WO2017155053A1 - Therapeutic agent for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis - Google Patents

Abstract

The present invention provides a prophylactic and/or therapeutic agent for nonalcoholic fatty liver disease (NAFLD) and nonacoholic steatohepatitis (NASH) obtained by including lansoprazole. Lansoprazole has been used safely as a therapeutic agent for gastric ulcers and duodenal ulcers, and so may be a safe and useful drug for treating NAFLD and NASH as well.

Description

Non-alcoholic fatty liver disease / non-alcoholic steatohepatitis therapeutic agent

The present invention relates to a novel pharmaceutical use of lansoprazole known as a proton pump inhibitor. More specifically, the present invention relates to a preventive and / or therapeutic agent for nonalcoholic fatty liver disease / nonalcoholic steatohepatitis containing lansoprazole.

In recent years, non-alcoholic steatohepatitis (hereinafter referred to as “NASH”), which exhibits liver tissue findings such as fat accumulation, inflammatory necrosis, and fibrosis despite the absence of alcohol consumption due to increased obesity and insulin resistance accompanying lifestyle changes. ”May be abbreviated as“. ”An increase in patients has been reported (Non-patent Document 1). Chronic progressive liver disease of non-drinkers who have fatty liver and whose cause is unknown is collectively called non-alcoholic fatty liver disease (hereinafter sometimes abbreviated as “NAFLD”), of which liver lesions are highly active and cirrhosis. NASH is defined as one that has a high frequency of transition to liver disease or death related to liver disease (Japanese Society of Gastroenterology NAFLD / NASH Clinical Practice Guidelines 2014; hereinafter, in this specification, general NAFLD typified by NASH is shown. (In some cases, “NAFLD / NASH” may be used as an abbreviation.) The main features of the clinical pathology of NAFLD / NASH include an increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. NASH patients have a high risk of progressing to fatal diseases such as cirrhosis and liver cancer, and NAFLD / NASH prevention, improvement, and development of treatment methods are important medical issues. However, the onset mechanism of NAFLD / NASH is still unclear, and safe and highly effective drugs and treatment methods for NAFLD / NASH have not yet been established.

Lansoprazole (trade name: Takepron (registered trademark)) has a proton pump inhibitory action on gastric wall cells (Patent Document 1) and is a highly safe compound that is commercially available as a therapeutic agent for peptic ulcer. In addition, the present inventors have induced oxidative stress tolerance of organs by inducing lansoprazole in the first phase drug metabolism system (Ahr-Cyp1a1) and the second phase drug metabolism system (Nrf2-HO-1). It has been reported that it has a therapeutic effect on drug-induced acute hepatitis (Patent Document 2, Non-Patent Document 2). However, there is no report that lansoprazole is effective for the prevention and / or treatment of NAFLD / NASH.

In addition, since NAFLD / NASH is pathologically formed by the progression of two parameters of inflammation and fibrosis, the importance of drugs that suppress fibrosis is recognized. Although these drugs are being developed all over the world, there are no reports of successful development yet. Here, although transforming growth factor-β (TGF-β) has been reported to be an important regulator of fibroblast activation in fibrotic diseases (Non-Patent Documents 3 and 4), lansoprazole is It has not been reported to suppress fibrosis or participate in the expression of TGF-β.

JP 61-50978 JP-A-2015-145343

Arun, J., Gastroenterology, 123 (5): 1705-1725 (2002) Yamashita, Y., Ueyama, T., Nishi, T., Yamamoto, Y., Kawakoshi, A., Sunami, S., Iguchi, M., Tamai, H., Ueda, K., Ito, T., Tsuo, Y, Ichinose, M., PLoS ONE, 9 (5): e97419 (2014) Robert, M., Borna, M., Chest, 136: 1364-1370 (2009) Verrecchia, F., Mauviel A., World J. Gastroenterol., 13: 3056-3062 (2007)

An object of the present invention is to provide a safe and useful preventive and / or therapeutic agent for nonalcoholic fatty liver disease / nonalcoholic steatohepatitis for which no effective drug therapy is currently known.

As a result of intensive studies to achieve the above object, the present inventors have confirmed that lansoprazole suppresses liver fibrosis and suppresses the progression of NAFLD / NASH by subcutaneous administration to NAFLD / NASH model animals. I found. Interestingly, it was found that this suppressive effect was not due to the action of lansoprazole to induce the expression of antioxidant stress gene.
Hepatic fibrosis is deeply involved in the accumulation of extracellular matrix (ECM) such as type I collagen α1 chain (Col1a1), which is a downstream factor of TGF-β. ECM is lysed by matrix metalloproteinases (MMPs), but when the activity of MMPs is inhibited by a tissue inhibitor of MMPs (Timp1), liver fibrosis occurs. Therefore, the present inventors examined whether lansoprazole suppresses the expression of these liver fibrosis-related genes, and none of the gene expression was significantly suppressed. It was found that protein expression (ie, activation of TGF-β) was remarkably suppressed. Although the involvement of reactive oxygen species (ROS), MMP2 and MMP9 has been reported as a mechanism of TGF-β activation, lansoprazole did not increase HO-1 expression in NAFLD / NASH model animals. It was suggested that TGF-β activation was inhibited by inhibiting.
In addition, since NASH is caused by inflammation following fat accumulation, the present inventors examined the influence of lansoprazole administration on the expression of proinflammatory cytokines in NAFLD / NASH model animals. As a result, it was revealed that lansoprazole suppresses IL-6 gene and protein expression.
From the above, the present inventors have found that lansoprazole is useful for the treatment of NAFLD / NASH, and that the therapeutic effect involves cytokines, not antioxidant stress proteins, and completed the present invention. I came to let you.

That is, the present invention is as follows.
[1] A preventive and / or therapeutic agent for NAFLD / NASH comprising lansoprazole.
[2] The agent according to [1], which is used so that 5 mg to 150 mg of lansoprazole is administered per day for an adult.
[3] The agent according to [1], which is used so that 30 mg of lansoprazole is administered per day for an adult.
[4] A TGF-β expression inhibitor comprising lansoprazole.
[5] An IL-6 expression inhibitor comprising lansoprazole.

According to the present invention, NAFLD / NASH can be prevented and / or treated by administering lansoprazole at a dose that has been confirmed to be safe as an existing drug.

It is a figure which shows that lansoprazole suppresses the fibrosis in NAFLD / NASH. A: shows the tissue of the liver of a NAFLD / NASH model rat in which collagen (I and III) fibers are stained with Picrosirus® Red. B: shows the result of scoring using the NAFLD / NASH stage criteria (Brunt criteria). The result of having investigated the density | concentration of AST and ALT in the serum of a NAFLD / NASH model rat by lansoprazole administration is shown. The result of having investigated the expression change of Nrf2, HO-1, Ahr, and Cyp1a1 gene in the liver of a NAFLD / NASH model rat by lansoprazole administration by RT-PCR is shown. The black bar shows the results of the CDAA 5w + LAP 4w group administered with lansoprazole, and the white bar shows the results of the control CDAA 5w group. The result of having investigated the expression change of the liver fibrosis related gene (Timp1, Col1a1, Tgfb1) in the liver of a NAFLD / NASH model rat by lansoprazole administration is shown. The result of having investigated the expression change of TGF- (beta) protein in the liver of a NAFLD / NASH model rat by lansoprazole administration is shown. Results of examination of pro-inflammatory cytokine gene (IL-6, IL-1β) expression changes (A) and IL-6 protein expression changes (B, C) in the liver of NAFLD / NASH model rats by administration of lansoprazole Indicates.

The present invention provides a prophylactic and / or therapeutic agent for NAFLD / NASH (hereinafter sometimes abbreviated as “the prophylactic and / or therapeutic agent of the present invention”) comprising lansoprazole.
Lansoprazole, which is an active ingredient in the present invention, is a compound represented by the following structural formula (2-[[[3-methyl-4- (2,2,2-trifluoroethoxy) -2-pyridinyl] methyl] sulfinyl] -1H-benzimidazole).

Lansoprazole in the present invention may be a racemate or an optically active form such as an R-form or S-form, but (R) -2-[[[3-methyl-4- (2 , 2,2-trifluoroethoxy) -2-pyridinyl] methyl] sulfinyl] -1H-benzimidazole and the like are preferred.
In addition, lansoprazole in the present invention may be a compound represented by the above structural formula or a pharmaceutically acceptable salt of an optically active substance thereof. Examples of such salts include salts with inorganic bases, salts with organic bases, salts with basic amino acids, and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salt and the like. Suitable examples of salts with organic bases include, for example, alkylamines (trimethylamine, triethylamine, etc.), heterocyclic amines (pyridine, picoline, etc.), alkanolamines (ethanolamine, diethanolamine, triethanolamine, etc.), dicyclohexylamine. And salts with N, N′-dibenzylethylenediamine and the like. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ornithine and the like. Of these salts, alkali metal salts or alkaline earth metal salts are preferable. The sodium salt is particularly preferable.

Since lansoprazole is unstable to acids, it can be provided in the form of an acid-stable prodrug to prevent degradation by gastric acid when administered orally. Examples thereof include prodrugs described in US Pat. No. 6,093,734.

Lansoprazole can be produced by a method known per se, for example, the method described in JP-A-61-50978, US Pat. No. 4,628,098, JP-A-10-195068, WO98 / 21201, or the like. Manufactured by. The optically active substance can be obtained by an optical resolution method (fractional recrystallization method, chiral column method, diastereomer method, method using a microorganism or enzyme, etc.), asymmetric oxidation, and the like. In this case, it can also be produced according to the methods described in WO00 / 78745, WO01 / 83473, WO01 / 87874 and WO02 / 44167.

As described above, lansoprazole is expressed in TGF-β involved in promoting fibrosis in the NAFLD / NASH pathology (in the present specification, “TGF-β ()) expression is mature unless otherwise specified”. TGF-β, meaning the expression of active TGF-β protein.) And the pro-inflammatory cytokine IL-6 gene expression and protein expression (in this specification, “IL-6 ()) expression” Unless otherwise noted, it is used to include both IL-6 gene expression and protein expression.), Liver fibrosis and inflammation are suppressed, and AST / ALT levels are lowered to improve liver function. Therefore, it is effective for the prevention and / or treatment of NAFLD / NASH.

Lansoprazole has low toxicity and is a pharmaceutical composition mixed with a pharmacologically acceptable carrier as it is or according to a method known per se, such as tablets (including sugar-coated tablets and film-coated tablets), powders, granules and capsules. (Including soft capsules), orally disintegrating tablets, liquids, injections, suppositories, sustained-release agents, patches, etc., for humans or other mammals, orally or parenterally (eg, Topical, subcutaneous, rectal, intravenous administration, etc.) can be safely administered. As shown in Examples described later, lansoprazole can exert an action of suppressing the pathological condition of NAFLD / NASH at a dose that has already been confirmed in animal experiments as a therapeutic agent for gastric ulcer and duodenal ulcer. Therefore, the preventive and / or therapeutic agent of the present invention can also be effectively administered to NAFLD / NASH patients in an amount of active ingredient equivalent to the dose as an existing therapeutic agent for gastric ulcer and duodenal ulcer.

The content of lansoprazole in the preventive and / or therapeutic agent of the present invention is about 0.01% to 100% by weight of the total composition.
The dose of the preventive and / or therapeutic agent of the present invention varies depending on the administration subject, administration route, severity of disease, etc., but for example, when administered orally to an adult (60 kg), the amount of active ingredient About 0.5 to 1500 mg / day, preferably about 5 to 150 mg / day, and most preferably 30 mg / day. When administered parenterally (for example, subcutaneously), the amount of the active ingredient is about 0.5 to 1500 mg / day, preferably about 5 to 150 mg / day, and most preferably 30 mg / day. Lansoprazole may be administered once a day or divided into 2-3 times.

Examples of the pharmacologically acceptable carrier that may be used for the production of the preventive and / or therapeutic agent of the present invention include various organic or inorganic carrier substances that are commonly used as pharmaceutical materials. Agents, lubricants, binders, disintegrants, water-soluble polymers, basic inorganic salts; solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, etc. in liquid preparations It is done. Further, if necessary, additives such as ordinary preservatives, antioxidants, colorants, sweeteners, sour agents, foaming agents, and fragrances can be used.

Examples of the “excipient” include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, titanium oxide and the like.
Examples of the “lubricant” include magnesium stearate, sucrose fatty acid ester, polyethylene glycol, talc, stearic acid and the like.

Examples of the “binder” include hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, starch, polyvinylpyrrolidone, gum arabic powder, gelatin, pullulan, low-substituted hydroxypropylcellulose, and the like.

Examples of the “disintegrant” include (1) crospovidone, (2) disintegrants called super disintegrants such as croscarmellose sodium (FMC-Asahi Kasei), carmellose calcium (Gotoku Pharmaceutical), (3) carboxymethyl Examples include starch sodium (eg, Matsutani Chemical Co., Ltd.), (4) low-substituted hydroxypropylcellulose (eg, Shin-Etsu Chemical Co., Ltd.), (5) corn starch and the like. The “crospovidone” is crosslinked with a chemical name of 1-ethenyl-2-pyrrolidinone homopolymer, including those called polyvinylpolypyrrolidone (PVPP) and 1-vinyl-2-pyrrolidinone homopolymer. Specific examples include Kollidon CL (manufactured by BASF), Polyplastidone XL (manufactured by ISP), Polyplaston XL-10 (manufactured by ISP), and Polyplastidone. INF-10 (manufactured by ISP).

Examples of the “water-soluble polymer” include ethanol-soluble water-soluble polymers [for example, cellulose derivatives such as hydroxypropylcellulose (hereinafter sometimes referred to as HPC), polyvinylpyrrolidone, etc.], ethanol-insoluble water-soluble polymers Molecules [for example, hydroxypropylmethylcellulose (hereinafter sometimes referred to as HPMC), cellulose derivatives such as methylcellulose, sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum, etc.] and the like.

Examples of the “basic inorganic salt” include basic inorganic salts of sodium, potassium, magnesium and / or calcium. Preferred is a basic inorganic salt of magnesium and / or calcium. More preferred is a basic inorganic salt of magnesium. Examples of the basic inorganic salt of sodium include sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate and the like. Examples of the basic inorganic salt of potassium include potassium carbonate and potassium hydrogen carbonate. Examples of the basic inorganic salt of magnesium include heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg ₆ Al ₂ ( OH) ₁₆ · CO ₃ · 4H ₂ O] and alumina hydroxide / magnesium, preferably heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide and the like. Examples of the basic inorganic salt of calcium include precipitated calcium carbonate and calcium hydroxide.

Examples of the “solvent” include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
Examples of the “dissolution aid” include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

Examples of the “suspending agent” include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; And hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

Examples of the “isotonic agent” include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.
Examples of the “buffering agent” include buffer solutions of phosphate, acetate, carbonate, citrate, and the like.

Examples of the “soothing agent” include benzyl alcohol and the like.
Examples of the “preservative” include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Examples of the “antioxidant” include sulfite, ascorbic acid, α-tocopherol and the like.
Examples of the “colorant” include edible pigments such as edible yellow No. 5, edible red No. 2, and edible blue No. 2; edible lake pigments, bengara and the like.

Examples of the “sweetening agent” include saccharin sodium, dipotassium glycyrrhizin, aspartame, stevia, thaumatin and the like.
Examples of the “sour agent” include citric acid (anhydrous citric acid), tartaric acid, malic acid and the like.

Examples of the “foaming agent” include sodium bicarbonate.
The “fragrance” may be a synthetic product or a natural product, and examples thereof include lemon, lime, orange, menthol, and strawberry.

Lansoprazole is compression-molded by adding a carrier such as an excipient, a disintegrant, a binder or a lubricant according to a method known per se such as JP-A-62-277322 and JP-A-63-301816. Then, if necessary, a preparation for oral administration can be prepared by coating by a method known per se for the purpose of taste masking, enteric property or persistence. In the case of an enteric preparation, an intermediate layer may be provided between the enteric layer and the drug-containing layer by a method known per se for the purpose of separating both layers.

For example, when lansoprazole is used as an orally disintegrating tablet, for example, a core containing crystalline cellulose and lactose is coated with lansoprazole and, if necessary, a basic inorganic salt in accordance with WO 99/59544, WO 00/00126, etc. Further, a composition is obtained by coating with a coating layer containing a water-soluble polymer, and the resulting composition is coated with an enteric coating layer containing polyethylene glycol, and then coated with an enteric coating layer containing triethyl citrate, It can be produced by a method of coating with a polyethylene glycol-containing enteric coating layer and finally coating with mannitol to obtain fine particles, mixing the obtained fine particles and additives, and molding.

Examples of the “enteric coating layer” include cellulose acetate phthalate (CAP), hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, methacrylic acid copolymer [for example, Eudragit® L30D-55 (trade name; Rame Co., Ltd.), Kollicoat MAE30DP (trade name; manufactured by BASF Corp.), Polykid PA30 (trade name; manufactured by Sanyo Kasei Co., Ltd.), etc.], carboxymethyl ethyl cellulose, shellac and other water-based enteric polymer bases; Sustained release bases such as coalescent [eg Eudragit NE30D (trade name), Eudragit RL30D (trade name), Eudragit RS30D (trade name), etc.]; water-soluble polymer; triethyl citrate, polyethylene glycol, acetylated monoglyceride De, triacetin, include one or consists like a mixture of two or more layers such as a plasticizer such as castor oil.

Examples of the “additives” include water-soluble sugar alcohols (eg, sorbitol, mannitol, maltitol, reduced starch saccharified product, xylitol, reduced palatinose, erythritol, etc.), crystalline cellulose (eg, Theolas KG 801, Avicel PH 101) , Avicel PH 102, Avicel PH 301, Avicel PH 、 302, Avicel RC-591 (crystalline cellulose, carmellose sodium), etc., low-substituted hydroxypropylcellulose (eg, LH-22, LH-32, LH-23, LH) -33 (Shin-Etsu Chemical Co., Ltd.) and mixtures thereof, etc.), binders, acidulants, foaming agents, sweeteners, fragrances, lubricants, colorants, stabilizers, excipients, Disintegrants are also used.

Lansoprazole can be used in combination with other drugs. Examples of concomitant drugs include liver protectants, hypoglycemic agents, antihypertensive agents, antioxidants, anti-inflammatory agents and the like. Examples of the liver protectant include ursodeoxycholic acid and betaine. Examples of hypoglycemic agents include insulin and insulin derivatives, sulfonylureas such as tolbutamide, gliclazide, glibenclamide and glimepiride, fast-acting insulin secretagogues such as nateglinide, repaglinide and mitiglinide, and α such as acarbose, voglibose and miglitol. -Glucosidase inhibitors, biguanides such as metformin and phenformin, and thiazolidines such as pioglitazone, rosiglitazone and troglitazone. Examples of the antihypertensive agent include an angiotensin converting enzyme inhibitor, an angiotensin receptor inhibitor, and a calcium antagonist. Examples of the antioxidant include vitamins such as vitamin C and vitamin E, N acetylcysteine, probucol, icosapentaenoic acid and esters thereof. Examples of the anti-inflammatory agent include cytokine production inhibitors such as pentoxifylline. In combination, lansoprazole is mixed according to a method known per se, and in one pharmaceutical composition (for example, tablet, powder, granule, capsule (including soft capsule), liquid, injection, suppository, sustained release etc.) In addition, both may be formulated separately, and each may be formulated separately and administered to the same subject simultaneously or with a time difference.

Hereinafter, the present invention will be described more specifically with reference to examples. However, these are merely examples and do not limit the scope of the present invention.

(Method)
Preparation of NAFLD / NASH model animals using CDAA Six-week-old male Wistar rats (Kiwa experimental animals) were raised for one week on a high-fat choline-deficient methionine-reduced diet (A06083101, EPS Masin Co., Ltd.) (CDAA). Fatty liver rats were prepared (CDAA 1w) (n = 6). In another group, the animals were reared for 4 weeks under the same conditions. During this period, lansoprazole (AG-1749, Takeda Pharmaceutical Co., Ltd.) was ground in a mortar and the solvent solution (0.5% carboxymethylcellulose solution) was used. A 0.67 ml suspension prepared to a final dose of 30 mg / ml was added in small portions and administered subcutaneously on the back of the back once daily with a 26 G tuberculin needle. The body weight was measured every day and the dose was adjusted to 30 mg / kg. Sampling was performed 4 weeks after the start of administration (CDAA 5w + LAP 4w). Control animals received an equal volume of solvent transdermally once daily (CDAA 5w) (n = 7 each).

Collection and Analysis of Blood Sample and Liver Tissue Rats fed with CDAA for 1 or 5 weeks were anesthetized with isoflurane after body weight measurement, and thoracotomy was performed to collect blood directly from the heart. After collecting the liver and measuring the weight, a part of the liver is fixed with 4% formalin aqueous solution for tissue analysis, or immersed in RNAlator solution for gene expression analysis, or liquid for protein expression analysis. Rapid freezing was performed with nitrogen. The collected blood was centrifuged, serum was collected, and the concentrations of AST and ALT were measured by a conventional method.

Evaluation of liver fibrosis The fixed tissue was embedded in paraffin and sliced, and collagen (I and III) fibers were stained using Picroserius Red Stain Kit (Polyscienes). Hepatic fibrosis scoring was performed using the criteria of Brunt (1) (Brunt, EM. Et al., Seminar Liv. Dis., 21: 3-16 (2001)).

Examination of gene expression Gene expression was measured by Real time RT-PCR. RNA samples were extracted from tissues soaked in RNAlator using RNeasy mini kit, concentration was measured using Nano Drop 1000 (Thermo Fischer), and then reverse transcription reaction was performed using AMV reverse transcriptase (Roche). As an internal control, the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used. The reverse-transcribed sample was quantified by real-time PCR using the primers shown in Table 1 and LightCycler FastStart DNA Master SYBR Green I (Roche) or KAPA SYBR FAST qPCR Kit (KAPA Biosystems). The amplification protocol was 45 cycles of 95 ° C. 5 seconds-60 ° C. 5 seconds-72 ° C. 12 seconds. A calibration curve was prepared by amplifying a series of 10-fold diluted standard cDNA samples using the same protocol.

Examination of protein expression level The amount of TGF-β protein was measured by Western blotting. The cryopreserved sample was homogenized in PRO-PREP ^™ (INTRON) buffer and incubated on ice for 20 minutes. Centrifuged at 13,000 xg for 5 minutes at 4 ° C to extract soluble protein. Electrophoresis on SDS-polyacrylamide gel (12.5%), transferred to PVDF membrane (Millipore), and reacted with rabbit anti-TGFβ antibody (CST, 3711S) or rabbit anti-IL-6 antibody (Abnova, PAB16165) as the primary antibody It was. Subsequently, after reaction with an HRP-labeled anti-rabbit IgG antibody (Thermo, 31458), detection was performed by the ECL method. After reprobing, a rabbit anti-actin antibody (Sigma-Aldrich, A5060) was reacted as a primary antibody, then reacted with a peroxidase-labeled anti-rabbit antibody (NA934, Amersham), and then detected by the ECL method. The quantification of the TGF-β immunopositive signal was performed with Lumino analyzer LAS-1000 (Fuji Film) and corrected with the actin immunopositive signal.

(result)
1. Effects on NAFLD / NASH In rats administered CDAA for 1 week, fatty liver was observed. By continuing CDAA administration for 4 weeks, the findings of bridging fibrosis, a characteristic of NAFLD / NASH, were observed (CDAA 5w) (left side of FIG. 1A). In contrast, fibrosis was suppressed by subcutaneous administration of lansoprazole at 30 mg / kg for 4 weeks from the second week after the start of CDAA administration (CDAA 5w + LAP 4w) (right in FIG. 1A). When fibrosis scoring was performed, the fibrosis score (NAFLD / NASH stage) was significantly suppressed by lansoprazole administration (*: P = 0.01, analyzed by Mann-Whitney test) (FIG. 1B) .
In rats administered CDAA (CDAA 5w), serum AST and ALT concentrations increased compared to control rats (Normal) fed on a normal diet. In contrast, administration of lansoprazole (CDAA 5w + LAP 4w) significantly reduced the serum AST concentration (#: P <0.05) and showed a decreasing trend for serum ALT (P = 0.10) ( Figure 2). Statistical analysis was performed by Holm's multiple test.
Therefore, lansoprazole was shown to significantly suppress the progression of NAFLD / NASH.

2. Expression analysis of downstream genes of Nrf2 and Ahr The expression levels of the HO-1 gene induced by Nrf2 and the Cyp1a1 gene induced by Ahr, which have antioxidant stress action, were examined using RT-PCR. In NAFLD / NASH model rats, lansoprazole administration significantly increased Nrf2 and Ahr mRNA levels (*: P <0.05, analyzed by Student's t test), but downstream HO-1 and Ahr mRAN levels No significant change was observed (FIG. 3). Therefore, it was shown that the prevention and / or treatment effect of lansoprazole on NAFLD / NASH is due to an action different from the antioxidant action of lansoprazole.

3. Expression analysis of liver fibrosis- related genes In order to investigate the mechanism of liver fibrosis, the expression levels of three genes (Timp1, Col1a1, Tgfb1) associated with liver fibrosis were measured in the liver of NAFLD / NASH model rats. As a result, the expression of any gene was not significantly changed by lansoprazole administration (FIG. 4).

4). Expression analysis of TGF-β protein In NAFLD / NASH model rats, lansoprazole significantly suppressed the expression of mature TGF-β protein in the liver (*: P <0.05, analyzed by Student's t test) (FIG. 5). On the other hand, the expression of TGF-β precursor protein was not suppressed by lansoprazole administration. Since TGF-β protein involved in the promotion of fibrosis is suppressed by lansoprazole, the effect of lansoprazole on liver fibrosis on NAFLD / NASH is the expression of mature TGF-β (ie, activation of TGF-β) It was suggested that it was due to a route through inhibition.

5). Expression analysis of proinflammatory cytokines In order to investigate the mechanism of the anti-inflammatory effect of lansoprazole administration, the amount of gene expression of proinflammatory cytokines IL-6 and IL-1β in the liver of NAFLD / NASH model rats was measured. As a result, IL-1β gene expression was not changed by lansoprazole administration, but IL-6 expression was significantly reduced (FIG. 6A). Similarly, lansoprazole administration also reduced IL-6 protein expression (FIGS. 6B and C).

While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

This application is based on Japanese Patent Application No. 2016-046340 filed in Japan on March 9, 2016, the contents of which are incorporated herein by reference.

Since lansoprazole has been used safely as a therapeutic agent for peptic ulcer, the prophylactic and / or therapeutic agent of the present invention is useful as a safe prophylactic and / or therapeutic agent for NAFLD / NASH.

Claims (5)

A preventive and / or therapeutic agent for nonalcoholic fatty liver disease / nonalcoholic steatohepatitis comprising lansoprazole. The agent according to claim 1, which is used so that 5 mg to 150 mg of lansoprazole is administered per day for an adult. The agent according to claim 1, which is used so that 30 mg of lansoprazole is administered per day for an adult. An inhibitor of TGF-β expression comprising lansoprazole. An IL-6 expression inhibitor comprising lansoprazole.

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