ABSTRACT:Itraconazole (ITZ) is a potent inhibitor of CYP3A in vivo. However, unbound plasma concentrations of ITZ are much lower than its reported in vitro K i , and no clinically significant interactions would be expected based on a reversible mechanism of inhibition. The purpose of this study was to evaluate the reasons for the in vitro-in vivo discrepancy. The metabolism of ITZ by CYP3A4 was studied.

All-trans-retinoic acid (atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower Km (19nM) and Vmax (0.8pmol/min/pmol) than CYP26A1 (Km=50nM and Vmax=10pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10 fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure.

The decline in bone mineral density that occurs after longterm treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D 3 metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23-and 24-hydroxylation of 1,25(OH) 2 D 3 . No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH) 2 D 3 hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH) 3 D 3 , 1,24S,25(OH) 3 D 3 , and 1,23S,25(OH) 3 D 3 . Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH) 2 D 3 23-and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH) 2 D 3 metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Itraconazole (ITZ) is metabolized in vitro to three inhibitory metabolites: hydroxy-ITZ (OH-ITZ), keto-ITZ, and N-desalkyl-ITZ (ND-ITZ). The goal of this study was to determine the contribution of these metabolites to drug-drug interactions caused by ITZ. Six healthy volunteers received 100 mg ITZ orally for seven days and pharmacokinetic analysis was conducted at day 1 and day 7 of the study. The extent of CYP3A4 inhibition by ITZ and its metabolites was predicted using this data. ITZ, OH-ITZ, keto-ITZ and ND-ITZ, were detected in plasma samples of all volunteers. A 3.9-fold decrease in the hepatic intrinsic clearance of a CYP3A4 substrate was predicted using the average unbound steady-state concentrations (C ss,ave,u ) and liver microsomal inhibition constants for ITZ, OH-ITZ, keto-ITZ, and ND-ITZ. Accounting for circulating metabolites of ITZ significantly improved the in vitro to in vivo extrapolation of CYP3A4 inhibition compared to a consideration of ITZ exposure alone.

Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor been characterized kinetically. We expressed the rCYP26A1 in baculovirus infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K m 9.4 ± 3.3 nM and V max 11.3 ± 4.3 pmoles/min/pmole P450) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxoatRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARb were increased 300-to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, .60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD 1 , suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (K m 5 63 nM and intrinsic clearance 5 90 ml/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.

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All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC 50 values for 10 inhibitors were determined. The imidazole-and triazole-containing inhibitors [S- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC 50 values of 3.7, 4.2, and 8.6 M, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear receptor agonists as inhibitors.