Toward a systems approach to the human cytochrome P450 ensemble: interactions between CYP2D6 and CYP2E1 and their functional consequences.

Functional cross-talk among human drug-metabolizing cytochrome P450 through their association is a topic of emerging importance. Here, we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase. The potential of CYP2D6 to form oligomers in the microsomal membrane is among the highest observed with human cytochrome P450 studied up to date. We also observed the formation of heteromeric complexes of CYP2D6 with CYP2E1 and CYP3A4, and found a significant modulation of these interactions by 3,4-methylenedioxymethylamphetamine, a widespread drug of abuse metabolized by CYP2D6. Our results demonstrate an ample alteration of the catalytic properties of CYP2D6 and CYP2E1 caused by their association. In particular, we demonstrated that preincubation of microsomes containing co-incorporated CYP2D6 and CYP2E1 with CYP2D6-specific substrates resulted in considerable time-dependent activation of CYP2D6, which presumably occurs via a slow substrate-induced reorganization of CYP2E1-CYP2D6 hetero-oligomers. Furthermore, we demonstrated that the formation of heteromeric complexes between CYP2E1 and CYP2D6 affects the stoichiometry of futile cycling and substrate oxidation by CYP2D6 by means of decreasing the electron leakage through the peroxide-generating pathways. Our results further emphasize the role of P450-P450 interactions in regulatory cross-talk in human drug-metabolizing ensemble and suggest a role of interactions of CYP2E1 with CYP2D6 in pharmacologically important instances of alcohol-drug interactions.

Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver.

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.

Mapping the genetic architecture of gene expression in human liver.

Genetic variants that are associated with common human diseases do not lead directly to disease, but instead act on intermediate, molecular phenotypes that in turn induce changes in higher-order disease traits. Therefore, identifying the molecular phenotypes that vary in response to changes in DNA and that also associate with changes in disease traits has the potential to provide the functional information required to not only identify and validate the susceptibility genes that are directly affected by changes in DNA, but also to understand the molecular networks in which such genes operate and how changes in these networks lead to changes in disease traits. Toward that end, we profiled more than 39,000 transcripts and we genotyped 782,476 unique single nucleotide polymorphisms (SNPs) in more than 400 human liver samples to characterize the genetic architecture of gene expression in the human liver, a metabolically active tissue that is important in a number of common human diseases, including obesity, diabetes, and atherosclerosis. This genome-wide association study of gene expression resulted in the detection of more than 6,000 associations between SNP genotypes and liver gene expression traits, where many of the corresponding genes identified have already been implicated in a number of human diseases. The utility of these data for elucidating the causes of common human diseases is demonstrated by integrating them with genotypic and expression data from other human and mouse populations. This provides much-needed functional support for the candidate susceptibility genes being identified at a growing number of genetic loci that have been identified as key drivers of disease from genome-wide association studies of disease. By using an integrative genomics approach, we highlight how the gene RPS26 and not ERBB3 is supported by our data as the most likely susceptibility gene for a novel type 1 diabetes locus recently identified in a large-scale, genome-wide association study. We also identify SORT1 and CELSR2 as candidate susceptibility genes for a locus recently associated with coronary artery disease and plasma low-density lipoprotein cholesterol levels in the process.

Lack of effect of aprepitant on hydrodolasetron pharmacokinetics in CYP2D6 extensive and poor metabolizers.

To prevent chemotherapy-induced nausea and vomiting, aprepitant is given with a corticosteroid and a 5-hydroxytryptamine type 3 antagonist, such as dolasetron. Dolasetron is converted to the active metabolite hydrodolasetron, which is cleared largely via CYP2D6. The authors determined whether aprepitant, a moderate CYP3A4 inhibitor, alters hydrodolasetron pharmacokinetics in CYP2D6 poor and extensive metabolizers. Six CYP2D6 poor and 6 extensive metabolizers were randomized in an open-label, crossover fashion to treatment A (dolasetron 100 mg on day 1) and treatment B (dolasetron 100 mg plus aprepitant 125 mg on day 1, aprepitant 80 mg on days 2-3). For hydrodolasetron area under the concentration-versus-time curve (AUC0-infinity) and peak plasma concentration (Cmax), geometric mean ratios (B/A) and 90% confidence intervals (CIs) fell below the predefined limit (2.0) for clinical significance (AUC0-infinity, 1.09 [90% CI, 1.01-1.18], Cmax, 1.08 [90% CI, 0.94-1.24]). Aprepitant did not affect the pharmacokinetics of hydrodolasetron, regardless of CYP2D6 metabolizer type, and was generally well tolerated when coadministered with dolasetron in volunteers.

Development and characterization of LLC-PK1 cells containing Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport to human and mouse.

INTRODUCTION: P-glycoprotein is localized in numerous tissues throughout the body and plays an important role in the disposition of many xenobiotics. The contribution of P-glycoprotein-mediated drug transport is being evaluated in early drug discovery stages, particularly for compounds targeted to the central nervous system, using in vitro tools including cell lines expressing P-glycoprotein. Previous work in our laboratory suggests there are species differences in P-glycoprotein transport activity between humans and animals. The rat Abcb1a form of P-glycoprotein (formerly known as Mdr1a), the predominate isoform in the brain, has not been described in a functional cell system. Here, we describe the development and characterization of LLC-PK1 cells expressing rat Abcb1. METHODS: We cloned rat Abcb1a and generated a stable LLC-PK1 cell line. Expression and function of the cells were evaluated by immunoblot analysis, cytotoxicity analysis, cellular accumulation assays, and transcellular transport of probe substrates. The transport ratios of structurally diverse compounds obtained from parental cells or cells stably transfected with human ABCB1, mouse Abcb1a or rat Abcb1a were compared. RESULTS: Two forms of rat Abcb1a were cloned from Sprague-Dawley cDNA that differ by six amino acids and a base pair deletion. The intact form was stably transfected in LLC-PK1 cells. Immunoblot analysis demonstrated expression of the protein. The cells demonstrated P-glycoprotein-mediated function by directional transport of dexamethasone, ritonavir, and vinblastine in a transwell assay that was inhibited in the presence of cyclosporin A, verapamil, or quinidine. Likewise, the cells showed reduced cellular accumulation of Rh123 by FACS analysis that was reversed in the presence of cyclosporin A. These cells showed >or=350-fold resistance to colchicine, doxorubicin, vinblastine, and taxol and were sensitized in the presence of verapamil or cyclosporin A. Of 179 chemically diverse compounds evaluated, approximately 20% of the compounds evaluated were predicted to be substrates in one species but not in other species. DISCUSSION: Taken together, these data suggest these cells will be useful for evaluation of rat Abcb1a-mediated transport and for evaluation of species-specific P-glycoprotein-mediated transport.

Cytochrome P450 pharmacogenetics in drug development: in vitro studies and clinical consequences.

Members of the human cytochrome P450 (CYP) superfamily play a role in the metabolism of many drugs and several of them, CYP2D6, CYP2C9 and CYP2C19, have been shown to be polymorphic as a result of single nucleotide polymorphisms (SNPs), gene deletions, and gene duplications. These polymorphisms can impact the pharmacokinetics (PK), metabolism, safety and efficacy of drugs, and because of the availability of automation, genotyped human tissue, recombinant CYP preparations (rCYPs) and reagents, most pharmaceutical companies have increasingly screened out compounds that are metabolized solely by polymorphic CYPs. In the absence of suitable animal models, it has been widely accepted that such in vitro data are useful because one can obtain information prior to dosing in man and select the most appropriate clinical studies with prospectively genotyped and phenotyped subjects. Overall, current trends in the industry have been fueled by increased managed healthcare, the desire to minimize the need for therapeutic drug monitoring and CYP genotyping in medical practice, and a very competitive market place. In the past, such paradigms have not been as influential and there are numerous examples of marketed drugs that are metabolized by polymorphic CYPs.

Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes.

Drug or xenobiotics metabolizing enzymes (DMEs or XMEs) play central roles in the biotransformation, metabolism and/or detoxification of xenobiotics or foreign compounds, that are introduced to the human body. In general, DMEs protect or defend the body against the potential harmful insults from the environment. Once in the body, many xenobiotics may induce signal transduction events either specifically or non-specifically leading to various cellular, physiological and pharmacological responses including homeostasis, proliferation, differentiation, apoptosis, or necrosis. For the body to minimize the insults caused by these xenobiotics, various tissues/organs are well equipped with diverse DMEs including various Phase I and Phase II enzymes, which are present in abundance either at the basal level and/or increased/induced after exposure. To better understand the pharmacogenomic/gene expression profile of DMEs and the underlying molecular mechanisms after exposure to xenobiotics or drugs, we will review our current knowledge on DNA microarray technology in gene expression profiling and the signal transduction events elicited by various xenobiotics mediated by either specific receptors or non-specific signal transduction pathways. Pharmacogenomics is the study of genes and the gene products (proteins) essential for pharmacological or toxicological responses to pharmaceutical agents. In order to assess the battery of genes that are induced or repressed by xenobiotics and pharmaceutical agents, cDNA microarray or oligonucleotide-based DNA chip technology can be a powerful tool to analyze, simultaneously, the gene expression profiles that are induced or repressed by xenobiotics. The regulation of gene expression of the various phase I DMEs such as the cytochrome P450 (CYP) as well as phase II DMEs generally depends on the interaction of the xenobiotics with the receptors. For instance, the expression of CYP1 genes can be induced via the aryl hydrocarbon receptor (AhR) which dimerizes with the AhR nuclear translocator (ARNT), in response to many polycyclic aromatic hydrocarbon (PAHs). Similarly, the steroid family of orphan receptors, the constitutive androstane receptor (CAR) and pregnane X receptors (PXR), heterodimerize with the retinoid X receptor (RXR), transcriptionally activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as phenobarbital-like compounds (CAR) and dexamethasone and rifampin-type of agents (PXR). The peroxisome proliferator activated receptor (PPAR) which is one of the first characterized members of the nuclear hormone receptor, also dimerizes with RXR and it has been shown to be activated by lipid lowering agent fibrate-type of compounds leading to transcriptional activation of the promoters on the CYP4A genes. The transcriptional activation of these promoters generally leads to the induction of their mRNA. The physiological and the pharmacological implications of common partner of RXR for CAR, PXR, and PPAR receptors largely remain unknown and are under intense investigations. For the phase II DMEs, phase II gene inducers such as phenolic compounds butylated hydroxyanisol (BHA), tert-butylhydroquinone (tBHQ), green tea polyphenol (GTP), (-)-epicatechin-3-gallate (EGCG) and the isothiocyanates (PEITC, sulforaphane) generally appear to be electrophiles. They can activate the mitogen-activated protein kinase (MAPK) pathway via electrophilic-mediated stress response, resulting in the activation of bZIP transcription factors Nrf2 which dimerizes with Mafs and binds to the antioxidant/electrophile response element (ARE/EpRE) enhancers which are found in many phase II DMEs as well as many cellular defensive enzymes such as thioredoxins, gammaGCS and HO-1, with the subsequent induction of gene expression of these genes. It appears that in general, exposure to phase I or phase II gene inducers or xenobiotics may trigger a cellular "stress" response leading to the increase in the gene expression of these DMEs, which ultimately enhance the elimination and clearance of the xenobiotics e xenobiotics and/or the "cellular stresses" including harmful reactive intermediates such as reactive oxygen species (ROS), so that the body will remove the "stress" expeditiously. Consequently, this homeostatic response of the body plays a central role in the protection of the organism against environmental insults such as xenobiotics. Advances in DNA microarray technologies and mammalian genome sequencing will soon allow quantitative assessment of expression profiles of all genes in the selected tissues. The ability to predict phenotypic outcomes from gene expression profiles is currently in its infancy, however, and will require additional bioinformatic tools. Such tools will facilitate information gathering from literature and gene databases as well as integration of expression data with animal physiology studies. The study of pharmacogenomic/gene expression profile and the understanding of the regulation and the signal transduction mechanisms elicited by pharmaceutical agents can be of potential importance during drug discovery and the drug development.

Cytotoxicity of 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) and analogues in wild type and CYP3A4 stably transfected HepG2 cells.

The thiazolidinedione (TZD) ring is a constituent of the glitazones that are used to treat type II diabetes. Liver injury has been reported following chronic glitazone use; however, they do not produce hepatic damage in common laboratory animal species. In contrast, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) causes hepatotoxicity in rats. DCPT toxicity is dependent upon the presence of an intact TZD ring and cytochrome P450 (CYP)-mediated biotransformation. To further investigate TZD ring-induced toxicity, DCPT and several structural analogues or potential metabolites were tested in vitro using wild type human hepatoma HepG2 and HepG2 cells stably transfected with the CYP3A4 isozyme. CYP3A4 activity was confirmed by measuring testosterone 6ß-hydroxylation. Both cell lines were treated with 0-250 µM of the compounds in Hanks' balanced salt solution. Cell viability was measured after 24 h. DCPT and S-(3,5-dichlorophenyl)aminocarbonyl thioglycolic acid (DCTA) were the most toxic compounds of the series. Furthermore, DCPT was significantly more toxic in transfected cells (LC50=160.2±5.9 µM) than in wild type cells (LC50=233.0±19.7 µM). Treatment with a CYP3A4 inhibitor or inducer attenuated or potentiated DCPT cytotoxicity, respectively. These results suggest that DCPT-induced cytotoxicity in the transfected HepG2 cells is partially dependent on CYP3A4.

Selective inhibition of dog hepatic CYP2B11 and CYP3A12.

In the present study, N-(alpha-methylbenzyl-)-1-aminobenzotriazole (MBA) and ketoconazole (KET) were identified as the inhibitors with selectivity toward dog CYP2B11 and CYP3A12, respectively. Their selectivity was evaluated using phenacetin O-deethylation (CYP1A), diazepam (DZ) N1-demethylation (CYP2B11), diclofenac 4'-hydrxylation (CYP2C21), bufuralol 1'-hydroxylation (CYP2D11), and DZ C3-hydroxylation (CYP3A12) activities in dog liver microsomes (DLM). MBA exhibited potent mechanism-based inhibition of DZ N1-demethylase activity catalyzed by both baculovirus-expressed CYP2B11 and DLM. In both cases, inhibition was characterized by a low K(I) (0.35 and 0.46 microM, respectively) and high k(inact) (1.5 and 0.56 min(-1), respectively). Despite complete loss of DZ N1-demethylase activity in the presence of MBA, there was no significant loss of cytochrome P450 (P450) CO-binding spectrum. These data suggest that the inactivation involved covalent modification of P450 apoprotein, instead of the prosthetic heme moiety. A homology model of CYP2B11 was constructed, based on the crystal structure of rabbit CYP2C5, for docking the substrate (DZ) and the inhibitor (MBA), respectively. The model, within the limits of our approximations, helped explain the substrate specificity and inhibitor selectivity of CYP2B11. In contrast to MBA, KET was identified as a potent and selective reversible (competitive) inhibitor of CYP3A12 (K(I) = 0.13-0.33 microM). In fact, complete inhibition of CYP3A12-dependent DZ C3-hydroxylation was possible at a low KET concentration (1 microM). Therefore, it is concluded that one can attempt to conduct P450 reaction phenotype studies with DLM using MBA and KET as selective inhibitors of CYP2B11 and CYP3A12, respectively.

Induction of CYP1A in the beagle dog by an inhibitor of kinase insert domain-containing receptor: differential effects in vitro and in vivo on mRNA and functional activity.

Compound I [3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one] is a potent inhibitor of human kinase insert domain-containing receptor (KDR kinase), which is under investigation for the treatment of cancer. Bile duct-cannulated male beagle dogs were administered 6 mg/kg compound I q.d. for 14 days. There was an approximately 2.5-fold decrease in the mean plasma area under the curve of I on days 7 and 14 (approximately 11.3 microM . h), relative to day 1 (28.2 microM . h). In the dog, compound I was eliminated by metabolism, with a major pathway being aromatic hydroxylation and subsequent sulfation to form the metabolite M3. Metabolic profiling suggested that the pathway leading to the formation of the sulfated conjugate M3 was induced upon multiple dosing of I. Studies conducted in vitro suggested that CYP1A1/2 was responsible for the formation of the hydroxylated metabolite, which is sulfated to yield M3. Additional studies confirmed induction of CYP1A protein and activity in the livers of dogs treated with I. However, studies in a dog hepatocyte model of induction showed a surprising decrease both in CYP1A mRNA and enzymatic activity in the presence of I, emphasizing the need to consider the results from a variety of in vitro and in vivo studies in deriving an understanding of the metabolic fate of a drug candidate. It is concluded that the autoinduction observed after multiple treatments with compound I occurs since compound I is both an inducer and a substrate for dog CYP1A.

Inhibition kinetics of monoclonal antibodies against cytochromes P450.

Monoclonal antibodies (MAbs) inhibitory to individual cytochromes P450 (P450s) are of tremendous utility in identification of P450s responsible for the metabolism of a given drug or drug candidate in pharmaceuticals. In the present study, two inhibitory MAbs against CYP2D6 (MAb(2D6-50,) IgG(2b) and MAb(2D6-184), IgG(2a)) were developed by hybridoma technology to exhibit their high specificity and potency. The MAbs were further employed to assess the quantitative role (47-93%) of CYP2D6 to the metabolism of bufuralol in human liver microsomes from seven donors. Together with the MAb inhibitory to CYP3A4 as previously reported (Mei et al., 1999), the MAbs were used to study the inhibition kinetics of dextromethorphan O-demethylation (CYP2D6), testosterone 6beta-hydroxylation (CYP3A4) and aflatoxin B1 3-hydroxylation (CYP3A4), respectively, with an adequate size of sample measurement. A kinetic model was proposed to fit the experimental observations with three-dimensional nonlinear regression, thereby resulting in a solution of kinetic parameters, i.e., K(I), K(S), V(max), alpha, and beta (changes in K(I) or K(S) and V(max) in the presence of the MAb). As a result, dissociation constants (K(I)) of the MAbs for the enzymes and the maximal inhibition (beta) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 microM and > or =94%, respectively. The results have demonstrated that the model can accurately predict the kinetic parameters and provide some insights into the understanding of the mechanism of MAb interaction with P450 enzyme in nature and the applications of the MAbs in qualitative and quantitative identification of P450s involved in drug metabolism.

Stereo- and regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450.

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.

Sulfotransferase 1E1 is a low km isoform mediating the 3-O-sulfation of ethinyl estradiol.

Sulfation of ethinyl estradiol (EE) is a major pathway of first pass metabolism in both the intestine and liver. Consequently, we sought to identify the human sulfotransferases (SULTs) involved in the 3-O-sulfation of EE (EE-SULT). Based on the results described herein, cDNA-expressed human cytosolic SULT1A3 and SULT1E1 were identified as low Km isoforms (18.9 and 6.7 nM, respectively) mediating the sulfation of EE. In contrast, the EE-SULT catalyzed by other recombinant SULTs (SULT1A1 and 2A1) was a relatively high Km process (Km > or = 230 nM). The kinetics of EE-SULT in human intestine (Km1 = 24 nM; Km2 = 1206 nM) and liver (Km1 = 8 nM; Km2 = 2407 nM) cytosol was biphasic and conformed to a two-Km model with both low and high Km components. At a low EE concentration (3 nM), inhibition of EE-SULT activity (intestinal) was characterized with 2,6-dichloro p-nitrophenol (DCNP) (IC50 = 15.6 microM) and quercetin (IC50 = 0.4 microM). When these IC50 values were compared with those derived from expressed enzyme, inhibition of EE-SULT was consistent with the SULT1E1 (DCNP, IC50 = 20 microM; quercetin, IC50 = 0.6 microM), but not SULT1A3 (DCNP, IC50 = 12.4; quercetin, IC50 = 7 microM). Moreover, when estrone (which selectively inhibits expressed SULT1E1 and SULT1A3) was included in intestinal incubations, the high-affinity component of the Eadie-Hofstee plot for EE sulfation was inhibited, converting the plot from biphasic to monophasic. Collectively, these data are consistent with SULT1E1 as the primary sulfotransferase involved in EE sulfation at clinically relevant concentrations (<10 nM).

Substrate specificity and kinetic properties of seven heterologously expressed dog cytochromes p450.

Seven dog cytochromes p450 (p450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 microM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 microM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 microM), similar to that in dog liver microsomes (Km = 2.3 microM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 microM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16alpha-, 2alpha/2beta-, and 6beta-positions. Selectivity of CYP3A12 was observed toward testosterone 6beta-(Km = 83 microM) and 2alpha/2beta-hydroxylations (Km = 154 microM). However, the 16alpha-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 microM for CYP2C21). Therefore, the 6beta- and 16alpha-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the p450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog p450s and the development of dog p450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.

Activators of the rat pregnane X receptor differentially modulate hepatic and intestinal gene expression.

Ligand-mediated activation of the pregnane X receptor (PXR, NR1I2) is postulated to affect both hepatic and intestinal gene expression, because of the presence of this nuclear receptor in these important drug metabolizing organs; as such, activation of this receptor may elicit the coordinated regulation of PXR target genes in both tissues. Induction of hepatic and intestinal drug metabolism can contribute to the increased metabolism of drugs, and can result in adverse or undesirable drug-drug interactions. 2(S)-((3,5-bis(Trifluoromethyl)benzyl)-oxy)-3(S)phenyl-4-((3-oxo-1,2,4-triazol-5-yl)methyl)morpholine (L-742694) is a potent activator of the rat PXR and was characterized for its effects on hepatic and intestinal gene expression in female Sprague-Dawley rats by DNA microarray analysis. Transcriptional profiling in liver and small intestine revealed that L-742694 and dexamethasone (DEX) induced the prototypical battery of PXR target genes in liver, including CYP3A, Oatp2, and UGT1A1. In addition, both DEX and L-742694 induced common gene expression profiles that were specific to liver or small intestine, but there was a distinct lack of coordinated gene expression of genes common to both tissues. This pattern of gene regulation occurred in liver and small intestine independent of PXR, constitutive androstane receptor, or hepatic nuclear factor-4alpha expression, suggesting that other factors are involved in controlling the extent of coordinated gene expression in response to a PXR agonist. Overall, these results suggest that ligand-mediated activation of PXR and induction of hepatic, rather than small intestinal, drug metabolism genes would contribute to the increased metabolism of orally administered pharmaceuticals.

Effect of quinidine on the 10-hydroxylation of R-warfarin: species differences and clearance projection.

Stimulation by quinidine of warfarin metabolism in vitro was first demonstrated with liver microsomal preparations. We report herein that this drug interaction is reproducible in an animal model but that it exhibits profound species differences. Thus, using rabbit liver microsomes and a kinetic model incorporating two binding sites, the hepatic intrinsic clearance of R-warfarin via the 10-hydroxylation pathway (CL(int)(W)) was projected to be 6 +/- 1 and 128 +/- 51 microl/min/g liver, respectively, in the absence and presence of 21 microM unbound quinidine. These estimates were consistent with the results from studies in which rabbit livers (n = 5) were perfused in situ with R-warfarin or R-warfarin plus quinidine. The CL(int)(W) increased from 7 +/- 3 to 156 +/- 106 microl/min/g liver after increasing the hepatic exposure of unbound quinidine from 0 to 21 microM. In contrast, when liver microsomes or intact livers from rats were examined, R-warfarin metabolism was inhibited by quinidine, the CL(int)(W) decreasing to 26% of the control value after exposure of perfused rat livers (n = 5) to 22 microM unbound quinidine. The third example involved monkey liver microsomes, in which the rate of 10-hydroxylation of R-warfarin was little affected in the presence of quinidine (<2-fold increase). In all three species, the 10-hydroxylation of R-warfarin was catalyzed primarily by members of CYP3A, based on immuno- and chemical inhibition analyses. These findings not only highlight the variability of drug interactions among different species but also suggest that changes in hepatic clearance resulting from stimulation of cytochrome P450 activity may be projected based on estimates generated from corresponding liver microsomal preparations.