WO2001058448A1 - Inhibiteur d'apoptose - Google Patents

Inhibiteur d'apoptose Download PDF

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Publication number
WO2001058448A1
WO2001058448A1 PCT/JP2001/000935 JP0100935W WO0158448A1 WO 2001058448 A1 WO2001058448 A1 WO 2001058448A1 JP 0100935 W JP0100935 W JP 0100935W WO 0158448 A1 WO0158448 A1 WO 0158448A1
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Prior art keywords
apoptosis
pirfenidone
lps
production
tnf
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PCT/JP2001/000935
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English (en)
Japanese (ja)
Inventor
Ryuji Suzuki
Hisashi Oku
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Shionogi & Co., Ltd.
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Priority to AU2001230605A priority Critical patent/AU2001230605A1/en
Publication of WO2001058448A1 publication Critical patent/WO2001058448A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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.
  • 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.
  • FasL Fas ligand
  • a compound capable of inhibiting the function of factors involved in apoptosis and controlling apoptosis it would be useful as a new therapeutic agent for diseases suspected of being involved in apoptosis.
  • various factors that induce apoptosis have been isolated, and their entire contents are being gradually revealed.
  • cell damage Sex lymphocytes induce apoptosis of target cells from outside the cells.
  • FasL The factor that acts at this time. If these factors can be effectively blocked, apoptosis can be controlled.
  • FasL FasL
  • pirfenidone Suppresses the synthesis and release of tumor necrosis factor a (TNF- ⁇ ) (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 pipressiveidone on liver fibrosis This might be explained by suppression of spawn production.
  • 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.
  • 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.
  • an acute hepatitis model of mouse liver induced by bacterial lipopolysaccharide hereinafter abbreviated as LPS
  • 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.
  • 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.
  • 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.
  • a therapeutic agent for a disease caused by apoptosis comprising the apoptosis inhibitor according to [1] as a main component.
  • a therapeutic agent for hepatitis containing 5-methyl-1-phenyl-2- (1H) -pyridone represented by the formula (1) as a main component.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • pyrphenidone US Pat. No. 3,839,346
  • Japanese Patent Application Laid-Open No. 49-8776 Japanese Patent Application Laid-Open No. 49-8776
  • 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 ⁇ Kupffer cells
  • IFN- ⁇ is an inflammatory cytokine mainly produced by T cells and NK cells.
  • IFN-H, IFN- ?, TNF-H, TNF- ?, IL-1H, IL- ?, I5, IL-6, or IL-10 are known as inflammatory cytokines. Have been.
  • TNF-string production occurs first.
  • TNF-hi leads to the production of IL-12 and IL-18, and these two cytokines induce IFN- ⁇ production.
  • IFN- ⁇ 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- ⁇ - and Fas Ligand -mediated Hepatotoxic Pathways in Endotoxin- Induced Liver Injury in Mice. J.
  • 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).
  • 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.
  • TNF-H is located at the most upstream in the sequence of site power-in described here.
  • the inhibitory effect of pirfenidone on inflammatory cytokines may appear as a result of the suppression of TNF-hi production.
  • inflammatory sites caused by pirfenidone The effect of suppressing the production of force-in is not indirect.
  • 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 can protect against lethality (Example 3).
  • pirfenidone suppresses the production of inflammatory site such as IFN- ⁇ even after the production of TNF- ⁇ 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).
  • 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- ⁇ . 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.
  • 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- ⁇ (Clinical Immunity, Vol. 30, No. 11, ⁇ 1471-1478, 1998).
  • 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.
  • organ-specific autoimmune diseases are thought to be caused by a bias toward the helper 1 type immune response.
  • 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.
  • GVHD graft-versus-host disease
  • the inflammatory cytokine inhibitory agent of the present invention can be used for the prevention or treatment of these diseases.
  • TNF-strings 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.
  • GVHD graft-versus-host disease
  • arthritis thyroiditis
  • Hashimoto's disease Hashimoto's disease
  • exocrine adenitis intracellular infections
  • 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.
  • IL-18 which is one of the inflammatory site power-in of the present invention, is associated with miscarriage or premature birth.
  • 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.
  • 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).
  • PARP poly-ADP-ribose-polymerase
  • 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.
  • 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. .
  • 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.
  • a disease includes acute hepatitis. That is, the present invention relates to a therapeutic agent for acute hepatitis containing pirfenidone as a main component.
  • 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.
  • NGF nerve growth factor
  • 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.
  • 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
  • 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.
  • 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.
  • apoptosis inhibitor containing 5-methyl-1-phenyl-12- (1H) -pyridone represented by the above formula (1), that is, pyrphenidone as a main component.
  • vipraidone has an apoptosis inhibitory action as shown in the Examples.
  • 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.
  • Cis inhibitors can inhibit ongoing apoptosis.
  • 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.
  • the apoptosis inhibitor of the present invention relates to a medicament used for treating ongoing apoptosis.
  • 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.
  • 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.
  • Apoptosis is generally called clean cell death without inflammation.
  • 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).
  • 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.
  • JNK inhibition and p38 inhibition are associated with anti-inflammatory effects including suppression of TNF- ⁇ production.
  • JNK inhibitors Swantek 3 JL et al., Mol. Cell. Biol., Vol. 17, No. 11, 6274-6282
  • p38 inhibitors Youngng, P. et al., Agents Actions, 1993) , 39, C67-C69; Prichett 3 W. et al, J. Infla Marauder, 1995, 45, 97-105
  • TNF- ⁇ acts on the TNF- ⁇ translation process (biosynthesis process). Production of It is said to suppress.
  • TNF- production of mRNA and secretion of TNF-hi are not indicated for suppression.
  • pirfenidone 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- ⁇ production (Tokuheihei 11-512699) is considered to be a phenomenon resulting from JNK inhibition and p38 MAPK inhibition.
  • 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
  • GVHD graft-versus-host disease
  • PTCA graft-versus-host disease
  • 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.
  • 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. .
  • apoptosis inhibitor of the present invention 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. .
  • injections 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.
  • FIG. 1 is a graph showing the inhibitory effect of pirfenidone on the production of TNF-H, IL-12 and IFN- ⁇ .
  • 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 pyblaidone on TNF-splen production from THP-1 cells.
  • FIG. 7 is a graph showing the inhibitory effect of pirfenidone on IFN- ⁇ 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.
  • 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 ⁇
  • 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 ⁇
  • 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 ⁇ Yuichi), and the horizontal axis shows the size of cells (body) (by forward scans ⁇ 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 pyblaidone on JNK.
  • FIG. 33 shows the effect of bilfenidone on p38 MAPK.
  • FIG. 34 is a diagram showing the effect of pyblaidone on PARP. BEST MODE FOR CARRYING OUT THE INVENTION
  • Example 1 Inhibitory effect of pirfenidone on TNF-H, IL-12 and IFN- ⁇ production
  • mice 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.
  • the inhibitory effect of pirfenidone on the production of TNF-o :, IL-12 and IFN- ⁇ was examined.
  • mice C57BL / 6, 8-week-old female
  • anti-Fas mAb Jo2, 100 zg / kg i.v.
  • 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.
  • 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.
  • Example 5 Effect of pirfenidone by in sitine production system
  • Monosite ⁇ -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 pyblaidone. 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 ⁇ l) 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 py physicianidone 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- ⁇ in NK3.3 cells in a dose-dependent manner (FIG. 7). IC 50 was calculated to be 51 ⁇ g / 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.
  • IC 5Q for suppressing the production of TGF- ⁇ and PDGF-AB was calculated to be 70 g / ml and 50 ⁇ g / 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.
  • mice The inhibitory effect of pirfenidone on blood TNF- production in LPS shock model mice (ac / zes killed bacteria sensitized) was examined.
  • 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.
  • pirfenidone suppressed TNF-hi production in mice inoculated with LPS with i / 2 FJ'TO (Fig. 8).
  • 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.
  • 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.
  • mice C57BL / 6, female, about 8 weeks
  • healthy mice control
  • 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)).
  • HE hematoxylin and eosin
  • the Tunel method was carried out using Oncore's Apop Tag //? Apoptosis Detection Kit—Peroxidase (Catalog No. S7100—KIT) according to the attached protocol.
  • 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
  • DAK0 Regeneration Cell Nuclear Antigen
  • HRP-Universal Catalog No.K0677
  • 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.
  • LPS mice administered with pirfenidone the basic findings were almost the same as in healthy mice, and no abnormalities were observed.
  • 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).
  • 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).
  • pirfenidone has a protective effect against tissue damage due to anti-inflammatory and anti-apoptotic effects.
  • Apoptosis was induced by adding etoposide to the human monocytic cell line THP-1, and the effect of pirfenidone administration was examined.
  • 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).
  • THP-1 THP-1 at ⁇ ⁇ ⁇ ⁇ ⁇ , spread 100 ⁇ 1 each on a 96-well culture plate, and add pi crizidone 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).
  • 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.
  • detection was performed by Western blotting using an anti-poly (ADP-Ribose) polyclonal anti body (rabbit, polyclonal).
  • 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 ⁇ -well was used for -etoposide and 5 ⁇ -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.
  • 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.
  • 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).
  • 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.
  • pirfenidone 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 ⁇ g / 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 ⁇ ImM MgCl 2 , 1 mM EGTA ⁇ 10% Glycerol ⁇ 1% Triton X-100, 100 mM NaF ⁇ 1 mM PMSFs 10 mg / ml Aprotinin 0 TNF- ⁇ : R & D mouse TNF-a immunoassay.
  • 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.
  • RAW264.7 cells are confluently cultured in a 6-well culture plate, and final l / g / ml LPS and pirfenidone (300 ⁇ g / 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.
  • 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.
  • 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 Stress-Activated Protein Kinase
  • SAPK Stress-Activated Protein Kinase
  • 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.
  • stress responses such as ultraviolet light, oxidative stress, ischemia reperfusion, damaged DNA, osmotic stimulation, heat stress, protein synthesis inhibition, inflammatory cytokines, and apoptosis.
  • the above results suggest that JM inhibition is involved in the main activity of pyblaidone.
  • 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.
  • 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 vinialidone are in good agreement, suggesting that the inhibition of both contributes to the efficacy of pirfenidone.
  • ERK Upstate Biotechnology MAPKl / Erk1, MAPK2 / Erk2 Sampler Pack.
  • MAPK Upstate Biotechnology MAPKl / Erk1, MAPK2 / Erk2 Sampler Pack.
  • 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 ⁇ g / ml. Omitted).
  • 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.
  • PARP activity was measured using Trevigen Poly (ADP-ribose) Polymerase assay Kit. According to the attached protocol.
  • pirfenidone inhibited the enzyme reaction of PARP in a concentration-dependent manner, At a concentration of 500 ⁇ g / 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.
  • PARP 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.
  • 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
  • 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.
  • 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.
  • 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.

Abstract

L'invention concerne un inhibiteur d'apoptose contenant une 5-méthyl-1-phényl-2-(1H)-pyridone en tant que principe actif de formule (1) ; et un agent régulant la production de cytokine inflammatoire, un inhibiteur de la poly-ADP-ribose-polymérase et un inhibiteur de JNK et/ou p38 MAPK, chacun contenant ce composé faisant office de principe actif.
PCT/JP2001/000935 2000-02-09 2001-02-09 Inhibiteur d'apoptose WO2001058448A1 (fr)

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