JP2010168283A - Inhibitor of cirrhosis and precancerous lesion using natriuretic peptide as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition for inhibiting hepatic fibrosis, serving to prevent or treat cirrhosis or precancerous lesion by using a natriuretic peptide as an active ingredient. <P>SOLUTION: It is discovered that the natriuretic peptide has the inhibiting effects against the hepatic fibrosis by examining the in-vivo effects of the natriuretic peptide to an active stellate cell of the liver by paying attention to the stellate cell of the liver which can be said as a "target cell" in the prevention or treatment of the hepatic fibrosis, and considering that the inhibition of the activation of the stellate cell of the liver is important, and the invention is completed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition for the prevention or treatment of cirrhosis or precancerous lesions comprising a natriuretic peptide as an active ingredient.

  Fibrosis in the liver (liver fibrosis) is caused by chronic liver damage due to various causes such as viral hepatitis and alcoholic hepatitis. In chronic liver disease, for example, in the progression from viral hepatitis to cirrhosis Liver fibrosis plays an important role. However, at present, removing the cause of liver damage is the first treatment to prevent liver fibrosis. Therefore, the development of treatments such as drugs that effectively suppress fibrosis has been developed in current hepatology. This is an extremely important research topic.

  Liver fibrosis is a response to hepatocyte necrosis and damage and is associated with a complex number of factors. For example, it may also be caused by liver inflammation or poisoning damage, changes in liver blood flow, or infections caused by liver viruses or bacteria. In addition, various storage abnormalities in hepatocytes are often associated with fibrosis. Furthermore, chemical substances and drugs such as alcohol and methotrexate, and impaired circulation to the liver such as chronic heart failure also cause liver fibrosis.

  The liver is not composed solely of liver parenchymal cells that are responsible for liver function; 40% of the total number of cells that make up the liver is non-parenchymal cells, and non-parenchymal cells are approximately 48% sinusoidal endothelium. It consists of about 39% Kupffer cells and about 13% hepatic stellate cells. Hepatic stellate cells are derived from fibroblasts and belong to the myofibroblast family. Liver myofibroblasts are cells that differentiate from undifferentiated mesenchymal cells, produce collagen and form a matrix during the organogenesis phase, have α-actin in the cytoskeleton, and when grown, the cell morphology is Changes to inactive hepatic stellate cells. Hepatic stellate cells usually have lipid droplets containing vitamin A in the cytoplasm, and are located in the sinusoidal cavity in the liver between hepatocytes and sinusoidal endothelial cells to support the construction of sinusoids In addition, it has a long branched branch, surrounds sinusoidal endothelial cells like so-called vascular pericytes, contracts in response to endothelin, or relaxes by nitric oxide (NO), It is known as a cell that acts to regulate blood flow in sinusoids. Furthermore, hepatic stellate cells are the main producers of extracellular matrix (ECM) such as collagen, laminin, and proteoglycan in the liver, and their functions are cytokines and platelet-derived growth factors (Kupffer cells and hepatic macrophages). It is regulated by growth factors such as PDGF.

  On the other hand, hepatic stellate cells are stimulated by cytokines such as TGF-β during liver injury or inflammation, or hepatic stellate cells are activated by endotoxemia caused by a large intake of alcohol, resulting in excessive production of ECM. As a result, liver fibrosis occurs. In other words, liver fibrosis is a pathological condition in which ECM has accumulated excessively as a result of wound healing mechanism for tissue injury in the liver. Normally, ECM such as collagen in the living body maintains a certain balance while repeating production and degradation, but under pathological conditions, the balance is lost and fibrosis occurs. Liver fibrosis occurs mainly with inflammation due to hepatitis virus and alcohol, and becomes prominent particularly when inflammation becomes chronic.

  In the process of liver fibrosis, hepatic stellate cells are activated by cytokines produced by Kupffer cells and liver macrophages and transformed into activated cells to produce a large amount of ECM such as collagen and fibronectin. It also plays a central role in fibrosis by producing matrix-degrading enzyme (MMP), its inhibitor (TIMP), cytokines such as TGF-β and PDGF, and growth factors such as HGF. Activated hepatic stellate cells have enhanced contractility and are involved in the regulation of hepatic blood flow, and also increase the expression of various cytokine receptors, making them highly sensitive to cytokines.

  Collagen production and degradation are complexly regulated by various cytokines, MMPs and TIMPs. TGF-β is the cytokine that most promotes liver fibrosis and promotes collagen production. On the other hand, TNF-α and IFN-γ suppress collagen production. TGF-β increases during hepatic fibrosis, activates hepatic stellate cells by paracrine and autocrine action, promotes ECM production, enhances TIMP production in hepatic stellate cells, and causes relative degradation of ECM Fibrosis progresses in order to reduce it. PDGF is also considered the most important cytokine that promotes the proliferation of hepatic stellate cells, and is involved in the promotion of liver fibrosis.

  Thus, cytokines associated with hepatic injury are important for the activation of hepatic stellate cells, and the activation is further enhanced through proliferation, contraction, ECM production, cytokine production, and the like.

  Liver fibrosis is a symptom resulting from chronic liver damage caused by various causes as described above, and if the symptom continues, it progresses to cirrhosis, and further progresses to liver cancer. Therefore, in order to prevent and treat progression to precancerous lesions at the stage of transition to liver cirrhosis or liver cancer, development of pharmaceuticals that can be used for the prevention or treatment of liver fibrosis is expected. Regarding suppression of hepatic stellate cell activation, PPARgamma-ligand (diabetic drug) acts on hepatic stellate cells to suppress not only activation but also proliferation (Non-Patent Document 1: Kawaguchi K et al. Biochem). Biophys. Res. Commun. 2004; 315: 187-95.) And angiotensin-type II receptor antagonists (hypertensive drugs) suppress hepatic stellate cell activation and induce suppression of ECM production (non-patented) Reference 2: Kurihara N et al. Br J Pharmacol 2003; 139: 1085-94.) Therapies using these have been attempted, but no clinically effective prevention or treatment method has been found at present. .

  In advanced decompensated cirrhosis, hepatic encephalopathy, ascites, rupture of esophageal gastric varicose veins occur, and quality of life (QOL) is extremely lowered. There is currently no fibrinolytic agent for the liver once it has led to cirrhosis, and there are only passive treatments that prevent and delay the progression of fibrosis. In other words, there are currently no drugs for treating cirrhosis.

  On the other hand, natriuretic peptide (NP) having diuretic action is known as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). In addition, receptors for these peptides are known as diuretic peptide receptor-A (NPR-A), diuretic peptide receptor-B (NPR-B), etc., which have a membrane-bound guanylyl cyclase structure. It is a guanylyl cyclase-coupled receptor.

  In addition, ANP and BNP are specific ligands for NPR-A, CNP is a specific ligand for NPR-B, and they are diuretic by increasing intracellular cGMP after binding to each receptor. It is considered to exhibit biological activities such as action and vasodilatory action. (Non-Patent Document 3: Rosenzweig A, and Seidman CE., Annu. Rev. Biochem., 60: 229-255, 1991). Natriuretic peptides have been reported to play an important role in controlling body fluid homeostasis and blood pressure (Non-patent Document 4: Ogawa Y, et al., J. Clin. Invest., 93 : 1911-1921, 1994), expression in various tissues other than the cardiovascular system and its physiological activity are also known (Non-Patent Document 5: Komatsu Y., et al., Endocrinology, 129: 1104-1106, 1991; Non-Patent Document 6: Chinkers M. and Garbers DL., Annu. Rev. Biochem., 60: 553-575, 1991).

  ANP is a peptide hormone that is secreted from the heart and plays an important role in the regulation of water electrolyte metabolism and blood pressure. In humans and model animals, it is known that blood ANP levels increase with the severity of cardiac hypertrophy and heart failure, and it is thought to compensate for the pathophysiology of heart failure. In fact, vasodilatory and diuretic effects are manifested by ANP administration in patients with heart failure, the preload and postload of the heart are reduced, and a hemodynamic improvement effect is recognized (Non-patent Document 7: Suzuki T., et al ., Cardiovasc. Res. 51: 489-494, 2001). It has already been used clinically as an acute heart failure drug.

  BNP is a hormone found in the brain, but it is secreted mainly from the heart rather than the brain, and has a vasodilatory action and diuretic action and plays an important role in regulating body fluid volume and blood pressure. is there. Plasma BNP levels in healthy individuals are extremely low, but increase in patients with heart failure according to their severity (Non-Patent Document 8: Mukoyama M., et al., J. Clin. Invest., 87: 1402-1412, 1991 ). BNP in blood is already high in asymptomatic heart failure, and increases significantly depending on the severity, so it is important as a method for evaluating heart failure function. BNP measurement is important for understanding the pathogenesis of heart failure ( Non-Patent Document 7: Suzuki T., et al., Cardiovasc. Res. 51: 489-494, 2001). BNP is also already approved for the treatment of acute heart failure, such as in the United States.

  CNP is known as a bone growth factor, and CNP significantly promotes long bone growth in mouse fetal tibial organ culture (Non-Patent Document 9: Yasoda A., et al., J. Biol. Chem). , 273: 11695-11700, 1998). In addition, organ culture of tibia of mouse fetus, chondrocyte culture cell, and osteoblast cell culture cell have higher cGMP production ability than ANP and BNP (Non-patent Document 10: Hagiwara H., et al., J. Biol Chem., 269: 10729-10733, 1994; Non-Patent Document 11: Suda M., et al., Biochem. Biophys. Res. Commun., 223: 1-6, 1996; Non-Patent Document 12: Inoue A. , et al., Biochem. Biophys. Res. Commun., 215: 1104-1110, 1995). In addition, it is known to have an inhibitory effect on hepatic stellate cell proliferation in an in vitro experiment (Non-patent Document 13: Gorbig M.N., Hepatology, 30: 501-509, 1999).

  Natriuretic peptide has a fibrosis-inhibiting action in the heart and kidney (Non-patent Document 14: Calderone A, et al. J. Clin. Invest., 101: 812-818 .; Non-patent Document 15: Suganami T et al. 12: 2652-2663, 2001), to date, natriuretic peptides have actually been used to treat liver fibrosis, which causes progression to cirrhosis and precancerous lesions, a process of transition to canceration. There has been no report on the in vivo hepatic fibrosis inhibitory effect and whether it is effective in inhibiting the activation of the cell by acting on hepatic stellate cells. Does not know the existence of the report.

Kawaguchi K et al. Biochem. Biophys. Res. Commun. 2004; 315: 187-95. Kurihara N et al. Br J Pharmacol 2003; 139: 1085-94. Rosenzweig A, and Seidman CE., Annu. Rev. Biochem., 60: 229-255, 1991 Ogawa Y, et al., J. Clin. Invest., 93: 1911-1921, 1994 Komatsu Y., et al., Endocrinology, 129: 1104-1106, 1991 Chinkers M. and Garbers DL., Annu. Rev. Biochem., 60: 553-575, 1991 Suzuki T., et al., Cardiovasc. Res. 51: 489-494, 2001 Mukoyama M., et al., J. Clin. Invest., 87: 1402-12, 1991 Yasoda A., et al., J. Biol. Chem., 273: 11695-11700, 1998 Hagiwara H., et al., J. Biol. Chem., 269: 10729-10733, 1994 Suda M., et al., Biochem. Biophys. Res. Commun., 223: 1-6, 1996 Inoue A., et al., Biochem. Biophys. Res. Commun., 215: 1104-1110, 1995 Gorbig M.N., Hepatology, 30: 501-509, 1999 Calderone A, et al. J. Clin. Invest., 101: 812-818. Suganami T. et al. 12: 2652-2663, 2001

  An object of the present invention is to provide a pharmaceutical composition for inhibiting liver fibrosis, which is used for the prevention or treatment of cirrhosis or precancerous lesions, which comprises a natriuretic peptide as an active ingredient.

  Based on the mechanism of liver fibrosis described above, (1) suppression of hepatic stellate cell activation, (2) control of cytokines important for liver fibrosis, (3) Inhibition of hepatic stellate cell growth, (4) control of the ECM degradation system, and (5) hepatocyte proliferation and hepatic stem cell transplantation are considered. Therefore, the present inventors focused on hepatic stellate cells, which can be said to be “target cells”, particularly in the prevention or treatment of liver fibrosis, and considered that it is important to suppress the activation of hepatic stellate cells. As a result, the present inventors have found that it has an inhibitory effect on liver fibrosis, and has completed the present invention.

That is, the present invention relates to the following matters.
(1) A pharmaceutical composition for inhibiting liver fibrosis, comprising a natriuretic peptide or a pharmaceutically acceptable salt thereof as an active ingredient, and inhibiting liver fibrosis induced by activation of hepatic stellate cells.
(2) The pharmaceutical composition for inhibiting liver fibrosis according to (1) above, wherein the natriuretic peptide is any one of atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide.
(3) The pharmaceutical composition for inhibiting liver fibrosis according to (2) above, wherein the natriuretic peptide is an atrial natriuretic peptide.
(4) A method for preventing or treating cirrhosis or precancerous lesions caused by liver fibrosis, comprising administering a natriuretic peptide or a pharmaceutically acceptable salt thereof.
(5) The treatment method according to (4) above, wherein the natriuretic peptide is any one of atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide.
(6) The treatment method according to the above (4), wherein the natriuretic peptide is an atrial natriuretic peptide.
(7) Use of natriuretic peptide or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for inhibiting liver fibrosis.
(8) The use according to (7) above, wherein the natriuretic peptide is any of atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide.
(9) The use according to (7) above, wherein the natriuretic peptide is an atrial natriuretic peptide.

  The pharmaceutical composition for suppressing liver fibrosis comprising the natriuretic peptide according to the present invention as an active ingredient has the special effect of acting on hepatic stellate cells to suppress its activation and further suppress the progression of liver fibrosis. It is what you play. Suppression of liver fibrosis has increased the possibility of suppressing or treating the progression to liver cirrhosis or precancerous lesions that are transitioning to canceration.

  The pharmaceutical composition for suppressing liver fibrosis comprising the natriuretic peptide according to the present invention as an active ingredient exerts a special effect of acting on hepatic stellate cells to suppress its activation and suppress the progression of liver fibrosis. Is. By suppressing liver fibrosis, it has become possible to suppress or treat progression to liver cirrhosis or precancerous lesions, which are the process of transition to canceration.

BEST MODE FOR CARRYING OUT THE INVENTION

  Liver fibrosis is a response to hepatocyte necrosis or damage and refers to changes in liver tissue that occur in a complex manner involving many factors. Liver fibrosis can be caused by, for example, liver inflammation or damage caused by toxins, changes in liver blood flow, or infections caused by liver viruses or bacteria, various storage abnormalities in hepatocytes, or chemicals or drugs such as alcohol or methotrexate. And circulatory disturbances to the liver such as chronic heart failure.

  Hepatic stellate cells are stimulated by excessive production and accumulation of ECM due to cytokines such as TGF-β produced during liver damage and inflammation, and endotoxemia caused by a large intake of alcohol. This causes a pathology of liver fibrosis. On the other hand, hepatic stellate cells produce matrix degrading enzyme (MMP) and its inhibitory factor (TIMP), cytokines such as TGF-β and PDGF, and growth factors such as HGF, and promote liver fibrosis.

  In the present invention, by administering a natriuretic peptide (NP) to an animal, the type I collagen gene involved in promoting liver fibrosis and the matrix degrading enzyme (MMP) suppressor (TIMP) gene It was found that the expression in the animal body was significantly reduced, and that the liver fibrosis of the animals that actually caused liver fibrosis was suppressed / improved.

  The substance that can be used as the active ingredient of the pharmaceutical composition according to the present invention may be any substance that has the property of enhancing cGMP production via NPR-A, which is a natriuretic peptide (NP) receptor. The substance is preferably a natriuretic peptide, and examples of the natriuretic peptide include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP).

  As ANP in the present invention, natriuretic peptide (NP) such as human-derived ANP (SLRRSSCFGG RMDRIGAQSG LGCNSFRY: SEQ ID NO: 1) and rat-derived ANP (SLRRSSCFGG RIDRIGAQSG LGCNSFRY: SEQ ID NO: 3) consisting of 28 amino acids. Those having a characteristic capable of enhancing cGMP production via the receptor NPR-A can be used. Since these peptides, which are active ingredients according to the present invention, exhibit the property of enhancing cGMP production via NPR-A, which is a natriuretic peptide (NP) receptor, at least the ring structure of the ANP (for example, human In the case of the amino acid sequence of ANP, a peptide having a C-terminal part following a ring structure (that is, a ring structure based on the formation of a disulfide bond based on 7-position Cys and 23-position Cys of SEQ ID NO: 1) (ie, human) In the case of ANP, SEQ ID NO: 2) corresponding to positions 7 to 28 of SEQ ID NO: 1 may be used. Examples of the peptide having such structural characteristics include an ANP itself described in SEQ ID NO: 1, or a peptide having a partial amino acid sequence thereof and consisting of amino acids at positions 7 to 28 of the human ANP. Examples of the peptide to be encapsulated, for example, the peptide consisting of amino acids at positions 7 to 28 of the human ANP (SEQ ID NO: 2) itself.

  As BNP in the present invention, human-derived BNP consisting of 32 amino acids (SPKMVQGSGC FGRKMDRISS SSGLGCKVLR RH: SEQ ID NO: 4), porcine-derived BNP (SPKTMRDSGC FGRRLDRIGS LSGLGCNVLR RY: SEQ ID NO: 6), rat-derived BNP (SQDSAFRIQE Those having the property of enhancing cGMP production via NPR-A, which is a natriuretic peptide (NP) receptor, such as RLRNSKMAHS SSCFGQKIDR IGAVSRLGCD GLRLF: SEQ ID NO: 7) can be used. Since these peptides as active ingredients according to the present invention exhibit the property of enhancing cGMP production via NPR-A, which is a natriuretic peptide (NP) receptor, at least the ring structure of the BNP (for example, human In the case of the amino acid sequence of BNP, a peptide having a C-terminal part following a ring structure (that is, a ring structure based on the formation of a disulfide bond based on Cys 10 and 26 Cys of SEQ ID NO: 4) (ie, human) In the case of BNP, SEQ ID NO: 5 corresponding to positions 10-32 of SEQ ID NO: 4 may be used. Examples of the peptide having such a structural feature include a BNP itself described in SEQ ID NO: 4, or a peptide having a partial amino acid sequence thereof and consisting of amino acids at positions 10 to 32 of the human BNP. Examples of the peptide to be encapsulated, for example, the peptide consisting of amino acids at positions 10 to 32 of human BNP (SEQ ID NO: 5) itself.

  As CNP in the present invention, human-derived CNP consisting of 22 amino acids (GLSKGCFGLK LDRIGSMSGL GC: SEQ ID NO: 8, pig and rat also have the same amino acid sequence), chicken-derived CNP (GLSRSCFGVK LDRIGSMSGL GC: SEQ ID NO: 10) Frog-derived CNP (GYSRGCFGVK LDRIGAFSGL GC: SEQ ID NO: 11), etc., can be used that have the property of enhancing cGMP production via NPR-A, a natriuretic peptide (NP) receptor . Since these peptides, which are active ingredients according to the present invention, exhibit the property of enhancing cGMP production via NPR-A, which is a natriuretic peptide (NP) receptor, at least the CNP ring structure (for example, human In the case of the amino acid sequence of CNP, any peptide having a ring structure based on the formation of a disulfide bond based on 6-position Cys and 22-position Cys of SEQ ID NO: 8 (ie, SEQ ID NO: 9) may be used. . Examples of the peptide having such structural characteristics include CNP itself described in SEQ ID NO: 4, or a peptide having a partial amino acid sequence thereof and consisting of amino acids at positions 6-22 of the human CNP. Examples of the peptide to be encapsulated, such as the peptide consisting of amino acids at positions 6-22 of the human CNP (SEQ ID NO: 9) itself.

  Furthermore, the natriuretic peptide according to the present invention may be one that has been isolated and purified purely from nature, or one that has been produced by a chemical synthesis method or a gene recombination method. ) Can be obtained by appropriately modifying the amino acid residues in the sequence by deletion, substitution, addition, insertion, or the like by a known method as appropriate. Any substance can be used as long as the substance obtained by the method can act on NP receptor NPR-A and enhance cGMP production.

  Whether or not the obtained substance can act on NPR-A, which is an NP receptor, to enhance cGMP production can be easily measured by those skilled in the art by conventional methods. Specifically, it is possible by adding a substance to cultured cells in which NPR-A (Chinkers M et al. Nature 338; 78-83, 1989) is forcibly expressed and evaluating cGMP production ability.

  A substance that can be used as an active ingredient of the pharmaceutical composition according to the present invention is a pharmaceutically acceptable substance having a property capable of enhancing cGMP production via the above-described natriuretic peptide (NP) receptor NPR-A. Or a pharmaceutically acceptable salt of a natriuretic peptide. That is, in the present invention, an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or an organic acid such as formic acid, acetic acid, butyric acid, succinic acid or citric acid is used as an active ingredient. You can also Alternatively, in the present invention, metal salts such as sodium, potassium, lithium and calcium, and salts with organic bases of the above-mentioned substances can be used as active ingredients. In addition, the pharmaceutical composition according to the present invention may be a free form of the substance relating to the active ingredient or a pharmaceutically acceptable salt thereof.

  Administration of the above-described composition of the present invention significantly reduces the expression of type I collagen gene and matrix degrading enzyme (MMP) suppressor (TIMP) gene by hepatic stellate cells, and activation of hepatic stellate cells Can suppress liver fibrosis induced by. In the present invention, it is also possible to prevent or treat cirrhosis or precancerous lesions caused by liver fibrosis by administering the above-described composition of the present invention and suppressing liver fibrosis as described above. it can.

  A substance that can be used as an active ingredient of the pharmaceutical composition according to the present invention or a pharmacologically acceptable salt thereof is mixed with a known pharmacologically acceptable carrier, excipient, diluent, etc. into a medicine. It is preferable to administer by a commonly used administration method, that is, an oral administration method or a parenteral administration method such as intravenous administration, intramuscular administration or subcutaneous administration.

  When the active ingredient is a peptidic substance, it is possible to orally administer it as a microcapsule in which the active ingredient peptide is encapsulated in a ribosome, such as a preparation that is not easily degraded in the digestive tract. Moreover, the administration method which absorbs from mucous membranes other than digestive tracts, such as a rectum, a nose, and sublingual, is also possible. In this case, it can be administered in the form of suppositories, nasal sprays, sublingual tablets.

  The dose of a substance that can be used as an active ingredient of the pharmaceutical composition according to the present invention varies depending on the type of disease, patient age, body weight, degree of symptom, administration route, etc., but is generally 0.1 μg / kg per day. It can be administered in a range of ˜100 mg / kg, and it is preferably administered at 0.5 μg / kg to 5 mg / kg.

  The frequency of administration of the pharmaceutical composition according to the invention will also vary depending on the active ingredient used, the route of administration, and the particular disease being treated. For example, when natriuretic peptide is administered orally, it is preferably formulated with a frequency of not more than 4 times per day, and when administered intravenously, it is preferably administered continuously using an infusion pump.

Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1 Examination of the Effect of Natriuretic Peptide on Liver Fibrosis In this example, the effect of atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) on liver fibrosis was determined using N-diethylnitrosamine. (N-diethylnitrosamine, hereinafter referred to as DEN) was examined using a rat liver injury model induced by rat.

  A persistent liver injury model was prepared by intraperitoneally administering DEN (Wako Pure Chemical; 200 mg / kg) twice a week for 4 weeks to Wistar male rats (Japan SLC). In the ANP administration group or CNP administration group, one week after the start of DEN administration, human ANP (Asbiophoma Co., Ltd.) and human CNP (Asbiophoma Co., Ltd.), 1.0 μg / kg / min each for 2 weeks Continuous intravenous administration.

  Continuous intravenous administration was performed as follows. That is, the animal was anesthetized with sodium pentobarbital and then held in the supine position. An incision was made in the midline of the neck, and a hydrocoat catheter (AccessTechnology, catalog number: CNC-3H) was inserted into the jugular vein. The catheter was subcutaneously led out of the body from the back of the neck and connected to an infusion pump (US Medellell, Inc., trade name: Infu-Disk, catalog number: 05-0030-S) fixed to the back to administer the drug. In the control group, no drug administration was performed.

  After 4 weeks of DEN administration (ie, 3 weeks after the start of administration of ANP or CNP in the case of ANP administration group or CNP administration group), blood sampling and liver extraction, serum biochemistry, and liver Histopathological examination was performed to evaluate liver function and liver fibrosis.

  Alanine aminotransferase (ALT; also called GPT (glutamate pyruvate transaminase)), aspartate aminotransferase (AST; GOT (glutamate oxaloacetate transaminase), which are serum biochemical markers that are indicators of liver function in serum biochemical tests )) And total bilirubin concentrations were measured, and serum ALT, AST, and total bilirubin concentrations were all significantly lower in the ANP administration group and the CNP administration group than in the control group (FIGS. 1A to 1). C). Furthermore, when serum total protein concentration and serum albumin concentration, which are indicators of liver function, were measured in serum biochemical tests, serum total protein concentration and serum albumin concentration were maintained at higher levels in both treatment groups than in the control group. (FIGS. 2A-B). From these data, it was shown that the decrease in liver function due to DEN was suppressed in the ANP administration group and the CNP administration group.

  On the other hand, in the histopathological examination of the liver tissue, liver function and liver fibrosis were evaluated from the examination by Azan staining or Sirius Red staining that can stain collagen fibers. In Azan staining (Fig. 3) or Sirius Red staining (Fig. 4), significant fibrosis of the liver was confirmed in the control group (described as "DEN"), whereas liver fibrosis was observed in the ANP and CNP administration groups. It was shown that conversion was significantly suppressed.

  From these results (FIGS. 1 to 4), it was found that administration of ANP and CNP suppressed fibrosis and improved liver function in a rat liver fibrosis model.

  Furthermore, expression of mRNA of each gene of type 1 procollagen, MMP-2, MMP-13, TIMP-1, and TIMP-2, which are indicators of liver fibrosis, was determined by a conventional method (Kawaguchi K. et al. al., Biochem. Biophys. Res. Commun., 2004; 315: 187-95).

  Briefly, total RNA from rat liver was isolated using RNeasy-kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's instructions. Β-actin was used as an internal standard.

Based on the total RNA thus prepared, Taqman reverse transcriptase reagent (Roche Diagnostics, Indianapolis, IN, USA) was used for cDNA synthesis as described in the manufacturer's instructions. β-actin was used as an internal control to perform specific quantification of gene expression as described in the manual. To calculate the relative amount of target RNA, a method based on threshold cycle number and a standard curve was used. Light Cycler Q-PCR (Roche Diagnostics) using RT-PCR synthesis kit AMV (Roche Diagnostics, Indianapolis, IN, US) and 2 μl light cycler fast start DNA SYBR Green I (Roche Diagnostics), It was performed in 13 μl of a mixture containing 25 mM MgCl ₂ , 10 μM of each primer, 5 μl of extracted DNA, and Advantage PCR polymerase (Clontech Laboratories, Palo Alto, Calif.) (Operating system version 3.0). The reaction conditions are as follows: preliminary denaturation at 95 ° C for 10 minutes (temperature gradient 20 ° C / s), denaturation at 95 ° C for 15 seconds (temperature gradient 20 ° C / s), annealing at 68 ° C for 5 seconds ( Temperature gradient 20 ° C / s), primer extension for 10 seconds at 72 ° C (temperature gradient 20 ° C / s) for 40 cycles, and purified product detection at 72 ° C for 60 seconds (temperature gradient 20 ° C / s) )

The following primers were used as primers for real-time PCR:
Type 1 procollagen:
Sense: 5'-agcggtgaagaaggaaagagagg-3 '(SEQ ID NO: 12); and Antisense: 5'-caataggaccagaaggaccagca-3' (SEQ ID NO: 13)
MMP-2:
Sense: 5'-gccctcccctgatgctgata-3 '(SEQ ID NO: 14); and Antisense: 5'-gtcactgtccgccaaataaacc-3' (SEQ ID NO: 15)
TIMP-1:
Sense: 5'-ccccaacccacccacagacagc-3 '(SEQ ID NO: 16); and Antisense: 5'-cgctgcggttctgggacttgtg-3' (SEQ ID NO: 17)
TIMP-2:
Sense: 5'-cagggccaaagcagtgagcgagaa-3 '(SEQ ID NO: 18); and Antisense: 5'-tcttgccatctccttccgccttcc-3' (SEQ ID NO: 19).

  The insert was verified by DNA sequencing and used as a probe for real-time PCR analysis.

  As a result of this real-time PCR analysis, the expression level of type 1 collagen in the liver and tissue inhibitor of metalloproteinase-1 is a factor that inhibits extracellular matrix degradation and promotes fibrosis. The gene expression levels of (TIMP-1) and TIMP-2 were significantly suppressed in the ANP administration group and the CNP administration group as compared to the control group (described as “DEN”) (FIGS. 5A to 5C).

Example 2 Effect of Natriuretic Peptide on Hepatic Stellate Cells Since fibrosis of the liver is thought to involve activation of hepatic stellate cells, in this example, next, isolated liver of ANP and CNP The direct effect on stellate cells was examined.

Hepatic stellate cells were isolated from the liver of Wistar male rats (Japan SLC) by conventional methods (Kawaguchi K., et al., Biochem. Biophys. Res. Commun., 2004; 315: 187-95) . Specifically, the rat liver was perfused sequentially with Krebs-Ringer solution not containing Ca 2+ and Mg 2+ maintained at 37 ° C., 0.1% pronase E (Merck, Germany), 0.032% collagenase (Wako Pure Chemicals, Osaka). After the enzyme-treated liver was isolated and minced, it was incubated at 37 ° C., 30 ° C. in Krebs-Ringer solution (pH 7.3) containing 0.08% pronase E, 0.04% collagenase, and 20 μg / mL DNase (Boehringer-Mannheim, Germany). Incubated for minutes. Insoluble matter was removed through a nylon mesh, and the filtrate was centrifuged at 450 g for 8 minutes. The precipitate was dispersed in 8.2% Nycodenz (Nycomed Pharma AS, Norway) solution, and centrifuged at 1400 g for 20 minutes at 4 ° C. The hepatic stellate cell fraction contained in the upper white layer after centrifugation was collected and centrifuged at 450 g for 8 minutes to collect cells. This is suspended in Dulbecco's modified Eagle medium (Nissui Pharmaceutical, Tokyo) containing 10% fetal bovine serum (Commonwealth Serum Laboratories, Australia), 100 U / mL penicillin, 100 U / mL streptomycin (Gibco Laboratories, Life Technologies, USA). By becoming cloudy, hepatic stellate cells were obtained. HSC-T6 cells (Vogel S., et al., J. Lipid Res. 2000. 41: 882-893), an established cell line of isolated hepatic stellate cells or rat hepatic stellate cells, 5.0 × 10 ⁵ cells / After seeding in a plastic culture dish at a density of mL and culturing at 37 ° C. for 4 hours, non-adherent cells were removed. ANP or CNP was added so that the concentration in the culture solution would be 0, 10, 50, or 100 μg / mL, and cultured at 37 ° C. under 5% CO ₂ condition. Cells were collected 24 hours after the addition, and after thawing, the expression level of α-smooth muscle actin (α-SMA) was measured by Western blotting as an index of hepatic stellate cell activation. Western blotting was performed using an anti-αSMA monoclonal antibody (Dako Japan) and a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Amersham).

  The results when ANP was added to the culture medium of isolated primary hepatic stellate cells or HSC-T6 cells are shown in FIG. In all cells, ANP suppressed the protein expression level of α-SMA in a concentration-dependent manner. Similar results were obtained with CNP, indicating that ANP and CNP have the effect of suppressing hepatic stellate cell activation.

  These results indicate that ANP and CNP act on hepatic stellate cells to suppress liver fibrosis and maintain liver function, and that these actions involve suppression of hepatic stellate cell activation. did.

  The pharmaceutical composition for suppressing liver fibrosis comprising the natriuretic peptide according to the present invention as an active ingredient exerts a special effect of acting on hepatic stellate cells to suppress its activation and suppress the progression of liver fibrosis. Is. By suppressing liver fibrosis, it has become possible to suppress or treat progression to liver cirrhosis or precancerous lesions, which are the process of transition to canceration.

FIG. 1 is a graph showing the effects of ANP and CNP on serum ALT (A), serum AST (B), and serum total bilirubin (T-bil) (C) in DEN-induced liver fibrosis model rats. Each value on the bar graph represents the average value of 6 cases, and the graph represents the average value ± standard error. **; p <0.01, t test against control group (DEN). FIG. 2 is a graph showing the effects of ANP and CNP on serum total protein concentration (A) and serum albumin concentration (B) in DEN-induced liver fibrosis model rats. Each value on the bar graph represents the average value of 6 cases, and the graph represents the average value ± standard error. **; p <0.01, t test against control group (DEN). FIG. 3 is a photograph showing an Azan-stained image of a liver tissue specimen of a DEN-induced liver fibrosis model. A control group (DEN), an ANP administration group (ANP), and a CNP administration group (CNP) are shown as typical examples. Compared to the control group, the ANP or CNP-administered group has fewer collagen fiber regions stained blue than the control group (DEN). FIG. 4 is a photograph showing a Sirius Red stained image of a liver tissue specimen of a DEN-induced liver fibrosis model. A control group (DEN), an ANP administration group (ANP), and a CNP administration group (CNP) are shown as typical examples. Compared to the control group, the ANP or CNP administration group has fewer collagen fiber regions that are stained reddish brown. FIG. 5 is a graph showing expression of type 1 procollagen (collagen I) gene (A), TIMP-1 gene (B), and TIMP-2 gene (C) in the liver of a DEN-induced liver fibrosis model. Each value represents the mean value ± standard error of 6 cases. **; p <0.01, t test against control group (DEN). Fig. 6 shows that ANP suppresses α-SMA protein expression in a dose-dependent manner in either hepatic stellate cells or HSC-T6 cells (established rat hepatic stellate cell lines) isolated from rats. It is a figure which shows the result of a Western blot which shows.

Claims (3)

  A pharmaceutical composition for inhibiting liver fibrosis, comprising natriuretic peptide or a pharmaceutically acceptable salt thereof as an active ingredient, and inhibiting liver fibrosis induced by activation of hepatic stellate cells.   2. The pharmaceutical composition for inhibiting liver fibrosis according to claim 1, wherein the natriuretic peptide is any of atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide.   3. The pharmaceutical composition for inhibiting liver fibrosis according to claim 2, wherein the natriuretic peptide is an atrial natriuretic peptide.