Cyclosporin A (CsA) inhibits the in vitro replication of HCV subgenomic replicons. We here report on the potent anti-HCV activity of the non-immunosuppressive cyclosporin DEBIO-025. The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by DEBIO-025 was 0.27 ุ 0.03 g/mL and for CsA 2.8 ุ 0.4 g/mL. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 27 g/mL for DEBIO-025 and 12 ุ 6 g/mL for CsA, resulting in a selectivity index of approximately 900 for DEBIO-025 and 40 for CsA. The superior activity of DEBIO-025, as compared with CsA, was corroborated by monitoring HCV RNA levels in Huh 5-2, two other HCV subgenomic replicon-containing cell lines, and by monitoring the luciferase signal and viral antigen production in hepatoma cells that had been infected with an infectious fulllength chimeric HCV construct. The combination of interferon alpha 2a with either CsA or DEBIO-025 resulted in an additive to slightly synergistic antiviral activity. DEBIO-025, at concentrations of 0.5 and 1 g/mL, was able to clear cells from their HCV replicon within three to four passages, whereas treatment with CsA at the same concentrations for seven consecutive passages did not result in clearance of the HCV replicon. In conclusion, DEBIO-025, a compound that is also endowed with potent anti-HIV activity and is well tolerated in animals and humans, may form an attractive new option for the therapy of HCV infections, particularly in HCV/HIV co-infected patients. (HEPATOLOGY 2006;43:761-770.) H epatitis C virus (HCV) is an enveloped singlestranded (ฯฉ) RNA virus that belongs to the separate genus Hepacivirus of the family Flaviviridae. 1 HCV causes acute and chronic liver disease, in-cluding chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 2 Worldwide more than 170 million people are chronically infected with HCV and are thus at increased risk of developing serious life-threatening liver disease. Current standard therapy for chronic hepatitis C consists of the combination of pegylated interferon alpha (IFN-โฃ) 2a in combination with ribavirin. 3 Cyclosporin A (CsA) (Fig. 1) is an 11-amino acid (AA) cyclic peptide from which seven AA are N-methylated and three AA are uncommon, that is, (4R)-4-[(E)-2-butenyl-4-methyl-L-threonine] in position 1, (L)-alpha-amino-butyric acid in postion 2, and (D)-alanine in position 8. CsA, a secondary metabolite of Tolypocladium sp., exerts potent immunosuppressive activity and is used to prevent organ transplant rejection. 4 In addition to its immunosuppressive effect, CsA has been reported to be a potent inhibitor of the replication of HIV-1. 5,6 The compound is also endowed with anti-schistosomal activity [7][8][9][10][11][12] and is an inhibitor of Plasmodium falciparum [13][14][15] and Plasmodium vivax. 16 Recently CsA was shown to exhibit anti-HCV activity