ABSTRACT:The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K m or S 50 values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 Ϸ CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.

ABSTRACT:The biotransformation of prasugrel to R-138727 (2- [

ABSTRACT:Studies were performed to determine the human enzymes responsible for the biotransformation of atomoxetine to its major metabolite, 4-hydroxyatomoxetine, and to a minor metabolite, N-desmethylatomoxetine. Utilizing human liver microsomes containing a full complement of cytochrome P450 (P450) enzymes, average K m and CL int values of 2.3 M and 103 l/min/mg, respectively, were obtained for 4-hydroxyatomoxetine formation. Microsomal samples deficient in CYP2D6 exhibited average apparent K m and CL int values of 149 M and 0.2 l/min/mg, respectively. In a human liver bank characterized for P450 content, formation of 4-hydroxyatomoxetine correlated only to CYP2D6 activity. Of nine expressed P450s examined, 4-hydroxyatomoxetine was formed at a rate 475-fold greater by CYP2D6 compared with the other P450s. These results demonstrate that CYP2D6 is the enzyme primarily responsible for the formation of 4-hydroxyatomoxetine. Multiple P450s were found to be capable of forming 4-hydroxyatomoxetine when CYP2D6 was not expressed. However, the efficiency at which these enzymes perform this biotransformation is reduced compared with CYP2D6. The formation of the minor metabolite Ndesmethylatomoxetine exhibited average K m and CL int values of 83 M and 0.8 l/min/mg, respectively. Utilizing studies similar to those outlined above, CYP2C19 was identified as the primary enzyme responsible for the biotransformation of atomoxetine to Ndesmethylatomoxetine. In summary, CYP2D6 was found to be the primary P450 responsible for the formation of the major oxidative metabolite of atomoxetine, 4-hydroxyatomoxetine. Furthermore, these studies indicate that in patients with compromised CYP2D6 activity, multiple low-affinity enzymes will participate in the formation of 4-hydroxyatomoxetine. Therefore, coadministration of P450 inhibitors to poor metabolizers of CYP2D6 substrates would not be predicted to decrease the clearance of atomoxetine in these individuals.Atomoxetine (Fig. 1) (formally known as tomoxetine; LY139603) is under development as a therapeutic agent for the treatment of attention deficit hyperactivity disorder in children and adults. Atomoxetine enhances norepinephrine function through a highly selective blockade of the presynaptic norepinephrine transporter and has low affinities for other neuronal transporters or neurotransmitter receptor sites (Wong et al., 1982;Gehlert et al., 1993). This is an interesting and potentially important new drug since it is likely to be the first approved treatment for attention deficit hyperactivity disorder that is not a psychostimulant. Studies with this compound in healthy human volunteers (Farid et al., 1985) showed that the clearance of atomoxetine exhibited a bimodal distribution, suggesting that an enzyme that exhibits a genetic polymorphism was involved in the metabolism of atomoxetine. The study further reported that in both extensive and poor metabolizers of atomoxetine, para-hydroxyatomoxetine (later definitively identified as 4-hydroxyatomoxetine) was the major oxidative metabo...

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Uridine diphosphate glucuronosyltransferases (UGTs) catalyze the glucuronidation of a wide range of xenobiotics and endogenous substrates. However, there is a lack of information concerning the response of human UGTs to inducers, and this observation prompted the current investigation. The glucuronidation of estradiol (3-and 17-positions), naphthol, propofol, and morphine (3-and 6-positions) was assessed against a battery of recombinant human UGTs to determine selective glucuronidation reactions for induction studies. The potential induction of the glucuronidation of estradiol at the 3-position, naphthol, propofol, and morphine at the 3-position was subsequently investigated in cultured primary human hepatocytes against a range of prototypic inducers including dexamethasone, 3-methylcholanthrene (3-MC), phenobarbital, rifampicin, and omeprazole. Treatment with 3-MC induced estradiol-3-glucuronidation (up to 2.5-fold) in four of five donors investigated. Statistically significant increases in naphthol glucuronidation (up to 1.7-fold) were observed following treatment with carbamazepine. UGT1A9-mediated propofol glucuronidation was induced by phenobarbital (up to 2.2-fold) and rifampicin (up to 1.7-fold). However, treatment with ␣-naphthoflavone and tangeretin resulted in a decrease in propofol glucuronidation (30% of control values). Statistically significant induction of morphine-3-glucuronidation was observed in at least three donors following treatment with phenobarbital, rifampicin, and carbamazepine. Each UGT isoform investigated displayed a distinct induction profile. Although statistically significant increases in glucuronidation were observed for each reaction studied, the level of induction was less than that observed for CYP1A2 or CYP3A4 and exhibited a large interdonor variability. The clinical relevance of the induction responses obtained in this study is unclear.

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol-or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC͞MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methylfecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by 1 H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.fungi ͉ sterol biosynthesis

In vitro results suggested that tadalafil would have little effect on the pharmacokinetics of drugs metabolized by CYP3A. Clinical studies demonstrated that the pharmacokinetics of 2 different CYP3A substrates, midazolam and lovastatin, were virtually unchanged after tadalafil coadministration. Thus therapeutic concentrations of tadalafil do not produce clinically significant changes in the clearance of drugs metabolized by CYP3A.

For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.