Anti-ulcer agent teprenone inhibits hepatitis C virus replication: potential treatment for hepatitis C.

BACKGROUND: Previously we reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, inhibited hepatitis C virus (HCV) RNA replication. Furthermore, recent reports revealed that the statins are associated with a reduced risk of hepatocellular carcinoma and lower portal pressure in patients with cirrhosis. The statins exhibited anti-HCV activity by inhibiting geranylgeranylation of host proteins essential for HCV RNA replication. Geranylgeranyl pyrophosphate (GGPP) is a substrate for geranylgeranyltransferase. Therefore, we examined the potential of geranyl compounds with chemical structures similar to those of GGPP to inhibit HCV RNA replication. METHODS: We tested geranyl compounds [geranylgeraniol, geranylgeranoic acid, vitamin K(2) and teprenone (Selbex)] for their effects on HCV RNA replication using genome-length HCV RNA-replicating cells (the OR6 assay system) and a JFH-1 infection cell culture system. Teprenone is the major component of the anti-ulcer agent, Selbex. We also examined the anti-HCV activities of the geranyl compounds in combination with interferon (IFN)-α or statins. RESULTS: Among the geranyl compounds tested, only teprenone exhibited anti-HCV activity at a clinically achievable concentration. However, other anti-ulcer agents tested had no inhibitory effect on HCV RNA replication. The combination of teprenone and IFN-α exhibited a strong inhibitory effect on HCV RNA replication. Although teprenone alone did not inhibit geranylgeranylation, surprisingly, statins' inhibitory action against geranylgeranylation was enhanced by cotreatment with teprenone. CONCLUSIONS: The anti-ulcer agent teprenone inhibited HCV RNA replication and enhanced statins' inhibitory action against geranylgeranylation. This newly discovered function of teprenone may improve the treatment of HCV-associated liver diseases as an adjuvant to statins.

Plural assay systems derived from different cell lines and hepatitis C virus strains are required for the objective evaluation of anti-hepatitis C virus reagents.

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are widely used as the only cell-based HCV replication system for HCV research, including drug assays. Recently, using different hepatoma Li23-derived cells, we developed an HCV drug assay system (ORL8), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently. In this study, using the HuH-7-derived OR6 assay system that we developed previously and the ORL8 assay system, we evaluated 26 anti-HCV reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than OR6. The results revealed that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities evaluated by the OR6 and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which was developed using the AH1 strain of genotype 1b, several reagents showed different anti-HCV activities in comparison with those evaluated by the OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents.

Serum-free cell culture system supplemented with lipid-rich albumin for hepatitis C virus (strain O of genotype 1b) replication.

HuH-7 is a highly differentiated hepatoma cell line and the only cell line that supports robust RNA replication of the hepatitis C virus (HCV). HuH-7 cells cause cell death in serum-free culture condition. However, the effect is reversed by supplementation with selenium. Serum-free cell cultures are advantageous for vaccine development and experimental reproducibility. However, HCV RNA replication in HuH-7 cells in serum-free medium had not yet been achieved. Therefore, we tried to develop a system for robust HCV RNA replication in a serum-free cell culture. Although HuH-7 cells grew in serum-free medium in the presence of selenium, HuH-7 cells under these conditions did not support HCV RNA replication in long-term culture. Among the supplements tested, serum-free medium with lipid-rich albumin (LRA) was found to yield robust HCV RNA replication. HCV proteins were detected for more than 9 months in serum-free medium supplemented with LRA. This is the first report to demonstrate a long-term, serum-free cell culture that successfully maintained robust HCV RNA replication. This cell culture system is expected to be a useful tool for vaccine development, as well as for further investigation of cellular factors that are essential for HCV RNA replication.