WO2001058448A1 - Apoptosis inhibitor - Google Patents

Abstract

An apoptosis inhibitor which contains as the active ingredient 5-methyl-1-phenyl-2-(1H)-pyridone represented by the formula (1); and an inflammatory cytokine production regulating agent, a poly-ADP-ribose-polymerase inhibitor and a JNK and/or p38 MAPK inhibitor each containing this compound as the active ingredient.

Description

 Description Apoptosis inhibitor

Technical field

 The present invention relates to an apoptosis inhibitor, an inhibitor of inflammatory site force in production, an inhibitor of poly-ADP-ribose-polymerase, and an inhibitor of Jun-quinase and Z or MAP kinase. Background art

 Apoptosis is cell death caused by activation of caspases. It is known that apoptosis is involved in many diseases, in addition to programmed cell death observed during development and homeostasis. For example, cancer can be described as a condition resulting from the breakdown of the apoptosis-inducing mechanism of abnormal cells. Conversely, the pathological enhancement of apoptosis leads to tissue atrophy As the mechanism of apoptosis has been revealed, the link between apoptosis and various diseases has been suspected. It is speculated that the control and control of apoptosis will enable treatment and prevention of these diseases. For example, in hepatitis symptoms and graft-versus-host disease (GVHD), perforin and Fas ligand (hereinafter abbreviated as FasL) have been implicated in the pathology. In other words, there is a report that inhibition of the binding between Fas and FasL leads to a reduction in hepatic inflammation. In addition, the survival rate of GVHD model animals was improved by administration of anti-FasL antibody (Experimental Medicine vol. 17, No. 13, pp. 1635-1641, 1999).

Thus, if a compound capable of inhibiting the function of factors involved in apoptosis and controlling apoptosis is provided, it would be useful as a new therapeutic agent for diseases suspected of being involved in apoptosis. It can be said that various factors that induce apoptosis have been isolated, and their entire contents are being gradually revealed. For example, cell damage Sex lymphocytes induce apoptosis of target cells from outside the cells. The factor that acts at this time is FasL. If these factors can be effectively blocked, apoptosis can be controlled. However, there have not been many reports of drug candidate compounds having apoptosis-related factors as action points.

 On the other hand, it is known that 5-methyl-2-1-phenyl-2- (1H) -pyridone represented by formula (1) has a therapeutic effect on fibrosis in lungs and atherosclerotic lesions. (Tokuhei 5-52814). This compound was named generically as "pirfenidon". A similar effect has also been reported for its derivatives (Tokuheihei 8-510251). Pirfenidone, a compound of formula (1), was previously reported as a useful compound for the treatment of inflammatory conditions in the respiratory tract and skin

(USP3974281, USP404Z699, USP4052509). Since then, it has been focused on its antifibrotic effect, and is a compound being developed as a drug for pulmonary fibrosis (Nicod, LP. Lancet, Vol. 354, July 24, 1999, p268-269).

Equation (1):

 The following findings have been obtained regarding the antifibrotic effect of pirfenidone.

 • Suppresses the action of cytokines that promote fibrosis (Lurton JM et al., Am J Research Crit Care Med. 153: A403.1996),

 • Reduces mouse lung fibrosis induced by cyclophosphamide and suppresses bleomycin-induced lung changes in hamsters (Kehrer and Margolin (1977) Toxicol. Lett. 90 125; Schelegle et al (1997) Proc. Soc. Exp. Biol. Med.

216 392), ■ Suppresses excessive production of collagen (Iyer et al (1999) J. Pharmacol. Exp. Ther. 289 211)

 • Suppresses the synthesis and release of tumor necrosis factor a (TNF-α) (Tokuhei 11-512699). Based on these effects, pirfenidone is currently being developed as a treatment for pulmonary fibrosis, sclerosing peritonitis, scleroderma, and uterine leiomyoma. However, the relationship between the antifibrotic effect of pirfenidone and apoptosis-related factors is unknown.

TNF-hi is positioned as an apoptotic inducer. It has also been suggested that the role of TNF- in hepatitis-induced liver tissue damage is important (Guidotti L., et al., Immunity, 4: 25-36, 1996, Kondo T., et al., Nature Med., 3: 409-413, 199 7, Seino K., et al. Gastroenterology, 113: 1315-1322, 1997) ₀ Based on these reports, the inhibitory effect of pirufenidone on liver fibrosis This might be explained by suppression of spawn production. However, the known pharmacological effects, including the antifibrotic effect of pirfenidone, cannot be explained solely by the effect of inhibiting the production of TNF-hi. Therefore, pirfenidone has a mechanism of action that needs to be further elucidated. Disclosure of the invention

 An object of the present invention is to provide a novel apoptosis inhibitor, an inflammatory cytokine inhibitor, an inhibitor of poly-ADP-ribose-polymerase, and a Jun-kinase and / or MAP kinase inhibitor.

We hypothesized that fibrosis was the result of chronic inflammation. According to this assumption, the antifibrotic effect of pirfenidone can be attributed to the result of the effect of suppressing inflammatory symptoms. Based on these assumptions, the mechanism of action of pirfenidone will be analyzed in more detail by administering pirfenidone to an artificially induced inflammatory condition and analyzing the effects in detail. Think you can Was. As a model for artificial inflammation, an acute hepatitis model of mouse liver induced by bacterial lipopolysaccharide (hereinafter abbreviated as LPS) was selected. This model is useful as a model for acute hepatitis because severe hepatic inflammation appears rapidly. Through such analysis, the present inventors have confirmed the therapeutic and preventive effects of LPS-induced hepatitis on pirfenidone. Furthermore, the present inventors have searched for the mechanism of action of pirfenidone, which has such an effect, and clarified the target molecule. The present inventors have found that all of these target molecules are apoptosis-related factors, and have completed the present invention.

 That is, the present invention provides an inhibitor of the following apoptosis-related factors, an inhibitor of inflammatory cytokine production, an inhibitor of poly-ADP-ribose-polymerase, and an inhibitor of Jun-kinase and / or MAP kinase. The provision of agents.

 [1] An apoptosis inhibitor containing 5-methyl-11-phenyl-2- (1H) -pyridone represented by the formula (1) as a main component.

Equation (1):

 [2] Inuichi Leukin 12, Interleukin 18, and Inu Yuichi containing 5-methyl-1-phenyl-1- (1H) -pyridone represented by the above formula (1) as a main component. An inhibitor of the production of inflammatory cytokines selected from the group consisting of ferrona.

 [3] An inhibitor of poly-ADP-ribose-polymerase, containing 5-methyl-1-phenyl 2- (1H) -pyridone as a main component represented by the formula (1).

[4] A Jun-quinase and / or p38 MAP kinase inhibitor containing 5-methyl-11-phenyl-2_ (1H) -pyridone as the main component represented by the formula (1). [5] A therapeutic agent for a disease caused by apoptosis, comprising the apoptosis inhibitor according to [1] as a main component.

 [6] A therapeutic agent for hepatitis, containing 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1) as a main component.

 [7] The therapeutic agent according to [6], wherein the hepatitis is fulminant hepatitis.

 [8] A therapeutic agent for a disease caused by necrosis, comprising the inhibitor of poly-ADP-ribose-1 polymerase according to [3] as a main component.

 [9] The therapeutic agent according to [8], wherein the disease is acute hepatitis.

 Also, the present invention provides a method comprising administering a pharmaceutical preparation comprising 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1) to a human or a non-human animal, The present invention relates to a method for treating and / or preventing the following diseases in animals other than humans. In particular, the present invention comprises the step of, after the onset of the following disease, administering a pharmaceutical preparation containing 5_methyl-1-monophenyl-2- (1H) -pyridone represented by the formula (1), And a method for treating the disease of Alternatively, the present invention provides the above formula

The present invention relates to the use of 5-methyl-1-phenyl-2- (1H) -pyridone represented by (1) in the manufacture of a therapeutic agent for the following diseases.

Diseases caused by apoptosis

Hepatitis

Fulminant hepatitis

Diseases caused by necrosis

Acute hepatitis

Further, the present invention provides a human or a human, comprising a step of administering a pharmaceutical preparation containing 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1) to a human or a non-human animal. Alternatively, the present invention relates to a method for suppressing the production of inflammatory cytokines selected from the group consisting of interleukin 12, inleuine leukin 18, and ineluene ferrona in animals other than humans. Alternatively, the present invention provides the above-mentioned formula (1) 5 —Methyl-1 —Phenyl— 2— (1 H) —Inflammation of pyridone in nonhuman animals selected from the group consisting of interleukin 12, inuichi leukin 18 and inuichi ferurona The present invention relates to the use in the production of an inhibitor of the production of sex cytokines.

 Further, the present invention provides a human or a human, which comprises the step of administering a pharmaceutical preparation containing 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1) to a human or a non-human animal. The present invention also relates to a method for inhibiting the activity of the following enzymes in animals other than humans, or the present invention relates to a method for inhibiting 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1), human or human The invention relates to the use of the following enzymes in the production of inhibitors in animals other than animals.

Poly-ADP-ribose-polymerase

 Jun-kinase and no or p38 MAP kinase

 The compound of the above formula (1) used as an active ingredient in the present invention is known under the common name pyrphenidone (US Pat. No. 3,839,346). For example, Japanese Patent Application Laid-Open No. 49-8776

It can be synthesized according to the method described in JP-A-77.

 [1] Inflammatory cytokine production inhibitor

 The present invention firstly comprises a 5-methyl-1-phenyl-2- (1H) -bilidon represented by the formula (1), viridone, ie, pyrphenidone, as a main component. 12), Interleukin 18 (hereinafter abbreviated as IL-18), and Inuichiferona (hereinafter abbreviated as IFN-a) Agent. IL-12 is mainly monocytes and macrophages, IL-18 is mainly macrophage ゃ Kupffer cells, and IFN-α is an inflammatory cytokine mainly produced by T cells and NK cells. In addition, IFN-H, IFN- ?, TNF-H, TNF- ?, IL-1H, IL- ?, I5, IL-6, or IL-10 are known as inflammatory cytokines. Have been.

It is well known that mammals can cause severe acute inflammatory symptoms with LPS. You. When LPS is administered to the abdominal cavity of mammals, TNF-string production occurs first. TNF-hi leads to the production of IL-12 and IL-18, and these two cytokines induce IFN-α production. IFN-α induces Fas and FasL, resulting in rapid hepatocyte apoptosis in the liver, leading to hepatic failure due to congestive necrosis with apoptosis (Tsutsui, H., et al. 1997. IL-18 Accounts for Both TNF- α- and Fas Ligand -mediated Hepatotoxic Pathways in Endotoxin- Induced Liver Injury in Mice. J. Immunol. 159: 3961.) ₀ Has been reported to be more important than elevations of IL-1, IL-6, IL-8, GM-CSF, etc., which are known to be elevated in Ozman, L., et al. 1994. Interleukin 12

J. Exp.Med. 180: 907, Wysocka, M., et al. 1995.Interleukin 12 is required for interferon r roduction a nd lethality, interferon y, and tumor necrosis factor a are the key cytokines of the generated Shwarzman reaction. in lipopolysaccharide-induced shock in mice. Eur. J.I thigh u nol. 25: 672).

 As mentioned above, pirfenidone has already been reported to inhibit the production of TNF-hi, but the present inventors have found that pirfenidone not only inhibits TNF-hi, but also IL-12, IL-18, and IFN. -Has also been shown to have an inhibitory effect on its production. Moreover, according to the findings obtained by the present inventors, pirfenidone has a weak inhibitory effect on IL-1, IL-6, and the like, which are also inflammatory site power-ins. For example, compared to IL-12 production suppression production, it is 50% or less. Therefore, it is considered that the inhibitory effect of pirfenidone on acute inflammation is due to the inhibitory effect on the production of these four types of inflammatory cytokines, TNF-, IL-12, IL-18, and IFN-a.

Now, it should be noted that TNF-H is located at the most upstream in the sequence of site power-in described here. In other words, the inhibitory effect of pirfenidone on inflammatory cytokines may appear as a result of the suppression of TNF-hi production. However, according to the findings of the present inventors, inflammatory sites caused by pirfenidone The effect of suppressing the production of force-in is not indirect. For example, the peak of LPS-administered TNF-spleen production peaked at 1.25 hours after LPS administration and was not detected in the blood after 3 hours. -Administration even after sperm production has passed (eg 3 and 4 hours after LPS administration) can protect against lethality (Example 3).

 Further, pirfenidone suppresses the production of inflammatory site such as IFN-α even after the production of TNF-α has ended (Example 9). In addition, pirfenidone inhibits necrosis and apoptosis separately from TNF-H (Example 10). Thus, pirfenidone has the characteristic that it can suppress acute inflammatory shock even after the release of TNF-. Further, pirfenidone suppresses poly-ADP-ribosylation and a decrease in NAD amount even after 4 hours from LPS administration (Example 11). These effects of pirfenidone are in contrast to the fact that protection, such as with anti-TNF-ligand antibodies, cannot suppress the reaction that proceeds automatically after large quantities of TNF-ligand are released into the blood. is there. From the above, it is clear that these actions by pirfenidone are not merely the result of suppressing the action of.

All cytokines whose production is suppressed by the inflammatory cytokine production inhibitor of the present invention are T helper 1 type cytokins. Therefore, the inflammatory cytokine inhibitory agent of the present invention is a disease caused by an increase in the production of T helper 1 type cytokines, and particularly the production of IL-12, IL-18 and IFN-α. It is effective for those caused by an increase in The T helper 1 type cytokine is a general term for a group of site force proteins that are involved in the induction of a T helper 1 type immune response among inflammatory cytokines. T helper 1 type cytodynamics include cytokines that induce differentiation of T helper-1 cells and cytokines that T helper 1 cells produce. Specific cytokines that induce the differentiation of T helper 1 cells include IL-12 and IL-18. To these site power in, Involved in the induction of one type of immune response (Xu, B. et al. J. Exp. Med., 188: 1485, 1998, and Takeda, K. et al. Immunity., 8: 383 -390, 199 8). The cytokines produced by T helper 1 cells specifically include IL-2 and IFN-α (Clinical Immunity, Vol. 30, No. 11, ρ1471-1478, 1998).

 Τ Imbalance in helper 1 / T helper 2 balance is thought to contribute to the development of immune disease.偏 When biased toward helper type 1 immunity, cell-mediated immunity is enhanced and the immune response to cancer and infectious diseases is increased, but at the same time, self-injury is also increased, which is one of the causes of the onset of immune diseases. Therefore, the inflammatory cytokine production inhibitor according to the present invention can be expected to be effective against all diseases that are said to be caused, for example, by an excessive immune response of a helper. Generally, organ-specific autoimmune diseases are thought to be caused by a bias toward the helper 1 type immune response. Specifically, diabetes, hepatic disorder, autoimmune myelitis, ulcerative colitis, graft-versus-host disease (GVHD), arthritis, thyroiditis, Hashimoto's disease, exocrine glanditis, intracellular infections (Leusmannia) , Mycobacterium tuberculosis, Rye), delayed-type hypersensitivity, scleroderma, or Beety's disease. The inflammatory cytokine inhibitory agent of the present invention can be used for the prevention or treatment of these diseases.

 Among the above-mentioned diseases, the following diseases are considered to be caused not solely by TNF-strings, and are unlikely to be indicated for TNF-string production inhibitors. That is, diabetes, liver damage, autoimmune myelitis, graft-versus-host disease (GVHD), arthritis, thyroiditis, Hashimoto's disease, exocrine adenitis, intracellular infections (Leusmania, Mycobacterium tuberculosis, Rye), Delayed type hypersensitivity, Behcet's disease and the like can be said to be novel indications specific to the inflammatory cytokine production inhibitor according to the present invention.

Furthermore, it has been pointed out that IL-18, which is one of the inflammatory site power-in of the present invention, is associated with miscarriage or premature birth. In other words, the development of inflammatory symptoms in the cervix through the production of IL-18 is thought to lead to dog opening of the cervix and contraction of the uterine muscle. You. Therefore, the inflammatory site force-in production inhibitor of the present invention is effective for treating or preventing miscarriage or premature birth through suppression of IL-18 production.

 [2] PARP inhibitor

 Next, the present invention relates to a poly-ADP-ribose-polymer comprising, as a main component, 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1). Related to inhibitors of poly-ADP-ribose-polymerase (hereinafter abbreviated as PARP).

 PARP is an enzyme that catalyzes the reaction of poly-ADP-ribosylation of proteins near the damaged DNA strand (histone, PARP itself, etc.). This enzymatic activity depends on the cleaved DNA and has the property of being activated by caspase-3 cleavage (116KD to 85KD) during apoptosis. PARP substrates are receptor-side proteins for NAD and poly-ADP-ribosylation. Another important role of this enzyme is to consume large amounts of intracellular NAD through poly-ADP-ribosylation. That is, it is thought that the rapid reduction of NAD and ATP in cells and tissues promotes apoptosis and necrosis in cells and tissues (Ha, HC and Snyder, SH Proc. Natl. Acad. Sci. Vol. 96, No. 24, 13978-13982, 1999). Therefore, PARP enzyme inhibition is expected to have therapeutic effects on arthritis, type I diabetes, diseases caused by various types of neuronal cell death (cerebral ischemia / reperfusion, Alzheimer's disease, Parkinson's disease, etc.), retrovirus infection, etc. . Further, the PARP inhibitor of the present invention is useful as a therapeutic agent for a disease associated with necrosis. It is particularly effective for diseases caused by necrosis caused by rapid apoptosis. Such a disease includes acute hepatitis. That is, the present invention relates to a therapeutic agent for acute hepatitis containing pirfenidone as a main component.

 [3] Jun-kinase and / or p38 MAP kinase inhibitor

Further, the present invention provides a method for preparing a Jun-quina containing, as a main component, 5-methyl-1-phenyl-12- (1H) -pyridone represented by the formula (1), ie, pyrphenidone. — And / or p38 MAP kinase inhibitors.

 Jun-kinase (JNK) and p38 MAP kinase (MAPK) have been shown to have very similar properties in the cascade of apoptosis (Experimental Medicine, 17, No. .2, 1999, p96; Experimental Medicine, 14, No.19, 1996, p27; Xia, Z. et al., Opp osing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science, 270: 1326-1331 , 1995). All are known to be activated against stimuli that induce apoptosis. PC12 cells differentiated like nerves by treatment with nerve growth factor (NGF) undergo apoptosis by removing NGF from the culture medium. At this time, activation of JNK and p38 was observed prior to apoptosis. This apoptosis was suppressed by expressing dominant negative SEK1 / MKK4 or dominant negative MKK3. Furthermore, apoptosis was induced by expressing the active form of MEKK1 or MKK3 and p38 in these PC12 cells. This has revealed that JNK and p38 are necessary and sufficient for apoptosis by removing NGF (Ayumi Kagaku, Vol. 187 No. 5, 1998, 10.31. P348-353). Other stress stimuli, DNA damage, ceramides, ultraviolet rays, y-rays, IgM, anoikis, etc., have also been implicated in both pathways.

 JNK is known to be elevated by various stresses such as oxidation, ultraviolet light, radiation, ischemia / reperfusion, DNA damage, osmotic stimulation, heat, inhibition of protein synthesis, heavy metals, arsenite, and inflammatory cytokines. I have. Therefore, the JNK and / or p38 MAPK inhibitor of the present invention is effective for treating or preventing all diseases in which these stresses are involved in the progression and formation of the disease state. It is effective in treating and preventing diseases caused by fever, trauma, hypertrophy, bacterial infection (mycobacteria), DNA virus infection, oxidative stress, inflammation, ischemia, and glucose starvation.

In particular, JNK has been suggested to be associated with signaling pathways of cell apoptosis. From that perspective, for example, to prevent or treat the following diseases: You can wait. That is, there may be mentioned hypertrophic cardiomyopathy, ischemia / reperfusion, myocardial infarction, radiation injury, or side effects due to anticancer drugs.

 When c-jun, a substrate of JNK, is phosphorylated together with c-fos, it forms AP-1, and this transcription factor associates with various growth factor receptors to signal growth factor signaling. Has also been shown to be involved. Therefore, treatment or prevention of a disease caused by cell overproliferation can be achieved by inhibiting JNK.

 Analysis of p38 MAPK-specific inhibitors and substrate enzymes downstream of the cascade belonging to the p38 pathway using knockout mice has already been performed. The results indicate that inhibition of p38 MAPK suppresses the production of IL-12 and IFN-α (Lu. HT et al., Defective IL-12 production in 'mitogen-activated protein). (MAP) kinase kinase3 (MKK3) -deficient mice, EMBO J 1999, 18 (7): 1 845-5.7; Rincon-M. Et al., Interferon-gamma expression by Thl effector T cells mediated by the p38 MAP kinase signaling pathway, EMBO J., 199 8, 17 (10): 2817-29). Therefore, it cannot be ruled out that IL-12 and IFN-a among the inflammatory cytokines may be the result of an inhibitory effect on p38 MAPK.

 [4] Inhibitor of apoptosis

Further, the present invention provides an apoptosis inhibitor containing 5-methyl-1-phenyl-12- (1H) -pyridone represented by the above formula (1), that is, pyrphenidone as a main component. provide. It is a novel finding that virufenidone has an apoptosis inhibitory action as shown in the Examples. It should be noted that pirfenidone administration after LPS has a therapeutic effect, especially in acute hepatitis symptoms caused artificially by LPS intraperitoneal administration. Many known anti-apoptotic active compounds cannot be expected to have a sufficient apoptotic inhibitory effect unless administered prophylactically before apoptosis occurs. In other words, although effective as a prophylactic agent, its effectiveness as a therapeutic agent could not be expected. On the other hand, the apototo according to the present invention Cis inhibitors can inhibit ongoing apoptosis. In other words, therapeutic effects can be expected. Pirfenidone, which has this mechanism of action against apoptosis, is a prophylactic agent against, for example, internal hemorrhagic necrosis of the liver (with rapid pathological apoptosis) and chronic rejection after transplantation caused by acute hepatitis. In addition, it is suitable for post-onset administration as a therapeutic agent. Accordingly, the apoptosis inhibitor of the present invention relates to a medicament used for treating ongoing apoptosis.

 Other indications involving apoptosis include, for example, glomerulonephritis, acute lung injury, interstitial pneumonia, cardiac hypertrophy, cardiomyopathy, retinal detachment, autoimmune disease, myocardial infarction ischemia, diabetes, inflammation Inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis, AIDS, pancytopenia, refractory anemia, aplastic anemia, virulent hepatitis, fulminant hepatitis, cirrhosis, brain Ischemia, cerebral infarction, Siegren's syndrome, salivary glanditis, severe myeloma, atherosclerosis, Behcet's disease, multiple sclerosis, glaucoma, cataract, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis, radiation Injury, sepsis and the like.

 It was mentioned earlier that bilfenidone was effective in inhibiting the apoptosis-related factors IL-12, IL-18, IFN-A, PARP, JNK, or p38 MAPK. Since all of these apoptosis-related factors are closely related to apoptosis based on the mechanism described below, pirfenidone is useful as an inhibitor of apoptosis.

 1) Suppression of IL-12 / IL-18 / IFN-α production and anti-apoptosis

 As shown in the experiments in the i / 2 FO LPS-administered mouse experiments, the suppression of the production of the above-mentioned inflammatory cytokine by pirfenidone is considered to lead to the suppression of the production of Fas and FasL. As a result, hepatocyte apoptosis is effectively suppressed.

2) Anti-PARP and anti-apoptosis Apoptosis is generally called clean cell death without inflammation. However, in the case of pathological apoptosis such as hepatitis due to intraperitoneal administration of LPS, it has been reported that necrosis occurs extensively in the liver tissue with rapid apoptosis ₍ this phenomenon was observed in the pathological findings in the examples). One of the possible causes is thought to be the rapid decrease in NAD in the tissue In cells that have undergone apoptosis, the ADP-ribosylation reaction by PARP proceeds rapidly, As noted earlier, rapid apoptosis is accompanied by massive consumption of NAD in the tissue, and necrosis is caused by a decrease in ATP in the tissue with a decrease in the NAD and a turnover of the tissue. It can be explained that this is caused by falling into a failure state (Ha, HC and Snyder, SH Proc. Natl. Acad. Sci. Vol. 96, No. 24, 13978-13982, 1999).

 By the way, as shown in the examples, pirfenidone surely suppresses necrosis in hepatitis caused by intraperitoneal administration of LPS. This effect is thought to be the result of pirfenidone suppressing the consumption of NAD, which causes necrosis, through the inhibition of PARP activity. Therefore, it can be said that pirfenidone has a pharmacological effect on inhibiting the internal hemorrhagic necrosis of the liver (with rapid pathological apoptosis) caused by acute hepatitis through PARP inhibitory action.

3) JNK inhibition, p38 MAPK inhibition and anti-apoptosis

 It was mentioned earlier that both JNK and p38 MAPK are activated in response to stimuli that induce cell apoptosis. Therefore, compounds having an inhibitory effect on these kinases are effective as apoptosis inhibitors.

Furthermore, it is known that JNK inhibition and p38 inhibition are associated with anti-inflammatory effects including suppression of TNF-α production. Known JNK inhibitors (Swantek ₃ JL et al., Mol. Cell. Biol., Vol. 17, No. 11, 6274-6282) and p38 inhibitors (Young, P. et al., Agents Actions, 1993) , 39, C67-C69; Prichett ₃ W. et al, J. Infla Marauder, 1995, 45, 97-105) reported that TNF-α acts on the TNF-α translation process (biosynthesis process). Production of It is said to suppress. In other words, TNF- production of mRNA and secretion of TNF-hi are not indicated for suppression. Since the analysis of the present inventors has revealed that pirfenidone also has such characteristics, it inhibits the production of TNF-hi by a mechanism similar to that of known JNK inhibitors and p38 MAPK inhibitors. I do. Therefore, the previously reported inhibitory effect of pirfenidone on TNF-α production (Tokuheihei 11-512699) is considered to be a phenomenon resulting from JNK inhibition and p38 MAPK inhibition.

 As described above, clarifying the action point of the apoptosis-inhibiting action of pirfenidone has great significance for the present invention. Apoptosis is a complex system initiated by a variety of mechanisms, including physical effects. Therefore, it goes without saying that the causes of diseases caused by the same apoptosis are various. Therefore, an apoptosis inhibitor that is effective for one disease does not necessarily show the same effect for another apoptosis-causing disease. In order for an apoptosis inhibitor to be used effectively in treatment and prevention, it is reasonable to clarify its point of action and then select an apoptosis inhibitor that is effective against the cause of apoptosis to be treated. For these reasons, the utility of the present invention for finding out which apoptosis-related factor is affected by the inhibitory effect of pirfenidone is apparent.

 “Apoptosis inhibitors” are effective in treating or preventing diseases caused by apoptosis, including the following diseases. Therefore, it can be used as a therapeutic or prophylactic agent for these diseases.

Fulminant hepatitis, Viral hepatitis C and B, Primary biliary cirrhosis, Secondary cirrhosis Glomerulonephritis, Tubular and interstitial nephritis, Focal glomerulosclerosis, Renal sclerosis, Peritoneal sclerosis, Nephrosis Syndrome, diabetic nephropathy

Acute lung injury, interstitial pneumonia, radiation lung injury

Acute and chronic graft-versus-host disease (GVHD), postoperative adhesions Dilated cardiomyopathy, restenosis after PTCA

Ulcerative colitis, Crohn's disease

Acquired immunodeficiency syndrome (AIDS)

Rheumatoid arthritis

Systemic lupus erythematosus, Behcet's disease, Siedalen's syndrome, dermatomyositis, sarcoidosis, myasthenia gravis, amyotrophic lateral sclerosis, muscular dystrophy, SLE Parkinson's disease, Alzheimer's disease, multiple sclerosis, Huntington's disease

Aplastic anemia, sepsis

Fever, keloid hypertrophic scar, psoriasis

Retinal detachment

 In particular, in the present invention, the following diseases can be newly listed as indications by elucidating the target factor. Hepatitis (including fulminant, chronic, alcoholic, hepatitis C and B virus), toxic or metabolic liver damage, Behcet's disease, aplastic anemia, AIDS, pancytopenia, Refractory anemia, cerebral infarction, glaucoma, cataract, salivary glanditis, radiation disorder, ultraviolet ray injury, sun dermatitis, erythema multiforme, fixed drug eruption, GVHDs TEN, flat warts, herpes simplex, lupus erythematosus, lichenified tissue Reactions, tubular injury, respiratory infections, diabetes, arteriosclerosis, cerebral ischemia, myocardial infarction, dilated and hypertrophic cardiomyopathy, alopecia areata, or drug-induced alopecia are clearly evident in the present invention. It is a new indication for anti-apoptotic drugs. Among them, fulminant hepatitis and secondary cirrhosis can be treated after the onset by administration of the apoptosis inhibitor of the present invention, and thus achieve a high therapeutic effect that cannot be expected with known drugs. .

When the inflammatory cytokine production inhibitor, PARP inhibitor, JNK and / or p38 MAPK inhibitor, or apoptosis inhibitor of the present invention is administered to humans for the purpose of treating or preventing the above diseases, , Powders, granules, tablets, capsules, pills, liquids, etc., orally, or as injections, suppositories, transdermal absorbents, inhalants, etc. Parenteral administration. It can also be used externally as ointments and cream preparations. An effective amount of the compound can be mixed with excipients, binders, wetting agents, disintegrants, lubricants, and other pharmaceutical additives as required to form a pharmaceutical formulation. . In the case of injections, they should be sterilized with a suitable carrier to produce the preparation. Dosage also depends on disease state, route of administration, age or weight of patient, and is ultimately left to the discretion of a physician, but if administered orally to adults, usually 10 to 40 mg / kg / day Can be administered. This may be administered once or in several divided doses.

 All prior art documents cited in the present specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing the inhibitory effect of pirfenidone on the production of TNF-H, IL-12 and IFN-α.

 FIG. 2 shows the effect of pretreatment with pirfenidone on the survival of LPS shock model mice.

 FIG. 3 shows the therapeutic effect of virfenidone on the survival of LPS shock model mice.

 FIG. 4 shows the therapeutic effect of pirfenidone on the survival of LPS shock model mice.

 FIG. 5 is a graph showing the effect of pirfenidone on plasma G0T and GPT values in a mouse fulminant hepatitis model administered with anti-Fas antibody.

 FIG. 6 is a diagram showing the inhibitory effect of pyrufenidone on TNF-splen production from THP-1 cells.

FIG. 7 is a graph showing the inhibitory effect of pirfenidone on IFN-α production from NK3.3 cells. FIG. 8 is a graph showing the inhibitory effect of pirfenidone on TNF-splen production in LPS shock model mice.

 FIG. 9 is a graph showing the inhibitory effect of pirfenidone on IL-18 production in LPS shock model mice.

 FIG. 10 is a photograph showing a liver 6 hours after LPS administration.

 FIG. 11 is a photograph showing the time course of the liver after LPS administration.

 FIG. 12 is a photograph showing the effect of pirfenidone administration on congestive necrosis of the liver of LPS shock model mice.

 FIG. 13 is a photograph showing a hematoxylin-eosin-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A: control without LPS inoculation, B: mouse inoculated with LPS.

 FIG. 14 is a photograph showing a hematoxylin-eosin-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A: Pirfenidone was administered 5 minutes before LPS inoculation, and B: Pirfenidone was administered 4 hours after LPS inoculation.

 FIG. 15 is a photograph showing a Tunel-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A and B are the same as in Fig. 13.

 FIG. 16 is a photograph showing a Tunel-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A and B can be the same as in Fig. 14.

 FIG. 17 is a photograph showing an ssDNA-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A and B can be the same as in Fig. 13. ,

FIG. 18 is a photograph showing an ssDNA-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A and B are the same as in Fig. 14. is there.

 FIG. 19 is a photograph showing a PCNA-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A and B are the same as in Fig. 13 ο

 FIG. 20 is a photograph showing a PCNA-stained image of a tissue section in which the effect of pirfenidone administration on the liver of an LPS shock model mouse was examined. A and B are the same as in Fig. 14 ο

 FIG. 21 is a diagram and a photograph showing the production of inflammatory cytokines and the formation of DNA ladder by liver apoptosis in LPS-administered mice.

 FIG. 22 is a diagram and a photograph showing the effect of pretreatment with pirfenidone on inflammatory cytokine development and LPS apoptosis in LPS shock model mice. FIG. 23 is a diagram and a photograph showing the effects of post-treatment with pirfenidone on inflammatory cytokine production and apoptosis of the liver in LPS shock model mice. FIG. 24 is a diagram and a photograph showing changes over time in DNA ladder formation, poly-ADP-ribosylation, and NAD amount in LPS shock model mice.

 Figure 25 is a diagram and a photograph showing the effects of pirfenidone administration on DNA ladder formation, poly-ADP-ribosylation, and NAD levels in LPS shock model mice.o

 FIG. 26 is a diagram and a photograph showing the effect of pirfenidone on apoptosis of THP-1 induced by etoposide. The vertical axis shows the number of cells (number of small bodies) (side scans ヅ Yuichi), and the horizontal axis shows the size of cells (body) (by forward scans ヅ Yuichi).

 FIG. 27 is a graph showing the effect of pirfenidone on the viability of THP-1 in which apoptosis of THP-1 was induced by etoposide.

FIG. 28 is a diagram and a photograph showing the time course of DNA ladder, poly-ADP-ribosylation, and NAD amount of THP-1 cells in which apoptosis was induced by etoposide. FIG. 29 is a diagram and a photograph showing the effect of pyruvidone on the time-dependent changes in DNA ladder, poly-ADP-ribosylation, and NAD amount of THP-1 cells in which apoptosis was induced by etoposide.

 FIG. 30 is a diagram showing the inhibitory effect of pirfenidone on intracellular caspase-3 activity of THP-1, which increased with apoptosis by etoposide. The vertical axis represents the fluorescence intensity.

 FIG. 31 is a diagram and a photograph showing the effect of pirfenidone on TNF-hi production and TNF-mRNA expression in and out of cells in RAW264.7 cells stimulated with LPS.

 FIG. 32 is a view showing the effect of pyrufenidone on JNK.

 FIG. 33 shows the effect of bilfenidone on p38 MAPK.

 FIG. 34 is a diagram showing the effect of pyrufenidone on PARP. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to Examples and Reference Examples, but the present invention is not limited thereto.

 [Example 1] Inhibitory effect of pirfenidone on TNF-H, IL-12 and IFN-α production

Using a mouse acute inflammation model (mainly LPS-administered mice), the effects of pyruvidone were evaluated mainly from the suppression of inflammatory cytokines and the protective activity against shock death in mice. First, the inhibitory effect of pirfenidone on the production of TNF-o :, IL-12 and IFN-α was examined. Using mice (C57BL / 6, 8-week-old female) (n = 4), only pirfenidone or a carrier (0.5% CMC) was administered 5 minutes before LPS inoculation. LPS (50 μg / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and mice were sacrificed 75 minutes after TNF and 5 hours after IL-12 and INF, and serum was collected. did. TNF, IL-12 and IFN-α in serum were assayed by ELISA. As a result, Pilfenidon Was found to suppress the production of a series of inflammatory cytokines, TNF-H, IL-12, and IFN-a, which appear in serum during LPS shock in in J'TO mice in a dose-dependent manner (Fig. 1). ).

 [Example 2] Preventive effect of pirfenidone on survival of LPS shock model mice

 The effect of pretreatment with pirfenidone on the survival of LPS shock model mice was investigated. Using a mouse (C57BL / 6, 8-week-old female) (n = 8), a carrier or pirfenidone (200-1 p.o.) was administered 5 minutes before LPS injection. LPS (E. coli, 50 g / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and the mice that survived over time were counted. As a result, it was found that pirfenidone exerts a protective effect against LPS-induced shock death (Fig. 2).

 [Example 3] Effect of treatment with pirfenidone on survival of LPS shock model mouse

 The effect of post-LPS inoculation of pirfenidone on the survival of LPS shock model mice was examined.

Using a mouse (C57BL / 6, 7-week-old female) (n = 6), LPS [(E. coli, 10 / g / kg) + D-gal (250 mg / kg)] was used in the same manner as in Example 2. After intraperitoneal administration, vehicle or pirpenidone 500 mg / kg (200 1 .0.) Was administered at the times indicated in the figure. Thereafter, mice that survived over time were counted. As a result, it was found that the therapeutic effect of pirfenidone administration was obtained even 5 hours after LPS inoculation (Fig. 3). Using mice (C57BL / 6, U-week-old female) (n = 5), LPS [(E. coli, 50 zg / kg) + D-gal (250 mg / kg)] was intraperitoneally administered to the mice. Vehicle (0.5% CMC) or pirfenidone 500 mg / kg (200 zl po) was administered at the times indicated in the figure. Thereafter, mice that survived over time were counted. Similar to the above results, it was found that the therapeutic effect of pirfenidone administration was obtained even 5 hours after LPS inoculation (Fig. 4). [Example 4] Preventive effect of pirufenidone on hepatic function decline in mouse fulminant hepatitis model

 The effect of pirfenidone administration on plasma G0T and GPT values in a mouse fulminant hepatitis model administered with anti-Fas antibody was examined. Mice (BALB / C, 10-week-old male) (n = 6 or 5) were administered with a carrier (0.5% CMC) or bilfenidone. Immediately thereafter, anti-Fas mAb (Jo2, 100 zg / kg i.v.) was injected. Twenty-four hours later, mice were sacrificed and plasma and liver were collected. Plasma GOT and GPT values were determined using GOT-UV Test Wako and GPT-UV Test Wako, respectively. As a result, it was found that mice administered with pirfenidone suppressed increases in plasma GOT and GPT levels, which are markers of liver destruction (Fig. 5). Furthermore, in mice administered with pirfenidone, tissue destruction due to apoptosis of hepatocytes was suppressed even at the tissue level.

 From the results of LPS-treated mice and anti-Fas antibody-treated mice shown in Examples 1-4, pirfenidone not only suppressed inflammatory cytokine production in blood of LPS-treated mice, but also accompanied hepatocellular apoptosis. It is suggested that direct death of liver tissue damage suppresses shock death.

[Example 5] Effect of pirfenidone by in sitine production system Monosite ΤΗΡ-1 cells (5 × 10 ⁵ I ml) derived from human were prepared in the presence or absence of LPS (10 // g / ml). Incubated for 3 hours with the indicated concentrations of pyrufenidone. After incubation, TNF-A in the culture supernatant was quantified by ELISA. As a result, pirfenidone dose-dependently suppressed TNF-sporule production in THP-1 cells (FIG. 6). IC _M was calculated to be 45 g / ml.

Human-derived NK cells (NK3.3) (5 × 10 ⁵ / well) (96-well plates, total volume 20 μl) were transfected in the presence or absence of IL-12 (10 ng / ml) with 10% FCS and The cells were incubated for 48 hours at 37 ° C in AIM-V medium containing 5% human serum and the concentration of pyrufenidone shown. After the incubation, human interferon 7 (IFN-a) in the culture supernatant was quantified by ELISA (Quantikine). As a result, pilfenide Inhibited the production of IFN-α in NK3.3 cells in a dose-dependent manner (FIG. 7). IC ₅₀ was calculated to be 51 μg / ml.

THP-1 cells (lx10 ⁶ I ml) were incubated for 24 hours with 0, 10, 30, 100, 300 ^ g / ml pirfenidone in the presence or absence of LPS (10 ^ g / ml) . After the incubation, TGF-? In the culture supernatant was quantified by ELISA. Similarly, THP-1 cells (lx10 ⁶ I ml) were transformed with 0,1,3,10,30,100,300 / g / ml of pilphene in the presence or absence of LPS (10 zg / ml). Incubated with Don for 24 hours. After the incubation, PDGF-AB in the culture supernatant was quantified by ELISA. As a result, pirfenidone dose-dependently suppressed TGF-5 and PDGF-AB production in THP-1 cells. IC _{5Q for} suppressing the production of TGF- ^ and PDGF-AB was calculated to be 70 g / ml and 50 μg / ml, respectively.

The macrophage cell line P388.D1 was incubated with various concentrations of pirfenidone in the presence or absence of LPS. After incubation, TNF- and IL-6 in the culture supernatant were quantified by ELISA. As a result, Pirufuenido emissions is, P388. D1 TNF-non-producing in cells was dose-dependently inhibited (IC _5Q was calculated to 90〃G / ml). However, it did not suppress IL-6 production.

 [Example 6] Effect of pretreatment with pirfenidone on survival of LPS shock model mice

 The effect of pretreatment with pirfenidone on the survival of LPS shock model mice was examined at various times. Using a mouse (C57BL / 6, 7-week-old female) (n = 5), 24 hours, 3 hours, and 30 minutes before LPS injection, vehicle or pirpenidone 500 mg / kg (200 / 1.o.) Was administered. LPS (E. coli, 50 zg / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and the mice that survived over time were counted. The results showed that pirfenidone had a protective effect on shock death even when administered at least 3 hours before LPS vaccination.

[Example 7] Effect of bilfenide on TNF-hi production in LPS shock model mice Suppression effect

 The inhibitory effect of pirfenidone on blood TNF- production in LPS shock model mice (ac / zes killed bacteria sensitized) was examined. Mice (C57BL / 6, 15-week-old female) (n = 5) were intraperitoneally administered with heat-inactivated P. araes at 2 mg / mouse. Ten days later, vehicle (0.5% CMC) or pirfenidone 500 mg / kg (200 1 p.o.) was administered, and 5 minutes later, LPS inoculation (100 / g / kg) was performed. Sera were collected at 1.5 hours after LPS inoculation and sacrificed. TNF-A in serum was assayed by ELISA. As a result, it was found that pirfenidone suppressed TNF-hi production in mice inoculated with LPS with i / 2 FJ'TO (Fig. 8).

 [Example 8] Inhibitory effect of pirfenidone on IL-18 production in LPS shock model mice

 The inhibitory effect of bilfenidone on blood IL-18 production in LPS shock model mice (sensitized with killed aczzes) was examined. Mice (C57BL / 6, 15-week-old female) (n = 5) were intraperitoneally administered with heat-inactivated acnes at 2 mg / mouse. Ten days later, vehicle (0.5% CMC) or pirfenidone 500 mg / kg (200 il p.o.) was administered, and 5 minutes later, LPS inoculation (100 mg / kg) was performed. Sera were collected 1.5 hours after LPS inoculation and sacrificed. IL-18 in serum was assayed by ELISA. As a result, it was found that Bilfenidone suppressed IL-18 production of LPS-inoculated mice by J '/ 2F / TO (FIG. 9).

 [Example 9] Congestive necrosis of liver of LPS-administered mice and effect of administration of pirfenidone LPS ((E. coli, 50 g / kg) + D-gal (250 mg / kg)), and macroscopic findings of the liver 6 hours after administration are shown in FIG. Liver hypertrophy and significant congestion are observed. FIG. 11 shows the time course of the liver after LPS administration.

 The effect of pirfenidone on congestive necrosis of the liver after LPS administration, and macroscopic observation of the liver 6 hours after LPS administration were observed. Pirfenidone was administered 5 minutes before or 4 hours after LPS administration. The results are shown in FIG. Lesions were alleviated both before and after administration of pirfenidone.

In addition, histopathological examination of LPS-inoculated and liver treated with pirfenidone Was done. Using mice (C57BL / 6, female, about 8 weeks), healthy mice (control) (vehicle (0.5% CMC) administration) and LPS-administered mice (50 g / kg + D-galactosamine 250 mg / kg, i (p. (Volume 200 zl)), the liver was excised 5.5 hours after administration (outer left lobe (three intermittent sections) (three of each three, three in total, nine in total)). After preparing tissue sections, hematoxylin and eosin (HE) staining, Tunel method, PCNA method, or ssDNA method were performed. The Tunel method was carried out using Oncore's Apop Tag //? Apoptosis Detection Kit—Peroxidase (Catalog No. S7100—KIT) according to the attached protocol. For the ssDNA method, anti-Single Stranded DNA (ssDN A) · Egret polyclonal antibody (DAK0 product code A4506) :! The antibody was used as the secondary antibody, and the detection was performed using DAK0 LSAB2 kit / Dish P-Universal (Catalog No. K0677) according to the attached protocol.

 PCNA (Proliferation Cell Nuclear Antigen) is a nuclear protein with a molecular weight of 36 KD that is synthesized maximally from the Gl phase to the S phase of the cell cycle. In this method, PCNA antibody (DAK0, Rikiyu Log NO.M879) was used as the primary antibody, and detection was performed using DAK0 LSAB2 kit / HRP-Universal (Catalog No.K0677) according to the attached protocol. .

 From the histological findings, no abnormalities were found in the liver tissue of healthy mice, as shown in FIG. 13A. No abnormalities were found in the Tunel section (Fig. 15A). Positive cells were found in a small number of hepatocytes only by the PCNA method. Many of the dinuclear hepatocytes were negative, and some of the mononuclear hepatocytes were positive. However, the total number of sections is extremely small. These findings are reconstructed images of normal hepatocytes (nuclear fission).

In contrast, in the liver tissue of LPS-administered mice (vehicle-administered group), almost all items were positive as shown in Table 1, and nuclear changes were particularly marked (+ to + + to + + +). In the Tunnel method and the ssDNA method, almost the same positive cells were observed. Positive findings were also found in the nuclei stained deeply in the nuclear wall, and some cells showed small amounts of microspherical granules in the cytoplasm. there were. The PCNA method also showed positive substances in a small number of nuclei, but the staining image was completely different from that of the Tunel method or ssMA method, and the number of positive cells was slightly higher than that in the control group of healthy mice. However, since normal nuclei were hardly seen in the vehicle-administered group, they were often found in deformed nuclei and in nuclei that were necrotic. In other words, “apoptosis” and “nucleoprotein synthesis” do not appear to occur in the same hepatocyte. In addition, the most prominent finding in the LPS-treated group was hemorrhage, and large and small hemorrhage spots were found on almost all leaves. Along with this bleeding, changes in hepatocyte nuclei—concentration, disintegration, nuclear wall staining, etc. were observed in various places. Furthermore, the loss of the nucleus, that is, cell necrosis occurred, and this was found as a so-called “acid body” (FIG. 13B). The fact that positive nuclei were found in various places in the Tunel method also suggests that even hepatocytes that seemingly normal by HE staining have started to undergo degeneration by apoptosis (Fig. 15B). . .

 Pirfenidone (500 mg / kg) (or vehicle) was orally administered to the LPS-inoculated mice 5 minutes before and 4 hours after the inoculation, and the effect on histological findings was examined. In LPS mice administered with pirfenidone, the basic findings were almost the same as in healthy mice, and no abnormalities were observed. Compared to healthy mice, the number of small cell aggregates was slightly higher, and nonspecific positive nuclei were found more frequently in the periphery of the section and in the lining of the portal vein by the Tunel method and ssDNA method (Fig. 16, Fig. 16). 18 and Figure 20). In addition, very few eosinophils exhibiting single cell necrosis were found, and other positive cells were found to be barely found by the Tunel method and the ssDNA method. In the PCNA method, positive cells comparable to those in the healthy mouse group were observed.

In summary, administration of LPS resulted in punctate-ecchymosis in the liver of mice and associated degeneration and necrosis of a wide range of hepatocytes, but these lesions were reduced by administration of pirufenidone. It was almost completely protected and had the same findings as in the liver of healthy mice. Tunel and ssDNA methods revealed that a large number of apoptosis-positive hepatocytes were observed in LPS-administered liver, but almost no positive hepatocytes were observed in drug-administered mice. Table 1 shows histopathological findings. 鹉

[Example 10] Inflammatory cytokine production and liver apoptosis in LPS-administered mice

MA cells induced by inflammatory cytokine production and liver apoptosis in LPS-treated mice In the evening, the formation of “I was examined. LPS (E. coli, 50 / g / kg) + D-gal (250 mg / kg) was intraperitoneally administered to mice (C57BL / 6 10-week-old female), and blood was collected over time. Inflammatory cytokines were quantified by ELISA TNF-, IL-12 and IFN-α production induction was observed (top of Fig. 21) Genomic DNA was prepared from the liver of mice after LPS administration. DNA ladder formation was detected using the Apoptosis Ladder Detection Kit Wako from Co. As a result, significant DNA ladder formation was observed 5 hours after LPS administration (Fig. 2 1 below).

 The effects of pretreatment with pirfenidone on inflammatory cytokine production and liver apoptosis in LPS shock model mice were examined. Using a mouse (C57BL / 6 10-week-old female), a carrier or pirpenidone (500 mg / kg P.O.) was administered 5 minutes before LPS injection. LPS (E. coli, SO jug / g) + D-gal (250 mg / kg) was intraperitoneally administered, and inflammatory site production and liver apoptosis (DNA ladder formation) were measured over time. . As a result, it was found that pirfenidone dramatically inhibited LPS-induced site force production and MA ladder formation (Fig. 22).

 The effects of post-administration of pirfenidone on inflammatory cytokine production and hepatic apoptosis in LPS-treated mice were examined. 3 hours after LPS [(E. coli, 50 zg / kg) + D-gal (250 mg / kg)] was intraperitoneally administered to mice using mice (C57BL / 6 10-week-old female), 500 mg / kg of don was administered (p.o.). After that, inflammatory site force production and liver apoptosis (DNA ladder formation) were measured over time. As a result, it was found that bilfenidone suppressed the induction of IFN-α production and the formation of DNA ladder (Fig. 23). These results and the results of Example 9 indicate that pyrufenidone exerts a direct inhibitory effect on apoptosis and necrosis independently of the induction of TNF-hi production.

 [Example 11] Apoptosis and biochemical changes in liver of LPS-administered mice

The time course of DNA ladder formation, poly-ADP-ribosylation, and NAD levels in the liver tissue of mice induced with congestive necrosis by LPS were examined. Mouse (C57BL / 6 11 week old female) LPS [(E. coli, 50 / g / kg) + D-gal (250mg / kg)] was intraperitoneally administered to mice, and liver apoptosis (2, 3, 4, 5, and 6 hours later) DNA ladder formation), poly-ADP-ribosylation, and NAD levels were measured. MA ladder formation was detected using liver samples according to the Apoptosis Ladder Detection Kit Wako. Poly-ADP-ribosinorei-dani was detected by Western blot using Anti-poly (ADP-Ribose) polyclonal antibody (rabbit, polyclonal). The NAD level was determined by the method of Nisselbaum (Nisselbaum, JS, and S. Green. A simple ultramicro method for determination of pyridine nucleotides in tissues. Anal. Biochem. 27, 212-217 (1969)). As a result, it was found that administration of LPS induced DNA ladder formation, poly-ADP-ribosylation, and reduction of NAD level (Fig. 24).

 The effects of pirfenidone administration on DNA ladder formation, poly-ADP-ribosylation, and NAD levels in liver tissues of mice induced with LPS-induced congestive necrosis were examined before and after administration. 5 min before intraperitoneal administration of LPS [(E. coli, 50 / ^ g / kg) + D-gal (250 mg / kg)] to mice using mice (C57BL / 6, 10-week-old female) Four hours later, vehicle or pirpenidone 500 mg / kg was administered (po) (n = 3). Six hours after LPS administration, liver apoptosis (DNA ladder formation), poly-ADP-ribosylation, and NAD levels were measured as described above. As a result, it was found that administration of pyrufenidone suppressed DNA ladder formation, poly-ADP-ribosylation, and reduction in NAD level (Fig. 25). From these results, it is concluded that pirfenidone has a protective effect against tissue damage due to anti-inflammatory and anti-apoptotic effects.

 [Example 12] Inhibition of apoptosis by pirfenidone in iniiio culture system

 Apoptosis was induced by adding etoposide to the human monocytic cell line THP-1, and the effect of pirfenidone administration was examined.

Spread 1.5 x 10 ⁶ pieces (/ 2ml) of THP-1 on a 6-well culture plate, and pill first. Preincubation was performed for 1 hour after adding 10 mM fenidone (the control was not added), and etoposide was added at 100〃M for 3 hours. The formation of induced and apoptotic bodies was analyzed by photography and Selso Ichiichi (Epix). As a result, it was revealed that pirfenidone significantly inhibited the formation of apoptotic bodies by etoposide (FIG. 26).

 THP-1 apoptosis was induced by etoposide, and the effect of pirfenidone on cell viability (survival) 6 hours after treatment was examined using WST-l (an assay based on the same principle as the MTT assay).

 Prepare THP-1 at δ χ ΐθ ^ ηιΐ, spread 100 〃1 each on a 96-well culture plate, and add pirufenidone 1, 3, 10 mM (final concentration) (no addition, control port) , Etoposide 100 (M (final concentration) added (no addition, control), prepared in each well, cultured for 6 hours (total volume 200 l), and after 6 hours, the following WST-1 (Dojin (Cell Counting kit) was added at 10/1 each, and the mixture was further reacted for 3 hours. The culture supernatant that had developed due to the viability of the cells was transferred to another 96-well plate at a time, and the absorbance was measured at 450 nm. As a result, it was found that pirfenidone inhibited etoposide-induced apoptosis in a dose-dependent manner (Fig. 27).

 [Example 13] Effect of pirfenidone on DNA ladder formation and biochemical changes in in culture system

 The time course of MA ladder, poly-ADP-ribosylation, and NAD levels of THP-1 cells that induced apoptosis with etoposide were examined.

Adjust THP-1 to IX 10 ⁶ / ml, spread 1 ml on a 24-well culture plate, add 100 μM etoposide (final concentration), and mix for 0, 1, 2, 3, 4, 5, and 6 hours. Later, the cell suspensions were sequentially collected, and 700/1 minutes were divided for observing DNA ladder formation, and 300 zl for ADP-ribosylation observation. After pelleting down cells from 700 1 min After extracting the DNA according to the protocol of the Apoptosis Ladder Detection Kit Wako, the DNA was quantified, the concentration was adjusted, agarose gel electrophoresis was performed, and the time course of DNA ladder formation due to apoptosis was observed. The cell suspension for ADP-ribosylation observation at 300/1 min was pelleted, dissolved in SDS-PAGE sample buffer, denatured at 100 ° C, subjected to SDS-PAGE, and transferred to a membrane for production. On the other hand, detection was performed by Western blotting using an anti-poly (ADP-Ribose) polyclonal anti body (rabbit, polyclonal). Further, the time-dependent change in the amount of NAD was detected as follows. THP-1 was prepared to lOVml and spread on a 24-well culture plate, of which 5 ゥ -well was used for -etoposide and 5 ゥ -well was used for + etoposide. After addition of etoposide (final concentration 100〃M), cells were collected at 0, 1, 2, 4, and 6 hours, respectively, and NAD was quantified by the Nisselbaum method described above. Was observed over time.

 As a result, DNA ladder formation and poly-ADP-ribosylation were detected 3 hours after etoposide treatment. In addition, etoposide treatment significantly reduced the NAD value (Fig. 28).

Next, we investigated the inhibitory effects of pirfenidone on DNA ladder, poly-ADP-ribosylation, and NAD levels in THP-1 cells that induced apoptosis with etoposide. To observe the inhibition of DNA ladder formation by pirfenidone, prepare THP-1 at 1 × 10 V ml, spread 1 ml each on a 24-well culture plate, add 100 μM (final concentration) etoposide, and add 10 mM pirfenidone. After 3 hours, collect the cells, pellet down, extract the DNA according to the protocol of Apoptosis Ladder Detection Kit Wako, and quantify the DNA, adjust the concentration, perform agarose gel electrophoresis, and perform agarose gel electrophoresis. The inhibition of DNA ladder formation by one cis was observed. To observe the inhibition of caspase-3 activation by pirfenidone, adjust THP-1 to lxlOVml, spread 2 ml on a 6-well culture plate, add etoposide 100 / M (final concentration), and add pirfenidone. 3 hours after adding lOmM, collect the cells and pellet down. Cells were lysed according to the protocol of Caspase-3 Assay Kit from mingen, and activated Caspase-3 activity in Lysate was measured using a fluorescent substrate. ADP - observation of ribosylation inhibition, THP-1 to ⁵ x l0 5 / ml in seeded by l ml in 24 Ueru culture plates were prepared, etoposide 100〃M (final concentration) was added, Pirufue two Don 10 mM After 4 hours, collect the cells, pellet down, dissolve in SDS-PAGE sample buffer, heat denature at 100 ° C, perform SDS-PAGE, transfer to SDS-PAGE membrane, transfer Anti-poly ( ADP-Ribose) was detected by Western blot using a polyclonal antibody (rabbit, polyclonal). NAD reduction curbing observations, THP-1 and 2 x l0 ⁶ / ml in seeded by, 1 ml of 24 Ueru culture plates were prepared, added to E Bok Poshido 100 zM (final concentration), Pirufue two Don 10 mM added After 4 hours, the cells were collected and NAD was quantified by the method of Nisselbaum described above.

 As a result, it was found that pirfenidone significantly inhibited etoposide-induced DNA ladder formation, poly-ADP-ribosylation, reduction of NAD level, and activation of Gaspase-3 (Fig. 29, Fig. 3). 0). These results in the in system indicate that virfenidone has a direct cytoprotective effect by anti-apoptotic action. Pirfenidone suppresses acute inflammation through anti-inflammatory actions such as suppression of site force production and suppresses tissue damage (such as apoptosis penecrosis) by cytoprotective activity through anti-apoptosis. It is suggested that

Caspase-3 is a protease involved in the process of apoptosis, and its activation (the 32kD pro form is processed during the apoptosis to become a 17kD form) is an indicator of cell apoptosis. It is one. Since pirfenidone suppresses the activity of this enzyme, it is clear that pirfenidone has an apoptosis inhibitory effect. The concentration dependence of pirpenidone for this enzyme was consistent with the concentration for inhibition of other parameters (such as inhibition of MA ladder formation, poly-ADP-ribosylation, and inhibition of NAD reduction). Western plots also confirmed that the production of the 17 kD activator was suppressed in a similar concentration-dependent manner. Also, When activated caspase-3 was used as an enzyme source and pirfenidone was added as a cell-free enzyme reaction system, pirfenidone showed no inhibitory effect on the activity of caspase-3 itself. Thus, it was thought that pirfenidone inhibited apoptosis, but caspase-3 was not the target molecule.

 [Example 14] Effect of pirfenidone on TNF-sporin production and mRNA expression inside and outside cells in splenic cell culture system

The inhibitory effect of pirfenidone on the production of TNF-intracellular and extracellular cells during LPS stimulation in RAW264.7 cells was examined as follows. RAW264.7 cells were seeded in a 6-well plate at 4 x lO / 2 ml RPMI 1640 per well, and cultured for 4 days.Confluent _c 2 μg / ml 2 ml RPMI 1640 containing LPS was added to each well. Pirfenidone was then added at the desired concentration of X2 and cultured for 8 hours. The culture supernatant was taken, and extracellular TNF-hi was measured by ELISA. TNF-intracellular cells were treated as follows. The recovered culture supernatant was pelleted down, the cells were lysed with 200 zl of Lysis buffer (described below), and TNF- contained in the cells was measured by ELISA.

The composition of the Lysis buffer is as follows. ELISA kit using 50 mM HEPES (H 7.5), 150 mM NaCl ヽ ImM MgCl ₂ , 1 mM EGTA ヽ 10% Glycerol ヽ 1% Triton X-100, 100 mM NaF ヽ 1 mM PMSFs 10 mg / ml Aprotinin ₀ TNF-α: R & D mouse TNF-a immunoassay.

 As a result of the measurement of TNF-string production, it was found that pirfenidone inhibited TNF-string production in RAW264.7 cells and in the supernatant in a dose-dependent manner (FIG. 31). Pirfenidone was found to inhibit TNF- and sperm production in culture supernatant (extracellular secretion) and cell lysate (intracellular) of LPS-stimulated THP-1 and P388.D1 cells.

In addition, changes in the expression of TNF-H mRNA by pirfenidone were examined as follows. RAW264.7 cells were cultured in a confluent state on a 10-cm culture plate, and final \ g / ml LPS, each concentration of pyrufenidone were added and cultured for 4 hours. culture After removing the supernatant, total RNA was purified from the cells using the Qiagen RNeasy Mini Kit, 2 g of MA was denatured with formamide, electrophoresed on a MOPS gel, and Northern hybridization was performed using the gel. Was. As a result, pirfenidone did not change the amount of TNF-mRNA (Figure 31, bottom).

Next, the effect of pirfenidone on TNF-H mRNA stability was examined. RAW264.7 cells are confluently cultured in a 6-well culture plate, and final l / g / ml LPS and pirfenidone (300 μg / ml) at each concentration are added. After 4 hours of culture, actinomycin D is added. (Sigma) was added at 5 g / ml of Final, and after 0, 1, and 3 hours, Total RNA was purified and RT-PCR was performed. Cells to which nothing was added and cells to which only LPS was added were used as controls. As a reverse transcriptase, Gibco BRL Superscript RT was used. PCR is used ExTaq to Taq polymerase Ichize by primer one 1 / M, it was carried out 29 cycles of ^{"95 ° C 1 min-55 p} C 1 min-72 ° C 1 min ." After electrophoresis of the PCR product on a gel, the band derived from TNF-a mRNA was detected to evaluate the stability of TNF-a mRNA.

 In cells to which nothing was added, almost no band corresponding to TNF-α was detected. Still, in the cells to which only LPS was added, a dark band corresponding to TNF-hi was detected at 0 hours after the addition of actinomycin D, and then gradually decreased. Each band corresponding to TNF-hi in the cells to which LPS + pyrufenidone was added was similar to that in the cells to which only LPS was added. This result indicates that pirfenidone does not promote the degradation of TNF-amRNA. This demonstrates that pyrphenidone does not suppress TNF-α production by affecting TNF-a mRNA stability (by promoting instability). . From these results, it is concluded that pirfenidone suppresses TNF-string production at the translational level, not at the secretory or transcriptional level.

[Example 15] Effect of pirfenidone on Jun N-terminal Kinase (JNK) and p38 MAP LPS-induced TNF-hi biosynthesis signals include the pathway leading to TNF-hi transcription induction via C-Hafl → MKKl, Z → U1, 2 and the LPS signal from MEKK1 MKK4 → JNK (SAP K) and MKK3, A pathway leading to the translation induction of TNF-hi via 6 → p38 is known (Jennifer et al., Mol. Cell. Biol. 17: 6274-6283, 1997). As described above, pirfenidone was found to suppress TNF- production at the translational level. In the mechanism of suppression of TNF-production, the one that inhibits production at the translational level is typically a p38 MAPK (p38 MAPK) inhibitor involved in the MAPK cascade or inhibition of c-Jun N-termina 1 kinase (JM) (Newton, RC and CPDecicco, T herapeutic potential and strategies for inhibiting tumor necrosis fact or- alpha.Journal of Medicinal Chemistry., 1999, Vol .42, No.13, 2295-2 314; Swantek, JL et al., Jun N-terminal kinase / stress-activated (JNK / SAPK) is required for lipopolysacc aride stimulation of tumor necrosis factor alpha (TNF -hi) translation: Glucocorticoids inhibit TNF- at ran slation by blocking JNK / SAPK. Molecular and Cellular Biology, Vol. 17, No. 11, 6274-6282). Therefore, next, the effect of pirfenidone on EM, JNK and p38 MAPK involved in the MAPK cascade was examined.

 First, the autophosphorylation of Jun N-terminal Kinase (JNK = Stress-Activated Protein Kinase; SAPK) and the phosphorylation of c-Jun were examined in a cell free enzyme reaction system. Phosphorylation was measured using Stratagene c-Jun N-Terminal Kinase (the contents of which were kitted) according to the attached protocol.

As a result, pirfenidone inhibited JNK phosphorylation of c-Jun in a concentration-dependent manner (Fig. 32). JNK is suggested to be important in the signal transduction system for various stress responses such as ultraviolet light, oxidative stress, ischemia reperfusion, damaged DNA, osmotic stimulation, heat stress, protein synthesis inhibition, inflammatory cytokines, and apoptosis. ing. The above results suggest that JM inhibition is involved in the main activity of pyrufenidone. Next, the inhibitory effect on p38 MAPK was examined in a cell free enzyme reaction system. The measurement was performed using Upstate Biotechnology SAPK2 «/ p38 / RK Assay Kit according to the attached protocol.

 As a result, pirfenidone inhibited the in vitro cell-free p38 MA PK enzyme reaction in a concentration-dependent manner (Fig. 33). Although it is slightly weaker than the effective concentration of RAW264.7 in suppressing LPS-stimulated TNF-splenid production, it is considered that p38 MAPK inhibition is at least included in the active form. This indicates that pirfenidone inhibits at least the p38 MAPK pathway in the MAPK cascade, thereby suppressing the activity of this group of enzymes at the onset of various stresses and inflammations and at the induction of apoptosis. Therefore, it is considered that the drug effect is exerted. The effective concentrations of p38 MAPK inhibition and JNK inhibition of virufenidone are in good agreement, suggesting that the inhibition of both contributes to the efficacy of pirfenidone.

 In addition, ERK (Upstate Biotechnology MAPKl / Erk1, MAPK2 / Erk2 Sampler Pack.) Was used as a protocol for ERK, which is a central kinase in the classical MAPK pathway. The inhibitory effect of pyruvidone on E1 and ERK2 in a cell-free in vitro enzyme reaction system was examined, but no inhibition was observed up to a concentration of 1000 μg / ml. Omitted). Based on these findings, pirfenidone inhibits the activity of this group of enzymes during the induction of various stresses and inflammations and the induction of apoptosis by inhibiting the JNK and p38 MAPK pathways of the MAPK cascade. It is considered that the drug is exerting its medicinal effect.

 [Example 16] Effect of pirfenidone on poly-ADP-Ribose-Polymerase (PARP)

 PARP activity was measured using Trevigen Poly (ADP-ribose) Polymerase assay Kit. According to the attached protocol.

As a result, pirfenidone inhibited the enzyme reaction of PARP in a concentration-dependent manner, At a concentration of 500 µg / ml, it was found to suppress about 45% (Fig. 34). ADP-ribosylation is closely linked to the reduction of NAD in apoptosis, and it is possible that inhibition of PARP may be part of the anti-apoptotic effect of pirfenidone. poly-ADP-Ribose-Polymerase (PARP) mediates the reaction of proteins near the damaged DNA strand (histone, PARP itself, etc.) to undergo poly-ADP-ribosylation by this enzyme. The enzyme activity of PARP depends on the cleaved DNA and is activated by caspase-3 cleavage (from 116Kd to 85KD) during apoptosis. The substrate is NAD and the protein on the receptor side, and it is important to note that a large amount of intracellular NAD is consumed during poly-ADP-ribosylation. There are various theories about the physiological significance of this reaction. PARP is mainly involved in the repair of damaged DNA, and by promoting a rapid decrease in the amount of NAD and ATP in cells and tissues, it promotes apoptosis and necrosis in cells and tissues. Is also considered. Specific diseases to which this enzyme inhibition can be applied include arthritis, type I diabetes, diseases derived from nerve cell death (cerebral ischemia / reperfusion, Alzheimer's disease, Parkinson's disease, etc.), retrovirus infection, etc. No. Industrial applicability

 According to the present invention, the following apoptosis-related factor-inhibiting effects of pirfenidone have been clarified.

Production of inflammatory cytokines

Poly-ADP-ribose-polymerase

JNK and / or p38 MAPK

All of these apoptosis-related factors are closely related to each other and play important roles in the apoptotic execution cascade. Therefore, by inhibiting these factors, apoptosis can be effectively inhibited. Apoptosis is thought to be responsible for various disease states Have been. For example, fulminant hepatitis is caused by hepatocyte apoptosis. Therefore, inhibitors of apoptosis-related factors may be useful in treating these conditions.

 Apoptosis is a phenomenon that occurs as a result of the collective action of complex cellular regulatory mechanisms. On the other hand, the causes of apoptosis that cause disease are diverse. Therefore, in the future, efforts will be required to identify the causes of apoptosis and to increase the therapeutic effect by selecting drugs that directly act on the causes. The apoptosis-related factor inhibitor of the present invention is required. Since the target factor is clear, it enables such an effective treatment after identifying the target factor.

 The apoptosis-related factor inhibitor of the present invention is not only useful as an inhibitor of apoptosis, but is expected to have new utility by clarifying its target factor. That is, for example, the inflammatory cytokines (IL-12, IL-18, and IFN-a) of the present invention are not only apoptotic factors but also play an important role in various inflammatory conditions. Therefore, the inflammatory cytokine inhibitor of the present invention can also be used for alleviating the inflammatory symptoms associated with these cytokines. Also, since PARP has an action of inducing tissue necrosis, for example, by consuming NAD, an inhibitor of PARP may be used as a necrosis inhibitor. In addition, JNK and / or p38 MAPK contribute to inflammation and immune abnormalities by participating in TNF-synthesis. Therefore, its inhibitors include anti-inflammatory agents, therapeutic agents for immunological disorders, Abdominal cavity inflammation (peritonitis, etc.), rheumatoid arthritis, arthritis, Crohn's disease, cancer cachexia, diabetic retinopathy, psoriasis, ischemic disease It is also useful as a drug for the prevention and treatment of illness, Alzheimer's disease and the like. As described above, the apoptosis-related factor inhibitor of the present invention has a more rational and effective therapeutic means for diseases associated with apoptosis because its target factor is clear. Or provide preventive measures.

Claims

The scope of the claims 1. An apoptosis inhibitor containing 5-methyl-1-phenyl-12- (1H) -pyridone as the main component represented by the formula (1). Equation (1):

 2. Inyuichi Leukin-12, Inyuichi Leukin 18, and Inyuichii which contain 5-methyl-1-phenyl-2- (1H) -pyridone as the main component represented by the formula (1) An inhibitor of the production of inflammatory cytokines selected from the group consisting of ferrona. Equation (1):

 3. An inhibitor of poly-ADP-ribose-polymerase containing 5-methyl-11-phenyl-2- (1H) -pyridone as a main component represented by the formula (1). Equation (1):

 4. A Jun-kinase and / or p38 MAP kinase inhibitor containing, as a main component, 5-methyl-11-phenyl-12- (1H) -pyridone represented by the formula (1). Equation (1):

 5. A therapeutic agent for a disease caused by apoptosis, which comprises the apoptosis inhibitor according to claim 1 as a main component. 6. A therapeutic agent for hepatitis, containing 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1) as a main component. Equation (1):

 7. The therapeutic agent according to claim 6, wherein the hepatitis is fulminant hepatitis.  8. A therapeutic agent for a disease caused by necrosis, comprising the poly-ADP-ribose-polymerase inhibitor according to claim 3 as a main component.  9. The therapeutic agent according to claim 8, wherein the disease is acute hepatitis.