In Vitro Assessment of Retreatment Options for Pa

Authors: Jacques Friborg Nannan Zhou Zhou Han Xiaoyan Yang Paul Falk Patricia Mendez Fiona McPhee

Publish Date: 2014/12/17

Volume: 4, Issue: 1, Pages: 137-144

PDF Link

Abstract

Daclatasvir is a nonstructural protein 5A NS5A inhibitor with activity against hepatitis C virus HCV genotypes 1–6 in vitro and asunaprevir is a nonstructural protein 3 NS3 protease inhibitor with activity against genotypes 1 4 5 and 6 This study evaluates potential options for the retreatment of HCV genotype 1binfected patients who have failed combination therapy with daclatasvir plus asunaprevirThe antiviral activity of drug combination regimens in HCV subgenomic replicon cell lines representing genotype 1b Con1 strain wildtype or a variant with specific NS5A and NS3 amino acid substitutions conferring resistance to daclatasvir and asunaprevir were compared using replicon elimination assays Drug concentrations representing multiple 50 effective concentrations EC50 derived in vitro and trough plasma concentrations observed in a clinical setting were utilizedAt multiple EC50 values of each drug 3× 10× and 30× EC50 combinations of daclatasvir plus sofosbuvir sofosbuvir plus ledipasvir sofosbuvir plus simeprevir and sofosbuvir plus either a nextgeneration NS3 or NS5A inhibitor demonstrated comparable activity in wildtype and daclatasvir/asunaprevirresistant cell lines At clinically relevant drug trough concentrations combination regimens of daclatasvir plus asunaprevir plus beclabuvir ±ribavirin and daclatasvir plus asunaprevir plus beclabuvir plus sofosbuvir efficiently cleared daclatasvir + asunaprevirresistant replicons from cells within 5 days of treatmentOur in vitro results highlight a number of potential alloral treatment options for patients who do not achieve a sustained virologic response following therapy with daclatasvir plus asunaprevir These results require further evaluation in clinical studiesCurrent options for the treatment of hepatitis C virus HCV infection are evolving rapidly with the recent approval of several directacting antiviral DAA agents Daclatasvir DCV is a nonstructural protein 5A NS5A inhibitor with activity against HCV genotypes 1–6 in vitro 1 Asunaprevir ASV is a nonstructural protein 3 NS3 protease inhibitor with activity against genotypes 1 4 5 and 6 2 The alloral interferonfree combination of DCV + ASV provided high rates of sustained virologic response and was well tolerated in genotype 1binfected patients in global and Japanese Phase III studies 3 4 Among genotype 1binfected patients who experience virologic escape with DCV + ASV the most common resistanceassociated variants RAVs detected together after HCV RNA rebound occur at NS5A positions L31 and Y93 and NS3 position D168 Here we aim to evaluate potential retreatment options for genotype 1binfected patients who have previously failed combination therapy with DCV + ASV using the in vitro HCV replicon systemHCV subgenomic replicon cell lines representing genotype 1b Con1 strain wildtype or a variant with specific NS5A and NS3 amino acid substitutions conferring resistance to DCV and ASV NS5AL31MY93H and NS3D168V respectively were established as previously described 5 Peginterferon alfa2a PEGASYS® was purchased from Hoffman–La Roche Inc Nutley NJ USA and ribavirin RBV was purchased from Sigma–Aldrich Co St Louis MO USA Simeprevir SMV NS3 inhibitor sofosbuvir SOF NS5B inhibitor and ledipasvir LDV NS5A inhibitor were synthesized at BristolMyers Squibb and have been described previously 6 7 8 DCV ASV beclabuvir BCV BMS791325 NS5B thumb 1 inhibitor BMS1 nextgeneration NS5A inhibitor and BMS2 nextgeneration NS3 inhibitor were also synthesized at BristolMyers Squibb The antiviral activities of the individual compounds and combination regimens were assessed using phenotypic analyses to determine 50 effective concentrations EC50 and replicon elimination assays as described previously 9 10 The ability of drug combinations to clear replicons was evaluated using two different approaches First wildtype and DCV + ASVresistant replicon cell lines with a neoselectable marker were incubated without G418 for 1 3 7 11 or 14 days with multiples of EC50 values for each drug 3× 10× and 30 × EC50 determined against wildtype replicon Combination regimens of DCV + ASV DCV + SOF SOF + LDV SOF + SMV DCV + ASV + BCV and DCV + ASV + BCV + SOF were assessed initially using multiple EC50 values of each agent estimated against wildtype replicon RBV was examined at 1 × EC50 concentration tenfold below any observed cell toxicity In another approach replicon cell lines were incubated without G418 for 1 2 3 5 and 7 days with drug concentrations representing trough plasma concentrations Ctrough observed in a clinical setting In both approaches the drug regimen was removed at the end of the incubation period and the cell cultures were further maintained for 2 weeks in growth medium supplemented with G418 05 mg/mL to monitor replicon elimination Surviving replicon colonies were fixed and stained with crystal violet as described previously 10

Keywords: