In vitro–in vivo correlations for drugs eliminated by glucuronidation: Investigations with the model substrate zidovudine

Aims To investigate the effects of incubation conditions on the kinetic constants for zidovudine (AZT) glucuronidation by human liver microsomes, and whether microsomal intrinsic clearance (CL int ) derived for the various conditions predicted hepatic AZT clearance by glucuronidation (CL H ) in vivo . Methods The effects of incubation constituents, particularly buffer type (phosphate, Tris) and activators (Brij58, alamethacin, UDP‐N‐acetylglucosamine (UDP‐NAcG)), on the kinetics of AZT glucuronidation by human liver microsomes was investigated. AZT glucuronide (AZTG) formation by microsomal incubations was quantified by h.p.l.c. Microsomal CL int values determined for the various experimental conditions were extrapolated to a whole organ CL int and these data were used to calculate in vivo CL H using the well‐stirred, parallel tube and dispersion models. Results Mean CL int values for Brij58 activated microsomes in both phosphate (3.66 ± 1.40 µl min −1 mg −1 , 95% CI 1.92, 5.39) and Tris (3.79 ± 0.74 µl min −1 mg −1 , 95% CI 2.87, 4.71) buffers were higher ( P < 0.05) than the respective values for native microsomes (1.04 ± 0.42, 95% CI 0.53, 1.56 and 1.37 ± 0.30 µl min −1 mg −1 , 95% CI 1.00, 1.73). Extrapolation of the microsomal data to a whole organ CL int and substitution of these values in the expressions for the well‐stirred, parallel tube and dispersion models underestimated the known in vivo blood AZT clearance by glucuronidation by 6.5‐ to 23‐fold (3.61–12.71 l h −1 vs 82 l h −1 ). There was no significant difference in the CL H predicted by each of the models for each set of conditions. A wide range of incubation constituents and conditions were subsequently investigated to assess their effects on GAZT formation, including alamethacin, UDP‐NAcG, MgCl 2 , d ‐saccharic acid 1,4‐lactone, ATP, GTP, and buffer pH and ionic strength. Of these, only decreasing the phosphate buffer concentration from 0.1 m to 0.02 m for Brij58 activated microsomes substantially increased the rate of GAZT formation, but the extrapolated CL H determined for this condition still underestimated known AZT glucuronidation clearance by more than 4‐fold. AZT was shown not to bind nonspecifically to microsomes. Analysis of published data for other glucuronidated drugs confirmed a trend for microsomal CL int to underestimate in vivo CL H . Conclusions AZT glucuronidation kinetics by human liver microsomes are markedly dependent on incubation conditions, and there is a need for interlaboratory standardization. Extrapolation of in vitro CL int underestimates in vivo hepatic clearance of drugs eliminated by glucuronidation.