the CON group, DM group, and DM + Tra group rat 8 weeks after drug administration. It is a photograph which shows the result of H & E dyeing | staining in the liver of the CON group (A), DM group (B), and DM + Tra group (C) rat 8 weeks after drug administration. It is a photograph which shows the result of the immuno-staining using ED-1 (macrophage marker) in the liver of the CON group (A), DM group (B), and DM + Tra group (C) rat 8 weeks after drug administration. It is a figure which shows the mRNA expression level of ICAM (A) and VCAM (B) which are the cell adhesion factors in the liver of the CON group, DM group, and DM + Tra group rat 8 weeks after drug administration. It is a figure which shows the result of the glucose tolerance test of the CON group, DM group, and DM + Tra group rat 8 weeks after drug administration. It is a figure which shows the result of the insulin tolerance test of the CON group, DM group, and DM + Tra group rat 8 weeks after drug administration. It is a figure which shows the blood glucose level (A), serum insulin level (B), and blood glucose level x insulin level (C) at any time 8 weeks after drug administration of the CON group, DM group and DM + Tra group rats. MRNA expression of G6PC (Glucose-6-phosphatase, catalytic subunit) (A) and IRS1 (Insulin receptor substrate-1) (B) in the liver of CON group, DM group and DM + Tra group rats 8 weeks after drug administration It is a figure which shows quantity.

Claims (8)

  A preventive or therapeutic agent for liver diseases comprising N- (3,4-dimethoxycinnamoyl) anthranilic acid or a pharmacologically acceptable salt thereof as an active ingredient.   The preventive or therapeutic agent for liver disease according to claim 1, wherein the liver disease is non-alcoholic fatty liver injury (NAFLD).   The preventive or therapeutic agent for liver disease according to claim 1, wherein the liver disease is non-alcoholic steatohepatitis (NASH).   The preventive or therapeutic agent for liver disease according to claim 1, wherein the liver disease is liver fibrosis associated with nonalcoholic steatohepatitis (NASH).   The preventive or therapeutic agent for liver disease according to claim 1, wherein the liver disease is hepatic steatosis accompanying nonalcoholic steatohepatitis (NASH).   The preventive or therapeutic agent for liver disease according to claim 1, wherein the liver disease is liver inflammation associated with nonalcoholic steatohepatitis (NASH).   An insulin resistance improving agent comprising N- (3,4-dimethoxycinnamoyl) anthranilic acid or a pharmacologically acceptable salt thereof as an active ingredient.   A metabolic syndrome-improving agent comprising the insulin resistance-improving agent according to claim 7.

Images