Prediction of Drug Clearance and Drug-Drug Interactions in Microscale Cultures of Human Hepatocytes

Abstract

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.

Genomic Characterization of Metformin Hepatic Response

“…Our assays were done in primary human hepatocytes, and not tissues, treated with 2.5 mM metformin in order to observe significant metformin response, which is higher than physiological conditions used to treat metformin in patients. Our cells also do not mimic as well hepatocytes in their in vivo environment compared to more complex culturing conditions such as micropatterned cocultures [62] or a collagen sandwich system [63]. Due to the large costs and limited hepatocyte material, the cells were only treated in a single concentration of compound C (40 μM) which was previously shown to be effective in suppressing metformin-stimulated AMPK phosphorylation and glucose production [11,64].…”

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

“…This dependency of the scaling approaches and their performance are widely discussed (27,(44)(45)(46). Recently, Lin et al (47) reported that HepatoPac® showed a better overall performance in predicting 73% of the drug clearances within twofold when applying conventional scaling for low-to very low-clearance compounds (≤1 mL min −1 kg −1…”

Long-term exposure to abnormal glucose levels alters drug metabolism pathways and insulin sensitivity in primary human hepatocytes

“…Indeed, micropatterned co-cultures (MPCCs) of PHHs and 3T3-J2 murine embryonic fibroblasts can maintain prototypical hepatic morphology, secrete albumin and urea, and display high levels of CYP450 activities for 4–6 weeks2123. In this model, PHHs are first organized onto collagen-coated circular domains of empirically optimized dimensions in 24- or 96-well plates using semiconductor-driven soft-lithographic tools and then surrounded by 3T3-J2 fibroblasts.…”