DMD #66027 TITLE PAGE Prediction of Drug Clearance and Drug-Drug Interactions in Microscale Cultures of Human Hepatocytes

Accurate prediction of in vivo hepatic drug clearance using in vitro assays is important to properly estimate clinical dosing regimens. Clearance of low turnover compounds is especially difficult to predict using short-lived suspensions of un-pooled primary human hepatocytes (PHHs) and functionally declining PHH monolayers. Micropatterned cocultures (MPCCs) of PHHs and 3T3-J2 fibroblasts have been previously shown to display major liver functions for several weeks in vitro. In this study, we first characterized long-term activities of major cytochrome P450 (CYP) enzymes in MPCCs created from un-pooled cryopreserved PHH donors. Then, MPCCs were utilized to predict the clearance of 26 drugs that exhibit a wide range of turnover rates in vivo (0.05-19.5 mL/min/kg). MPCCs predicted 73%, 92% and 96% of drug clearance values for all tested drugs within 2-fold, 3-fold and 4-fold of in vivo values, respectively. There was good correlation (R2=0.94, slope=1.05) of predictions between the two PHH donors. On the other hand, suspension hepatocytes and conventional monolayers created from the same donor had significantly reduced predictive capacity (i.e. 30-50% clearance values within 4-fold of in vivo), and were not able to metabolize several drugs. Finally, we modulated drug clearance in MPCCs by inducing or inhibiting CYPs. Rifampin-mediated CYP3A4 induction increased midazolam clearance by 73%, while CYP3A4 inhibition with ritonavir decreased midazolam clearance by 79%. Similarly, quinidine-mediated CYP2D6 inhibition reduced clearance of dextromethorphan and desipramine by 71% and 22%, respectively. In conclusion, MPCCs created using cryopreserved un-pooled PHHs can be utilized for drug clearance predictions and to model drug-drug interactions. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on October 9, 2015 as DOI: 10.1124/dmd.115.066027 at A SPET Jornals on O cber 2, 2015 dm d.aspurnals.org D ow naded rom