Wendel L. Nelson

ROLE OF ITRACONAZOLE METABOLITES IN CYP3A4 INHIBITION

Itraconazole (ITZ) is a potent inhibitor of CYP3A in vivo. However, unbound plasma concentrations of ITZ are much lower than its reported in vitro Ki, 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. Three metabolites were detected: hydroxy-itraconazole (OH-ITZ), a known in vivo metabolite of ITZ, and two new metabolites: keto-itraconazole (keto-ITZ) and N-desalkyl-itraconazole (ND-ITZ). OHITZ and keto-ITZ were also substrates of CYP3A4. Using a substrate depletion kinetic approach for parameter determination, ITZ exhibited an unbound Km of 3.9 nM and an intrinsic clearance (CLint) of 69.3 ml·min-1·nmol CYP3A4-1. The respective unbound Km values for OH-ITZ and keto-ITZ were 27 nM and 1.4 nM and the CLint values were 19.8 and 62.5 ml·min-1·nmol CYP3A4-1. Inhibition of CYP3A4 by ITZ, OH-ITZ, keto-ITZ, and ND-ITZ was evaluated using hydroxylation of midazolam as a probe reaction. Both ITZ and OH-ITZ were competitive inhibitors of CYP3A4, with unbound Ki (1.3 nM for ITZ and 14.4 nM for OH-ITZ) close to their respective Km. ITZ, OH-ITZ, keto-ITZ and ND-ITZ exhibited unbound IC50 values of 6.1 nM, 4.6 nM, 7.0 nM, and 0.4 nM, respectively, when coincubated with human liver microsomes and midazolam (substrate concentration < Km). These findings demonstrate that ITZ metabolites are as potent as or more potent CYP3A4 inhibitors than ITZ itself, and thus may contribute to the inhibition of CYP3A4 observed in vivo after ITZ dosing.

Intestinal and Hepatic CYP3A4 Catalyze Hydroxylation of 1α,25-Dihydroxyvitamin D3: Implications for Drug-Induced Osteomalacia

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D3 metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] 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)2D3. 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)2D3 hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)3D3, 1,24S,25(OH)3D3, and 1,23S,25(OH)3D3. 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)2D3 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)2D3 metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Tian ma, an ancient Chinese herb, offers new options for the treatment of epilepsy and other conditions

Our purpose is to bring attention to the antiepileptic properties of the Chinese herb tian ma and its constituents, as well as to suggest the potential for the development of new antiepileptic drugs (AEDs) related to this herb. All available literature regarding the chemistry, pharmacology, animal data, and clinical use of tian ma and its constituents are reviewed, showing that tian ma, its constituents, and its symbiotic fungus Armillaria mellea have antiepileptic properties in in vitro and in vivo models. One clinical study reportedly demonstrated the AED effects of a component of tian ma, vanillin. Thus, tian ma, its constituent vanillin, and its symbiotic fungus armillaria hold promise as cost-effective and less toxic alternatives to standard AEDs. In addition, similar chemical compounds may be developed as AEDs.

Contribution of Itraconazole Metabolites to Inhibition of CYP3A4 in vivo

Itraconazole (ITZ) is metabolized in vitro to three inhibitory metabolites: hydroxy‐itraconazole (OH‐ITZ), keto‐itraconazole (keto‐ITZ), and N‐desalkyl‐itraconazole (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 7 days, and pharmacokinetic analysis was conducted at days 1 and 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 (Css,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.

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.

In vivo interaction of the enantiomers of disopyramide in human subjects

Disopyramide, an antiarrhythmic agent, is marketed as a racemic mixture of two enantiomers. The racemic drug has unusual pharmacokinetic properties because of its concentration-dependent binding to plasma proteins in the therapeutic plasma concentration range. This study examined, in healthy subjects, the individual pharmacokinetic properties of both total and unbound d-and ldisopyramide in plasma after intravenous administration of each enantiomer separately (1.5mg/kg).Also investigated is the pharmacokinetics of total d-and l-disopyramide in plasma after intravenous administration of a pseudoracemate. Both d-and l-disopyramide are found to exhibit concentration-dependent binding to plasma proteins, with d-disopyramide being more avidly bound at lower concentrations. The stereoselective, concentration-dependent binding to plasma proteins resulted in distinct pharmacokinetic properties when the enantiomers were given together as the pseudoracemate. d-Disopyramide had a lower plasma clearance and renal clearance, a longer half-life, and a smaller apparent volume of distribution than l-disopyramide. However, when the enantiomers were administered separately, there were no differences in the clearance, renal clearance, and volume of distribution between enantiomers calculated from either total or unbound drug concentrations. The results reveal an important pharmacokinetic interaction between the enantiomers of disopyramide when given as a racemic mixture, which may be dose-dependent and is not apparent upon administration of the enantiomers separately.

Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases

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 K(m) (19nM) and V(max) (0.8 pmol/min/pmol) than CYP26A1 (K(m)=50 nM and V(max)=10 pmol/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 cardiac effects of d- and l-disopyramide in normal subjects: a noninvasive study.

Commercially available disopyramide is a racemic mixture of equal parts of dextrorotatory (d‐) and levorotatory (1‐) optical isomers. We studied the cardiac effects of i.v. adnministration of each isomer and the racemic mixture (dl‐) in six normal males by digitized echocardiography, systolic time intervals and ECG. Both isomers and the racemic mixture produced equally marked dose‐dependent negative inotropic effects (28.1 11.8% mean maximal reduction in fractional shortening of left ventricular dimension) and diastolic effects (28.6 ± 24.1% mean maximal reduction in peak left ventricular filling rate). Howevet, only the d‐isomer prolonged QTc duration (by 13.6 5.2% at maximum, p < 0.001 vs 1‐isomer). We conclude that disopyramide, in the doses used, produces marked adverse effects on left ventricular systolic and diastolic function in normal subjects independent of optical rotation. The production of these effects bEy the 1‐isomer without affecting QTc duration suggests different subcellular mechanisms for the

Clonidine Pharmacokinetics in Pregnancy

The objective of this study was to determine the pharmacokinetic parameters of clonidine during pregnancy compared with previously published data in nonpregnant subjects. Serial blood and urine samples were collected in 17 women during mid to late pregnancy over one steady-state dosing interval to determine clonidine noncompartmental pharmacokinetic parameters (n = 17) and creatinine clearance. In six of these pregnant subjects, maternal and umbilical cord (venous and arterial) plasma samples were collected at the time of delivery for measurement of clonidine concentrations. Clonidine apparent oral clearance was found to be 440 ± 168 ml/min during pregnancy compared with 245 ± 72 ml/min as previously reported in nonpregnant subjects (p < 0.0001) (Cunningham et al., 1994). There was a strong correlation (r = 0.82, p < 0.001) between clonidine renal clearance, adjusted for variation in glomerular filtration rate, and urine pH. Umbilical cord to maternal plasma clonidine concentration ratios were 1.0 ± 0.1 (arterial) and 1.0 ± 0.1 (venous). In conclusion, clonidine is cleared more rapidly in pregnant women than in nonpregnant subjects. At the time of delivery, the fetus is exposed to similar plasma clonidine concentrations as the mother.

STEREOCHEMICAL ASPECTS OF ITRACONAZOLE METABOLISM IN VITRO AND IN VIVO

Itraconazole (ITZ) has three chiral centers and is administered clinically as a mixture of four stereoisomers. This study evaluated stereoselectivity in ITZ metabolism. In vitro experiments were carried out using heterologously expressed CYP3A4. Only (2R,4S,2′R)-ITZ and (2R,4S,2′S)-ITZ were metabolized by CYP3A4 to hydroxy-ITZ, keto-ITZ, and N-desalkyl-ITZ. When (2S,4R,2′R)-ITZ or (2S,4R,2′S)-ITZ was incubated with CYP3A4, neither metabolites nor substrate depletion were detected. Despite these differences in metabolism, all four ITZ stereoisomers induced a type II binding spectrum with CYP3A4, characteristic of coordination of the triazole nitrogen to the heme iron (Ks 2.2–10.6 nM). All four stereoisomers of ITZ inhibited the CYP3A4-catalyzed hydroxylation of midazolam with high affinity (IC50 3.7–14.8 nM). Stereochemical aspects of ITZ pharmacokinetics were evaluated in six healthy volunteers after single and multiple oral doses. In vivo, after a single dose, ITZ disposition was stereoselective, with a 3-fold difference in Cmax and a 9-fold difference in Cmin between the (2R,4S)-ITZ and the (2S,4R)-ITZ pairs of diastereomers, with the latter reaching higher concentrations. Secondary and tertiary ITZ metabolites (keto-ITZ and N-desalkyl-ITZ) detected in plasma were of the (2R,4S) stereochemistry. After multiple doses of ITZ, the difference in Cmax and Cmin decreased to 1.5- and 3.8-fold, respectively. The initial difference between the stereoisomeric pairs was most likely due to stereoselective metabolism by CYP3A4, including stereoselective first-pass metabolism as well as stereoselective elimination. However, stereoselective elimination was diminished after multiple dosing, presumably as a result of CYP3A4 autoinhibition. In conclusion, the metabolism of ITZ is highly stereoselective in vitro and in vivo.