ABSTRACT:Several human immunodeficiency virus (HIV) protease inhibitors, including atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, were tested for their potential to inhibit uridine 5-diphospho-glucuronosyltransferase (UGT) activity. Experiments were performed with human cDNA-expressed enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) as well as human liver microsomes.All of the protease inhibitors tested were inhibitors of UGT1A1, UGT1A3, and UGT1A4 with IC 50 values that ranged from 2 to 87 M. The IC 50 values found for all compounds for UGT1A6, 1A9, and 2B7 were >100 M. The inhibition (IC 50 ) of UGT1A1 was similar when tested against the human cDNA-expressed enzyme or human liver microsomes for atazanavir, indinavir, and saquinavir (2.4, 87, and 7.3 M versus 2.5, 68, and 5.0 M, respectively). By analysis of the double-reciprocal plots of bilirubin glucuronidation activities at different bilirubin concentrations in the presence of fixed concentrations of inhibitors, the UGT1A1 inhibition by atazanavir and indinavir was demonstrated to follow a linear mixed-type inhibition mechanism (K i ‫؍‬ 1.9 and 47.9 M, respectively). These results suggest that a direct inhibition of UGT1A1-mediated bilirubin glucuronidation may provide a mechanism for the reversible hyperbilirubinemia associated with administration of atazanavir as well as indinavir. In vitro-in vivo scaling with [I]/K i predicts that atazanavir and indinavir are more likely to induce hyperbilirubinemia than other HIV protease inhibitors studied when a free C max drug concentration was used. Our current study provides a unique example of in vitro-in vivo correlation for an endogenous UGT-mediated metabolic pathway.The HIV protease is an essential enzyme that cuts the viral gag-pol polyprotein into its functional subunits. Atazanavir, indinavir, saquinavir, lopinavir, ritonavir, and nelfinavir are HIV protease inhibitors. The structures of these HIV protease inhibitors are shown in Fig. 1. As opposed to atazanavir, which is an azapeptide protease inhibitor (Goldsmith and Perry, 2003), other listed HIV protease inhibitors are peptidomimetics sharing the same structural determinant, i.e., a hydroxyethylene or a hydroxyethylamine moiety, which makes them nonscissile HIV protease substrate analogs. These HIV protease inhibitors are metabolized primarily by hepatic CYP3A enzymes, and they are also inhibitors of CYP3A enzymes (Flexner, 2000;de Maat et al., 2003;Goldsmith and Perry, 2003;Ernest et al., 2005). All of these HIV protease inhibitors have a high protein binding (Ͼ98%), except indinavir and atazanavir, which have a protein binding of 60 and 86%, respectively. Most of the HIV protease inhibitors are bound to ␣1-acid glycoprotein instead of albumin (de Maat et al., 2003). There are no literature reports indicating that HIV protease inhibitors are good substrates for human UGT enzymes, although there is evidence to support indinavir as a substrate of UGTs (Balani et al., 1996).Glucuronidation represents a major pathway for the elimin...

We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE) with estrogen receptor alpha (ERalpha), ERbeta, and the androgen receptor (AR). In this study, we characterize the ERalpha, ERbeta, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERalpha, ERbeta, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17beta-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2, 2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERalpha agonist activity and ERbeta and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERalpha agonist activity and did not alter ERbeta or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERalpha and ERbeta agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERalpha and ERbeta activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.