JP4825977B2 - Cholesterol homeostasis-related gene transcriptional activity regulator through FXR activation - Google Patents

Description

  The present invention relates to a FXR (farnesoid X receptor) transcriptional activity regulator useful as a therapeutic agent for hyperlipidemia and a therapeutic agent for intrahepatic cholestasis.

  Hyperlipidemia is caused by hereditary or inappropriate diet and lack of exercise, resulting in excess blood lipids, causing arteriosclerosis and causing various adult diseases such as ischemic heart disease.

  On the other hand, nuclear receptors are transcription factors that are activated by the binding of ligands and control the expression of target genes, and play an important role in various physiological phenomena. In 1999, the ligand of FXR, a member of the nuclear receptor, was a bile acid molecule such as chenodeoxycholic acid (CDCA), and it was shown that the transcriptional activity of FXR is enhanced by CDCA (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, but this conversion is inhibited by the end product bile acids. FXR activated by bile acid is responsible for this feedback control by suppressing gene expression of cholesterol 7α hydroxylase (CYP7A1), which is a rate-limiting enzyme. Mice deficient in FXR were found to increase blood cholesterol, bile acids, triglycerides and the like (Sinal et al., Cell 102, pp731-744, 2000). Bile acid promotes cholesterol absorption from the intestinal tract. In FXR-deficient mice, the conversion of cholesterol to bile acid is promoted, but the increase in bile acid biosynthesis promotes cholesterol absorption from the intestinal tract. Conceivable. It has also been reported that FXR activation causes a reduction in serum triglycerides (Maloney et al., J. Med. Chem. 43, pp.2971-2974, 2000). Therefore, the activator of FXR is expected to have an effect of lowering serum triglyceride and cholesterol and is a promising candidate for a prophylactic and therapeutic drug for hyperlipidemia.

  FXR activators are also useful as therapeutic agents for intrahepatic cholestasis (Liu et al., J. Clin. Invest, pp.1678-1687, 2003). Intrahepatic cholestasis is a disease in which the flow of bile is stagnant in the liver and hepatocytes are destroyed. FXR is responsible for excretion of bile acids into bile and promotes the expression of bile salt export pump gene (BSEP), an important gene for enterohepatic circulation. It is thought that it promotes the discharge of blood and improves intrahepatic cholestasis.

  Since chenodeoxycholic acid (CDCA), which is known as a strong FXR physiological ligand, is metabolized to toxic lithocholic acid, it is difficult to use it as a medicine. However, compounds with a structure different from bile acids are expected to prevent or treat hyperlipidemia that is not metabolized to toxic substances.

  As a cholesterol lowering agent, an HMG-CoA reductase inhibitor is highly evaluated clinically. However, for patients with familial hypercholesterolemia with high serum cholesterol levels or patients with coronary artery disease, it is not effective enough to lower the serum cholesterol level to the target low level. There is a need for a more potent antihyperlipidemic agent that is effective in such patients.

  It is considered that the main mechanism of action of the HMG-CoA reductase inhibitor is an indirect action of enhancing expression of the LDL receptor. However, its action is limited, and it is also true that the serum cholesterol concentration has stopped dropping. Therefore, by developing a pharmaceutical having a mechanism of action different from that of an HMG-CoA reductase inhibitor, it can be expected to show a strong serum cholesterol lowering effect alone or in combination with an HMG-CoA reductase inhibitor. An activator of FXR has a mechanism of action different from that of HMG-CoA, and thus can be a useful cholesterol-lowering drug. Furthermore, since the activator of FXR has a function of lowering serum triglycerides, it is expected as a prophylactic and therapeutic agent for hyperlipidemia.

The present invention has been completed by discovering that ginkgolic acid has an FXR activating effect using a reporter gene assay. That is, the present invention is based on the fact that the compound (1) represented by the following formula (1) has an FXR activating action.

In the formula, R ₁ represents a functional group selected from a hydrogen atom, an acyl group, an alkyl group, and an aryl group; R ₂ represents a functional group selected from a hydrogen atom, an alkyl group, and an aryl group;
R ₃ represents a functional group selected from an alkyl group, an alkynyl group, and an alkenyl group.
This compound includes a case where it is a pharmaceutically acceptable salt such as sodium, potassium, magnesium, calcium and the like.

The result of the reporter gene assay which showed the concentration-dependent FXR activation effect of each test compound is shown. It is the figure which showed the influence on the gene expression which uses HepG2 cell and is transcription-controlled by FXR of each test compound. CYP7A1 and SHP mRNAs were measured as genes.

  The compound represented by the chemical formula and a salt thereof can be administered orally or parenterally when used as a medicine. That is, it can be administered orally in the form of commonly used dosage forms, such as tablets, capsules, syrups, suspensions, etc., or in the form of solutions such as solutions, emulsions, suspensions thereof It can be administered parenterally in the form of injections. It can also be administered rectally in the form of a suppository. The appropriate dosage forms described above can also be prepared by blending the active compound with the usual acceptable carriers, excipients, binders, stabilizers and the like. In addition, when used as an injection, an acceptable buffer, solubilizer, isotonic agent and the like can be added. The dosage of the compound of the present invention is usually 1-50 mg / kg body weight, preferably 5-30 mg / kg body weight. The subject of administration is a mammal, usually a human.

Evaluation of FXR activating compound by reporter gene assay FXR binds to a ligand and promotes transcription of a downstream gene by binding to an FXR binding sequence (FXRE). A plasmid (reporter plasmid) in which the 3'-side 201 bp of the CMV promoter and enhanced yellow fluorescent protein (EYFP) gene are linked under the FXR binding sequence (FXRE) and an enhanced cyan fluorescent protein (ECFP) gene in the SV40 promoter (Internal standard measurement plasmid), plasmids having RXR gene and plasmids having FXR gene as plasmids for expression of nuclear receptors were introduced into COS7 cells. The test compound was added to the culture medium of the cells, and after about 40 hours, the medium was removed and washed with PBS, and then the fluorescence of EYFP and ECFP was measured. In order to correct the efficiency of introducing a plasmid into a cell, the fluorescence value of EYFP was corrected with the fluorescence value of ECFP measured as an internal standard. That is, FXR transcriptional activity was evaluated based on the value of (EYFP fluorescence value) / (ECFP fluorescence value).

In formula (1), R ₁ = H, R ₂ = 8-pentadecenyl ginkgolic acid 15: 1 (compound of formula (2)), and in formula (1), R ₁ = H, R ₂ = 10 Treatment with -heptadecenyl, ginkgolic acid 17: 1 (compound of formula (3)), promoted FXR transcriptional activity (FIG. 1). These activations were comparable or higher than the same concentration of CDCA. These compounds increased the expression of the reporter gene by activating FXR without causing RXR activation.

Quantitative RT-PCR
The suppression of the expression level of CYP7A1 mRNA and the increase in the expression level of small heterodimer partner (SHP) mRNA were measured by quantitative PCR. The SHP gene has been shown to be transcriptionally promoted by FXR activation, and can be used as an index for FXR activation in the same way as CYP7A1. HepG2, a cultured cell derived from liver cancer, was cultured in DMEM medium containing 10% FCS. After exchanging the medium with phenol red-free DMEM containing 10% activated carbon-treated FCS, 6 hours later, the medium was replaced with phenol red-free DMEM containing 10% activated carbon-treated FCS and cultured. 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 the TaqMan PCR method. Each sample was corrected for RNA concentration using 18s rRNA.

  As a result of comparison with DMSO 0.1% treatment used as a control, ginkgolic acid suppressed the expression of CYP7A1 mRNA by about 70% at 30 νμM and increased the expression of SHP mRNA by about 2-fold. This revealed that these compounds cause FXR activation and regulate the expression level of downstream genes.

The above compounds are expected to lower serum triglycerides and cholesterol by regulating the transcriptional activity of FXR. Further, these compounds are compounds having a structure different from bile acids and are not metabolized to toxic lithocholic acid. Therefore, it can be expected as an effective prophylactic and therapeutic drug for hyperlipidemia. In addition, activation of FXR promotes transcription of BSEP and is effective as a preventive and therapeutic agent for intrahepatic cholestasis.

Claims (8)

An activator of FXR containing ginkgolic acid represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient .

In the formula, R ₁ represents hydrogen , R ₂ represents hydrogen , and R ₃ represents an alkyl group or an alkenyl group . The activator of FXR according to claim 1, wherein the ginkgolic acid is a compound represented by the following formula (2) or formula (3).

A cholesterol-lowering drug containing ginkgolic acid represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the formula, R ₁ represents hydrogen , R ₂ represents hydrogen , and R ₃ represents an alkyl group or an alkenyl group . The cholesterol-lowering drug according to claim 3, wherein the ginkgolic acid is a compound represented by the following formula (2) or formula (3).

A hyperlipidemia preventive or therapeutic agent comprising ginkgolic acid represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the formula, R ₁ represents hydrogen , R ₂ represents hydrogen , and R ₃ represents an alkyl group or an alkenyl group . 6. The hyperlipidemia preventive or therapeutic drug according to claim 5, wherein the ginkgolic acid is a compound represented by the following formula (2) or formula (3).

A therapeutic agent for preventing or treating intrahepatic cholestasis comprising ginkgolic acid represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the formula, R ₁ represents hydrogen , R ₂ represents hydrogen , and R ₃ represents an alkyl group or an alkenyl group . The intrahepatic cholestasis preventive or therapeutic drug according to claim 7, wherein the ginkgolic acid is a compound represented by the following formula (2) or formula (3).

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