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.