Integrated in vitro models for hepatic safety and metabolism: evaluation of a human Liver-Chip and liver spheroid.

Drug-induced liver injury remains a frequent reason for drug withdrawal. Accordingly, more predictive and translational models are required to assess human hepatotoxicity risk. This study presents a comprehensive evaluation of two promising models to assess mechanistic hepatotoxicity, microengineered Organ-Chips and 3D hepatic spheroids, which have enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Sensitivity of the models to two hepatotoxins, acetaminophen (APAP) and fialuridine (FIAU), was assessed across a range of cytotoxicity biomarkers (ATP, albumin, miR-122, α-GST) as well as their metabolic functionality by quantifying APAP, FIAU and CYP probe substrate metabolites. APAP and FIAU produced dose- and time-dependent increases in miR-122 and α-GST release as well as decreases in albumin secretion in both Liver-Chips and hepatic spheroids. Metabolic turnover of CYP probe substrates, APAP and FIAU, was maintained over the 10-day exposure period at concentrations where no cytotoxicity was detected and APAP turnover decreased at concentrations where cytotoxicity was detected. With APAP, the most sensitive biomarkers were albumin in the Liver-Chips (EC50 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC50 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC50 126 µM) and miR-122 (15-fold) in the liver spheroids, both on day 7. In conclusion, both models exhibited integrated toxicity and metabolism, and broadly similar sensitivity to the hepatotoxicants at relevant clinical concentrations, demonstrating the utility of these models for improved hepatotoxicity risk assessment.

An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CLint) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CLint u) for each compound. This was compared with observed unbound intrinsic clearance (observed CLint u) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CLint u using hepatocytes (R2 = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained (R2 = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates.

Industrial Approach to Determine the Relative Contribution of Seven Major UGT Isoforms to Hepatic Glucuronidation.

The pharma industry designs increasingly less cytochrome P450 dependent and more metabolically stable drugs, and consequently UGT-metabolism becomes more frequently involved. This study compares 2 glucuronidation RAF-scaling approaches, product formation and substrate depletion, regarding their potential for prediction of in vivo DDI and the relative contribution of UGT-mediated phase II reactions in an industrial setting. RAFs were developed for UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15 recombinant UGT isoforms and a large 150-donor pooled human liver microsome batch. The RAF-values ranged from small values of 0.06 (UGT1A3), over 0.24 and 0.48 (UGT1A9 and UGT1A4), to values around 1 (1.11 for UGT2B7, 1.14 for UGT1A1), and high RAFs of 4.8 (UGT1A6) and 6.57 (UGT2B15). Both approaches identified the same primarily involved isoforms (≥75% relative contribution) of 5 clinical reference compounds (raloxifene, haloperidol, laropiprant, telmisartan and naloxone), in concordance with reported in vitro (R2 = 0.65) and clinical results for UGT1A1, 1A3, 1A4, 1A9, 2B7 and 2B15. This study is distinctive in that it is reporting the glucuronide formation in addition to substrate depletion. The product formation approach proved more sensitive and enables UGT phenotyping of slowly metabolized drugs, additionally it allows identification of structurally different glucuronides.