WO2005032576A1

WO2005032576A1 - Therapeutic agent for hepatitis c

Info

Publication number: WO2005032576A1
Application number: PCT/JP2004/014901
Authority: WO
Inventors: Kunitada Shimotono; Michinori Kohara
Original assignee: Astellas Pharma Inc.; Tokyo Metropolitan Organization For Medical Research
Priority date: 2003-10-06
Filing date: 2004-10-01
Publication date: 2005-04-14
Also published as: TW200514553A; JP2007015926A

Abstract

A preventive and/or therapeutic agent for hepatitis C which contains a compound (I) represented by the following general formula (I): or a salt thereof as an active ingredient.

Description

Specification

Hepatitis C therapeutic agent

The present invention relates to a therapeutic agent for hepatitis c. More specifically, the present invention relates to a hepatitis C virus (hereinafter referred to as HCV) replicon RNA replication inhibitor. 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. Background art

The estimated number of carriers of HCV is about 170 million (about 3%) worldwide, and about 1.5 million in Japan. Interferon (IFN) -ribavirin (Virazole) combination therapy, which is the first choice of treatment, has an effective rate of 40% through all types of HCV, especially in Japan; The effective rate for viruses (genotype lb) is only 15-20%, while the frequency of side effects is high. With current therapies, complete eradication of the virus is difficult. If chronic hepatitis cannot be cured, it will progress to cirrhosis (30%) and hepatocellular carcinoma (25%). In Europe and the United States, hepatitis C has become a major indication of moon-swallow transplantation. However, HCV reappears frequently in the liver after transplantation. Therefore, there is a great social need for new drugs that have improved efficacy and safety, have higher antiviral effects, and can suppress hepatitis C.

HCV is a virus that has a plus-strand RNA as a gene, and is classified into Flaviviridae based on the analysis of genetic base sequences. According to Fiel ds Virology fourth edition, D. Kme et al ed., Philadelphia, Lippincott Williams & Wilkins 2001, Ί 127-1161, the existence of HCV was assumed in the 1970s, but its discovery was extremely difficult. Non-A non-B hepatitis An era called virus lasted for a long time. In 1989, Choo QL et al., Science 244, 359-362 (1989) identified part of the virus gene from the serum of infected experimental animals and identified its cDNA sequence. Was identified as HCV. Genomic RNA is about 9,500 bases and has an open reading frame encoding a polyprotein of about 3,000 amino acid residues. In that polyprotein, the structural proteins that form the virus particles are located on the N-terminal side, and are arranged in the order of core protein C, two types of envelope proteins El, E2, and non-structural protein NS1. The polyprotein is processed, producing the proteins that make up the replication machinery necessary for the replication of genomic RNA. Disclosure of the invention

Cyclosporin A (Sandimmun) is used in organ transplantation as an immunosuppressant. M. Thali et al., Nature 372, 363-365 (1994) reported that cyclophilin A force S, a virus particle forming protein of Human Immunodeficiency Virus Type 1 (HIV-1), and cyclophilin A It reports that it inhibits the interaction and exhibits anti-HIV-1 activity. In addition, it has been reported that cyclosporin A has a direct anti-HCV action. K. Inoue et al, 6 ^l "International Symposium on Hepatitis C and Related Virus. 3-6 June (2000) Bethesda, MD, USA, etc. However, to date, no other group has supported this finding.

In the clinic, the use of the immunosuppressant cyclosporin A has been shown to promote the growth of HCV in transplanted patients, according to a review by M. Berenguer et al., J. Hepatol 32, 673-684 (2000). There are reports. G. Everson and Liver Tran showed no difference in the reinfection rate when comparing HCV reinfection with cyclosporine A and other immunosuppressants in liver transplantation. There is also a report on splantation 10 Suppl 1: S19-27 (2002).

Studies showing that combination therapy of IFN and cyclosporine A is more effective than IFN monotherapy are based on the aforementioned K. Inoue et al., Τ, Gastroenterol 38, 567-572 (2003). In a study by Cotler et al., J. Clin. Gastroenterol, b 352-35t> (2003), which supplements this report, the combination therapy of IFN and cyclosporin A is currently not available. It has been reported that the effect is inferior to the combined treatment of IFN and ribavirin. However, in this report, relatively few patients with high blood trough levels of cyclosporin A have shown relatively favorable treatment results. '

For this reason, there is a need for a therapeutic agent for hepatitis C which has improved activity, blood translocation, selectivity, reduced side effects, etc., as compared to cyclosporin A. Means for solving the problem

The compound (I) of the present invention is represented by the following formula, and is a compound having a different chemical structure from cyclosporin A, particularly a constituent amino acid. For example, the method described in JP-A-5-271267 Can be produced from mold (Stachybotrys chart aura No. 19392: accession number FERM BP-3364)

Suitable salts of compound (I) are the usual non-toxic salts that are pharmaceutically acceptable, and salts with inorganic bases (for example, alkali metal salts such as sodium salt and potassium salt, calcium salts, and the like). Alkaline earth metal salts such as magnesium salts, ammonium salts, and salts with organic bases (eg, triethylamine salt, diisopropylethylamine salt, pyridine salt, picolin salt, ethanolamine salt, Organic amine salts such as ethanolamine salt, dicyclohexylamine salt, Ν'Ν'-dibenzylethylenediamine salt), and inorganic acid addition salts (for example, hydrochloride, hydrobromide, Sulfates, phosphates, etc.), organic carboxylic or sulfonic acid addition salts (eg, formate, acetate, trifluoroacetate, maleate, tartrate, dalconate, fumaric acid) salt, Salts with bases such as methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), salts with basic or acidic amino acids (eg, arginine, aspartic acid, glutamic acid, etc.) or Acid addition salts.

In the present invention, the therapeutic agent for HCV containing compound (I) or a salt thereof as an active ingredient is orally; sublingually; 頰; nasal cavity; respiratory organ; extraintestinal (intradermal, intraorgan, subdermal, intradermal, intradermal, intramuscular). Intraocular, intraocular or periocular infusions, including injections into the joints, central vein, hepatic vein, peripheral vein, lysate, heart, artery, and peri-ocular); Ears, including tubing, nipples Air chamber, external and internal auditory canal, tympanic membrane, middle ear, cochlea spiral gandarian, inner ear including maze; intestinal tract; rectum; vagina; ureter; organic or inorganic carrier suitable for administration to the bladder It can be contained as a mixture with a formulation, for example, in the form of a solid, semi-solid, or liquid pharmaceutical formulation. For intrauterine and perinatal indications, also in the maternal blood vessels, or in maternal organs, including the uterus, offspring, and vagina; In a placenta-like space, parenteral is preferred, but these routes will vary depending on the condition of the patient.

Although it is possible to administer Compound (I) or a salt thereof alone as a therapeutic agent, it is also desirable to utilize this as part of a prescription preparation. The "therapeutic agent for HCV" of the present invention comprises at least one or several suitable organic or inorganic carriers or excipients or a mixture with other pharmacological therapeutic agents, for example, It can be used in the form of pharmaceutical preparations in solid, semi-solid or liquid form; the active ingredient can be mixed with, for example, pharmaceutically usual non-toxic carriers to give granules, tablets, pellets, troches, capsules Or liquid forms such as suppositories, glymes, ointments, aerosols, powders for insufflation, injections, emulsions or suspensions; ingestions; eye drops; any other suitable for use In the form of If necessary, auxiliary agents such as stabilizers, thickeners, wetting agents, curing agents, and coloring agents; fragrances or buffers; and any other conventional additives can be added to the above-mentioned preparations.

The compound (I) or a salt thereof is added to the “HCV therapeutic agent” of the present invention in an amount sufficient to obtain a desired HCV inhibitory effect depending on the progress or state 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 prescription administered with the therapeutic agent and the mode in which the administration is performed, and Therapeutic agents will usually be included in amounts from 0.1 to 90% by weight of the total composition, depending on the stage of the disease or the interval between administrations. Body weight for parenteral administration for HCV suppression 1 An amount of 0,000 lmg per day per kg, or 50 mg or preferably 0.001 to 25 mg per day, and O.OO'l or less per day per kg of body weight for enteral administration An amount of 100 mg, preferably 0.01, or 60 mg can be given. However, these dosages may need to exceed their upper limits in order to obtain therapeutic results.

The compound (I) or a salt thereof may contain stereoisomers such as optical isomers and geometric isomers due to one or more asymmetric carbon atoms and double bonds, and all such isomers. In general, mixtures thereof are also included in the present invention.

Compound (I) or a salt thereof also includes a solvate (eg, hydrate, ethanolate, etc.).

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

The compound (I) or a salt thereof also includes a prodrug form. The present invention relates to the use of HCV therapeutic agents under the conditions described throughout the specification. The invention includes all advertising, labeling, packaging, informational materials, inserts, product details, advertising materials, text, pamphlets, magazines, books, etc., or conversation, fax, telephone, photo, radio , Video, television, film, internet, e-mail, etc., information exchange media, computer-based information means, proposals for clinical trials, research projects for clinical trials using therapeutic agents for HCV suppression. Tokor etc. is also included.

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

V. Lohmann et al., Science 285, 110-113 (1999) produced a human hepatoma cell line (Huh7) transfected with a suogenomic HCV RNA molecule and introduced it into the cells. Report that ubgenomi c HCV RNA replicates at a high rate Tells. The replication mechanism of subgenomic HCV RNA in these cell lines is thought to be very similar to the replication of the full-length HCV RNA genome in HCV-infected hepatocytes. Therefore, the cell-level assay method using Huh-7 cells into which subgenomic HCV RNA has been introduced provides a basis for the activity evaluation method of compound (I) or a salt thereof that inhibits HCV replication according to the present invention. I have. '

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

HCV replicon replication inhibitory activity

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

Test compound

1) Drug addition to cells

HCV replicon-expressing cells (Kishine et al., Β. Β. R. C 293: 993-999 (20

02)) on a 6-well microtiter plate (manufactured by Corning) at 5 ° / per henole. (5) 1.5 x 10 (6) cells were prepared and seeded in 2 ml of D-MEM medium containing 300 µg / ml of fetal serum and G418. 37 ° C, 5 ° /. After culturing for 16 hours in the presence of C02, the medium was replaced with the D-MEM medium in which the test compound was dissolved.

2) RNA extraction from cells

After culturing for two more days, extract the cellular RNA according to the protocol of the RNA extraction column RNeasy Mini (Qiagen), and use the DU800 UV-Visible spectroscopy system (Beckman) to measure the absorbance. (260 nm / 280 nm) was measured to determine the total RNA amount.

3) Quantification of replicon RNA by Real-time RT-PCR

Real-time RT-PCR is a primer that amplifies part of the HCV gene sequence

This was performed by a combination of probes to be captured (all manufactured by Takara Shuzo). The RNA extracted in 2) was diluted with RNase inhibitor-containing NF water to 25 ng /// l, and 2 μl was dispensed into a 384 μl PCR plate. The reaction solution for RT-PCR was prepared by mixing TaqMan Ez RT-PCR Core Reagent (Applied Biosystems) according to the protocol and adding 8 μl each. RT-PCR was performed using the ABI PRISM 7900HT sequence detection system (Applied Biosystems) to quantify HCV replicon RNA. For the calibration curve, synthetic HCV RNA was diluted 10-fold and used. The negative control was reacted without the addition of RNA.

4) Quantification of endogenous control RNA

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same way as 3) as an intrinsic juo control! "eal—time RT—Quantified by PCR. Primer and probe used were TaqMan GAPDH control reagent from Applied Biosystems. The standard curve was prepared by diluting the control RNA attached to the kit 10-fold. Negative control was performed without the addition of RNA.

HCV replicon-expressing cells were cultured under the same conditions as in the evaluation system for replicon replication inhibitory activity, and the cells were dispersed with 0.05% Trypsin-0.5 mM EDTA, stained with 0.1% erythrocyte syn-PBS (-), and the number of live cells Was illuminated under a microscope.

Test results

The concentration of the drug showing 50% RNA level relative to the control (DMS0 only, no drug added) with respect to the value obtained by correcting the quantitative value of the replicon RNA with the quantitative value of the endogenous control (GAPDH). Was calculated from the plot and used as the EC50 value. The cytotoxicity was calculated by plotting the drug concentration at which the number of viable cells showed a 50% level relative to the control (only DMS0 without drug added group) as the CC50 value.

The results are shown in Table 1.

【table 1 】

HCV replicon replication inhibitory activity:

EC50 (μΜ) Test compound <0.5 The results of the above test examples showed that the compound (I) of the present invention or a salt thereof had anti-hepatitis C virus activity.

Claims

Scope of claim General formula (I) below

A preventive and / or therapeutic agent for hepatitis C or a therapeutic agent comprising the compound (I) or a salt thereof represented by the following formula:

2. Use of the compound (I) or a salt thereof according to claim 1 for the manufacture of a medicament for preventing or treating hepatitis C.

3. A method for preventing and / or treating hepatitis C, which comprises administering the compound (I) according to claim 1 or a salt thereof to a human or an animal.

4. A commercial package consisting of a functional book stating that the compound (I) or a salt thereof according to claim 1 is or is to be used for the prevention and / or treatment of hepatitis C. Goods.