Use of selective substrates and inhibitors to rapidly characterise batches of cryopreserved primary human hepatocytes for assessment of active uptake liability in drug discovery and development.

The use of hepatocytes to predict human hepatic metabolic clearance is the gold standard approach. However whilst enzymes are well characterised, knowledge gaps remain for transporters. Furthermore, methods to study specific transporter involvement are often complicated by overlapping substrate specificity. Selective substrates and inhibitors would aid investigations into clinically relevant pharmacokinetic effects. However, to date no consensus has been reached.This work defines selective hepatic uptake transporter substrates and inhibitors for the six main human hepatocyte transporters (OATP1B1, OATP1B3, OATP2B1, NTCP, OAT2 & OCT1), and demonstrates their use to rapidly characterise batches of human hepatocytes for uptake transporter activity. Hepatic uptake was determined across a range of substrate concentrations, allowing the definition of kinetic parameters and hence active and passive components. Systematic investigations identified a specific substrate and inhibitor for each transporter, with no overlap between the specificity of substrate and inhibitor for any given transporter.Early characterisation of compound interactions with uptake transporters will aid in early risk assessment and chemistry design. Hence, this work further highlights the feasibility of a refined methodology for rapid compound characterisation for the application of static and dynamic models, for early clinical risk assessment and guidance for the clinical development plan.

Drug clearance by aldehyde oxidase: can we avoid clinical failure?

Despite increased awareness of aldehyde oxidase (AO) as a major drug-metabolising enzyme, predicting the pharmacokinetics of its substrates remains challenging. Several drug candidates have been terminated due to high clearance, which were subsequently discovered to be AO substrates. Even retrospective extrapolation of human clearance, from models more sensitive to AO activity, often resulted in underprediction.The questions of the current work thus were: Is there an acceptable degree of in vitro AO metabolism that does not result in high in vivo human clearance? And, if so, how can this be predicted?We built an in vitro/in vivo correlation using known AO substrates, combining multiple in vitro parameters to calculate the blood metabolic clearance mediated by AO (CLbAO). This value was compared with observed blood clearance (CLb-obs), establishing cut-off CLbAO values, to discriminate between low and high CLb-obs. The model was validated using additional literature compounds, and CLb-obs was predicted in the correct category.This simple, categorical, semi-quantitative yet multi-factorial model is readily applicable in drug discovery. Further, it is valuable for high-clearance compounds, as it predicts the CLb group, rather than an exact CLb value, for the substrates of this poorly-characterised enzyme.

Evaluation of In Vitro Models for Assessment of Human Intestinal Metabolism in Drug Discovery.

Although intestinal metabolism plays an important role in drug disposition, early predictions of human outcomes are challenging, in part because of limitations of available in vitro models. To address this, we have evaluated three in vitro models of human intestine (microsomes, permeabilized enterocytes, and cryopreserved intestinal mucosal epithelium) as tools to assess intestinal metabolism and estimate the fraction escaping gut metabolism (f g) in drug discovery. The models were tested with a chemically diverse set of 32 compounds, including substrates for oxidoreductive, hydrolytic, and conjugative enzymes. Liquid chromatography-high-resolution mass spectrometry was used to quantify substrate disappearance [intrinsic clearance (CLint)] and qualify metabolite formation (quantitative-qualitative bioanalysis). Fraction unbound in the incubation (f u,inc) was determined by rapid equilibrium dialysis. Measured in vitro results (CLint and f u,inc) were supplemented with literature data [passive Caco-2 apical to basolateral permeability, enterocyte blood flow, and intestinal surface area (A)] and combined using a midazolam-calibrated Q gut model to predict human f g values. All three models showed reliable CYP and UDP-glucuronosyltransferase activities, but enterocytes and mucosa may offer advantages for low-clearance compounds and alternative pathways (e.g., sulfation, hydrolases, and flavin-containing monooxigenases). Early predictions of human f g values were acceptable for the high-f g compounds (arbitrarily f g > 0.7). However, predictions of low- and moderate-f g values (arbitrarily f g < 0.7) remain challenging, indicating that further evaluation is needed (e.g., saturation effects and impact of transporters) but not immediate compound avoidance. Results suggest that tested models offer an additional value in drug discovery, especially for drug design and chemotype evaluation. SIGNIFICANCE STATEMENT: We found that cellular models of the human gut (permeabilized enterocytes and cryopreserved intestinal mucosa) offer an alternative to and potential advantage over intestinal microsomes in studies of drug metabolism, particularly for low-clearance compounds and alternative pathways (e.g., sulfation, hydrolases, and flavin-containing monooxigenases). The predictivity of human fraction escaping gut metabolism for common CYP and UDP-glucuronosyltransferase substrates based on the Q gut model is still limited, however, and appropriate further evaluation is recommended.