JP2007015926A - Therapeutic agent for hepatitis c - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a therapeutic agent for hepatitis C, which has improved activity strength, migration in blood, selectivity, reduction in adverse effect, etc., in comparison with cyclosporine A. <P>SOLUTION: The therapeutic agent for hepatitis C comprises a compound (I) represented by general formula (I) or its salt as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for hepatitis C. More specifically, the present invention relates to an RNA replication inhibitor of hepatitis C virus (hereinafter referred to as HCV) replicon. In particular, the present invention relates to a therapeutic agent for hepatitis C comprising the following compound (I) or a salt thereof as an active ingredient.

  The estimated number of carriers of HCV is about 1.7 billion (about 3%) worldwide, and about 1.5 million in Japan. Even in the interferon (IFN) -ribavirin (Virazole) combination therapy, which is the first treatment option, the efficacy rate is 40% for all types of HCV, especially in IFN-resistant viruses (genotype 1b) that are common in Japan. The effective rate is only 15-20%, while the frequency of side effects is high. It is difficult to completely eliminate viruses with current therapies. If chronic hepatitis cannot be cured, it will certainly progress to cirrhosis (30%) and hepatocellular carcinoma (25%). In Europe and the United States, hepatitis C has become a major indication for liver transplantation. However, the frequency of HCV reappearance is not low in the liver after transplantation. Therefore, there is a great social need for new drugs that improve both efficacy and safety, have higher antiviral effects, and can suppress hepatitis C.

HCV is a virus having plus-strand RNA as a gene, and is classified into the Flaviviridae family from the analysis of the gene base sequence. According to Fields Virology fourth edition, D. Knipe et al ed., Philadelphia, Lippincott Williams & Wilkins 2001, 1127-1161, the existence of HCV was assumed in the 1970s. The era called hepatitis B virus lasted for a long time. In 1989 Choo QL et al., Science 244, 359-362 (1989) cloned a part of this viral gene from the serum of an experimental laboratory animal and named it HCV because its cDNA sequence was identified and confirmed. . Genomic RNA has an open reading frame encoding a polyprotein consisting of about 9,500 bases and about 3,000 amino acid residues. In the polyprotein, the structural protein forming the virus particle is located on the N-terminal side, and is arranged in the order of core protein C, two types of envelope proteins E1, E2, and nonstructural protein NS1. The polyprotein is processed, thereby creating the proteins that make up the replication equipment necessary for viral genomic RNA replication.

Cyclosporin A (Sandimmun) is used for organ transplantation as an immunosuppressant. M. Thali et al., Nature 372, 363-365 (1994) show that cyclosporin A inhibits the interaction between human immunodeficiency virus type 1 (HIV-1) virus particle-forming protein and cyclophilin A -1 activity is reported. As the reported that cyclosporin A has a direct anti-HCV activity, K. Inoue et al, 6 th International Symposium on Hepatitis C and Related Virus. 3-6 June (2000) Bethesda, MD , USA. Et al. However, to date, no other group has supported the findings.

There are reports from M. Berenguer et al., J. Hepatol 32, 673-684 (2000) et al. That clinical use of cyclosporin A, an immunosuppressive agent, promotes the growth of HCV in transplant patients. In addition, G. Everson, Liver Transplantation 10 Suppl 1: S19-27 (2002) reports that there was no difference in the reinfection rate when cyclosporin A and other immunosuppressive agents were compared for reinfection in liver transplantation. There is also.

The above-mentioned K. Inoue et al., J. Gastroenterol 38, 567-572 (2003) suggests that IFN and cyclosporin A combination therapy is more effective than IFN monotherapy On the other hand, in the study of S. Cotler et al., J. Clin. Gastroenterol , 36 352-355 (2003) that conducted a follow-up of this report, the combination therapy with IFN and cyclosporin A is the current pegylated IFN There is a report that the effect is inferior compared with the combination treatment with Ribavirin. However, this report shows relatively good treatment results in cases with high blood trough levels of cyclosporin A, although only a few cases.

  For these reasons, there has been a demand for a therapeutic agent for hepatitis C that is improved in comparison with cyclosporin A, for example, in terms of activity, blood translocation, selectivity, and reduction of side effects.

  The compound (I) of the present invention is represented by the following formula, and is a compound having a chemical structure different from that of cyclosporin A, particularly constituent amino acids. For example, according to the method described in JP-A-5-271267, a mold (Stachybotrys chartaum No. 19392: It can be produced from the deposit number FERM BP-3364).

  Suitable salts of compound (I) are pharmaceutically acceptable ordinary non-toxic salts, such as salts with inorganic bases (for example, alkali metal salts such as sodium salt and potassium salt, alkaline earth such as calcium salt and magnesium salt). Metal salts, ammonium salts), salts with organic bases (for example, triethylamine salt, diisopropylethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N′N′-dibenzylethylenediamine) Organic amine salts such as salts), inorganic acid addition salts (eg, hydrochloride, hydrobromide, sulfate, phosphate, etc.), organic carboxylic acids or sulfonic acid addition salts (eg, formate, acetate, Trifluoroacetate, maleate, tartrate, gluconate, fumarate, methanesulfonate, benzenesulfonate, Toluenesulfonic acid salt, etc.), basic or acidic amino acid (e.g., arginine, aspartic acid, such as salts with glutamic acid and the like), and salts or acid addition salts with bases.

  In the present invention, the HCV therapeutic agent comprising compound (I) or a salt thereof as an active ingredient is oral; sublingual; buccal; nasal cavity; respiratory organ; extraintestinal (intradermal, organ, subcutaneous, intradermal, intramuscular, Intraocular or intraocular infusion including intraarticular, central vein, hepatic vein, peripheral vein, lymph, heart, artery, periocular injection); intraocular, ocular structure, ocular drip; eustachian tube Inner ear including ear, papillary air chamber, external and internal auditory canal, tympanic membrane, middle ear, cochlear spiral ganglion, labyrinth, etc .; intestine; rectum; vagina; ureter; organic or inorganic carrier suitable for administration to the bladder or It can be contained as a mixture with excipients and used, for example, in the form of pharmaceutical preparations in solid, semi-solid or liquid form. For intrauterine and perinatal indications, such as maternal blood vessels or maternal organs including the uterus, cervix, vagina, fetus, fetus, newborn and allied tissue and amniotic membrane, umbilical cord, umbilical artery and vein, placenta, etc. Within the space and preferably outside the intestine, these pathways vary depending on the patient's condition.

  While it is possible for Compound (I) or a salt thereof to be administered alone as a therapeutic agent, it is also desirable to utilize this as part of a prescription formulation. The “HCV therapeutic agent” of the present invention contains at least one, or some suitable organic or inorganic carrier or excipient, or a mixture with other pharmacological therapeutic agents, eg, solid, semi-solid, The active ingredient can be used in the form of a pharmaceutical preparation in the form of a solid or liquid, and the active ingredient can be mixed with, for example, a pharmaceutically normal non-toxic carrier to prepare granules, tablets, pellets, troches, capsules, suppositories. Liquid form such as powders, creams, ointments, aerosols, powders for injection, liquids for injections, emulsions or suspensions; oral ingestants; eye drops; any other form suitable for use Can do. Then, if necessary, adjuvants such as stabilizers, thickeners, wetting agents, curing agents, coloring agents, fragrances or buffering agents, and any other conventional additives can be added to the above preparation.

  In the “HCV therapeutic agent” of the present invention, Compound (I) or a salt thereof is added in an amount sufficient to obtain a desired HCV inhibitory effect depending on the course or condition of the disease.

  The therapeutically effective dose of compound (I) or a salt thereof will vary depending on the age and condition of each patient to be treated, the type of formulation to which the therapeutic agent is administered, the mode in which the administration takes place, and the stage or administration of the disease Usually, the therapeutic agent is included in an amount of 0.1 to 90% of the weight of the total composition, depending on the interval. For HCV suppression, an amount of 0.0001 mg to 50 mg, or preferably 0.001 to 25 mg per kg body weight per day for parenteral administration and 0.001 per day per kg body weight for enteral administration An amount of from 100 to 100 mg, preferably from 0.01 to 60 mg can be given. However, these dosages may have to exceed their upper limits in order to obtain a therapeutic result.

  Compound (I) or a salt thereof may contain stereoisomers such as optical isomers and geometric isomers by one or more asymmetric carbon atoms and double bonds, and all such isomers. And mixtures thereof are also included in the present invention.

  Compound (I) or a salt thereof also includes solvates (eg, hydrates, ethanolates, etc.).

  Compound (I) or a salt thereof includes both crystalline and amorphous forms.

  Compound (I) or a salt thereof includes a prodrug form.

  The present invention relates to the use of HCV therapeutic agents in the conditions described throughout the specification. To this end, the present invention includes all advertisements, labeling, packaging, informational materials, merged advertisements, product details, advertising materials, text, brochures, magazines, books, etc., or conversation, fax, telephone, photography, radio, video , Information exchange media by television, film, Internet, e-mail, etc., information means by computer, proposals for clinical trials, research protocols for clinical trials using therapeutic agents for HCV inhibitory action, etc.

  The patent specifications and publications cited herein are hereby incorporated by reference.

V. Lohmann et al., Science 285,110-113 (1999) created a human hepatoma cell line (Huh7) into which subgenomic HCV RNA molecules were introduced and introduced it into the cells, but subgenomic HCV RNA replicated at a high rate. Have reported that. The replication mechanism of subgenomic HCV RNA in these cell lines is thought to be very close to that of full-length HCV RNA genome in hepatocytes infected with HCV. Therefore, the assay method at the cell level using Huh-7 cells into which subgenomic HCV RNA has been introduced is the basis of the method for evaluating the activity of compound (I) or a salt thereof that inhibits HCV replication according to the present invention.

  In order to show the usefulness of the compound (I) of the present invention or a salt thereof, test examples thereof are shown below.

HCV replicon replication inhibitory activity
The inhibitory effect on HCV replicon replication was evaluated by directly quantifying replicon RNA purified from cells using an RNA extraction column by real-time RT-PCR using Taq-Man chemistry. The evaluation method is a modification of Lohmann et al., Science 285: 110 (1999) and Takeuchi et al, Gastroenterology 116: 636-642 (1999). Details are described below.

Test compound

1) Addition of drugs to cells
HCV replicon-expressing cells (Kishine et al., BBRC 293: 993-999 (2002)) in a 6-well microtiter plate (Corning) D-MEM medium containing 5% fetal calf serum and G418 300 μg / ml per well It was prepared and seeded at 1.5 × 10 (6) in 2 ml. After culturing at 37 ° C. in the presence of 5% CO 2 for 16 hours, the medium was replaced with the D-MEM medium in which the test compound was dissolved.

2) RNA extraction from cells After 2 days of culture, total RNA was extracted from the cells according to the protocol of RNA extraction column RNeasy Mini (Qiagen), and the absorbance ratio (by Beckman) was used for the absorbance ratio (Beckman). 260 nm / 280 nm) was measured to determine the total RNA amount.

3) Quantification of replicon RNA content by real-time RT-PCR
Real-time RT-PCR was performed using a combination of a primer that amplifies part of the sequence of the HCV gene and a complementary probe (all manufactured by Takara Shuzo).
The RNA extracted in 2) was diluted with RNase inhibitor-containing NF water to 25 ng / μl and dispensed in 2 μl aliquots onto a 384 well PCR plate. The reaction solution for RT-PCR was mixed with TaqMan Ez RT-PCR Core Reagent (Applied Biosystems) according to the protocol, and 8 μl was added.
RT-PCR was performed using an ABI PRISM 7900HT sequence detection system (Applied Biosystems) to quantify HCV replicon RNA. For the calibration curve, synthetic HCV RNA was diluted 10 times. As a negative control, the reaction was carried out without addition of RNA.

4) Quantification of endogenous control RNA As an endogenous control, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was quantified by real-time RT-PCR as in 3). Primer and probe used TaqMan GAPDH control reagent of Applied Biosystems. The calibration curve was used after diluting the control RNA attached to the kit 10 times. As a negative control, the reaction was carried out without addition of RNA.

Cytotoxicity assay
HCV replicon-expressing cells are cultured under the same conditions as the evaluation system for replicon replication inhibition activity, the cells are dispersed with 0.05% Trypsin-0.5 mM EDTA, stained with 0.1% erythrosin-PBS (-), and the number of living cells is counted under a microscope. I counted.

Test result For the value obtained by correcting the quantitative value of the replicon RNA with the quantitative value of the endogenous control (GAPDH), the concentration of the drug showing 50% RNA level is calculated by plotting with respect to the control (drug-free group-DMSO only) The EC50 value was used. The cytotoxicity was calculated by plotting the concentration of the drug showing a 50% level with respect to the control of the number of viable cells (no drug added group-DMSO only), and was defined as the CC50 value.
The results are shown in Table 1.

From the results of the above test examples, it was shown that the compound (I) of the present invention or a salt thereof has anti-hepatitis C virus activity.

Claims (4)

The following general formula (I):
The therapeutic agent for hepatitis C which uses the compound (I) shown by these, or its salt as an active ingredient.   Use of compound (I) or a salt thereof according to claim 1 for the manufacture of a medicament for preventing and / or treating hepatitis C.   A method for preventing and / or treating hepatitis C, which comprises administering the compound (I) or a salt thereof of claim 1 to a human or an animal. A commercially available packaged product consisting of a written statement describing that the compound (I) or a salt thereof according to claim 1 is or should be used for the prevention and / or treatment of hepatitis C.