Leslie J. Dickmann

Expression and functional characterization of cytochrome P450 26A1, a retinoic acid hydroxylase

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 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+/-3.3nM and V(max) 11.3+/-4.3pmolesmin(-1)pmoleP450(-1)) 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.

An Inducible Cytochrome P450 3A4-Dependent Vitamin D Catabolic Pathway

Vitamin D3 is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D3 (25OHD3), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD3 was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD3 hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] dominating (Vmax/Km = 0.85 ml · min−1 · nmol enzyme−1). 4β,25(OH)2D3 was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D3, and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)2D3 in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4β,25(OH)2D3, but not CYP24A1-dependent 24R,25-dihydroxyvitamin D3 formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD3 metabolism may play an important role in the regulation of vitamin D3 in vivo and in the etiology of drug-induced osteomalacia.

Functional expression and comparative characterization of nine murine cytochromes P450 by fluorescent inhibition screening.

The increasing number of transgenic or gene knockout mouse models generated for use in drug metabolism studies has meant that a greater understanding of the function and substrate specificities of murine cytochromes P450 (P450s) has become essential, particularly with the recent advances in “humanized” mouse models. In this study, we have heterologously expressed nine murine P450s—Cyp1a1, Cyp1a2, Cyp1b1, Cyp2a4, Cyp2b20, Cyp2c29, Cyp2d22, Cyp2e1, and Cyp3a11—individually with human P450 oxidoreductase to generate functional monooxygenase systems in Escherichia coli. We have identified a suitable fluorogenic probe for each P450 and determined the apparent kinetic parameters. These probes have enabled the screening of a panel of 31 test compounds classified as “drugs,” “natural compounds,” “endogenous compounds,” and “pesticides” by measurement of IC50, thus allowing the comparison of binding affinities. Human P450s CYP2C9, CYP2D6, and CYP3A4 were also included in the study to enable direct comparisons to be made with the mouse enzymes. Although there were general similarities between human and mouse P450s, perhaps the most significant finding in this study was the observation that, despite 77% amino acid identity, Cyp2d22 and CYP2D6 were remarkably dissimilar in a range of enzymatic properties, with potentially serious implications for pharmacokinetic studies using CYP2D substrates. The data presented in this study provide a solid foundation with which to assess the degree of similarity (or difference) between mouse and human P450s involved in xenobiotic metabolism and can be used as a basis for further studies.

Quantitative Prediction of CYP2B6 Induction by Estradiol During Pregnancy: Potential Explanation for Increased Methadone Clearance During Pregnancy

There is considerable evidence that pregnancy changes the disposition of drugs in an enzyme- and gestational stage–specific manner. On the basis of probe drug studies, the activity of CYP3A4 and CYP2D6 increases and CYP1A2 decreases during human pregnancy. However, no studies of CYP2B6 activity during human pregnancy have been conducted. In rodent models and in HepG2 cells, CYP2B enzymes have been shown to be regulated by estradiol. Because estradiol concentrations increase by ∼50-fold during human pregnancy, it was hypothesized that the increasing estradiol concentrations during human pregnancy would result in induction of CYP2B6 activity. Hepatocytes from three female donors were treated with estradiol, and the EC50 and Emax were measured for CYP2B6 mRNA and bupropion hydroxylation activity. The measured values were used to predict the magnitude of CYP2B6 induction during human pregnancy. At 100 nM total estradiol, a concentration achievable during the third trimester of pregnancy, CYP2B6 activity was predicted to increase by 1.5–3-fold, based on increased CYP2B6 activity and mRNA. When the Emax and EC50 values were compared with those for carbamazepine and rifampin, estradiol was found to be as potent an inducer of CYP2B6 as rifampin and carbamazepine. These data suggest that, during human pregnancy, the increasing estradiol concentrations will result in increased clearance of drugs that have CYP2B6-mediated clearance pathways. This could in part explain the observed increase in methadone clearance during pregnancy.

Effects of Oral Vitamin K on S‐ and R‐Warfarin Pharmacokinetics and Pharmacodynamics: Enhanced Safety of Warfarin as a CYP2C9 Probe

Evidence for the selectivity of S‐warfarin metabolism by CYP2C9 is substantial, suggesting that warfarin may be a potential CYP2C9 phenotypingprobe. It is, however, limited by its ability to elevate the international normalized ratio (INR) and potentially cause bleeding. The effect of vitamin K to attenuate the elevation of INR may enable the safe use of warfarin as a probe. The objective of this study was to investigate the pharmacokinetics and pharmacodynamics of S‐ and R‐warfarin in plasma following the administration of warfarin alone versus warfarin and vitamin Kin CYP2C9 homozygotes. Healthy adults received, in a randomized crossover fashion in a fasted state, warfarin 10 mg orally or warfarin 10 mg plus vitamin K10 mg orally. Blood samples were obtained over 5 days during each phase. INR measurements were obtained at baseline and day 2 in each phase. INR, AUC0‐∞, and t1/2 of plasma S‐ and R‐warfarin were examined. Eleven CYP2C9*1 homozygotes (3 men, 8 women) were enrolled. INR at day 2 following warfarin 10 mg was 1.18 0.19, which differed significantly from baseline (INR = 1.00 0.05) and warfarin with vitamin K (INR = 1.06 0.07).INR at baseline was not significantly different from warfarin with vitamin K. t1/2 and AUC0 of both enantiomers did not significantly differ between the phases. It was concluded that INR is apparently attenuated by concomitant administration of a single dose of vitamin K without affecting the pharmacokinetics of either warfarin stereoisomer. Warfarin 10 mg may be safely used as a CYP2C9 probe in homozygotes when given concomitantly with 10 mg of oral vitamin K.

CYP2C9 Genetic Polymorphisms and Warfarin

The objective of this study was to report 2 cases of CYP2C9 genetic polymorphism and elevated warfarin S: R ratios in patients taking low doses of warfarin, and compare the observed characteristics with those in published reports. Two patients of different age groups and races were evaluated for CYP2C9 genotype and warfarin S: R ratios. The patients had been stabilized on weekly warfarin doses of 10.5 mg and 10 mg, respectively. Each patient was found to have at least 1 variant CYP2C9 allele. Elevated warfarin S: R ratios in both patients provided evidence for impaired metabolism of S-warfarin. This report of a CYP2C9*3 heterozygous individual taking a low dose of warfarin is consistent with previous reports in the literature. This summary of a CYP2C9*6 homozygous individual taking a low dose of warfarin is the first such published report. CYP2C9 genotyping in these patients provided a likely explanation for their continued low warfarin dosage requirements. Awareness of a patient’s CYP2C9 genotype may provide an explanation for low warfarin dosage requirements in stable patients and may help in determining the optimal dose in patients being initiated on warfarin.