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.

Discovery of selective glucocorticoid receptor modulator MK-5932.

A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists.

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.

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.

Pharmacokinetics of raltegravir in individuals with UGT1A1 polymorphisms.

Raltegravir is a human immunodeficiency virus-1 (HIV-1) integrase strand transfer inhibitor metabolized by glucuronidation via UDP-glucuronosyltransferase 1A1 (UGT1A1). In this study, 30 subjects with a UGT1A1*28/*28 genotype (associated with decreased activity of UGT1A1) and 27 UGT1A1*1/*1 control subjects (matched by race, age, gender, and body mass index) received a single 400-mg dose of raltegravir after fasting. No serious adverse experiences were reported, and there were no discontinuations due to adverse experiences.

Cytochrome P450 2C9 type II binding studies on quinoline-4-carboxamide analogues.

CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient predrug screening of these potential type II binding compounds is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide analogues to study the factors that determine the structure-activity relationships.

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.

CYP2C75-involved autoinduction of metabolism in rhesus monkeys of methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), a bradykinin B1 receptor antagonist.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B(1) receptor antagonist methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways.

Modeling and synthesis of novel tight-binding inhibitors of cytochrome P450 2C9.

Cytochrome P450 2C9 (2C9) is one of the three major drug metabolizing cytochrome P450 enzymes in human liver. Although the crystal structure of 2C9 has been solved, the important physicochemical properties of substrate-enzyme interactions remain difficult to be determined. This is due in part to the conformational flexibility of mammalian P450 enzymes.

Diclofenac hydroxylation in monkeys: efficiency, regioselectivity, and response to inhibitors.

The catalytic efficiency, regioselectivity, and response to chemical inhibitors of diclofenac (DF) hydroxylation in three Old World monkey liver microsomes (rhesus, cynomolgus, and African green monkey) are different from those determined with human liver microsomes. In contrast to the high affinity-high capacity (low Km-high Vmax) characteristics of DF 4'-hydroxylation in humans, this reaction proceeded in all monkey species with catalytic efficiencies >20-fold lower. However, DF 5-hydroxylation, a negligible reaction in human liver microsomes, was kinetically favored in monkeys mainly due to the increased Vmax values.

Compendium of gene expression profiles comprising a baseline model of the human liver drug metabolism transcriptome.

Oligonucleotide microarrays were used to study the variability of pharmacokinetics and drug metabolism (PKDM)-related gene expression in 75 normal human livers. The objective was to define and use absorption, distribution, metabolism and excretion (ADME) gene expression variability to discern co-regulated genes and potential surrogate biomarkers of inducible gene expression. RNA was prepared from donor tissue and hybridized on Agilent microarrays against an RNA mass balanced pool from all donors.

Microarray-based compendium of hepatic gene expression profiles for prototypical ADME gene-inducing compounds in rats and mice in vivo.

To examine species-specific aspects of the induction of absorption, distribution, metabolism and excretion (ADME)-related genes, we used 25 000 gene oligonucleotide microarrays to construct a rodent gene-response compendium that compared hepatic gene expression profiles and developed consensus aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) ligand signatures relevant to drug clearance. Twenty-six inducer compounds were chosen from the literature. Rats and mice received one of six dose levels (log2 dose escalation, 32-fold dose range) of each compound daily for 3 days.

Characterization of the rhesus monkey CYP3A64 enzyme: species comparisons of CYP3A substrate specificity and kinetics using baculovirus-expressed recombinant enzymes.

The rhesus monkey (Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 (AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 (Komori et al.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.