Understanding biotransformation of slowly cleared chemicals in in vitro models including 3D liver Spheroid and HepatoPac.

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The transition to a pathway-based approach for risk assessment will require better understanding of exposure science. One of those approaches is to understand the bioavailability of chemicals in the human body and define toxicologically relevant approaches that permit the estimation of internal dose in humans. Physiologically-based biokinetic (PBBK) modelling is recognised as the means to achieve this, simulating whole-body toxicokinetic profiles by integrating chemical-independent (physiological) and chemical-dependent (absorption, deposition, metabolism and excretion, (ADME)) parameters. We present an evaluation of how well current non-animal methods can accurately predict human pharmacokinetics of topically applied compounds. Hepatic metabolism data was generated in different in vitro model systems using S9, microsomal fractions and primary human hepatocytes (suspensions, liver spheroid, and micropatterned co-cultured systems) with the aim of investigating the most appropriate system to study low clearance compounds. Early prediction of clearance in human is often challenging, in particular for the growing number of low-clearance compounds. Widely used microsomes or hepatocytes suspensions only allow short term metabolism studies due to drastically declined the activity of drug metabolizing enzyme (i.e. in hepatocyte assay the activity declines within 4 hours). Recent development of 3D cell culture models as well as micropatterned co-culture model (MPCC, HepatoPac) allow use for a wide range of biological applications and may potentially provide a useful tool for improving intrinsic clearance predictions of slow clearance chemicals. The aim of this work was to assess the potential application of 3D cellular models (such as InSphero’s primary liver microtissue and MPCC HepatoPac) for prediction of CLint for low clearance compounds in longer duration incubation, in comparison to other 5 in vitro systems. We’ve tested a range of substrates including CYP probe drugs, such as diclofenac and tolbutamide, measuring substrate depletion and metabolite profiles of each by LC-MS/MS. Rates of metabolism have been compared with those from other assays (e.g hepatocyte suspension, 2D culture). Predictions of plasma profiles following topical application were generated using Gastroplus 9.0 PBPK software for test chemicals. The predictions corresponded to published clinical studies. The combination of in vitro data generation and in silico PBPK modelling showed good accuracy for predicting human plasma maximum concentration and AUC values within an order of magnitude for all slowly cleared compounds. The micropatterned hepatocyte showed promise for measuring the clearance of chemicals that are metabolised too slowly to be detectable in a suspension assay.

 

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