WO2005097097A1 - Agent for controlling cholesterol homeostasis-associated gene transcription activity mediated by fxr activation - Google Patents

Agent for controlling cholesterol homeostasis-associated gene transcription activity mediated by fxr activation Download PDF

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WO2005097097A1
WO2005097097A1 PCT/JP2005/003602 JP2005003602W WO2005097097A1 WO 2005097097 A1 WO2005097097 A1 WO 2005097097A1 JP 2005003602 W JP2005003602 W JP 2005003602W WO 2005097097 A1 WO2005097097 A1 WO 2005097097A1
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group
alkyl
fxr
aryl
hydrogen
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PCT/JP2005/003602
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French (fr)
Japanese (ja)
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Toru Kawanishi
Takuo Suzuki
Tomoko Mogami
Kazuhide Inoue
Takao Hayakawa
Yoshinori Asakawa
Toshihiro Hashimoto
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Japan Health Sciences Foundation
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Priority to JP2006511917A priority Critical patent/JP4825977B2/en
Publication of WO2005097097A1 publication Critical patent/WO2005097097A1/en

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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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 a FXR (farnesoid X receptor) transcription activity regulator useful as a therapeutic agent for hyperlipidemia and intrahepatic cholestasis.
  • FXR farnesoid X receptor
  • Hyperlipidemia causes an excess of lipids in the blood due to hereditary or inadequate diet and lack of exercise, causing arteriosclerosis to lead to various adult diseases such as ischemic heart disease.
  • nuclear receptors are activated by the binding of ligands, are transcription factors that control the expression of target genes, and play an important role in various physiological phenomena.
  • it is a bile acid molecule, including the ligand cadenodeoxycholic acid (CDCA) of FXR, a member of the nuclear receptor, and it was shown that CDCA enhances the transcriptional activity of FXR (Makishima, M. et al "Science, 284, pp.1362—1365, 1999; Parks DJ et al., Science, 284, pp.1365-1368, 1999; Wang'H. Et al., Mol. Cell, 3, pp.543-553, 1999)
  • Bile acids are synthesized from cholesterol, and this conversion is suppressed by the final product, bile acids.
  • FXR which is activated by bile acids, controls this feedback by suppressing the gene expression of cholesterol 7a-hydroxylase (CYP7A1), the rate-limiting enzyme.
  • CYP7A1 cholesterol 7a-hydroxylase
  • FXR-deficient mice increased levels of blood cholesterol, bile acids, triglycerides, etc. became apparent (Sinai et al., Cell 102, pp731-744, 2000). Since bile acids promote intestinal cholesterol absorption, in FXR-deficient mice, the conversion of cholesterol to bile acids is promoted, but the increase in bile acid biosynthesis promotes intestinal cholesterol absorption. it is conceivable that.
  • FXR activator is expected to have serum triglyceride and cholesterol lowering effects, and is a promising candidate for the prevention and treatment of hyperlipidemia. It becomes.
  • FXR activators are also useful as therapeutic agents for intrahepatic cholestasis (Liu et al, J. Am.
  • Intrahepatic cholestasis is a disease in which the flow of bile in the liver is disrupted and hepatocytes are destroyed.
  • FXR promotes the expression of the bile salt export pump gene (BSEP), which is a gene responsible for the excretion of bile acids into bile and is important for enterohepatic circulation. It is thought to promote acid excretion and improve intrahepatic cholestasis.
  • BSEP bile salt export pump gene
  • an HMG-CoA reductase inhibitor As a cholesterol-lowering agent, an HMG-CoA reductase inhibitor has been highly evaluated clinically. However, in patients with familial hypercholesterolemia with high serum cholesterol levels or patients with coronary artery disease, they are not sufficiently effective to lower the target serum cholesterol level. There is a need for a more potent therapeutic agent for hyperlipidemia that is effective for such patients.
  • HMG-CoA reductase inhibitors are the indirect action of enhancing LDL receptor expression.
  • the effect is limited, and it is also true that the concentration of serum cholesterol stops decreasing. Therefore, by developing a drug with an action mechanism different from that of the HMG-CoA reductase inhibitor, it can be expected that a potent serum cholesterol-lowering effect will be exhibited when used alone or in combination with the HMG-CoA reductase inhibitor.
  • FXR activators may be useful cholesterol-lowering drugs because they have a different mechanism of action from HMG-CoA. Furthermore, since the activator of FXR has a function of lowering serum triglyceride, it is expected to be a therapeutic drug for preventing and treating hyperlipidemia.
  • gincholic acid has an FXR activating effect using a reporter gene atsey It was completed by discovering that. That is, the present invention is based on the fact that the compound (1) represented by the following formula (1) has FXR activity.
  • R is a hydrogen atom, an acyl group, an alkyl group, or an aryl group.
  • R represents a hydrogen atom, an alkyl group, or an aryl group.
  • R represents a functional group selected from an alkyl group, an alkynyl group, and an alkenyl group.
  • This compound is a pharmaceutical compound of sodium, potassium, magnesium, calcium, etc.
  • FIG. 1 shows the results of a reporter gene assay showing the concentration-dependent FXR activity of each test compound.
  • FIG. 2 is a diagram showing the effect of each test compound on the expression of a gene that is transcriptionally regulated by FXR using HepG2 cells.
  • BEST MODE FOR CARRYING OUT THE INVENTION In which mRNA of CYP7A1 and SHP were measured as genes
  • the compound represented by the chemical formula and a salt thereof can be administered orally or parenterally when using it as a medicine. That is, it can be orally administered in the form of commonly used dosage forms, for example, tablets, capsules, syrups, suspensions, etc., or in the form of solutions, emulsions, suspensions, etc. It can be administered parenterally in the form of injections. It can also be administered rectally in the form of suppositories.
  • the above-mentioned suitable dosage forms are prepared by mixing the active compound with an acceptable usual carrier, excipient, binder, stabilizer or the like. Can be manufactured. When used as an injection, an acceptable buffer, solubilizing agent, isotonic agent and the like can be added.
  • the dose of the compound of the present invention is generally 1 to 50 mg / kg of body weight, preferably 5 to 30 mg / kg of body weight.
  • the administration subject is a mammal, usually a human.
  • FXR promotes transcription of downstream genes by binding to ligand and binding to FXR binding sequence (FXRE).
  • FXRE FXR binding sequence
  • the plasmid RXD as the plasmid for nuclear receptor expression
  • a test compound was added to the culture medium of the cells, and about 40 hours later, the medium was removed and the cells were washed with PBS, and then the fluorescence of EYFP and ECFP was measured.
  • the fluorescence value of EYFP was corrected using the fluorescence value of ECFP measured as an internal standard. That is, the transcription activity of FXR was evaluated based on the value of (fluorescence value of EYFP) I (fluorescence value of ECFP).
  • R H-pentadecenyl, gincholic acid 15: 1 (compound of the formula (2)), and
  • CYP7A1 mRNA expression and the increase in expression of small heterodimer partner (SHP) mRNA were measured by quantitative PCR. It has been clarified that the transcription of the SHP gene is promoted by the activation of FXR, and it can be used as an index of FXR activation like CYP7A1.
  • HepG2 a cultured cell derived from liver cancer, was cultured in DMEM medium containing 10% FCS. The medium was replaced with phenol red-free DMEM containing 10% activated carbon-treated FCS, and after 6 hours, the medium was replaced with a test substance and phenol red-free DMEM containing 10% activated carbon-treated FCS, followed by culturing.
  • the above compounds are expected to reduce serum triglycerides and cholesterol by regulating the transcription activity of FXR.
  • these compounds are compounds having structures different from bile acids, and are not metabolized to toxic lithocholic acid. Therefore, it can be expected as an effective drug for preventing and treating hyperlipidemia.
  • the activation of FXR promotes the transcription of BSEP and is also effective as a preventive and therapeutic drug for intrahepatic cholestasis.

Abstract

It is intended to provide a preventive/remedy for hyperlipemia having an elevated efficacy. This object is achieved by using the following compound (1) having an FXR activity. wherein R1 represents a functional group selected from among hydrogen, acyl, alkyl and aryl; R2 represents a functional group selected from among hydrogen, alkyl and aryl; and R3 represents a functional group selected from among alkyl, alkynyl and alkenyl.

Description

FXR活性ィ匕を介したコレステロールホメォスタシス関連遺伝子転写活性 調節剤  Regulator of cholesterol homeostasis-related gene transcription activity through FXR activity
技術分野  Technical field
[0001] 本発明は、高脂血症治療剤及び肝内胆汁うっ滞症治療剤として有用な FXR ( farnesoid X receptor)転写活性調節剤に関する。  The present invention relates to a FXR (farnesoid X receptor) transcription activity regulator useful as a therapeutic agent for hyperlipidemia and intrahepatic cholestasis.
背景技術  Background art
[0002] 高脂血症は、遺伝性、もしくは不適切な食事や運動不足が原因で、血中の脂質が 過剰となり、動脈硬化を引き起こして虚血性心疾患などの様々な成人病をもたらす。  [0002] Hyperlipidemia causes an excess of lipids in the blood due to hereditary or inadequate diet and lack of exercise, causing arteriosclerosis to lead to various adult diseases such as ischemic heart disease.
[0003] 一方核内レセプターは、リガンドの結合により活性ィ匕され、標的遺伝子の発現を制 御する転写因子であり、様々な生理現象に重要な役割を果たしている。 1999年に、 核内レセプターの一員である FXRのリガンドカ ケノデォキシコール酸（CDCA)をは じめとする胆汁酸分子であり、 CDCAにより FXRの転写活性が増強されることが示さ れた（Makishima, M. et al" Science, 284, pp.1362— 1365, 1999; Parks D. J. et al., Science, 284, pp.1365- 1368, 1999; Wang'H. et al., Mol. Cell, 3, pp.543- 553, 1999)  [0003] On the other hand, nuclear receptors are activated by the binding of ligands, are transcription factors that control the expression of target genes, and play an important role in various physiological phenomena. In 1999, it is a bile acid molecule, including the ligand cadenodeoxycholic acid (CDCA) of FXR, a member of the nuclear receptor, and it was shown that CDCA enhances the transcriptional activity of FXR (Makishima, M. et al "Science, 284, pp.1362—1365, 1999; Parks DJ et al., Science, 284, pp.1365-1368, 1999; Wang'H. Et al., Mol. Cell, 3, pp.543-553, 1999)
[0004] 胆汁酸はコレステロールより合成されるが、この転換は最終産物である胆汁酸によ り抑制される。胆汁酸により活性ィ匕される FXRは律速酵素であるコレステロール 7 aヒ ドロキシラーゼ (CYP7A1)の遺伝子発現を抑制することにより、このフィードバック制 御を担っている。 FXRを欠損したマウスでは、血中コレステロール、胆汁酸、トリグリセ リド等の上昇をきたすことが明ら力となった (Sinai et al., Cell 102, pp731-744, 2000)。 胆汁酸は腸管力 のコレステロール吸収を促進するため、 FXR欠損マウスでは、コレ ステロールの胆汁酸への転換は促進されるものの、胆汁酸生合成量の増加により、 腸管からのコレステロール吸収が促進されたためと考えられる。また、 FXRの活性ィ匕 が血清トリグリセリドの低下を引き起こすという報告もされている (Maloney et al, J. Med. Chem. 43, pp.2971-2974, 2000)。したがって、 FXRの活性化剤には血清トリグ リセリド、コレステロール低下作用が期待され、高脂血症の予防治療薬の有力な候補 となる。 [0004] Bile acids are synthesized from cholesterol, and this conversion is suppressed by the final product, bile acids. FXR, which is activated by bile acids, controls this feedback by suppressing the gene expression of cholesterol 7a-hydroxylase (CYP7A1), the rate-limiting enzyme. In FXR-deficient mice, increased levels of blood cholesterol, bile acids, triglycerides, etc. became apparent (Sinai et al., Cell 102, pp731-744, 2000). Since bile acids promote intestinal cholesterol absorption, in FXR-deficient mice, the conversion of cholesterol to bile acids is promoted, but the increase in bile acid biosynthesis promotes intestinal cholesterol absorption. it is conceivable that. It has also been reported that the activation of FXR causes a decrease in serum triglyceride (Maloney et al, J. Med. Chem. 43, pp.2971-2974, 2000). Therefore, FXR activator is expected to have serum triglyceride and cholesterol lowering effects, and is a promising candidate for the prevention and treatment of hyperlipidemia. It becomes.
[0005] また、 FXR活性化剤は肝内胆汁うっ滞症の治療薬としても有用である (Liu et al, J.  [0005] Furthermore, FXR activators are also useful as therapeutic agents for intrahepatic cholestasis (Liu et al, J. Am.
Clin. Invest, pp.1678-1687, 2003)。肝内胆汁うっ滞症は肝臓内で胆汁の流れが滞 り、肝細胞が壊れていく病気である。 FXRは、胆汁酸の胆汁中への排泄を担い腸肝 循環に重要な遺伝子である bile salt export pump遺伝子 (BSEP)の発現を促進するこ とから、 FXRの活性化剤は肝内からの胆汁酸の排出を促し、肝内胆汁うっ滞を改善 すると考えられる。  Clin. Invest, pp. 1678-1687, 2003). Intrahepatic cholestasis is a disease in which the flow of bile in the liver is disrupted and hepatocytes are destroyed. FXR promotes the expression of the bile salt export pump gene (BSEP), which is a gene responsible for the excretion of bile acids into bile and is important for enterohepatic circulation. It is thought to promote acid excretion and improve intrahepatic cholestasis.
[0006] 強力な FXRの生理的リガンドとして知られるケノデォキシコール酸 (CDCA)は毒性 をもつリトコール酸に代謝されるため、医薬品としての活用は困難である。しかし、胆 汁酸とは異なる構造を持つ化合物は，毒性物質へ代謝されな!、高脂血症予防治療 薬として期待される。  [0006] Chenodeoxycholic acid (CDCA), which is known as a powerful physiological ligand of FXR, is metabolized to toxic lithocholic acid, making it difficult to use as a pharmaceutical. However, compounds with a structure different from bile acid are not metabolized to toxic substances! They are expected to be used as preventive and therapeutic drugs for hyperlipidemia.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0007] コレステロール低下剤としては、 HMG— CoA還元酵素阻害剤が臨床上高い評価 を受けている。しかしながら、高い血清コレステロール値を持つ家族性高コレステロ一 ル血症の患者、あるいは冠動脈疾患をもつ患者に対し、目標とする低レベルの血清 コレステロール値まで下げるには十分な効果を有しておらず、このような患者にも有 効な、より強力な高脂血症治療剤が望まれている。 [0007] As a cholesterol-lowering agent, an HMG-CoA reductase inhibitor has been highly evaluated clinically. However, in patients with familial hypercholesterolemia with high serum cholesterol levels or patients with coronary artery disease, they are not sufficiently effective to lower the target serum cholesterol level. There is a need for a more potent therapeutic agent for hyperlipidemia that is effective for such patients.
課題を解決するための手段  Means for solving the problem
[0008] HMG— CoA還元酵素阻害剤の主な作用メカニズムは間接的な LDL受容体の発現 増強作用にあると考えられている。し力しながら、その作用には限界があり、血清コレ ステロールの濃度に下げ止まりがあることも事実である。そこで HMG— CoA還元酵 素阻害剤とは異なる作用メカニズムの医薬品を開発する事により、単独または HMG CoA還元酵素阻害剤との併用により強力な血清コレステロール低下作用を示すこ とが期待できる。 FXRの活性化剤は HMG— CoAとは異なる作用メカニズムを持った め有用なコレステロール低下薬となりうる。さらに FXRの活性化剤には血清トリグリセ リドを低下させる働きがあることからも、高脂血症予防治療薬として期待が持たれる。 [0008] It is believed that the main mechanism of action of HMG-CoA reductase inhibitors is the indirect action of enhancing LDL receptor expression. However, the effect is limited, and it is also true that the concentration of serum cholesterol stops decreasing. Therefore, by developing a drug with an action mechanism different from that of the HMG-CoA reductase inhibitor, it can be expected that a potent serum cholesterol-lowering effect will be exhibited when used alone or in combination with the HMG-CoA reductase inhibitor. FXR activators may be useful cholesterol-lowering drugs because they have a different mechanism of action from HMG-CoA. Furthermore, since the activator of FXR has a function of lowering serum triglyceride, it is expected to be a therapeutic drug for preventing and treating hyperlipidemia.
[0009] 本発明はレポータージーンアツセィを用いギンコール酸が FXR活性化作用をもつ ことを発見する事により完成に至った。すなわち本発明は下記の式 (1)に示したィ匕合 物（1)が FXR活性ィ匕作用をもつことに基づくものである。 [0009] In the present invention, gincholic acid has an FXR activating effect using a reporter gene atsey It was completed by discovering that. That is, the present invention is based on the fact that the compound (1) represented by the following formula (1) has FXR activity.
[化 1]  [Chemical 1]
Figure imgf000004_0001
Figure imgf000004_0001
OR1  OR1
[0010] 式中、 Rは水素原子、ァシル基、アルキル基、ァリール基力 選ばれる官能  [0010] In the formula, R is a hydrogen atom, an acyl group, an alkyl group, or an aryl group.
基、 Rは水素原子、アルキル基、ァリール基力 選ばれる官能基を示し、  Group, R represents a hydrogen atom, an alkyl group, or an aryl group.
2  2
Rはアルキル基、アルキニル基、アルケニル基から選ばれる官能基を示す。  R represents a functional group selected from an alkyl group, an alkynyl group, and an alkenyl group.
3  Three
本化合物は、ナトリウム、カリウム、マグネシウム、カルシウム等の薬学的  This compound is a pharmaceutical compound of sodium, potassium, magnesium, calcium, etc.
に許容される塩である場合を含む。  Including the case where the salt is acceptable.
図面の簡単な説明  Brief Description of Drawings
[0011] [図 1]各被験化合物の濃度依存的 FXR活性ィ匕効果を示したレポータージーンアツセ ィの結果を示す。  FIG. 1 shows the results of a reporter gene assay showing the concentration-dependent FXR activity of each test compound.
[図 2]HepG2細胞を用い、各被験化合物の FXRにより転写制御を受ける遺伝子発現 への影響を示した図である。遺伝子として CYP7A1及び SHPの mRNAの測定を行った 発明を実施するための最良の形態  FIG. 2 is a diagram showing the effect of each test compound on the expression of a gene that is transcriptionally regulated by FXR using HepG2 cells. BEST MODE FOR CARRYING OUT THE INVENTION In which mRNA of CYP7A1 and SHP were measured as genes
[0012] 化学式で表される化合物およびその塩は、これを医薬として用いるにあたり経口的 または非経口的に投与することができる。すなわち通常用いられる投与形態、例えば 錠剤、カプセル剤、シロップ剤、懸濁液等の形で経口的に投与することができ、ある いはその溶液、乳剤、懸濁液等の液剤の形にしたものを注射の形で非経口投与する ことができる。坐剤の形で直腸投与することもできる。また、前記の適当な投与剤形は 許容される通常の担体、賦形剤、結合剤、安定剤などに活性化合物を配合すること により製造することができる。また、注射剤として用いる場合には許容される緩衝剤、 溶解補助剤、等張剤等を添加することもできる。また、本発明の化合物の投与量は、 通常 1一 50mg/体重 kgであり、好ましくは 5— 30mg/体重 kgである。投与対象は、哺乳 動物であり、通常ヒトである。 [0012] The compound represented by the chemical formula and a salt thereof can be administered orally or parenterally when using it as a medicine. That is, it can be orally administered in the form of commonly used dosage forms, for example, tablets, capsules, syrups, suspensions, etc., or in the form of solutions, emulsions, suspensions, etc. It can be administered parenterally in the form of injections. It can also be administered rectally in the form of suppositories. In addition, the above-mentioned suitable dosage forms are prepared by mixing the active compound with an acceptable usual carrier, excipient, binder, stabilizer or the like. Can be manufactured. When used as an injection, an acceptable buffer, solubilizing agent, isotonic agent and the like can be added. In addition, the dose of the compound of the present invention is generally 1 to 50 mg / kg of body weight, preferably 5 to 30 mg / kg of body weight. The administration subject is a mammal, usually a human.
実施例 1  Example 1
[0013] レポータージーンアツセィによる FXR活性化化合物の評価  [0013] Evaluation of FXR Activating Compounds by Reporter Gene Atsey
FXRはリガンドと結合し FXR結合配列 (FXRE)に結合することにより下流の遺伝子 の転写促進を行う。 FXR結合配列 (FXRE)下に CMV promoterの 3'側 201 bpと enhanced yellow fluorescent protein (EYFP)遺伝子をつな！/、だプラスミド (レポータ ~~プフス^ド)と SV40 promoter に ennanced cyan fluorescent protein (ECFP)遺 子 をつな!/、だプラスミド (内部標準測定用プラスミド)、核内受容体発現用プラスミドとして RXR遺伝子を有するプラスミド及び FXR遺伝子を有するプラスミドの 4種を COS7細 胞に導入した。この細胞の培養液中に被験化合物を添加し、約 40時間後に培地を 除き PBSで washした後 EYFPと ECFPの蛍光を測定した。細胞内へのプラスミドの導 入効率等の補正を行うために、内部標準として測定した ECFPの蛍光値で EYFPの 蛍光値の補正を行った。すなわち FXRの転写活性は (EYFPの蛍光値） I (ECFPの 蛍光値)の値により評価を行った。  FXR promotes transcription of downstream genes by binding to ligand and binding to FXR binding sequence (FXRE). Connect the 3'-side 201 bp of the CMV promoter and the enhanced yellow fluorescent protein (EYFP) gene under the FXR binding sequence (FXRE)! / Connect the ennanced cyan fluorescent protein (ECFP) gene to the SV40 promoter and the plasmid (reporter ~~ puffs ^)! /, The plasmid RXD as the plasmid for nuclear receptor expression Four types of plasmids, one having the gene and the other having the FXR gene, were introduced into COS7 cells. A test compound was added to the culture medium of the cells, and about 40 hours later, the medium was removed and the cells were washed with PBS, and then the fluorescence of EYFP and ECFP was measured. In order to correct the introduction efficiency of the plasmid into the cells, the fluorescence value of EYFP was corrected using the fluorescence value of ECFP measured as an internal standard. That is, the transcription activity of FXR was evaluated based on the value of (fluorescence value of EYFP) I (fluorescence value of ECFP).
[0014] 式 (1)中、 R =H - pentadecenylであるギンコール酸 15:1(式 (2)の化合物)、及び、  [0014] In the formula (1), R = H-pentadecenyl, gincholic acid 15: 1 (compound of the formula (2)), and
1 、 R =8  1, R = 8
2  2
式 (1)中、 R =H  In equation (1), R = H
1 、 R =10-heptadecenylであるギンコール酸 17:1(式 (3)の化合物)処理に 2  1, treatment with gincholic acid 17: 1 (compound of formula (3)) where R = 10-heptadecenyl
より、 FXR転写活性の促進が引き起こされた（図 1)。これらの活性化は同濃度の CDCAと同等もしくは高い率であった。なお、これらの化合物は RXRの活性ィ匕は引き 起こさず FXRを活性ィ匕する事により、レポータージーンの発現を上昇させた。  This led to the promotion of FXR transcriptional activity (Figure 1). These activations were at the same or higher rates as CDCA at the same concentration. In addition, these compounds did not cause activation of RXR but activated FXR, thereby increasing the expression of the reporter gene.
[化 2]  [Formula 2]
Figure imgf000005_0001
Figure imgf000005_0001
[化 3]
Figure imgf000006_0001
実施例 2 [Formula 3]
Figure imgf000006_0001
Example 2
[0015] 定量的 RT— PCR  [0015] Quantitative RT—PCR
CYP7A1 mRNAの発現量抑制及び small heterodimer partner(SHP) mRNAの発現 量増加を定量的 PCRで測定した。 SHP遺伝子は FXRの活性ィ匕により転写促進される ことが明らかとなっており、 CYP7A1と同様に FXR活性ィ匕の指標として使用できる。肝 癌由来培養細胞である HepG2を 10% FCSを含む DMEM培地で培養した。培地を 10% 活性炭処理 FCSを含むフエノールレッド不含 DMEMに交換後、さらに 6時間後に被験 物質及び 10%活性炭処理 FCSを含むフエノールレッド不含 DMEMに置換し培養を行 つた。 24時間後細胞を回収し、 RNeasy kit (QIAGEN)を用いて RNAの抽出を行った。 得られた RNAを用い、 TaqMan PCR法により CYP7A1及び SHP mRNAの定量を行った 。各試料は 18s rRNAを用いて RNA濃度の補正を行った。  The suppression of CYP7A1 mRNA expression and the increase in expression of small heterodimer partner (SHP) mRNA were measured by quantitative PCR. It has been clarified that the transcription of the SHP gene is promoted by the activation of FXR, and it can be used as an index of FXR activation like CYP7A1. HepG2, a cultured cell derived from liver cancer, was cultured in DMEM medium containing 10% FCS. The medium was replaced with phenol red-free DMEM containing 10% activated carbon-treated FCS, and after 6 hours, the medium was replaced with a test substance and phenol red-free DMEM containing 10% activated carbon-treated FCS, followed by culturing. After 24 hours, the cells were collected, and RNA was extracted using RNeasy kit (QIAGEN). Using the obtained RNA, CYP7A1 and SHP mRNA were quantified by TaqMan PCR. Each sample was corrected for RNA concentration using 18s rRNA.
[0016] コントロールとして使用した DMSO 0.1%処理との比較の結果、ギンコール酸は 30 υ μ Μで CYP7A1 mRNAの発現を約 70%抑制し、 SHP mRNAの発現を約 2倍に上昇させ た。これより、これら化合物が FXRの活性化を引き起こし下流の遺伝子の発現量を調 整することが明らかとなった。  [0016] As a result of comparison with DMSO 0.1% treatment used as a control, gincholic acid suppressed the expression of CYP7A1 mRNA by about 70% and increased the expression of SHP mRNA about 2-fold at 30 µm. This revealed that these compounds caused FXR activation and regulated the expression level of downstream genes.
産業上の利用可能性  Industrial applicability
[0017] 上記化合物は、 FXRの転写活性調節を行うことにより血清トリグリセリド、コレステロ ール低下作用が期待される。また、これら化合物は胆汁酸とは異なる構造をもつ化 合物であり、毒性を持つリトコール酸に代謝されることが無い。従って、効果的な高脂 血症予防治療薬として期待できる。また、 FXRの活性ィ匕は BSEPの転写を促進し肝 内胆汁うっ滞症の予防治療薬としても有効である。 [0017] The above compounds are expected to reduce serum triglycerides and cholesterol by regulating the transcription activity of FXR. In addition, these compounds are compounds having structures different from bile acids, and are not metabolized to toxic lithocholic acid. Therefore, it can be expected as an effective drug for preventing and treating hyperlipidemia. In addition, the activation of FXR promotes the transcription of BSEP and is also effective as a preventive and therapeutic drug for intrahepatic cholestasis.

Claims

請求の範囲  The scope of the claims
下記の式（1)で示されるギンコール酸又はその誘導体を用いることを特徴とする FX Rの活性化法。  A method for activating FX R, comprising using gincholic acid represented by the following formula (1) or a derivative thereof.
Figure imgf000007_0001
Figure imgf000007_0001
OR1  OR1
式中 Rは水素、ァシル基、アルキル基、ァリール基力 選ばれる官能基、 R Where R is hydrogen, acyl, alkyl, aryl. Functional group selected, R
1 2 は水素、アルキル基、ァリール基力 選ばれる官能基を示し、 Rはアルキル基、  1 2 represents hydrogen, an alkyl group, or an aryl group. R represents an alkyl group,
3  Three
アルキニル基、アルケニル基力 選ばれる官能基を示す。 Alkynyl group, alkenyl group Shows the functional group selected.
下記の式（1)で示されるギンコール酸又はその誘導体を有効成分として含有するコ レステロール低下薬。  A cholesterol-lowering drug containing, as an active ingredient, gincholic acid or a derivative thereof represented by the following formula (1)
[化 2]  [Formula 2]
Figure imgf000007_0002
Figure imgf000007_0002
OR1  OR1
式中 Rは水素、ァシル基、アルキル基、ァリール基力 選ばれる官能基、 R Where R is hydrogen, acyl, alkyl, aryl. Functional group selected, R
1 2 は水素、アルキル基、ァリール基力 選ばれる官能基を示し、 Rはアルキル基、  1 2 represents hydrogen, an alkyl group, or an aryl group. R represents an alkyl group,
3  Three
アルキニル基、アルケニル基力 選ばれる官能基を示す。 Alkynyl group, alkenyl group Shows the functional group selected.
下記の式（1)で示されるギンコール酸又はその誘導体を有効成分として含有する高 脂血症予防治療薬。
Figure imgf000008_0001
A preventive and therapeutic drug for hyperlipidemia, comprising as an active ingredient gincholic acid represented by the following formula (1) or a derivative thereof.
Figure imgf000008_0001
OR1  OR1
式中 Rは水素、ァシル基、アルキル基、ァリール基力 選ばれる官能基、 R Where R is hydrogen, acyl, alkyl, aryl. Functional group selected, R
1 2 は水素、アルキル基、ァリール基力 選ばれる官能基を示し、 Rはアルキル基、  1 2 represents hydrogen, an alkyl group, or an aryl group. R represents an alkyl group,
3  Three
アルキニル基、アルケニル基力 選ばれる官能基を示す。 Alkynyl group, alkenyl group Shows the functional group selected.
下記の式（1)で示されるギンコール酸又はその誘導体を有効成分として含有する 肝内胆汁うっ滞症予防治療薬。  An agent for preventing or treating intrahepatic cholestasis, comprising as an active ingredient gincholic acid or a derivative thereof represented by the following formula (1).
[化 4]  [Formula 4]
Figure imgf000008_0002
Figure imgf000008_0002
OR1  OR1
式中 Rは水素、ァシル基、アルキル基、ァリール基力 選ばれる官能基、 R Where R is hydrogen, acyl, alkyl, aryl. Functional group selected, R
1 2 は水素、アルキル基、ァリール基力 選ばれる官能基を示し、 Rはアルキル基、  1 2 represents hydrogen, an alkyl group, or an aryl group. R represents an alkyl group,
3  Three
アルキニル基、アルケニル基力 選ばれる官能基を示す。 Alkynyl group, alkenyl group Shows the functional group selected.
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