Patent TW-201103530-A (TW)
Title: The application for inhibiting HCV peplication by hypericin in Ava-5 cells
Inventor: KO JIUNN-LIANG
IPC: A61K31/122
Filed: 2009-07-24
Abstract: Hepatitis C virus (HCV) is a positive-sensed, single-stranded RNA virus that belong to the Flaviviridae family and Hepacivirus. World Health Organization(WHO) is estimated that there are more than 170 million, about 2.9% people infected by HCV in the world. About 80% of HCV infection results in chronic infection that can lead to severe liver diseases, such as cirrhosis and hepatocellular carcinoma. Recently, the combination therapy of peginterferon alpha-2a and ribavirin is recommended as a standard care for HCV infections in clinical, but chronic infection often leads to HCV can resistance interferon alpha-2a and escape the treatment. In clinical search, interferon alpha-2a and ribavirin had great side effects to patients, so we were eager to find drugs that have less side effects and good anti-viral effect to improve clinical treatment for HCV infection. In order to understand the pathway that HCV replicate in host cells, Scientists recently develop many type of cell lines that contain HCV subgenomic replicon system. A synthetic HCV subgenomic RNA has been replaced HCV structural protein region to the neomycin-phosphotransferase gene and encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES). HCV RNA 5'-untranslated region (UTR) is as an internal ribosomal entry site (IRES) and promoting HCV downstream gene, so that HCV replicon can be expressed from these special cell lines and has good expressed efficiency. we used the Ava.5 cell line that is expressed HCV1b replicon establish the platform for testing anti-HCV drugs, and studied the biochemical mechanism of anti-HCV drugs. First, we used MTS assay to measure cytotoxicity after the drugs treated cells. The treatment with the drugs at concentrations that did not induce cell death, and then we examined HCV subgenomic RNA by reverse transcription polymerase chain reaction and western blot to check the drugs whether inhibit HCV replication or not. Finally, we confirmed hypericin can inhibit HCV1b NS5A RNA and NS3 protein. In order to clarify the biochemical mechanisms that inhibit HCV replication, we combined treated hypericin and interferon alpha-2a for Ava.5 cells. In this study, we comfirm that there is no synergistic effect between hypericin and interferon alpha-2a to inhibit HCV1b NS3 protein. In the other hand, hypericin can inhibit Phospho- PKC α Thr638/β II Thr641, but interferon alpha-2a can't inhibit it. We think hypericin is different from the mechanism of interferon alpha-2a inhibited HCV NS3 protein. After treated Ca2+ -dependent PKC inhibitor Go 6976, HCV NS3 protein was inhibited by Go 6976 in Ava.5 cell line. According to this result, we expect that Ca2+ -dependent PKC signaling transduction regulated NS3 protein replicated. In our study, we comfirm that hypericin is a excellent pure compound to inhibit HCV replicon replicated in Ava.5 cells. In order to interferon alpha-2a has many side effects to HCV patients, we think that hypericin may be a good option to cure HCV patients in clinical. In the other hand, when the HCV patients resistance to IFN- α treatment, we have the option to cure the IFN- α resistanced HCV patients by hypericin.