Grazoprevir (GZR) is a hepatitis C (HCV) NS3/4A protease inhibitor which can be combined with antiretroviral drugs to treat HIV/HCV co-infected patients. Coadministration with boosted darunavir (DRV/RTV) is contraindicated as it increases GZR AUC and Cmax 7.5-fold and 5.3-fold. Although assumed to be caused by OATP1B1 and CYP3A4 inhibition, the mechanism of this drug-drug interaction (DDI) has not been fully elucidated. GZR also exhibits a 20% increase in AUC in elderly patients and the role of OATP1B1 remains unclear. The current study quantified OATP1B1-mediated transport of GZR in the presence of DRV and RTV in vitro and evaluated the expression of OATP1B1 in primary hepatocytes from both young and elderly donors.
Pooled human cryopreserved hepatocytes were suspended in Krebs-Henseleit buffer in 24-well cell culture plates and incubated with GZR (0.1µM) and either DRV or RTV (0.01-33µM) for 2 minutes at 37°C. Transporter uptake was terminated using ice cold phosphate buffered saline (PBS) followed by immediate centrifugation, washing with ice cold PBS and freeze thaw for cell lysis. GZR concentrations were quantified using LC-MS/MS and IC[sub]50[/sub] were calculated. The abundance of OATP1B1 in pooled human cryopreserved hepatocytes aged 18-60 years old and three individual elderly donors aged 74-80 years old were quantified using a sandwich ELISA. Statistical significance was assessed using an unpaired t-test.
DRV reduced OATP1B1-mediated uptake of GZR by 35% (maximal inhibition at 0.01µM; IC50 of 4.4×10-8µM) whilst RTV decreased uptake by 40% (maximal inhibition at 10µM; IC50 of 9.4µM) (see figure). When compared to pooled hepatocytes from donors aged 18-60 years old, abundance of OATP1B1 in three individual elderly donors aged 74-80 years old were between 35% and 56% lower with P values between 0.005 and 0.032.
The in vitro model identified that RTV does not inhibit OATP1B1-mediated transport in the range of physiologically relevant concentrations, unlike DRV which produced a moderate inhibition of OATP1B1. Our experimental approach represents an effective strategy to characterize the role of transporters in DDIs and may be useful to identify clinically relevant DDIs. Furthermore, the lower expression of OATP1B1 in hepatocytes from elderly donors provides a plausible mechanistic basis for the increased GZR AUC reported in this sub-population and justifies further investigation.