WO2002011725A1

WO2002011725A1 - Inflammatory cytokine production inhibitors

Info

Publication number: WO2002011725A1
Application number: PCT/JP2001/006814
Authority: WO
Inventors: Hisashi Oku; Ryuji Suzuki
Original assignee: Shionogi & Co., Ltd.
Priority date: 2000-08-08
Filing date: 2001-08-08
Publication date: 2002-02-14
Also published as: AU2001278699A1

Abstract

Inflammatory cytokine production inhibitors, IL-10 production promoters or apoptosis inhibitors containing as the active ingredient compounds relating to nicotinic acid. The compounds relating to nicotinic acid usable herein include nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, 1-methyl-2-pyridone and tryptophan. These drugs are useful in inhibiting the production of inflammatory cytokines such as IL-12, IFN-η or TNF-a. It is also possible to use these drugs for promoting the production of IL-10 which is an anti-inflammatory cytokine. Use of these drugs in treating hepatitis, etc.

Description

Description Inflammatory cytokine production inhibitor Technical field

The present invention relates to an agent for suppressing the production of inflammatory site force-in, an agent for enhancing the production of interleukin 10, a poly-ADP-ribose-polymerase inhibitor, or an apoptotic inhibitor.

In the immune system and the hematopoietic mechanism, a humoral factor responsible for signal transmission between cells that control these mechanisms is called a cytokine. Inflammatory cytokines are a collective term for cytokines that have been shown to particularly cause inflammatory symptoms. C For example, interleukin (hereinafter abbreviated as IL) -12, IL-18, and IL-1 Hue (hereinafter abbreviated as IFN)-A, tumor necrosis factor (hereinafter abbreviated as TNF)-Fibrils and the like are inflammatory cytokines that are closely related to inflammatory symptoms. IL-12 is mainly monocyte-macrophage, IL-18 is mainly macrophage cupper cells, and IFN-α is an inflammatory cytokine produced mainly by T cells and NK cells. It is. In addition, IFN-H, IFN- ヽ TNF- ^ sIL-Ια, IL-1 ?, IL-5, IL-6, IL-10, and the like are known as inflammatory cytokines. Suppressing the production of these inflammatory cytokines is considered an important tool for achieving control of inflammatory conditions. For example, it has been reported that the use of compounds such as benzamide-nicotinic acid amide to suppress TNF-synthesis production can be used to treat inflammatory conditions (W097 / 32582). In particular, apoptosis-inducing activity was confirmed for some of these compounds. Apoptosis-inducing activity has also made it possible to control inflammatory conditions through induction of apoptosis. This report specifically addresses systemic lupus erythematosus (Hereinafter abbreviated as SLE), nicotinic acid amide can be applied to the treatment of diseases such as rheumatoid arthritis, asthma, sepsis, ulcerative colitis, psoriasis, and ischemic disorders. In addition, 3,4,2-phenylsulfonamide compounds (W096 / 36595) and N-substituted nicotinamides (Japanese Unexamined Patent Publication No. 9-291032) also include substances having an inhibitory effect on TNF-hi production. Have been reported. Nicotinic acid has also been reported to have a therapeutic effect in hamster lung fibrosis models (Q. Wang et al., J. Biochem. Toxicology, 1990, Vol. 5, No. l, pl3-22., Niacin Attenuates Bleomycin- Induced Lung Fibrosis in the Hamster.). However, its efficacy for acute inflammation is unknown.

Inflammatory cytokines stimulate the production of Fas and Fas ligand (hereinafter abbreviated as Fas-L) and consequently induce apoptosis. Indeed, TNF-hi has been positioned as an apoptosis-inducing factor. Therefore, suppression of inflammatory cytokine production may lead to suppression of apoptosis. Thus, suppression of the production of inflammatory cytokines is considered to be useful in controlling the pathology of many diseases related to inflammatory symptoms and apoptosis.

Apoptosis is cell death caused by activation of caspases. It is known that apoptosis is involved in many diseases, as well as in 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, a pathological increase in apoptosis leads to tissue atrophy. As the mechanisms by which apoptosis occurs become apparent, the association of apoptosis with 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 the case of hepatitis symptoms and graft-versus-host disease (GVHD), it has been suggested that parforin and Fas-L are involved in the pathology. That is, there is a report that inhibition of the binding between Fas and Fas-L leads to a reduction in inflammation. In addition, the survival rate of GVHD model animals was lower than that of anti-Fas-L antibody. (Experimental Medicine vol.17, No.13, pp.1635-1641, 1999).

Thus, if a compound capable of inhibiting the action 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 revealed gradually. For example, cytotoxic lymphocytes extracellularly induce apoptosis of target cells. The site force acting at this time is Fas-L. If these factors can be effectively blocked, apoptotic control will be realized. However, there have not been many reports of drug candidate compounds that act on apoptosis-related factors.

On the other hand, it is known that 5-methyl-tophenyl-2- (1H) -pyridone has a therapeutic effect on fibrosis in the lungs, arterial sclerosis and the like (Japanese Patent Publication 5-52814). From the structure shown below, this compound can also be said to be a nicotinic acid-related compound. 5-Methyl-triphenyl-2- (1H) -pyridone was named generically as "pirfenidone". A similar effect has been reported for its derivatives (Tokuheihei 8-510251). Pirfenidone, a compound represented by the following formula, has long been reported as a compound useful for treating inflammatory symptoms in the respiratory tract and skin (USP3974281, USP4042699 USP4052509). Since then, it has been focused on its antifibrotic effect, and is a compound under development as a drug for pulmonary fibrosis (Nicod, LP. Lancet, Vol. 354, July 24, 1999, p268-269).

Pilfuenidon

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 hamster lung changes (Kehrer and Margolin (199

7) Toxicol. Lett. 90 125; Schelegle et al (.1997) Proc. Soc. Exp. Biol. Med. 216 392),.

• Suppresses overproduction of collagen (Iyer et al (1999) J. Pharmacol. Exp. Ther. 2

89 211),

• Suppresses the synthesis and release of tumor necrosis factor (TNF-hi) (Tokuhei 11-512699). Based on these effects, pirfenidone is now used in pulmonary fibrosis, sclerosing peritonitis, and scleroderma. Developed as a therapeutic agent for uterine leiomyomas.

TNF-α is positioned as an apoptosis inducer. It has also been suggested that the role of TNF-hi in hepatitis-induced liver tissue injury is important (Guidotti L., et a 1., Immunity, 4: 25-36, 1996, Kondo T., et al. , Nature Med., 3: 409-413, 1997, Seino K., et al., Gastroenterology, 113: 1315-1322, 1997) ₀ Further, the present inventors have added to pirfenidone an inflammatory site. We found a use for inhibiting the production of force-in and as an inhibitor of apoptosis, and filed a patent application (Japanese Patent Application No. 2000-38048). However, in addition to these reports, no nicotinic acid-related compounds that can be used as inhibitors of inflammatory cytokine production or as inhibitors of apoptosis have not been reported. Disclosure of the invention

An object of the present invention is to provide a novel inflammatory cytokine production inhibitor, an interleukin 10 production enhancer, a poly-ADP-ribose-polymerase inhibitor, or an apoptosis inhibitor.

The present inventors administer a nicotinic acid-related compound to an artificially caused inflammatory condition, and analyze the effect in detail to obtain a mechanism of action of the nicotinic acid-related compound. We thought that the structure could be analyzed in more detail. 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 an analysis, the present inventors confirmed that the nicotinic acid-related compound has a therapeutic effect or a preventive effect on LPS-induced hepatitis. Furthermore, the present inventors have searched for the mechanism of action of a nicotinic acid-related compound having such an effect, and have studied its anti-inflammatory action, poly-ADP-ribose-polymerase inhibitory action, and anti-inflammatory action. The present inventors have found a cytokine production promoting action or an anti-apoptotic action, and completed the present invention. Furthermore, the present inventors have paid attention to the fact that all of the nicotinic acid-related compounds that have found these effects are present in vivo. Then, it was clarified that the anti-inflammatory effect can be evaluated using the activity of increasing the amount as an index, and a method for screening a compound useful as an anti-inflammatory agent was established.

That is, the present invention provides the following inflammatory site force-in production inhibitor, interleukin-10 production enhancer, poly-ADP-ribose-polymerase inhibitor, or apoptosis inhibitor, and uses thereof. About.

[1] Any compound selected from the group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, and trimethyl-2-pyridone, or a pharmaceutically acceptable compound thereof An inhibitor of the production of insulin and leukin 12 and / or interferoner, which comprises a salt as a main component.

[2] A tumor necrosis factor production inhibitor comprising, as a main component, trimethyl-2-pyridone-5-carboxamide or a pharmaceutically acceptable salt thereof.

[3] A tumor necrosis factor α production inhibitor comprising nicotinamide or a pharmaceutically acceptable salt thereof and tributophan or a pharmaceutically acceptable salt thereof as a main component.

(4) nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, An interleukin 10 production enhancer containing, as a main component, any compound selected from the group consisting of 1-methyl-2-pyridone and tributophan, or a pharmaceutically acceptable salt thereof.

[5] Any compound selected from the group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, and trimethyl-2-pyridone, or a pharmaceutically acceptable compound thereof An inhibitor of apoptosis, comprising a salt as a main component.

[6] a compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and trimethyl-2-pyridone, or a pharmaceutically acceptable compound thereof Prophylactic or therapeutic agent for hepatitis, containing a salt as a main component

[7] Any compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and trimethyl-2-pyridone, or a pharmaceutically acceptable compound thereof An inhibitor of poly-ADP-ribose-polymerase, comprising a salt as a main component.

[8] A prophylactic or therapeutic agent for a disease caused by necrosis, comprising as an active ingredient the inhibitor of poly-ADP-ribose-polymerase according to [7].

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

[10] The prophylactic or therapeutic agent according to [8], wherein the hepatitis is fulminant hepatitis.

[11] A method for screening a compound useful as an anti-inflammatory agent, comprising the following steps. a) a step of administering a candidate compound to a test animal

b) from the group consisting of nicotinic acid, nicotine amide, 1-methyl-2-pyridone-5-carboxamide, and trimethyl-2-pyridone contained in the tissue and / or biological fluid of the test animal Measuring any of the selected compounds; and

c) selecting candidate compounds that increase the measured value of b) compared to the control

[12] An anti-inflammatory agent comprising, as a main component, a compound obtainable by the method according to [11].

The compounds utilized as active ingredients in the present invention are all known nicotinic acid Related compounds. The structures of these compounds are shown below.

Trimethyl-2-pyridone-5-carboxamide methyl-2-pyridone

Tribute fan;

[1] An agent for inhibiting the production of cinnamon-leukin 12 and / or cinnamon-ferrona The present invention relates to nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and trimethyl- The present invention relates to an interleukin 12 and / or interferona production inhibitor containing, as a main component, any compound selected from the group consisting of 2-pyridones, or a pharmaceutically acceptable salt thereof.

In the present invention, the term “pharmaceutically acceptable salt” includes hydrates thereof. For example, alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, salts with organic bases and amino acids, or inorganic acids (hydrochloric acid, hydrobromic acid, phosphorus) Acid, sulfuric acid, etc.) and organic acids (acetic acid, citric acid, maleic acid, fumaric acid, benzenesulfonic acid, P-toluene) Sulfonic acid and the like). These salts can be formed by a commonly used method. When forming a hydrate, any number of water molecules may be coordinated.

Alternatively, the present invention relates to a method for suppressing the production of interleukin 12 and / or insulin feron ", which comprises a step of administering an effective amount of the above compound. The present invention relates to the use of 12 and / or interferona in the production of a production inhibitor.

It is well known that mammals produce severe acute inflammatory symptoms with LPS administration. C When LPS is administered to a mammal's peritoneal cavity, first production of TNF-spin occurs. TNF- leads to the production of IL-12 and IL-18, and these two cytokines induce IFN-α production. IFN-a induces Fas and Fas-L, resulting in rapid induction of hepatocellular apoptosis in the liver and hepatic failure due to congestive necrosis with apoptosis (Tsutsui, H., et al. 1997). IL-18 Accounts for Both TNF -hi-and Fas Ligand- mediated Hepatotoxic Pathways in Endotoxin- Induced Liver Injury in Mice. J.I thigh u nol. 159: 3961.) O It has been reported that it is more important than the elevation of IL-1, IL-6, IL-8, GM-CSF, etc., which are also known to be elevated in acute inflammation (Ozman, L., et al. al. 1994.Interleukin 12, interferon 7, and tumor necrosis factor are the key cyto ines of the generated Sh arzman reaction.J. Exp.Med. 180: 907, Wysocka, M. et al. 1995.Int erleukin 12 is required for interferon r production and lethality in lip opolysaccharide-induced shock in mice.Eur.J. Immunol. 25: 67) ₀

As mentioned earlier, some nicotinic acid-related compounds have already been reported to suppress TNF-hi production. Further, it is also shown in Examples 1 and 7 described later that the production of TNF-hi increases by administration of the anti-niacin compound. This can be explained by the fact that endogenous niacin was competitively inhibited by the anti-niacin compound, and TNF-production was enhanced. In fact, anti-niacin compounds It has long been known that competitive inhibition of causative niacin causes symptoms similar to niacin deficiency. The present inventors have clarified that a specific nicotinic acid-related compound has an inhibitory effect not only on TNF-specific but also on IL-12 and IFN-y.

Now, it should be noted that T-is located at the uppermost stream in the sequence of site force-in flow described here. In other words, the inflammatory cytokine-suppressing effect of the nicotinic acid-related compound may appear as a result of suppressing the production of TNF-hi. However, according to the findings of the present inventors, the action of inhibiting the production of inflammatory cytokines by nicotinic acid-related compounds is not indirect. For example, the peak of TNF-spike production by LPS administration was 1.25 hours after LPS administration, and nicotinic acid-related compounds were not detected in blood 3 hours after LPS administration. Administration even after medium TNF-production has passed (eg, 3 hours after LPS administration) can protect against lethality.

The nicotinic acid-related compound also suppresses apoptosis even when administered 3 hours after LPS administration (Example 13). As described above, the nicotinic acid-related compound has a characteristic that it can suppress acute inflammatory shock even after the release of TNF-fiber. Furthermore, the nicotinic acid-related compound suppresses poly-ADP-ribosylation and a decrease in the amount of NAD even after 3 hours from LPS administration (Example 13). These effects of nicotinic acid-related compounds are in contrast to the fact that protection, such as with anti-TNF-antibodies, fails to suppress the response of TNF-antibodies to automatically evolve after they are released into the blood in large quantities. . From the above, it is clear that these effects of nicotinic acid-related compounds are not merely manifested as a result of suppressing the effects of TNF-H.

All of the cytokines whose production is suppressed by the inflammatory cytokine production inhibitor of the present invention are T helper 1 type site potentin. Therefore, the inflammatory cytokine inhibitory agent of the present invention is a disease caused by enhanced production of T helper 1 type cytokines, and particularly enhances the production of IL-12 and IFN-α. It is effective for the cause. T helper 1 type site force in is a general term for a group of inflammatory cytokines involved in the induction of T helper 1 type immune response among inflammatory cytokines. The T helper 1 type of site force-in includes a cytokine that induces the differentiation of T helper 1 cells and a cytokine that is produced by T helper 1 cells. Specific examples of the site that induces the differentiation of T helper 1 cells include IL-12 and the like. These site forces have been implicated in the induction of T helper 1 type immune responses (Xu, B. et al. J. Exp. Med., 188: 1485, 1998, and Takeda, K. et al. al. Immunity., 8: 383-390, 1998). In addition, the site force produced by T helper-1 cells specifically includes IFN-α and the like.

(Clinical Immunity, Vol. 30, No. 11, pl471-1478, 1998).

An imbalance in the T helper 1 / T helper 2 balance is thought to contribute to the development of immune disease. When biased toward T 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 inhibitor of the production of inflammatory cytokines according to the present invention can be expected to be effective against all diseases which are said to be caused, for example, by an excessive T helper 1 type immune response. Generally, organ-specific autoimmune diseases are thought to be caused by a bias toward T helper 1 type immune responses. Specifically, diabetes, hepatic disorder, autoimmune myelitis, ulcerative colitis, graft-versus-host disease (GV HD), arthritis, thyroiditis, Hashimoto's disease, exocrine glanditis, intracellular infection (Leishmani A. Tuberculosis, mycobacterium tuberculosis, rye bacteria), delayed-type hypersensitivity, scleroderma, or Behcet's disease. The inflammatory cytokine inhibitory agent of the present invention can be used for preventing or treating these diseases.

Among the above-mentioned diseases, it is considered that the etiology of the following diseases is not caused solely by TNF-α alone, and it is difficult to imagine the indication as an indication for a TNF-producing inhibitor. That is, diabetes, hepatic disorder, autoimmune myelitis, graft-versus-host disease (GVHD), arthritis, thyroiditis, Hashimoto's disease, exocrine glanditis, intracellular infection , Mycobacterium tuberculosis, Rye), delayed-type hypersensitivity, Behcet's disease, and the like can be said to be novel indications specific to the inflammatory cytokine inhibitory agent of the present invention.

The present invention also relates to a TNF-hi production inhibitor comprising trimethyl-2-pyridone-5-carboxamide or a pharmaceutically acceptable salt thereof as a main component. Alternatively, the present invention relates to an agent for inhibiting the production of TNF-hi, which comprises nicotinamide or a pharmaceutically acceptable salt thereof and tributophan or a pharmaceutically acceptable salt thereof as main components. Alternatively, the present invention relates to a method for suppressing the production of TNF-hi, comprising the step of administering an effective amount of the compound. Furthermore, the present invention relates to the use of the above compound in the production of a TNF- production inhibitor.

The apoptosis-inducing activity of benzamides and nicotinamide and the action of inhibiting TNF-production are known (W097 / 32582). In addition, it has been reported that administration of N-substituted nicotinamide inhibits the production of phosphodiesterase IV and TNF-hi and is effective for the treatment of chronic inflammatory diseases associated with TNF-hi (Japanese Patent Application Laid-Open No. -291032). Alternatively, in 7 iro, nicotinamide suppressed TNF-a production in LPS-stimulated human peripheral blood monosites, and in 'TO, TNF-hi production was suppressed in mice treated with LPS (I Lett. 1997 59/1, 7-11). In addition, nicotinamide is said to show a protective effect in mice administered with SEB, a superantigen. This report confirms the suppression of production of IL-1, TNF-, IL-2R, IL-2, and IFN-y (Toxicology, 1996, 107/1, 69-81). In addition, there is a report comparing the effects of nicotinamide with those of steroid, FK506, and cyclosporine on the suppression of TNF-proliferation in IL-15-stimulated mouse spleen / cells (Life Sci. , 1996, 59/17, 1423-1429).

However, there is no report on the inhibitory effect of trimethyl-2-pyridone-5-carboxamide on the production of TNF. It is also unknown that the combination of nicotinamide and tributophan achieves suppression of TNF-hi production. As mentioned earlier, TNF-hi is located upstream of the inflammatory cytokine signaling pathway. Therefore, Compounds that suppress its production are important in controlling inflammatory conditions. The inhibitor of TNF-hi production is useful for treating the above-mentioned diseases caused by the action of inflammatory cytokines. In the present invention, the interleukin-12, interferona, or TNF-hi production inhibitor may be simply referred to as an inflammatory site force-in production inhibitor.

(2) PMP inhibitor

Next, the present invention relates to any compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and 1-methyl-2-pyridone, Or an inhibitor of poly-ADP-ribose-polymerase (hereinafter abbreviated as PMP) containing a pharmaceutically acceptable salt thereof as a main component.

Alternatively, the present invention relates to a method for inhibiting PARP, comprising a step of administering an effective amount of the compound. The invention further relates to the use of said compounds in the manufacture of inhibitors of PMP.

PARP is an enzyme that catalyzes the reaction of poly-ADP-ribosylation of proteins near the damaged DNA strand (histone, PARP itself, etc.). This enzyme activity depends on the cleaved DNA, and is activated by caspase-3 cleavage (from 116KD to 85KD) during apoptosis. PMP 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 by poly-ADP-ribosylation. That is, it is considered that apoptosis and necrosis of cells and tissues are promoted by rapidly decreasing NAD and ATP of cells and tissues (Ha, HC and Snyder, SH Proc. Natl. Acad. Sci. Vol. 96, No. 24, 13978-13982, 1999).

Therefore, PMP enzyme inhibition is expected to have therapeutic effects on arthritis, type I diabetes, diseases derived from various types of neuronal cell death (cerebral ischemia, reperfusion, Alzheimer's disease, Parkinson's disease, etc.), retrovirus infection, and the like. The PARP inhibitor of the present invention, It is useful as a therapeutic or prophylactic agent for diseases associated with necrosis. It is particularly effective for diseases caused by necrosis caused by rapid apoptosis. Such diseases include acute hepatitis.

That is, the present invention relates to any compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-potassium lipoxamide, and methyl-2-pyridone, or a pharmaceutically acceptable compound thereof. The present invention relates to a prophylactic or therapeutic agent for hepatitis, comprising a salt as a main component. Alternatively, the present invention relates to a method for treating or preventing hepatitis, comprising administering an effective amount of the compound. The present invention also relates to the use of the compound in the manufacture of a prophylactic or therapeutic agent for hepatitis. The compounds are particularly useful for the prevention and treatment of acute hepatitis and fulminant hepatitis.

[3] Inhibitor of apoptosis

Further, the present invention provides a compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and 1-methyl-2-pyridone, or The present invention relates to an inhibitor of apoptosis, containing a pharmaceutically acceptable salt thereof as a main component.

Alternatively, the present invention relates to a method for inhibiting apoptosis, comprising a step of administering an effective amount of the compound. The invention further relates to the use of said compounds in the manufacture of inhibitors of apoptosis.

It is a novel finding that a nicotinic acid-related compound has an apoptosis inhibitory action as shown in the Examples. It should be noted that administration of nicotinic acid-related compounds after LPS has a therapeutic effect, especially in acute hepatitis symptoms induced artificially by LPS peritoneal administration. Many known anti-apoptotic active compounds cannot be expected to have a sufficient apoptosis inhibitory effect unless administered prophylactically before apoptosis occurs. That is, although effective as a prophylactic agent, its effectiveness as a therapeutic agent could not be expected. In contrast, the apoptosis inhibitor according to the present invention can inhibit apoptosis that is in progress. That is, therapeutic The effect can be expected. Nicotinic acid-related compounds that have this mechanism of action against apoptosis include, for example, prophylactic agents against internal hemorrhagic necrosis of the liver (with rapid pathological apoptosis) and chronic rejection after transplantation that occur in acute hepatitis. 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 glomerulonephritis, acute lung injury, interstitial pneumonia, cardiac hypertrophy, cardiomyopathy, retinal detachment, autoimmune disease, myocardial infarction ischemia, diabetes, inflammatory bowel Disease, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis, AIDS, pancytopenia, refractory anemia, aplastic anemia, viral hepatitis, fulminant hepatitis, cirrhosis, cerebral ischemia , Cerebral infarction, Cedaren syndrome, salivary glanditis, severe myeloma, arteriosclerosis, Peyette's disease, multiple sclerosis, glaucoma, cataract, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis, radiation injury, sepsis And so on.

We have already mentioned that nicotinic acid-related compounds are effective in inhibiting the production of apoptosis-related factors, IL-12, IFN-α, PP, or TNF-hi. Since all of these apoptosis-related factors are closely related to apoptosis based on the mechanism described below, nicotinic acid-related compounds are useful as inhibitors of apoptosis.

1) IL-12 / IFN-α production suppression and anti-apoptosis

As shown in the experiments of mice administered with FWLPS in the examples, it is considered that the suppression of the production of inflammatory cytokines by nicotinic acid-related compounds leads to the suppression of the production of Fas and Fas-L. As a result, hepatocyte apoptosis is effectively suppressed.

2) Anti-PARP and anti-apoptosis

—Apoptosis is generally referred to as clean cell death without inflammation. However 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 liver tissue with rapid apoptosis. Pathological findings in cases have also been observed. One of the causes is thought to be the rapid decrease in NAD in the organization. As mentioned earlier, ADP-ribosylation by PMP proceeds rapidly in apoptotic cells and consumes large amounts of NAD. Therefore, rapid apoptosis is accompanied by massive consumption of NAD in tissues. Necrosis can be explained by a decrease in ATP in tissues and a decrease in tissue turnover associated with a decrease in NAD (Ha, HC and Snyder, SH Proc. Natl. Acad. Sci. Vol. 96, No. 24, 13978-13982, 1999).

Nicotinic acid-related compounds are thought to suppress the consumption of NAD, which causes necrosis through inhibition of PMP activity. Therefore, it can be said that nicotinic acid-related compounds can effectively contribute to the suppression of internal hemorrhagic necrosis of the liver (with rapid pathological apoptosis) caused by acute hepatitis through PMP inhibitory action. .

As described above, clarifying the action point of the apoptosis-inhibiting action of the nicotinic acid-related compound has great significance of the present invention. Apoptosis is a complex system initiated by a variety of mechanisms, including physical effects. Therefore, it goes without saying that even the diseases caused by the same apoptosis have various causes. Therefore, an apoptosis inhibitor that is effective for a certain disease does not necessarily show the same effect for other apoptosis-causing diseases. In order to effectively utilize an apoptosis inhibitor for treatment or prevention, it is reasonable to clarify its action point and then select an apoptosis inhibitor having an action point at the cause of apoptosis. For these reasons, the utility of the present invention, which has found which apoptosis-related factor is affected by the inhibitory action of a nicotinic acid-related compound, is apparent.

“Apoptosis inhibitors” are caused by apoptosis containing the following diseases: It is effective for the treatment or prevention of diseases that occur. 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, Sjegren'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 viral), toxic or metabolic liver damage, Behcet's disease, aplastic anemia, AIDS, pancytopenia, Refractory anemia, cerebral infarction, glaucoma, cataract, salivary glanditis, radioactive disorder, ultraviolet ray injury, sun dermatitis, erythema multiforme, fixed drug eruption, GVHD, TEN, flat warts, herpes simplex, lupus erythematosus, lichen group Tissue reactions, tubular injury, respiratory infections, diabetes, arteriosclerosis, cerebral ischemia, myocardial infarction, dilated and hypertrophic cardiomyopathy, alopecia areata, or drug-induced alopecia are evident in the present invention. This is a new indication for antiapoptotic drugs that have been identified. Among them, fulminant hepatitis For secondary cirrhosis and secondary cirrhosis, post-onset treatment is possible by administration of the apoptosis inhibitor of the present invention, thereby achieving a high therapeutic effect that cannot be expected with known drugs.

Further, the present invention provides a compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, trimethyl-2-pyridone, and tributophan Or a pharmaceutically acceptable salt thereof as a main component. The present invention also relates to a method for promoting the production of IL-10, comprising a step of administering an effective amount of the compound. In addition, the present invention relates to the use of the compound in the production of an IL-10 production enhancer.

The present inventors have found that nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, tributofan, and methyl-2-pyridone further enhance IL-; 10 production in LPS-administered mice. It has an effect. In order for an anti-inflammatory drug to exert a strong anti-inflammatory effect, it must not only suppress the production of TNF-hi, which is an inflammatory cytokine, but also enhance the production of IL-10, which is an anti-inflammatory cytokine. Has been argued to be extremely effective. That is, the above-mentioned nicotinic acid-related compounds all have an anti-inflammatory drug, which has an action of promoting the production of an anti-inflammatory cytokine, IL-10, in addition to the action of producing inflammatory cytokines and the action of inhibiting PARP. It can be said that this is an ideal compound. In particular, trimethyl-2-pyridone had a strong effect on enhancing IL-10 production. The IL-10 production enhancer according to the present invention is useful for, for example, treating or preventing the following diseases.

Multiple sclerosis (TH1-dependent autoimmune disease), scleroderma, insulin-dependent diabetes mellitus, inflammatory bowel disease, Crohn's disease, sepsis, septic shock, hepatitis C, severe perforated abdominal smog, deformity Arthritis, rheumatoid arthritis

Administer the inflammatory site production inhibitor, PMP inhibitor, apoptosis inhibitor or IL-1.0 production enhancer of the present invention to humans for the treatment or prevention of the above-mentioned diseases. In the case of powders, granules, tablets, capsules, pills, liquids, etc. It can be administered orally or parenterally as injections, suppositories, transdermal absorbers, inhalants and the like. It can also be used externally as ointments and creams. Pharmaceutical preparations can be made by mixing, as necessary, pharmaceutical additives such as excipients, binders, wetting agents, disintegrating agents, and lubricants with the effective amount of the compound. . In the case of injections, they should be sterilized with a suitable carrier to produce the preparation. Dosage will also vary depending on disease state, route of administration, age or weight of patient, and will ultimately be at the discretion of a physician, but if administered orally to adults, usually 10 to 40ι ^ / 1 g / day Can be administered. It may be administered once or divided into several doses.

In the present invention, nicotinic acid-related compounds that have been shown to inhibit the production of inflammatory site power-in, inhibit apoptosis, or enhance the production of IL-10 are all compounds found in the living body. . The NAD cycle is known as a biosynthesis system for nicotinic acid-related compounds. That is, quinolinic acid obtained by synthesizing kynurenine from L-tributophan is converted to nicotinic acid mononucleotide. Further, the NAD cycle returns to nicotinic acid mononucleotide via nicotinic acid adenine nucleotide, NAD (nicotinamide adenine dinucleotide), nicotinamide and nicotinic acid. The following products are generated in the NAD cycle. These compounds are all metabolites of nicotinic acid found in urine and the like. ,

NAD: Produces NADP (nicotinamide adenine dinucleotide phosphate)

Nicotinic acid: produces trigonelline

Nicotinamide: 1-methylnicotinamide を Generates 1-methyl-2-pyridone-5-carboxamide

These compounds are regarded as having potential therapeutic effects on acute and chronic inflammation in vivo. Therefore, if the production of these nicotinic acid-related compounds in the living body can be promoted, the treatment of inflammatory symptoms in the living body Can be realized. Based on such a concept, the present inventors have clarified that a compound useful as an anti-inflammatory agent can be found in vivo by using the action of inducing the production of these nicotinic acid-related compounds as an index. did. That is, the present invention relates to a method for screening a compound useful as an anti-inflammatory agent, comprising the following steps.

a) a step of administering a candidate compound to a test animal

b) Any one selected from the group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, and 1-methyl-2-pyridone contained in the biological fluid of the test animal The step of measuring one compound

Hereinafter, the screening method according to the present invention will be specifically described. First, the screening of the present invention has a metabolic pathway similar to that of humans, and in the If process for nicotinic acid-related compounds, nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-potency lipoxamide, or 1 Use animals that produce -methyl-2-pyridone, etc. as test animals. For example, mice and rats are common experimental animals. You can also use egrets, dogs, dogs, bush, goats, goats, or sea lions. These test animals are given candidate compounds. Candidate compounds can be taken orally or parenterally, based on their absorption characteristics and the like. Parenteral administration includes intradermal injection and intraperitoneal administration. Usually, a suspension (or aqueous solution) of a candidate compound is prepared using a 0.5% aqueous solution of methylcellulose as a carrier (vehicle), and the suspension is orally administered to animals at the same dose. The dose of the candidate compound can be appropriately set, for example, in the range of generally 10 to 1000 mg, usually 50 to 500 mg per kg of body weight. As a control, a group to which only the carrier is administered is provided.

After the candidate compound is administered and breeding is continued, the target tissue is excised, and the change in the content of the nicotinic acid-related compound is observed over time. Alternatively, the candidate compound can be administered continuously and the effect can be observed. The target tissue means a tissue expected to have an anti-inflammatory action by the candidate compound. Specifically, in addition to organs such as the liver, lungs, and kidneys, body fluids such as serum and urine also contain the nicotinic acid-related compound. It is important as an object to be measured. Candidate compounds that effect changes in individual tissues are useful as compounds with specific anti-inflammatory activity in each tissue. On the other hand, compounds that increase the content of nicotinic acid-related compounds in body fluids can be expected to have systemic anti-inflammatory effects.

The measurement interval of the nicotinic acid-related compound after administration of the candidate compound can be appropriately set. For example, if rapid effects are expected, measurements should be taken at short time intervals immediately after administration. Conversely, if you want to confirm long-term effects, you should continue to observe changes in the levels of nicotinic acid-related compounds over time.

Samples for which nicotinic acid-related compounds are to be measured can be stored frozen. Therefore, samples collected over time can be frozen and stored, and after the collection is completed, the measurement of the nicotinic acid-related conjugate can be performed simultaneously.

Methods for measuring nicotinic acid-related compounds contained in various biological samples are known. For example, the NAD content is determined by the method of Nisselbaum (Nisselbaum, JS., And S. Green.As imle ultramicro method for determination of pyridine nucleotide in tissues.Anal.Biochem. 27, 212-217, 1969). can do. Measurement methods for other nicotinic acid-related compounds are also known (TA Tyler RR Shrago J. Liq. Chromatgr., 3,269, 1980, Zenzo Kitada, Masanari Inoue, Yoshihisa Tamaki, Makiko Imoda, Akiichi Hasuike, Michiko Sasaki, Kaoru Tanigawa, Nutrition and Food, 35, 121, 1982, K. ral, Presenilis A. Anal. Chem. 314, 479, 1983).

Candidate compounds that have significantly increased nicotinic acid-related compound concentrations compared to the respective nicotinic acid-related compound concentrations in each organ of the control group of mice without drug administration have anti-inflammatory effects Is selected as a compound having The selected candidate compounds are evaluated for their anti-inflammatory effect level and safety. For example, the level of the anti-inflammatory effect can be evaluated by comparing the anti-inflammatory effect in the acute inflammatory model of LPS-sensitized mice shown in the Examples.

For example, the following compound groups are used as candidate compounds for the screening of the present invention. Can be. Among them, compounds derived from the NAD cycle and compounds obtained by modifying a nicotinic acid-related compound by a synthetic method include many compounds capable of increasing the measured value of a nicotinic acid-related compound.

Cell extract,

Expression product of gene library,

Synthetic low molecular compounds,

Synthetic peptides,

Natural compounds

According to the screening method of the present invention, there is a possibility that a compound showing the same anti-inflammatory effect as a large dose of nicotinic acid can be obtained by a small dose. If such a compound is used as an anti-inflammatory drug, it can be replaced with a large dose of a nicotinic acid-related compound. Some nicotinic acid-related compounds may cause minor side effects, such as flushing of the face and pruritus, with large doses. The use of the compound of the present invention which can be obtained by screening makes it possible to avoid such side effects. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the inhibitory effect of various nicotinic acid-related compounds on TNF- production in blood of mice administered with LPS in vivo. Significant differences were tested by Student's t-test. p * 0.05 *, p * 0.01 **, p * 0.005 ***.

FIG. 2 is a graph showing the inhibitory effect of nicotinic acid on the production of TNF-, Mr and IL-12 in blood of LPS-administered mice.

FIG. 3 shows the effect of nicotinic acid and its derivatives on serum TNF-α levels in LPS shock mice.

FIG. 4 is a graph showing the inhibitory effect of trimethyl-2-pyridone-5-carboxamide on the production of TNF-spi in blood of LPS-inoculated mice. FIG. 5 is a graph showing the inhibitory effect of 1-methyl-2-pyridone-5-potorupoxamide on blood IL-12 production in LPS-inoculated mice. The column labeled 1M2P5C shows the results for trimethyl-2-pyridone-5-carboxamide.

FIG. 6 is a graph showing the inhibitory effect of methyl-2-pyridone-5-potassium lipoxamide on the production of IFNα in blood of LPS-inoculated mice. The column labeled 1M2P5C shows the results for 1-methyl-2-pyridone-5-carboxamide.

FIG. 7 is a graph showing the effect of 3-pyridinesulfonic acid on the production of TNF-spi in blood of LPS-inoculated mice.

FIG. 8 shows the effect of nicotinamide on the survival of LPS shock model mice.

FIG. 9 is a diagram showing the effects of nicotinamide and tritophan on survival of LPS shock model mice.

FIG. 10 is a graph showing the effect of post-treatment of nicotinamide on the survival of LPS shock model mice. The plot labeled NM shows the results for nicotinamide.

FIG. 11 shows the effect of pretreatment with trimethyl-2-pyridone-5-carboxamide on the survival of LPS shock model mice. The plot labeled 1M2P5C shows the results for 1-methyl-2-pyridone-5-carboxamide.

FIG. 12 is a diagram showing the therapeutic effect of trimethyl-2-pyridone-5-carboxamide on the survival of LPS shock model mice. The plot described as 1M2P5C shows the results for trimethyl-2-pyridone-5-carboxamide.

FIG. 13 is a photograph showing a hematoxylin-eosin-stained image of a tissue section in which the effect of nicotinamide or 1-methyl-2-pyridone-5-carboxamide administration on the liver of an LPS shock model mouse was examined. . A: control without LPS inoculation, B: mouse administered with carrier only 3 hours after LPS inoculation, C: nicotinamide administration 3 hours after LPS inoculation, D: methyl-2-pyridone- 3 hours after LPS inoculation. Administration of 5-carboxamide. FIG. 14 is a photograph showing an ssDNA-stained image of a tissue section in which the effect of nicotinamide or methyl-2-bilidon-5-carboxamide administration on the S pancreas of an LPS shock model mouse was examined. A ~! ) Is similar to Fig. 13.

Figure 15 shows the effects of nicotinamide and methyl-2-pyridone-5-carboxamide on DNA ladder formation, poly-ADP-ribosylation, and NAD levels in LPS shock model mice. It is. In the figure, NA indicates nicotinamide, and MPC indicates trimethyl-2-pyridone-5-carboxamide.

FIG. 16 shows the effect of nicotinic acid and its derivatives on serum IL-10 levels in LPS-administered mice. It shows the action of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide and tributofan to enhance IL-10 production.

FIG. 17 shows the effect of nicotinic acid and its derivatives on serum IL-10 levels in LPS-administered mice. In particular, the results for trimethyl-2-pyridone, whose action was strong, are shown. 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] Antiproducing action of nicotinic acid-related compound

The inhibitory effect of various nicotinic acid-related compounds on blood TNF-sporosis production was examined. A mouse (C57BL / 6, 8-week-old female) was orally administered with a carrier (0.5% CMC) (negative control) or each of the indicated compounds 5 minutes before LPS inoculation (300 mg / kg, n = 4). ).

LPS (dO / g / g) + D-gal (250 mg / kg) was intraperitoneally administered, and after 75 minutes, the mice were sacrificed and the serum was collected. TNF-serum in serum was assayed by ELISA. As a result, it was found that nicotinic acid, nicotinamide, 1-methyl-2-pyridone, and trimethyl-2-pyridone-5-carboxamide suppressed the production of TNF-hi (FIG. 1). In mice that were orally administered pyridine-3-sulfonic acid, an anti-niacin active compound, LPS administration increased the production of TNF- spleen. This can be regarded as a pseudo niacin-deficient animal model.

In addition, THP-1 cells were washed twice in a medium (deficient medium) containing 10% of dialyzed FCS (Gibco BRL) in RPMI1640 (Niken) excluding both nicotinamide and tributophan, and cultured in the same medium as it was did. Comparison of the amount of TNF- production in the culture supernatant 3.5 hours after the addition of 10 g / mL LPS gave the following results. Conversely, TNF-spleen production showed significantly higher secretion when cultured in a deficient medium.

Normal medium (without LPS) 15.7 ± 1.2 pg / mL

Normal medium (with LPS) 19 7 + 15 pg / mL

Defective medium (without LPS) 9 3 ± 2.9 pg / mL. Defective medium (with LPS) 7 2 7 ± 31 pg / mL

These facts suggest that nicotinamide and tributophan may have the effect of potentially suppressing cells from producing excessive TNF-spin in response to LPS stimulation.

From the above, it has been clarified that nicotinic acid-related compounds control site force-in production in acute inflammation and exert an anti-inflammatory effect. It is suggested that there is a mechanism in vivo that regulates the cytokine production response in acute inflammation by nicotinic acid-related compounds.

[Example 2] Anti-inflammatory action of nicotinic acid

The inhibitory effect of nicotinic acid on the blood TF-, IFN-, and IL-12 production in LPS-shock mice in vivo was examined. Using a mouse (C57BL / 6, 8-week-old female), a carrier (PBS) (P-aggressive control) or nicotinic acid was orally administered 5 minutes before LPS inoculation. LPS (50 g / kg) + D-gal (250 mg / kg) was intraperitoneally administered, the mice were sacrificed 75 minutes after TNF, and 5 hours after IL-12 and INF, and serum was collected. TNF, IL-12, and IFN in serum were assayed by ELISA. ELISA has the following The sales kit was used. As a result, nicotinic acid was found to suppress the production of a series of inflammatory cytokines, TNF-, IL-12, and IFNa, which appear in serum during LPS shock in in vivo mice ( Figure 2 ) .

Kit used for ELISA:

Mouse TNF-EUSA Kit (D Systems Cat. No.MTAOO)

Mouse IL-12 ELISA kit (Genzyme Cat. No. Part 80-4244-03)

Mouse IFN-EL ELISA kit (R & D Systems Cat. No.MIFOO)

[Example 3] Anti-TNF-α producing action of nicotinic acid and its derivatives

The inhibitory effect of nicotinic acid and its derivatives on blood TNF- production was examined. Mice (C57BL / 6, 9-week-old female) were orally administered with a carrier (0.5% CMC) (negative control) or each of the indicated compounds 5 minutes before LPS inoculation (300 mg / kg, n = 4) . LPS (10 mg / kg) and D-gal (250 mg / kg) were intraperitoneally administered, and the mice were sacrificed 75 minutes later, and the serum was collected. TNF-serum in serum was assayed by ELISA. Nicotinic acid, nicotine amide, and trimethyl-2-pyridone suppressed the production of TNF-hi (Figure 3).

[Example 4] Effect of urinary metabolites of nicotinic acid on TNF-producing activity

The inhibitory effect of methyl-2-pyridone-5-carboxamide on the production of TNF-blood in blood of LPS-shock mice in vivo was examined. Using mice (C57BL / 6, 9-week-old female) (n2-4), 5 minutes before LPS inoculation, carrier (0.5% CMC) (P-aggressive control) or methyl-2-pyridone-5-carboxami Was orally administered. LPS (50 g / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and the mice were sacrificed 75 minutes later, and the serum was collected. TNF-α in serum was analyzed by ELISA (R & D, Quantikine). As a result, it was found that trimethyl-2-pyridone-5-carboxamide suppressed the production of sperm expressed in serum upon LPS shock in in vivo mice (FIG. 4).

[Example 5] Inhibitory effect of nicotinic acid urinary metabolite on IL-12 production

The inhibitory effect of trimethyl-2-bilidon-5-carboxamide on blood IL-12 production in LPS shocked mice in vivo was examined. Mouse (C57BL / 6, 9-week-old female) (n = 4) The vehicle (0.5% CMC) (negative control) or trimethyl-2-pyridone-5-carboxamide was orally administered 5 minutes before LPS inoculation. LPS (50 / g / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and 5 hours later, the mice were sacrificed and serum was collected. Serum IL-12 was analyzed by ELISA. As a result, it was found that trimethyl-2-pyridone-5-carboxamide suppressed the production of IL-12 that appeared in serum in LPS shock of in vivo mice (FIG. 5).

[Example 6] Effect of urinary metabolite of nicotinic acid on IFN production

The inhibitory effect of 1-methyl-2-pyridone-5-carboxamide on blood IFN production in LPS shocked mice in vivo was examined. Using mice (C57BL / 6, 9-week-old female) (n2-4), 5 min before LPS inoculation, vehicle (0.5% CMC) (negative control) or 1-methyl-2-pyridone-5-carboxamide Was orally administered. LPS (50 zg / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and 5 hours later, the mice were sacrificed and serum was collected. IFN in serum was analyzed by ELISA. As a result, it was found that methyl-2-pyridone-5-carboxamide suppressed the production of IFNy appearing in the serum upon LPS shock in in vivo mice (FIG. 6).

[Example 7] TNF-production enhancement effect of anti-niacin compound

The effect of an anti-niacin compound on blood TNF-splen production of LPS-shock mice in vivo was examined. Mice (C57BL / 6, 7-week-old female) (n = 3) were orally administered vehicle (0.5% CMC) (negative control) or 3-pyridinesulfonic acid 1.5 hours before LPS inoculation. LPS (50 µg / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and after 75 minutes, the mice were sacrificed and serum was collected. Serum in serum was assayed by ELISA. As a result, 3-pyridinesulfonic acid enhanced the production of TNF o: expressed in serum during LPS shock in in vivo mice (FIG. 7).

[Example 8] Effect of nicotinamide on survival of LPS shock model mouse

The effect of nicotinamide on the survival of LPS shock model mice was examined. Five minutes before LPS administration, 500 mg / kg (200 ^ 1.o.) Orally (C57BL / 6, 12-week-old female) (n = 5). LPS [(E. coli, 50 ig / kg) + D-gal (250 mg / kg)] was administered intraperitoneally. Thereafter, mice that survived over time were counted. As a result, nicotinamide was found to protect against LPS shock (Figure 8).

[Example 9] Effect of nicotinamide and tributofan on survival of LPS shock model mouse

The effects of nicotinamide and tryptophan on the survival of LPS shock model mice were examined. 5 minutes before LPS administration, vehicle (0.5% CMC) or each compound (500 mg / kg for nicotinamide, lg / kg for tryptophan) (200〃1 po) mouse (C57BL / 6, 12 weeks) (N = 5). LPS [(E. coli, 50 g / kg) + D-gal (250 mg / kg)] was administered intraperitoneally. Subsequently, the mice that survived over time were counted. As a result, nicotinamide and tributophan were found to protect against LPS shock (Fig. 9).

[Example 10] Effect of post-treatment of nicotinamide on survival of LPS shock model mouse

The effect of nicotinamide (NM) after LPS inoculation on the survival of LPS shock model mice was examined. LPS [(E. coli, 50 g / kg) + D-gal (250 mg / kg)] is intraperitoneally administered to mouse (C57BL / 6, U-12 week old female) (n = 5) 5 min Before or 3 hours later, vehicle (0.5% CMC) or nicotinamide 1000 mg / kg (200/1 po) was orally administered. Thereafter, mice that survived over time were counted. As a result, nicotinamide was found to protect against LPS shock even when administered 3 hours after LPS inoculation (Fig. 10).

[Example 11] Effect of pretreatment with trimethyl-2-pyridone-5-carboxamide on survival of LPS shock model mice

The effect of pre-LPS inoculation of methyl-2-pyridone-5-carboxamide (1M2P5C) on the survival of LPS shock model mice in vivo was examined. LPS [(E. coli, 5 0 ig / kg) + D-gal (250 mg / kg)] intravenously to mice (C57BL / 6, 11-week-old female) (n = 5) 5 minutes before injection of carrier (0.5% CMC) or 1M2P5C was administered at 1000 mg / kg (200 zl po). Thereafter, mice that survived over time were counted. The results showed that 1M2P5C protected LPS shock even before LPS vaccination (Fig. 1

1) o

[Example 12] Therapeutic effect of trimethyl-2-pyridone-5-carboxamide on survival of LPS shock model mice

The therapeutic effect of trimethyl-2-pyridone-5-carboxamide (1M2P5C) on the survival of LPS shock model mice in vivo was examined. LPS [(E. coli, 50 zg / kg) + D-gal (250 mg / kg)] was intraperitoneally administered to M. us (C57BL / 6, 11-week-old female) (n = 5) for 3 hours Later, vehicle (0.5 ° / _D CMC) or 1M2P5C 1000 mg / kg (200/1 po) was administered. Thereafter, mice that survived over time were counted. As a result, it was found that 1M2P5C showed a therapeutic effect to protect against LPS shock even when administered after LPS inoculation (Fig. 1

2) o

Example 13: Effect of nicotinic acid-related compound administration on congestive necrosis and apoptosis in liver of mice treated with LPS

The effects of nicotinic acid-related compounds on congestive necrosis of the liver caused by LPS administration to mice were examined histopathologically and biochemically. Using mice (C57BL / 6 about 10-week-old female), healthy mice (control) (vehicle (0.5% CMC) administered) and LPS-administered mice, E. coli ζ0 μg / g + D-galactosamine 250 mg / kg, ip (volume 200〃1)), in the drug administration group, nicotinamide and methyl-2-pyridone-5-carboxamide were selected as nicotinic acid-related compounds, and nicotinamide lg / kg and 1-methyl, respectively. -Oral administration of 2-pyridone-5-carboxamide lg / kg 3 hours after LPS inoculation,

Approximately 5.5 hours after LPS inoculation, the animals were sacrificed and dissected, and the liver was collected. The cells were fixed with a formalin buffer (for histopathological examination), a portion was immediately frozen on dry ice, and then stored frozen at -80 ° C (for biochemical examination). Liver was cut out for histopathological examination (outside After (left lobe), tissue sections were prepared, and then hematoxylin and eosin (HE) staining and ssDNA staining were performed. In the ssDNA method, anti-Single Stranded DNA (ssDNA) and egret polyclonal antibody (DAK0 product code A4506) were used as the primary antibody, and detection was performed using the DAK0 LSAB2 kit / HRP-Universal (catalog NO.K0677). used. The protocol using the primary antibody was performed in the same manner as in the following literature.

Narese I, et al., Antibody against single-stranded DNA detects both pr ogranuned cell death and drug- induced apoptosis.Histochemistry 101: 73-7 8, 1994.

oba ashi S, et al., Detection of DNA fragmentation in human breast cancer tissue by an antibody specific to single stranded DNA.Breast Cance r 5: 47-52, 1998.

Detection was performed according to the attached protocol.

The findings are summarized in Table 1. No abnormalities were observed in the items in the table in healthy mice. On the other hand, in the vehicle (carrier) administration group, severe congestion (partially hemorrhage), eosinophils (unicellular necrosis), nucleus enrichment and disintegration, vacuoles, and some large and small glass droplets in hepatocytes were observed. In addition, ssDNA staining revealed a considerable number of hepatocytes that diffused brown. In the nicotinamide-administered group (M in the table), eosinophils and vitreous droplets were found in only a few places, and microvesicles and porosity were also found in some places, but almost all were “normal” (control) ) And the same. Although a very small number of positive cells were observed by ssDNA staining, they were significantly suppressed compared to the Vehicle group. The trimethyl-2-pyridone-5-carboxamide-administered group (MPC in the table) showed slightly more nuclear findings and glass droplets and a smaller number of ssDNA-positive cells than the nicotinamide group. In this group as well, the apoptosis is significantly suppressed as compared with the Vehicle group. The photographs of the above microscope are shown in Fig. 13 (stained by hematoxylin zeodin (HE)) and Fig. 14 (stained by the ssDNA method). table 1

[Mouse C57BL / 6]

Shower

Staining method H.E.ssDNA

Pathological findings a Clear picture of small vegetables e Nucleus enrichment h Porosity k Apotosis

b ¾g blood (bleeding) f Nuclear decay i Glass drop

c dark cells g vacuoles j cell infiltration

d Eosinophil (single cell necrosis) Overall judgment b c d e f h 1 j k

Normal mice ±

Carrier +++ one ++ ++ ++ + ++ ++ ++++

NA Sat-Ten + ± ± + + + +

PC ± + + + + + Sat + ++ Note) 1. [Degree of lesion] —: No physician: Extremely mild +: Mild ++: Moderate +++: 髙

++++: Extreme i¾ biochemical examination of these liver tissues was also performed. Items examined were liver MA ladder formation, poly-ADP-ribosylation, and changes in NAD content. The DNA ladder formation was detected by preparing genomic DNA from the moon spleen of each mouse and following the Apoptosis Ladder Detection Kit Wako of the mouse. Poly-ADP-ribosylation was detected by Western blot using an Anti-poly (ADP-Ribose) polyclonal antibody (rabMt, polyclonal). The NAD amount was determined by the method of Nisselbaum. (Nisselbaum, J.S., and S. Green. A simple ultramicro method for determination of pyridine nucleotides in tissues. Anal. Biochem. 27, 212-217 (1969)). Figure 15 summarizes the results of the biochemical studies.

Biochemical studies indicate that DNA ladder formation and poly-ADP-ribosylation are significantly suppressed by nicotine amide and 1-methyl-2-pyridone-5-carboxamide, and that apoptosis is suppressed It turned out that it was. The decrease in NAD content associated with apoptosis was also suppressed by administration of these drugs, but interestingly, nicotinamide increased NAD content. This is because nicotinamide is a precursor of NAD, This may be due to the large amount of NAD biosynthesized in the liver due to the administration of NAD ₍ considering that the drug was administered 3 hours after LPS inoculation, the above results indicate that nicotinamide and trimethyl-2 Nicotinic acid-related compounds, such as -pyridone-5-carboxamide, have an anti-apoptotic action in addition to an anti-inflammatory action, and are presumed to have a protective action against tissue damage by their combined actions.

[Example 14] Enhancing effects of various nicotinic acid-related compounds on IL-10 production The enhancing effects of various nicotinic acid-related compounds on blood IL-10 production were examined. The mice (C57BL / 6, 18-week-old female) were orally administered with a carrier (0.5% CMC) (または control) or various nicotinic acid-related compounds 5 minutes before LPS inoculation (300 mg / ml). kg, n = 3).

LPS (50 zg / kg) + D-gal (250 mg / kg) was intraperitoneally administered, and 3 hours later, the mice were sacrificed and serum was collected. Serum IL-10 was assayed by ELISA (Genzyme). As a result, all the compounds studied (nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, tributanphan, trimethyl-2-pyridone) further increased IL-10 production in mice treated with LPS. It was found that it had an effect of enhancing (Fig. 16). The effect of trimethyl-2-pyridone was particularly strong (Fig. 17). Kakura I for industrial use, raw

According to the present invention, the inhibitory effect of a nicotinic acid-related compound on the production of inflammatory cytokines associated with apoptosis associated with IL-12, IFN-α, or TNF-like activity was revealed. Also, the inhibitory effect of nicotinic acid-related compounds on PARP was clarified. Furthermore, it has been found that a nicotinic acid-related compound has an activity of enhancing the production of an anti-inflammatory cytokine, IL-10.

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 presumed to be responsible for various disease states Have been. For example, fulminant hepatitis is caused by hepatocyte apoptosis. Therefore, inhibitors of apoptosis-related factors are useful for 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 determine what is causing apoptosis and to select drugs that act directly on the cause to increase the therapeutic effect. Since the target factor is clear, it is possible to carry out an effective treatment after identifying such a target factor.

The apoptosis-related factor inhibitor of the present invention is not only useful as an apoptosis inhibitor, but is also expected to have new utility by clarifying its mechanism of action. That is, for example, the inflammatory site force-in (IL-12, IFN-α, and TNF-H) in the present invention is an apoptosis-related factor and also plays an important role in various inflammatory conditions. I have. Therefore, the inflammatory site force production inhibitor of the present invention can also be used for alleviating inflammatory symptoms associated with these cytokines. In addition, since PMP has an action of inducing tissue necrosis, for example, by consuming NAD, an inhibitor of PMP may be used as a necrosis inhibitor. In addition, it has been found that nicotinic acid-related compounds have an activity of enhancing the production of an anti-inflammatory cytokine, IL-10. This further enhances the significance of controlling inflammatory symptoms using nicotinic acid-related compounds. Therefore, these drugs include anti-inflammatory drugs, therapeutic drugs 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 prevention and treatment of Alzheimer's and the like.

As described above, the target factor of the inhibitor of the production of inflammatory cytokines, the promoter of the production of anti-inflammatory cytokines, or the inhibitor of apoptosis according to the present invention is clear. Therefore, it provides a more rational and effective treatment or prevention for apoptosis-related diseases.

Claims

Claims Any compound selected from the group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, and methyl-2-pyridone, or a pharmaceutically acceptable salt thereof. An agent for inhibiting the production of ink-leukin 12 and Z or inulin-feron y containing a salt as a main component. A tumor necrosis factor α production inhibitor containing 1-methyl-2-pyridone-5-carboxamide or a pharmaceutically acceptable salt thereof as a main component; A tumor necrosis factor α production inhibitor comprising nicotinamide or a pharmaceutically acceptable salt thereof, and tributophan or a pharmaceutically acceptable salt thereof as a main component. A compound selected from the group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, 1-methyl-2-pyridone, and tributophan, or a pharmaceutically acceptable compound thereof An agent for enhancing the production of hinyu leukin 10 containing a salt as a main component. A compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and 1-methyl-2-pyridone, or a pharmaceutically acceptable salt thereof. Inhibitor of apoptosis contained as an ingredient. Any compound selected from the group consisting of nicotinic acid, nicotinamide, trimethyl-2-pyridone-5-carboxamide, and 1-methyl-2-pyridone, or a pharmaceutically acceptable salt thereof. An agent for preventing or treating hepatitis, which is contained as a main component. A compound selected from the group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, and trimethyl-2-pyridone, or a pharmaceutically acceptable salt thereof. An inhibitor of poly-ADP-ribose-polymerase, which is contained as an ingredient. An agent for preventing or treating a disease caused by necrosis, comprising the inhibitor of poly-ADP-ribose-polymerase according to claim 7 as a main component. 9. The preventive or therapeutic agent according to claim 8, wherein the disease is acute hepatitis. 0. The preventive or therapeutic agent according to claim 8, wherein the hepatitis is fulminant hepatitis.

1. A method for screening a compound useful as an anti-inflammatory agent, comprising the following steps:

a) a step of administering a candidate compound to a test animal

b) a group consisting of nicotinic acid, nicotinamide, 1-methyl-2-pyridone-5-carboxamide, and 1-methyl-2-bilidon contained in the tissue and / or biological fluid of the test animal Measuring any compound selected from: and

• c) a step of selecting a candidate compound that increases the measured value of b) compared to a control 2. An anti-inflammatory agent comprising, as a main component, a compound obtainable by the method of claim 11.