The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K_m or S₅₀ (sigmoidal kinetics) and V_max values varied 525- (8–4204 μM) and 5535-fold, respectively, for 4MU glucuronidation, and 1360- (1.3–1768 μM) and 560-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 1A6, 2B15, and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K_i values, determined for selected isoforms, ranged from 11 to 60 μM and 96 to 2790 μM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

Intestinal first-pass metabolism may contribute to low oral drug bioavailability and drug-drug interactions, particularly for CYP3A substrates. The current analysis predicted intestinal availability (F_G) from in vitro metabolic clearance and permeability data of 25 drugs using the Q_Gut model. The drug selection included a wide range of physicochemical properties and in vivo F_G values (0.07–0.94). In vitro clearance data (CLu_int) were determined in human intestinal (HIM) and three liver (HLM) microsomal pools (n = 105 donors) using the substrate depletion method. Apparent drug permeability (P_app) was determined in Caco-2 and Madin-Darby canine kidney cells transfected with human MDR1 gene (MDCK-MDR1 cells) under isotonic conditions (pH = 7.4). In addition, effective permeability (P_eff) data, estimated from regression analyses to P_app or physicochemical properties were used in the F_G predictions. Determined CLu_int values ranged from 0.022 to 76.7 μl/min/pmol of CYP3A (zolpidem and nisoldipine, respectively). Differences in CLu_int values obtained in HIM and HLM were not significant after normalization for tissue-specific CYP3A abundance, supporting their interchangeable usability. The F_G predictions were most successful when P_app data from Caco-2/MDCK-MDR1 cells were used directly; in contrast, the use of physicochemical parameters resulted in significant F_G underpredictions. Good agreement between predicted and in vivo F_G was noted for drugs with low to medium intestinal extraction (e.g., midazolam predicted F_G value 0.54 and in vivo value 0.51). In contrast, low prediction accuracy was observed for drugs with in vivo F_G <0.5, resulting in considerable underprediction in some instances, as for saquinavir (predicted F_G is 6% of the observed value). Implications of the findings are discussed.

With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic anion-transporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under- and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.