Shangara S. Dehal

IN VITRO INHIBITION OF UDP GLUCURONOSYLTRANSFERASES BY ATAZANAVIR AND OTHER HIV PROTEASE INHIBITORS AND THE RELATIONSHIP OF THIS PROPERTY TO IN VIVO BILIRUBIN GLUCURONIDATION

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 IC50 values that ranged from 2 to 87 μM. The IC50 values found for all compounds for UGT1A6, 1A9, and 2B7 were >100 μM. The inhibition (IC50) 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 μM versus 2.5, 68, and 5.0 μM, 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 (Ki = 1.9 and 47.9 μM, 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]/Ki predicts that atazanavir and indinavir are more likely to induce hyperbilirubinemia than other HIV protease inhibitors studied when a free Cmax drug concentration was used. Our current study provides a unique example of in vitro-in vivo correlation for an endogenous UGT-mediated metabolic pathway.

HUMAN CYTOCHROME P450 INDUCTION AND INHIBITION POTENTIAL OF CLEVIDIPINE AND ITS PRIMARY METABOLITE H152/81

Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 μM. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 μM and 100 μM concentrations of clevidipine tested. Induction response for CYP3A4 to 100 μM clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC50 values below 10 μM, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC50 values >70 μM). The Ki values for CYP2C9 and CYP2C19 were 1.7 and 3.3 μM, respectively, and those for CYP3A4 were 8.3 and 2.9 μM, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC50 values ≥ 69 μM). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans.

TAMOXIFEN METABOLISM BY MICROSOMAL CYTOCHROME P450 AND FLAVIN-CONTAINING MONOOXYGENASE

Publisher Summary This chapter discusses the metabolism of tamoxifen by microsomal cytochrome P450 as well as flavin-containing monooxygenase. Tamoxifen (tam) is an antiestrogen and is developed as a therapeutic agent for hormone responsive (estrogen receptor-positive) breast tumors. Tamoxifen is metabolized by liver microsomes from animals and humans into a variety of compounds, such as tamoxifen N -oxide (tam- N -oxide), N -desmethyl ( N -desmethyl-tam), and 4-hydroxy (4-OH-tam) derivatives. Microsomal CYP3A catalyzes covalent binding of tamoxifen to proteins and tam- N -demethylation. This chapter describes the radiometric methods developed for detection, identification, and quantitation of several of the major tamoxifen metabolites. The chapter also describes the hepatic enzymes forming these tamoxifen metabolites. The amount of N-oxide observed in incubations of tamoxifen with liver microsomes represents the net result of the flavin-containing monooxygenase (FMO)-catalyzed N -oxide formation (forward reaction) and the N -oxide reduction (reverse reaction).

Differential Time-and NADPH-Dependent Inhibition of CYP2C19 by Enantiomers of Fluoxetine

Fluoxetine [±-N-methyl-3-phenyl-3-[(α, α, (-trifluoro-p-tolyl)oxy]-propylamine)] a selective serotonin reuptake inhibitor, is widely used in treating depression and other serotonin-dependent disease conditions. Racemic, (R)- and (S)-fluoxetine are potent reversible inhibitors of CYP2D6, and the racemate has been shown to be a mechanism-based inhibitor of CYP3A4. Racemic fluoxetine also demonstrates time- and concentration-dependent inhibition of CYP2C19 catalytic activity in vitro. In this study, we compared fluoxetine, its (R)- and (S)-enantiomers, ticlopidine, and S-benzylnirvanol as potential time-dependent inhibitors of human liver microsomal CYP2C19. In a reversible inhibition protocol (30 min preincubation with liver microsomes without NADPH), we found (R)-, (S)- and racemic fluoxetine to be moderate inhibitors with IC50 values of 21, 93, and 27 μM, respectively. However, when the preincubation was supplemented with NADPH, IC50 values shifted to 4.0, 3.4, and 3.0 μM, respectively resulting in IC50 shifts of 5.2-, 28-, and 9.3-fold. Ticlopidine showed a 1.8-fold shift in IC50 value, and S-benzylnirvanol shifted right (0.41-fold shift). Follow-up KI and kinact determinations with fluoxetine confirmed time-dependent inhibition [KI values of 6.5, 47, and 14 μM; kinact values of 0.023, 0.085, 0.030 min–1 for (R)-, (S)-, and racemate, respectively]. Although the (S)-isomer exhibits a much lower affinity for CYP2C19 inactivation relative to the (R)-enantiomer, it exhibits a more rapid rate of inactivation. Racemic norfluoxetine exhibited an 11-fold shift (18–1.5 μM) in IC50 value, suggesting that conversion of fluoxetine to this metabolite represents a metabolic pathway leading to time-dependent inhibition. These data provide an improved understanding of the drug-interaction potential of fluoxetine.

Selective time- and NADPH-dependent inhibition of human CYP2E1 by Clomethiazole

The sedative clomethiazole (CMZ) has been used in Europe since the mid-1960s to treat insomnia and alcoholism. It has been previously demonstrated in clinical studies to reversibly inhibit human CYP2E1 in vitro and decrease CYP2E1-mediated elimination of chlorzoxazone. We have investigated the selectivity of CMZ inhibition of CYP2E1 in pooled human liver microsomes (HLMs). In a reversible inhibition assay of the major drug-metabolizing cytochrome P450 (P450) isoforms, CYP2A6 and CYP2E1 exhibited IC50 values of 24 µM and 42 µM, respectively with all other isoforms exhibiting values >300 µM. When CMZ was preincubated with NADPH and liver microsomal protein for 30 minutes before being combined with probe substrates, however, more potent inhibition was observed for CYP2E1 and CYP2B6 but not CYP2A6 or other P450 isoforms. The substantial increase in potency of CYP2E1 inhibition upon preincubation enables the use of CMZ to investigate the role of human CYP2E1 in xenobiotic metabolism and provides advantages over other chemical inhibitors of CYP2E1. The KI and kinact values obtained with HLM-catalyzed 6-hydroxylation of chlorzoxazone were 40 µM and 0.35 minute−1, respectively, and similar to values obtained with recombinant CYP2E1 (41 µM, 0.32 minute−1). The KI and kinact values, along with other parameters, were used in a mechanistic static model to explain earlier observations of a profound decrease in the rate of chlorzoxazone elimination in volunteers despite the absence of detectable CMZ in blood.