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

Abstract

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 (CLint) derived for the various conditions predicted hepatic AZT clearance by glucuronidation (CLH) 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 CLint values determined for the various experimental conditions were extrapolated to a whole organ CLint and these data were used to calculate in vivo CLH using the well-stirred, parallel tube and dispersion models.

Results: Mean CLint values for Brij58 activated microsomes in both phosphate (3.66 +/- 1.40 micro l min-1 mg-1, 95% CI 1.92, 5.39) and Tris (3.79 +/- 0.74 micro l 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 micro l min-1 mg-1, 95% CI 1.00, 1.73). Extrapolation of the microsomal data to a whole organ CLint 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-1vs 82 l h-1). There was no significant difference in the CLH 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, MgCl2, 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 CLH 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 CLint to underestimate in vivo CLH.

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 CLint underestimates in vivo hepatic clearance of drugs eliminated by glucuronidation.

Figure 1

Representative Eadie-Hofstee plots for the conversion of AZT to GAZT using microsomes from a single liver. Incubations were performed at pH 7.4 and a buffer concentration of 0.1 m, with other conditions as described in Microsomal incubations. Panel a, native microsomes–phosphate buffer; Panel b, Brij58 activated microsomes–phosphate buffer; Panel c, native microsomes–Tris buffer; Panel d, Brij58 activated microsomes–Tris buffer.

Figure 2

Effects of incubation components on the rate of AZT glucuronidation by pooled human liver microsomes. Incubations were performed in phosphate buffer (0.1 m, pH 7.4) in the presence of AZT (1 mm), UDPGA (10 mm) and, unless indicated, MgCl₂ (4 mm). Abbreviations: Native = native microsomes; SAL, d-saccharic acid 1,4-lactone (8.5 mm); Brij58, microsomes activated with Brij58 (0.15 w/w); Alamethacin, microsomes activated with alamethacin (50 mg l⁻¹); UDP-NAcG, microsomes activated with UDP-NAcG (1 mm); UDP-NAcG*, microsomes activated with UDP-NAcG (1 mm) and UDPGA concentration reduced to 0.25 mm.

Figure 3

Effects of phosphate buffer (0.1 m) pH on the rate of AZT glucuronidation by Brij58 activated pooled human liver microsomes (1 mg ml⁻¹). Incubations contained AZT (1 mm), UDPGA (10 mm) and MgCl₂ (4 mm).

Figure 4

Effect of phosphate buffer (pH 7.4) concentration on the rate of AZT glucuronidation by pooled human liver microsomes. Native (▴) refers to native microsomes, and Activated (▪) to Brij58 treated microsomes (0.15 w/w). Incubations contained AZT (1 mm), UDPGA (10 mm) and MgCl₂ (4 mm).

Figure 5

Correlation between the in vivo blood clearances by glucuronidation and hepatic clearances predicted from published in vitro kinetic data for the drugs listed in Table 4.