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 unpooled primary human hepatocytes (PHHs) and functionally declining PHH monolayers. Micropatterned cocultures (MPCCs) of PHHs and 3T3-J2 fibroblasts have been shown previously to display major liver functions for several weeks in vitro. In this study, we first characterized long-term activities of major cytochrome P450 enzymes in MPCCs created from unpooled cryopreserved PHH donors. MPCCs were then used to predict the clearance of 26 drugs that exhibit a wide range of turnover rates in vivo (0.05-19.5 ml/min per kilogram). 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 (R(2) = 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 P450s. 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 unpooled PHHs can be used for drug clearance predictions and to model drug-drug interactions.

Long-term stability of primary rat hepatocytes in micropatterned cocultures.

Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac™). Here, we assess the long-term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96-well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel™ sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW-7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3-hydroxy-3-methylglutaryl-CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96-well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways.

Use of micropatterned cocultures to detect compounds that cause drug-induced liver injury in humans.

Because drug-induced liver injury (DILI) remains a major reason for late-stage drug attrition, predictive assays are needed that can be deployed throughout the drug discovery process. Clinical DILI can be predicted with a sensitivity of ~50% and a false positive (FP) rate of ~5% using 24-h cultures of sandwich-cultured primary human hepatocytes and imaging of four cell injury endpoints (Xu et al., 2008). We hypothesized that long-term drug dosing in a functionally stable model of primary hepatocytes (micropatterned cocultures [MPCCs]) could provide for increased predictivity over short-term dosing paradigms. We used MPCCs with either primary human or rat hepatocytes to understand possible species differences along with standard endpoints (glutathione levels, ATP levels, albumin, and urea secretion) to test 45 drugs either known or not known to cause clinical DILI. Human MPCCs correctly detected 23 of 35 compounds known to cause DILI (65.7% sensitivity), with a FP rate of 10% for the 10 negative compounds tested. Rat MPCCs correctly detected 17 of 35 DILI compounds (48.6% sensitivity) and had a higher FP rate than human MPCCs (20 vs. 10%). For an additional 19 drugs with the most DILI concern, human MPCCs displayed a sensitivity of 100% when at least two hepatocyte donors were used for testing. Furthermore, MPCCs were able to detect relative clinical toxicities of structural drug analogs. In conclusion, MPCCs showed superiority over conventional short-term cultures for predictions of clinical DILI, and human MPCCs were more predictive for human liabilities than their rat counterparts.