WO2008133349A1 - Agent for prevention of cirrhosis/precancerous lesion comprising natriuretic peptide as active ingredient - Google Patents

Abstract

Disclosed is a pharmaceutical composition for preventing hepatic fibrosis, which comprises a natriuretic peptide as an active ingredient and can be used for the prevention or treatment of cirrhosis or precancerous lesion. Attention is particularly focused on a hepatic stellate cell which is believed to be 'a target cell' for the prevention or treatment of hepatic fibrosis, and it is considered that the prevention of the activation of a hepatic stellate cell is critical for the prevention or treatment of hepatic fibrosis. Then, examination is made about the effect of a natriuretic peptide on an activated hepatic stellate cell in vivo, and it is found that a natriuretic peptide has an inhibitory effect on hepatic fibrosis.

Description

 Specification

 Cirrhosis and precancerous lesion inhibitor containing natriuretic peptide as active ingredient

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

Background art

 Fibrosis in the liver (liver fibrosis) is caused by chronic liver damage caused by 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, and therefore the development of treatments such as drugs that suppress effective fibrosis is the current treatment in liver disease. Is an extremely important research topic.

Liver fibrosis is a response to hepatocyte necrosis and damage, and it involves a complex number of factors. For example, it may be caused by liver inflammation or poisoning, 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. In addition, chemical substances such as alcohol and methotrexate, drugs, and circulatory disturbance to the liver such as chronic heart failure also cause liver fibrosis. The liver is not only composed of liver parenchymal cells that are responsible for liver function, but 40% of the total number of cells constituting the liver is non-parenchymal cells, and non-parenchymal cells are approximately 48% sinusoidal. It consists of endothelial cells, 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 differentiated from undifferentiated mesenchymal cells, and in the organogenesis stage, they produce collagen to form a matrix that has α-actin in the cytoskeleton. Changes to inactive hepatic stellate cells. Hepatic stellate cells usually have lipid droplets containing vitamin Α 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 long branched branches, surrounds sinusoidal endothelial cells like so-called vascular pericytes, and contracts in response to endothelin. It is known as a cell that regulates blood flow in sinusoids by relaxing with nitric oxide (NO). Furthermore, hepatic stellate cells are the main producers of extracellular matrix (extracelular matrix: ECM) such as collagen, laminin, and proteoglycan in the liver. And regulated by growth factors such as platelet-derived growth factor (PDGF).

 On the other hand, hepatic stellate cells are stimulated by cytokines such as TGF-0 during liver damage or inflammation, or hepatic stellate cells are activated by endotoxemia caused by a large intake of alcohol, resulting in excessive ECM. It is produced, and this causes liver fibrosis. In other words, liver fibrosis is a condition in which ECM has accumulated excessively as a result of wound healing mechanisms for tissue injury in the liver. Normally, ECM such as collagen in a 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 due to inflammation caused by hepatitis virus and alcohol, and becomes prominent especially 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-fibronectin. It also plays a central role in liver fibrosis by producing matrix-degrading enzyme (MMP), its inhibitory factor (TIMP), TGF_3, PDGF and other growth factors, and HGF. Activated hepatic stellate cells have enhanced contractility and are involved in the regulation of hepatic blood flow, and the expression of various site force-in receptors increases, making them highly sensitive to site force-in.

Collagen production and degradation are complexly regulated by various cytokines such as MMP and TIMP. TGF-; 3 is a site force-in that most promotes liver fibrosis and promotes collagen production. On the other hand, TNF-α and IFN-a suppress collagen production. TGF-jS increases during hepatic fibrosis, activates hepatic stellate cells by the action of paracrine and autocrine, promotes ECM production, enhances TIMP production in hepatic stellate cells, and degrades ECM. Fibrosis progresses in order to reduce it relatively. PDGF is also considered to be 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 activity is further enhanced through proliferation, contraction, ECM production, site force-in production, and the like. Liver fibrosis is a symptom that results from chronic liver damage caused by various causes as described above. If the symptom persists, 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 cirrhosis or transition to liver cancer, development of a drug that can be used for the prevention or treatment of liver fibrosis is expected. Regarding the suppression of hepatic stellate cell activation, PPARga 匪 a-ligand (diabetic drug) acts on hepatic stellate cells to suppress not only activation but also proliferation (Non-patent Document 1: Ka aguchi K et al. Biochem. Biophys. Res. Commun. 2004; 315: 187-95.) and angiotensin-type II receptor antagonist (hypertensive drugs) suppress the activation of hepatic stellate cells and In order to induce suppression (Non-Patent Document 2: Kurihara N et al. Br J Pharmacol 2003; 139: 1085-94.), Therapies using these have been attempted, but at present, clinically effective prevention or No cure has been found.

 In advanced decompensated cirrhosis, fl dry encephalopathy, ascites, rupture of esophageal gastric varicose veins occur and the quality of life (Q0L) is extremely reduced. 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). As receptors for these peptides, diuretic peptide receptor-A (NPR-A), diuretic peptide receptor-B (NPR-B), etc. are known. These are membrane-bound guanylyl cycler. It is a guanylyl cyclase co-receptor with a ze structure.

In addition, ANP and BNP are specific ligands of NPR-A, CNP is a specific ligand of NPR-B, and they increase intracellular cGMP after binding to each receptor. Therefore, it is considered to exhibit biological activities such as diuretic action and vasodilatory action. (Non-Patent Document 3: Rosenzweig A, and Seidman CE., Annu. Rev. Biochem., 60: 229-255, 1991). Natriuretic peptides are still reported to play an important role in controlling body fluid homeostasis and blood pressure (Non-Patent Document 4: OgawaY, et al., J. Clin. Invest., 93: 1911-1921, 1994), expression in various tissues other than the cardiovascular system and their physiological activities 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., A. 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, reducing the preload and afterload of the heart, and the hemodynamic improvement effect has been confirmed (Non-patent Document 7: Suzuki T., et al.) al., Cardiovasc. Res. 51: 489-494, 2001). It is already 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 and diuretic action and plays an important role in regulating body fluid volume and blood pressure. Rumon. The plasma BNP concentration in healthy individuals is extremely low, but increases with severity in patients with heart failure (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 according to the severity, so it is important as a method for evaluating heart failure function.BNP measurement is important for understanding the pathophysiology of heart failure. (Non-Patent Document 7: Suzuki L, et al., Cardiovasc. Res. 51: 489-494, 2001). BNP is also already approved for the treatment of acute heart failure in the United States and other countries.

CNP is known as a bone growth factor. In mouse fetal tibial organ culture, CNP significantly promotes long bone growth (Non-patent Document 9: Yasoda A., et al., J. Biol. Chem). , 273: 11695-11700, 1998). In addition, organ cultures of mouse fetus tibia, chondrocyte culture cells, and osteoblast cell culture cells have higher cGMP production capacity 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. Co. un., 223: 1-6, 1996; Non-Patent Document 12: Inoue A., et al., Biochem. Biophys. Res. : 1104-1110, 1995). In addition, it is known to have an inhibitory effect on hepatic stellate cell proliferation in in vitro experiments (Non-patent Document 13: Gorbig MN, Hepatology, 30: 501-509, 1999).

 5 Natrium diuretic 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) .So far, natriuretic peptides have become a cause of liver fibrosis that causes progression to liver cirrhosis and precancerous lesions, which are the transition process to canceration. On the other hand, there is no report on the fact that it actually has an inhibitory effect on liver fibrosis in vivo, and whether it is effective in inhibiting the activation of hepatic stellate cells 10. At least the present inventors do not know the existence of the report.

 Non-Patent Document 1 Ka aguchi K et al. Biochem. Biophys. Res. Commun. 2004; 315:

187-95.

 Non-Patent Document 2 urihara N et al. Br J Pharmacol 2003; 139: 1085-94. 15 Non-Patent Document 3 Rosenzweig A, and Seidman CE., A Awakening. Rev. Biochem.

60: 229-255, 1991

 Non-patent document 4 Ogawa Y, et al., J. Clin. Invest., 93: 1911-1921, 1994 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

20 1991

 Non-Patent Document 7 Suzuki T., et al., Cardiovasc. Res. 51: 489-494, 2001 Non-Patent Document 8 Mukoyama M., et al., J. Clin. Invest., 87: 1402-12, 1991 Patent Document 9 Yasoda A., et al., J. Biol. Chem., 273: 11695-11700, 1998 Non-patent Document 10 HagiwaraH., Et al., J. Biol. Chem., 269: 10729-10733, 1994 25 Non-Patent Document 1 1 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

 Non-Patent Literature 1 3: Gorb ig M. N., Hepatology, 30: 501-509, 1999

 Non-patent literature 14: Cal derone A, et al. J. Cl in. Invest., 101: 812-818. Non-patent literature 15: Suganami T. et al. 12: 2652-2663, 2001

Disclosure of the invention

Problems to be solved by the invention

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

Means for solving the problem

 As a method of preventing or treating liver fibrosis, (1) suppression of hepatic stellate cell activation, (2) control of site force in, which is important for liver fibrosis, ( 3) Suppression of hepatic stellate cell proliferation, (4) control of ECM degradation system, and (5) hepatocyte proliferation and hepatic stem cell transplantation. Therefore, the present inventors focused on hepatic stellate cells, which can also be called “target cells J”, 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 of examining the effect of the peptide on activated hepatic stellate cells in vivo, it was found that it has an inhibitory effect on liver fibrosis, and the present invention has been completed.

 That is, the present invention relates to the following matters.

 (1) 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 the above (1), 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) It is characterized by administering sodium diuretic peptide or pharmaceutically acceptable salt thereof. A method for preventing or treating cirrhosis or precancerous lesions caused by liver fibrosis.

 (5) The method according to (4) above, wherein the natriuretic peptide is any one of atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.

 (6) The method according to (4) above, wherein the natriuretic peptide is an atrial natriuretic peptide.

 (7) Use of a natriuretic peptide or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for inhibiting liver fibrosis.

 (8) The use described in (7) above, wherein the natriuretic peptide is any one of atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.

 (9) The use according to (7) above, wherein the natriuretic peptide is an atrial natriuretic peptide.

 (10) A sodium diuretic peptide or a pharmaceutically acceptable salt thereof for suppressing liver fibrosis.

 (11) The natriuretic peptide according to (10) above, wherein the natriuretic peptide is either atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide, or a pharmaceutically acceptable peptide thereof salt.

 (12) The sodium diuretic peptide or the pharmaceutically acceptable salt thereof according to (10), wherein the natriuretic peptide is an atrial natriuretic peptide.

 The pharmaceutical composition for inhibiting fibrosis of the liver comprising the sodium diuretic peptide according to the present invention as an active ingredient acts particularly on hepatic stellate cells to inhibit its activation, and further suppresses the progression of liver fibrosis. It plays. 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 invention's effect

Pharmaceutical composition for inhibiting liver fibrosis comprising the sodium diuretic peptide according to the present invention as an active ingredient The substance exerts a special effect when acting on hepatic stellate cells to suppress its activation and suppress the progression of liver fibrosis. Suppression of liver fibrosis has made it possible to suppress or treat progression to liver cirrhosis or precancerous lesions that are the transition to canceration.

Brief Description of Drawings

 FIG. 1 is a graph showing the effects of ANP and CNP on serum ALT (A), serum AST (B), and serum total pyrilvin (T-bi l) (C) in DEN-induced liver fibrosis model rats. Each value on the bar graph shows the average value of 6 cases, and the graph shows the average soil 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 shows the average value of 6 cases, and the graph shows the average soil standard error. : P 0.01, t test against control group (DEN).

 Figure 3 is a photograph (Figure 3A) showing an Azan-stained image of a liver tissue specimen of a DEN-induced liver fibrosis model and a graph (Figure 3B) showing the area ratio (%) of cells positive by Azan staining. In FIG. 3A, the control group (group administered with DEN only; DEN), ANP administration group (ANP), CNP administration group

(CNP), each showing a typical example. Compared with the control group (DEN), the ANP administration group (ANP) or the CNP administration group (CNP) has fewer collagen fiber regions stained blue than the control group (DEN). Fig. 3B also shows that Azan staining-positive cells are significantly reduced in both the ANP administration group (ANP) and the CNP administration group (CNP) compared to the control group (DEN) («: p <0. 01) o

Fig. 4 is a photograph (Fig. 4A) showing a Sirius Red stained image of a liver tissue specimen of a DEN-induced liver fibrosis model and a graph (Fig. 4B) showing the area ratio (%) of cells positive by Sirius Red staining. is there. A typical example is shown for each of the control group (DEN), ANP administration group (ANP), and CNP administration group (CNP). In FIG. 4A, the collagen fiber region stained in reddish brown is less in the ANP administration group (ANP) or CNP administration group (CNP) than in the control group (DEN). In FIG. 4B, both the ANP administration group (ANP) and the CNP administration group (CNP) compared to the control group (DEN). Showed a significant decrease in staining-positive cells (<<: p <0. 01).

 Fig. 5 is a photograph (Fig. 5Α) showing an a SMA antibody-stained image of a liver tissue specimen of a DE-induced liver fibrosis model and a graph (Fig. 5B) showing the area ratio (%) of cells positive by a SMA staining. . A control group (DEN;), an ANP administration group (ANP), and a CNP administration group (CNP) are shown as typical examples. In FIG. 5A, the smooth muscle region stained brown is less in the ANP administration group (ANP) or CNP administration group (CNP) than in the control group (DEN). In addition, in FIG. 5B, it was shown that anti-SMA antibody staining positive cells were significantly decreased in both the ANP administration group (ANP) and the CNP administration group (CNP) compared to the control group (DEN) (<< : P <0. 01).

 Figure 6 shows the type 1 procollagen (collagen I) gene (A), MMP2 gene (B), T IMP-1 gene (C), and TIMP-2 gene (D) in the liver of the DEN-induced liver fibrosis model ) It is a graph showing the expression. Each value represents the average soil standard error of 6 cases. : P <0. 01, t test against control group (DEN).

 Figure 7 shows that hepatic stellate cells isolated from rats (Figure 7A) or HSC-T6 cells (established cell lines of rat hepatic stellate cells) (Figure 7B). It is a figure which shows the result of a Western plot which shows suppressing protein expression level. BEST MODE FOR CARRYING OUT THE INVENTION

 Drosophila fibrosis is a response to hepatocyte necrosis and damage, and refers to changes in liver tissue that occur in complex ways involving many factors. Liver fibrosis is caused by, for example, liver inflammation or damage caused by poisons, 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 disturbance 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-j3 produced during liver damage and inflammation, and endotoxemia caused by high 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), site force-in such as TGF-3, PDGF, and growth factors such as HGF, and hepatic fibrosis. Facilitate.

In the present invention, natriuretic peptide (NP) is administered to animals. Found that the type I collagen gene opioid matrix degrading enzyme (MMP) suppressor (TIMP) gene, which is involved in promoting liver fibrosis, is significantly reduced in its animal expression, and indeed We found that liver fibrosis in animals with liver fibrosis was suppressed and 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 sodium diuretic peptide (NP) receptor. Natriuretic peptides, which are peptidic substances, are preferred, and examples of natriuretic peptides include atrial natriuretic peptide (ANP), brain sodium diuretic peptide (BNP), and C-type natriuretic peptide (CNP). It is

 As ANP in the present invention, a human-derived ANP (SLRRSSCFGG RMDRIGAQSG LGCNSFRY: SEQ ID NO: 1) consisting of 28 amino acids, a rat-derived ANP (SLRRSSCFGG RIDRIGAQSG LGCNSFRY: SEQ ID NO: 3), natriuretic peptide (NP) Those having a property 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 a property capable of enhancing cGMP production via NPR-A, which is a natriuretic peptide (NP) receptor, at least the ring structure of the MP (for example, In the case of the amino acid sequence of human ANP, a peptide having 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 and a C-terminal portion following the ring structure (that is, In the case of human ANP, SEQ ID NO: 2) corresponding to positions 7-28 of SEQ ID NO: 1 may be used. Examples of the peptide having such a structural feature include the ANP itself described in SEQ ID N0: 1, or a peptide having a partial amino acid sequence thereof, from amino acids at positions 7 to 28 of the human ANP. For example, a peptide (SEQ ID NO: 2) consisting of amino acids 7-28 of the above human ANP itself may be mentioned.

As BNP in the present invention, human-derived MP consisting of 32 amino acids (SPKMVQGSGC FGRKMDRISS SSGLGCKVLR RH: SEQ ID NO: 4), buyu-derived BNP (SPKTMRDSGC FGRRLDRIGS LSGLGCNVLR RY: SEQ ID NO: 6), rat-derived MP ( SQDSAFRIQE RLRNSKMAHS SSCFGQKIDR Those having the property of enhancing cGMP production via NPR-A, a natriuretic peptide (NP) receptor, such as IGAVSRLGCD GLRLF: SEQ ID NO: 7), 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, 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 ring structure based on the formation of a disulfide bond based on Cys 10 and 26 of SEQ ID NO: 4 and a C-terminal part following the ring structure (ie In the case of human BNP, SEQ ID NO: 5) corresponding to positions 10-32 of SEQ ID NO: 4 may be used. Peptides having such structural characteristics include, for example, the BNP itself described in SEQ ID N0: 4, or a peptide having a partial amino acid sequence thereof, from the amino acids at positions 10 to 32 of the above-mentioned BNP. A peptide encapsulating the peptide, for example, a peptide (SEQ ID NO: 5) itself consisting of amino acids at positions 10 to 32 of the above human BNP can be mentioned.

 As CNP in the present invention, human-derived CNP consisting of 22 amino acids (GLSKGCFGLK LDRIGSMSGL GC: SEQ ID NO: 8, BUTATA and rat also have the same amino acid sequence), birch-derived CNP (GLSRSCFGVK LDRIGSMSGL GC: SEQ ID NO: 10), CNP (GYSRGCFGVK LDRIGAFSGL GC: SEQ ID NO: 11), etc. that have the property of enhancing cGMP production through NPR-A, a natriuretic peptide (NP) receptor be able to. 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 CNP (for example, In the case of the amino acid sequence of human CNP, a 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 N0: 8 (ie, SEQ ID NO: 9) That's fine. Examples of the peptide having such a structural feature include the CNP itself described in SEQ ID N0: 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 encapsulating the peptide, such as the peptide consisting of amino acids at positions 6-22 of the human CNP (SEQ ID NO: 9) itself.

Further, the natriuretic peptide according to the present invention includes a purely isolated and purified natural product. For example, based on the amino acid sequence of the substance (ANP, etc.) The amino acid residue in the sequence can be obtained by modification such as deletion, substitution, addition, insertion, etc., and the substance obtained by any method acts on NP receptor NPR-A Any substance that can enhance cGMP production can be used.

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

 The 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 NPR-A, which is the above-mentioned sodium diuretic peptide (NP) receptor. Or a pharmaceutically acceptable salt of sodium diuretic 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, and succinic acid is added as an active ingredient. It can also be used as 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 It can suppress liver fibrosis induced by activation. In the present invention, the above-described composition of the present invention is administered to prevent or treat liver cirrhosis or precancerous disease caused by liver fibrosis by suppressing liver fibrosis as described above. You can also.

Substances that can be used as active ingredients of the pharmaceutical composition according to the present invention or pharmacologically acceptable substances thereof The acceptable salts are mixed with known pharmacologically acceptable carriers, excipients, diluents and the like, and are generally used in medicines, that is, oral administration methods, or intravenous administration, muscle It is preferably administered by a parenteral administration method such as internal administration or subcutaneous administration.

 When the active ingredient is a peptidic substance, it can be orally administered as a preparation that is not easily degraded in the digestive tract, for example, as a microcapsule encapsulating the active ingredient peptide in a ribosome. In addition, it can be administered by absorption through the mucosa other than the digestive tract, such as the rectum, intranasal, sublingually. 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 is generally 0.1 xg per day, although it varies depending on the type of disease, patient age, weight, symptom severity, and administration route. / kg to 100 ig / kg can be administered, preferably 0.5 g / kg to 5 mg / kg.

 The frequency of administration of the pharmaceutical composition according to the present invention will vary depending on the active ingredient used, the route of administration, and the particular disease being treated. For example, when sodium diuretic peptide is administered orally, it is preferable to prescribe it at a frequency of not more than 4 times per day, and when administered intravenously, it can be administered continuously using an infusion pump. preferable. Example

 Hereinafter, the present invention will be described more specifically with reference to examples.

 Example 1 Examination of the effect of sodium diuretic peptide on liver fibrosis

 In this example, the effect of atrial natriuretic peptide (ANP) and C-type sodium diuretic peptide (CNP) on liver fibrosis was determined as N-jethyl nitrosamine (hereinafter referred to as DEN).ラ ッ ト B, le 7 using the rat liver injury model induced by.

A persistent liver injury model was prepared by intraperitoneally administering DEN (Wako Pure Chemicals; 200 mg / kg) twice a week for 4 weeks to Wistar male rats (Japan SLC). In the ANP or CNP group, human MP (ASBIPHOMA Co., Ltd.) and human CNP (ASBIPHOMA Co., Ltd.), 1 week after the start of DEN administration, 1.0 ig / kg / min each for 3 weeks Continuous vein It was administered internally.

 Continuous intravenous administration was performed as follows. That is, the animals were anesthetized with pentobarbital sodium and then kept in the supine position. A midline cervical incision was made, and a Hyde-Coat catheter (AccessTechnology, catalog number: CNC-3H) was inserted into the jugular vein. The catheter was guided subcutaneously through the back of the neck through 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 was administered.

 Four weeks after the start of DEN administration (that is, three 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 A liver histopathological study was performed to evaluate liver function and liver fibrosis. Seran biochemical markers that are indicators of liver function in serum biochemical tests, alanin aminotransferase (ALT; also called GPT (glutamate pyruvate transaminase)), aspartate aminotransferase AST (also called GOT (Daltamate Oxaloacetate Transaminase)) and total pyrilrubin concentrations were measured, and serum ALT, AST, and total pyrilrubin concentrations were all compared to the control group. And significantly lower in the CNP-treated groups (Figures 1A-C). In serum biochemical tests, serum total protein concentration and serum albumin concentration, which are indicators of liver function, were measured. Serum total protein concentration and serum albumin concentration were higher in both treatment groups than in the control group. Maintained (Figures 2A-B). From these data, it was shown that the decrease in monthly 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 liver tissue, by examining the area ratio (%) of positive cells from the examination by Azan staining or Sirius Red staining, which can stain collagen fibers, liver function, And liver fibrosis was evaluated. In Azan staining (Fig. 3), Sirius Red staining (Fig. 4) or S-smooth muscle actin (a-SMA) antibody staining (Fig. 5), the control group ("DEN" and Marked) showed significant liver fibrosis, whereas in the group administered DEN and ANP (described as “ANP”) and in the group administered DEN and CNP (described as “CNP”) It was shown that fibrosis was significantly suppressed. Here, -a-smooth muscle actin (α-SMA) is known as an indicator of hepatic stellate cell activation, and as an antibody against α-smooth muscle actin, anti-aSMA monoclonal antibody (Dako Apan) was used.

 In addition, when the area ratio (%) of the cells stained by these staining methods was examined, the ANP-administered group (ANP) was compared with the control group (DEN), regardless of which staining method was used. ) And CNP-administered group (CNP), it was clearly shown that the number of staining positive cells decreased significantly (Fig. 3B, Fig. 4B and Fig. 5B).

 From these results (FIGS. 1-5), it was found that administration of ANP and CNP suppressed fibrosis and improved liver function in the rat liver fibrosis model.

 In addition, type 1 procollagen, which is an index of liver fibrosis, matrix metalloptidase-2 (Mtrix-2), matrix metallopeptidase_13 (matrix metallopeptidase-13; MMP-13), tissue metabolite MRNA of each gene of oral protease inhibitor-1 (tissue inhibitor of metalloproteinase-1; TIMP-1) and tissue metaprotease inhibitor-2 (Tissue inhibitor of metalloproteinase-2; TIMP-2) Expression in the liver was evaluated by real-time PCR according to a conventional method (Kawaguchi K. et al., Biochem. Biophys. Res. Com., 2004; 315: 187-95).

 Briefly, total RNA from rat liver was isolated using RNeasy-kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The internal standard was / 3_actin.

Based on total RNA thus prepared, Taqman reverse transcriptase reagent (Roche Diagnostics, Indianapolis, IN, USA) was used for synthesis of cDNA as described in the manufacturer's instructions. -Actin was used as an internal control to perform specific quantification of gene expression as described in the manual. A method based on the threshold cycle number and standard curve was used to calculate the relative amount of target RNA. Light Cycler Q-PC (Roche Diagnostics) using RT-PCR synthesis kit AMV (Roche Diagnostics, Indianapolis, IN, US) and 2 ^ 1 light cycler fast start DNASYBR Green I (Roche Diagnostics) Diagnostics), 25 mM MgCl ₂ , 10 M each primer, 5 1 extracted DNA, It was then performed in a 13 ^ 1 mixture containing Advantage PCR polymerase (Clontech Laboratories, Palo Alto, CA) (operating system version 3.0). Reaction conditions are 95 minutes at 95 ° C for 10 minutes (temperature gradient 20 ° C / s), followed by denaturation at 95 ° C for 15 seconds (temperature gradient 20 ° C / s) at 68 ° C. Annealed for 5 seconds (temperature gradient 20C / s), primer extension for 10 seconds at 72 ° C (temperature gradient 20 ° C / s) for one cycle, 40 cycles, and purified product at 72 ° C for 60 seconds Detection (temperature gradient 2 (TC / s)).

 The following primers were used as primers for real-time PCR: Type 1 procollagen:

 i: 5'-agcggtgaagaaggaaagagagg-3 '(SEQ ID NO: 12) ^ and

 Antisense: 5 '-caataggaccagaaggaccagca-3' (SEQ ID N0: 13)

 MMP-2:

 Sense: 5'- gccctcccctgatgctgata-3 '(SEQ ID NO: 14); and

 Antisense: 5'-gtcactgtccgccaaataaacc-3 '(SEQ ID NO: 15)

 Thunder-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 confirmed 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 collagen type 1 in the liver, the expression level of matrix metallopeptidase-2 (MMP-2), and the degradation of extracellular matrix Tissue inhibitor of metal loproteinase-2 (TIMP-1) and tissue inhibitor of metalloproteinase-2 (TIMP-1) The gene expression levels in 2) are all in the control group (described as “DEN”) Compared with the ANP group, it was significantly suppressed in the ANP-administered group and the CNP-administered group (Figs. 6A to D; «: p Example 2 Effect of sodium diuretic peptide on hepatic stellate cells

 Since hepatic fibrosis is thought to involve the activation of hepatic stellate cells, in this Example 5, we next examined the direct effects of ANP and CNP on isolated hepatic stellate cells.

 Hepatic stellate cells were isolated from the liver of Wistar male rats (Nihon SLS1) by conventional methods (Ka aguchi K., et al., Biochem. Biophys. Res. Commun., 2004; 315: 187-95) did. Specifically, rat liver was maintained at 37 ° C with Ca2 + and Mg2 + free Krebs-Ringer solution, 0.1% pronase E (Merck, Germany), 0.032% collagenase (sum)

10 Permeabilized in order by Mitsui Junyaku, Osaka). Enzyme-treated liver was isolated and minced in Krebs-Ringer solution (pH 7.3) containing 0.08% pronase Ε, 0.04% collagenase, and 20 zg / mL DNase (Boehringer-Mannheim, Germany). Incubated at 37 ° C for 30 minutes. The insoluble material was removed through a nylon mesh, and the filtrate was centrifuged at 450 g for 8 minutes. 3. Disperse the precipitate in 8.% Nycodenz (Nycomed Pharma AS, Norway) solution. 1400 g for C,

15 Centrifuged for 20 minutes. 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. Dulbecco's modified Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% urine fetal serum (Co 匪 onwealth Serum Laboratories, Australia), lOOU / mL penicillin, lOOU / mL streptomycin (Gibco Laboratories, Life Technologies, USA) ) To obtain hepatic stellate cells. Isolated hepatic stellate cells or rat

20 HSC-T6 cells (Vogel S., et al., J. Lipid Res. 2000.

41: 882-893) was seeded on a plastic culture dish at a density of 5. OX 10 ⁵ cells / mL and cultured at 37 ° C for 4 hours, and then non-adherent cells were removed. ANP or CNP was added at a concentration of 0, 10, 50 or 100 / xg / mL in the culture medium, and cultured at 37 ° C under 5% CO ₂ conditions. After 24 hours, the cells were collected, and after thawing, as an indicator of hepatic stellate cell activation

The expression level of 25 tin (-smooth muscle actin; -SUA) was measured by the Western plot method. Western plotting was performed using an anti-aSMA monoclonal antibody (Dako Japan) and a western rabbit sapiperoxidase-conjugated anti-mouse IgG secondary antibody (Amersham). It was.

 Figure 7 shows the results after 24 hours of addition of sputum to the culture medium of isolated primary hepatic stellate cells or HSC-T6 cells (Figures 7 and 7 respectively). In any cell, ΑΝΡ suppressed the protein expression level of a-SMA in a concentration-dependent manner. Similar results were obtained with CNP, indicating that sputum and CNP have the effect of suppressing hepatic stellate cell activation. These results indicate that sputum and CNP act on hepatic stellate cells to suppress liver fibrosis and maintain liver function, and these effects may involve inhibition of hepatic stellate cell activation. found.

Industrial applicability

 The pharmaceutical composition for inhibiting liver fibrosis comprising the sodium diuretic peptide according to the present invention as an active ingredient has a special effect of acting on hepatic stellate cells to suppress its activity and suppress the progression of liver fibrosis. It plays. Suppression of liver fibrosis has made it possible to suppress or treat progression to liver cirrhosis or precancerous lesions that are the transition to canceration.

Claims

The scope of the claims

 1. A pharmaceutical composition for inhibiting liver fibrosis, comprising sodium diuretic peptide or a pharmaceutically acceptable salt thereof as an active ingredient, and suppressing liver fibrosis induced by hepatic stellate cell activity. .

2. The pharmaceutical composition for inhibiting hepatic fibrosis according to claim 1, wherein the sodium diuretic peptide is any one of atrial sodium diuretic peptide, cerebral sodium diuretic peptide, or C-type sodium diuretic peptide. .

 3. The pharmaceutical composition for inhibiting liver fibrosis according to claim 2, wherein the sodium diuretic peptide is an atrial sodium diuretic peptide.