Asunaprevir

Huh 7.5.1 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The infectious JFH1 plasmid was obtained from Dr. Takaji Wakita and inoculated as previously described (Lin et al., 2010 (link)). DENV-2 PL046 (Genbank accession # AJ968413.1) isolated from patients with dengue fever was kindly provided by Lin et al. (1998 ). These viruses were propagated in mosquito cell line C6/36 (ATCC: CRL-1660) grown in RPMI 1640 medium containing 5% FBS. HepG2 cells were purchased from Bioresource Collection and Research Center (BCRC) and grown in DMEM supplemented with 10% FBS. Asunaprevir was obtained from the Bristol-Myers Squibb Company.

After a stable HCV infection was established in the mice, they were administered DAA therapy orally once per day as follows. Mice inoculated with HCV from patient serum were treated with a dual therapy of ledipasvir (LDV, 15 mg/kg)/GS-558093 (50 mg/kg, nucleotide analogue NS5B polymerase inhibitor) (LDV and GS-558093 were kindly provided by Gilead Sciences Inc., Foster City, CA). A mouse that experienced relapse after LDV/GS-558093 was re-treated with asunaprevir (40 mg/kg)/daclatasvir (30 mg/kg) (ASV and DCV were kindly provided by Bristol-Myers Squibb) for 4 weeks. A mouse inoculated with the full genome HCV RNA was treated with a monotherapy of LDV (15 mg/kg) for 4 weeks. All experimental protocols were approved by the Animal Care and Use Committee of Osaka University Medical School and the Animal Care Committee of Central Institute for Experimental Animals (CIEA), and all experiments were performed according to approved protocols.

Patients diagnosed with chronic hepatitis C and receiving a combination of asunaprevir (ASV) (Bristol-Myers Squibb, New York, NY, USA) and daclatasvir (DCV) (Bristol-Myers Squibb) for 24 weeks were included in the present study. Patients with decompensated liver cirrhosis and infected with hepatitis B or human immunodeficiency virus were excluded. Patients received an oral dose of 100 mg ASV twice daily and 60 mg DCV once daily for 24 weeks. During this period, basic liver function indexes, such as ALT and the amount of HCV RNA, were monitored. Serum HCV RNA levels were measured using a real-time PCR method with the lower quantification limit of 1.2 log IU/mL (COBAS TaqMan HCV Test 2.0; Roche Diagnostics, Tokyo, Japan). A sustained virologic response (SVR) was defined as undetectable HCV RNA at 24 weeks post-treatment. The human leukocyte antigen (HLA) typing of patients was performed using peripheral blood mononuclear cells (PBMCs) and the PCR–reverse-sequence-specific oligonucleotide method. HLA-A24-positive patients were included in the present study. All patients provided written informed consent to participate, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the regional Ethics Committee (Medical Ethics Committee of Kanazawa University, No. 1639).