5, 0.75, 1, 1.5, 2, 2.5, 3,

4, 6, 8, 12, 16, 24, 48, 72, and 96 hours postdose). For telaprevir concentration analysis, blood samples were drawn on day 1 and day 8, period 2 (sampling timepoints: predose, 0.5, 1, 2, 2.5, 3, 4, 6, and 8 hours post-morning dose). The effect of telaprevir on tacrolimus PK was studied at steady-state telaprevir. During period 1, volunteers were admitted to the CRU on day −1 and discharged on day 3. On day 1, a single 2-mg oral dose of tacrolimus (4 capsules Prograf, 0.5 mg) was administered 2.5 hours after the start of the standard, medium-fat breakfast. There was a minimum washout of 14 days between day 1, period 1 and day 1, period 2. During period 2, volunteers were admitted

to the CRU on day 7 and PF-562271 mw discharged on day 11. From days 1 to 13 of period 2, telaprevir 750 mg q8h was administered 0.5 hours after the start of a meal or snack. On day 8, a single 0.5-mg oral dose of tacrolimus (1 capsule Prograf, selleck screening library 0.5 mg) was administered 2.5 hours after the start of a standard, medium-fat breakfast (i.e., 2 hours post-telaprevir dose). Volunteers returned for a follow-up visit on day 23 (±3 days). Approximately 4 mL of blood was drawn by way of direct venipuncture or indwelling catheter at each timepoint and processed for analyzing whole blood tacrolimus concentrations and plasma telaprevir concentrations. When tacrolimus was administered alone, blood samples were collected for tacrolimus analysis on day 1, period 1 (sampling timepoints: predose, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, ID-8 24, 48, 72, 96, and

120 hours postdose). When tacrolimus was coadministered with telaprevir, blood samples were collected for tacrolimus analysis on day 8, period 2 (sampling timepoints: predose, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, 24, 48, 72, 96, 120, and 144 hours postdose). Similarly, for telaprevir concentration analysis, blood samples were drawn on day 8, period 2 (sampling timepoints: predose, 0.5, 1, 2, 2.5, 3, 4, 6, and 8 hours post-morning dose). Whole blood concentrations of both cyclosporine and tacrolimus and plasma telaprevir concentrations were analyzed using validated assay methods. Briefly, cyclosporine, telaprevir, and their internal standards were extracted from samples using liquid/liquid extraction. Tacrolimus and its internal standard were extracted from samples using protein precipitation followed by solid-phase extraction. After evaporation under nitrogen, the residue of each analyte was reconstituted and analyzed using liquid chromatography followed by tandem mass spectrometry with selected ion monitoring in the positive ion mode. Calibration curves for each analyte was generated using weighted (1/x2) linear least-squares regression. The lower limit of quantitation for the cyclosporine assay in whole blood was 0.