The Evolution of Drug Metabolism and Disposition: A Perspective From the Editors.

This article was solicited to commemorate the 50th anniversary of Drug Metabolism and Disposition (DMD) and features perspectives from five former editors spanning the years 1994 to 2020. During that time frame the journal underwent significant changes in manuscript submission and processing as well as multiple generational changes in the composition of the editorial board and associate editors. A constant, however, has been the commitment to be the premier journal for publications of articles in the areas of drug metabolism, absorption, distribution, excretion, and pharmacokinetics. Advances in some of those areas during the past 3 decades have been monumental. Two cases in point involve cytochromes P450 and drug transporters. In 1994 rigorous characterization of human cytochrome P450 enzymes was in its infancy, there were no proven selective inhibitors, and the idea of solving a human P450 X-ray crystal structure was just a fantasy. Likewise, little was known about individual drug transporters. Today, detailed knowledge of individual human P450 enzymes and drug transporters is integral in drug design and drug discovery and in avoiding drug interactions. In the face of these huge advances in knowledge, each editor has been charged with maintaining the caliber and significance of the journal and its financial solvency while serving the needs of individual authors. We present 5 individual perspectives on the challenges and rewards of serving as DMD editor and hope that, by humanizing the job, we will encourage others to assume positions of responsibility in publication of society journals. SIGNIFICANCE STATEMENT: The 5 most recent former editors of DMD describe their experiences and perspectives on the position in the context of constantly changing scientific emphases, technology, and publishing practices. The article offers subscribers, authors, and future editors and editorial board members valuable insights into the inner workings of the journal.

Four Decades of Cytochrome P450 2B Research: From Protein Adducts to Protein Structures and Beyond.

This article features selected findings from the senior author and colleagues dating back to 1978 and covering approximately three-fourths of the 60 years since the discovery of cytochrome P450. Considering the vast number of P450 enzymes in this amazing superfamily and their importance for so many fields of science and medicine, including drug design and development, drug therapy, environmental health, and biotechnology, a comprehensive review of even a single topic is daunting. To make a meaningful contribution to the 50th anniversary of Drug Metabolism and Disposition, we trace the development of the research in a single P450 laboratory through the eyes of seven individuals with different backgrounds, perspectives, and subsequent career trajectories. All co-authors are united in their fascination for the structural basis of mammalian P450 substrate and inhibitor selectivity and using such information to improve drug design and therapy. An underlying theme is how technological advances enable scientific discoveries that were impossible and even inconceivable to prior generations. The work performed spans the continuum from: 1) purification of P450 enzymes from animal tissues to purification of expressed human P450 enzymes and their site-directed mutants from bacteria; 2) inhibition, metabolism, and spectral studies to isothermal titration calorimetry, deuterium exchange mass spectrometry, and NMR; 3) homology models based on bacterial P450 X-ray crystal structures to rabbit and human P450 structures in complex with a wide variety of ligands. Our hope is that humanizing the scientific endeavor will encourage new generations of scientists to make fundamental new discoveries in the P450 field. SIGNIFICANCE STATEMENT: The manuscript summarizes four decades of work from Dr. James Halpert's laboratory, whose investigations have shaped the cytochrome P450 field, and provides insightful perspectives of the co-authors. This work will also inspire future drug metabolism scientists to make critical new discoveries in the cytochrome P450 field.

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship.

Preclinical and clinical evidence for the collaborative transport and renal secretion of an oxazolidinone antibiotic by organic anion transporter 3 (OAT3/SLC22A8) and multidrug and toxin extrusion protein 1 (MATE1/SLC47A1).

N-({(5S)-3-[4-(1,1-dioxidothiomorpholin-4-yl)-3,5-difluorophenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide (PNU-288034), an oxazolidinone antibiotic, was terminated in phase I clinical development because of insufficient exposure. Analysis of the drug pharmacokinetic and elimination profiles suggested that PNU-288034 undergoes extensive renal secretion in humans. The compound was well absorbed and exhibited approximately linear pharmacokinetics in the oral dose range of 100 to 1000 mg in human. PNU-288034 was metabolically stable in liver microsomes across species, and unchanged drug was cleared in the urine by an apparent active renal secretion process in rat and monkey (two to four times glomerular filtration rate) but not dog. In vitro studies conducted to characterize the transporters involved demonstrated PNU-288034 uptake by human organic anion transporter 3 (OAT3; K(m) = 44 +/- 5 microM) and human multidrug and toxin extrusion protein 1 (hMATE1; K(m) = 340 +/- 55 microM). The compound was also transported by multidrug resistance P-glycoprotein and breast cancer resistance protein. In contrast, human organic cation transporter 2, human OAT1, and hMATE2-K did not transport PNU-288034. Coadministration of PNU-288034 and the OAT3 inhibitor probenecid significantly increased PNU-288034 plasma area under the curve (170%) and reduced both plasma and renal clearance in monkey. Coadministration of PNU-288034 and cimetidine, a MATE1 inhibitor, also reduced plasma clearance in rat to a rate comparable with probenecid coadministration. Collectively, our results demonstrated a strong in vitro-in vivo correlation for active renal secretion coordinated through the vectorial transport process of OAT3 and MATE1, which ultimately resulted in limiting the systemic exposure of PNU-288034.

A humanized UGT1 mouse model expressing the UGT1A1*28 allele for assessing drug clearance by UGT1A1-dependent glucuronidation.

Humanized mice that express the human UDP-glucuronosyltransferase (UGT) 1 locus have been developed in a Ugt1-null background as a model to improve predictions of human UGT1A-dependent drug clearance. Enzyme kinetic parameters (K(m) and V(max)) and pharmacokinetic properties of three probe drugs were compared using wild-type and humanized UGT1 mice that express the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28)) Ugt1(-/-) mice]. The well characterized substrate for UGT1A1, 7-ethyl-10-hydroxy-camptothecin (SN-38), showed the greatest difference in parent drug exposure ( approximately 3-fold increase) and clearance ( approximately 3-fold decrease) in Tg(UGT1(A1*28)) Ugt1(-/-) mice after intravenous administration compared with wild-type and phenobarbital-treated animals. In contrast, the clearance of the UGT2B7 substrate (-)-17-allyl-4, 5alpha-epoxy-3, 14-dihydroxymorphinan-6-one (naloxone) was not altered in Tg(UGT1(A1*28)) Ugt1(-/-) mice. In addition, pharmacokinetic parameters with 1-(4-fluorophenyl)3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone (ezetimibe, Zetia; Merck & Co., Whitehouse Station, NJ), considered to be a major substrate for UGT1A1, showed small to no dependence on UGT1A1-directed glucuronidation. Enzyme kinetic parameters assessed for SN-38, ezetimibe, and naloxone using liver microsomes prepared from wild-type and Tg(UGT1(A1*28)) Ugt1(-/-) mice showed patterns consistent with the in vivo pharmacokinetic data. For SN-38 glucuronidation, V(max) decreased 5-fold in Tg(UGT1(A1*28)) Ugt1(-/-) mouse liver microsomes compared with microsomes prepared from wild-type mice, and decreased 10-fold compared with phenobarbital-treated Tg(UGT1(A1*28)) Ugt1(-/-) mice. These differences are consistent with SN-38 glucuronidation activities using HLMs isolated from individuals genotyped as UGT1A1*1 or UGT1A1*28. For ezetimibe and naloxone the differences in V(max) were minimal. Thus, Tg(UGT1(A1*28)) Ugt1(-/-) mice can serve as a pharmacokinetic model to further investigate the effects of UGT1A1 expression on drug metabolism.

Evaluation of drug transporter interactions in drug discovery and development.

Drug transporters play an important role in the absorption, distribution, excretion and toxicity of both endogenous and exogenous compounds. Transporters may act as physiological 'gatekeepers' in the regulation of the pharmacological and/or toxicological effects of drugs by limiting distribution to tissues responsible for their effect and/or toxicity. This review will first provide a brief outline of the characteristics of membrane bound drug transporter families and their respective roles in regulating drug pharmacokinetics. This background then provides the context for a discussion of the characterization of a drug candidate as a substrate, inhibitor and/or inducer of drug transporter(s), followed by an assessment of the in vitro and in vivo preclinical methods used in drug discovery and development for screening molecules to identify potential transporter interactions. Finally, specific examples of the translation of in vitro findings to the in vivo effects are discussed to link the current understanding of the impact of drug transporters to clinical pharmacology. Thus, the goal is to provide the drug discovery scientist with a cadre of concepts, strategies, and tools for ultimately making rational decisions in drug design and delivery resulting in the optimization of drug concentrations at the target of pharmacology.

Expression and characterization of functional dog flavin-containing monooxygenase 3.

Mammalian flavin-containing monooxygenase (FMO) enzymes catalyze oxidation at nucleophilic, heteroatom centers and are important for drug, xenobiotic, and endogenous substrate metabolism. In human liver, human FMO3 (hFMO3) is the most abundant FMO isoform and is known to contribute to the hepatic clearance of a variety of clinical drugs. The purpose of the current study was to express and compare the dog (beagle) FMO3 (dFMO3) to hFMO3. A full-length dFMO3 cDNA was obtained from liver by reverse transcription-polymerase chain reaction. Using a baculovirus expression system in Spodoptera frugiperda insect cells, dFMO3 was expressed to protein levels of 0.50 nmol/mg, as determined by liquid chromatography-fluorescence detection. Expressed dFMO3 displayed Michaelis-Menten kinetics, catalyzing NADPH-dependent N-oxidation of benzydamine, with K(m) and V(max) values of 18.6 microM and 0.63 nmol N-oxide formed/min/nmol of enzyme, respectively. Benzydamine N-oxidation catalyzed by hFMO3 showed values of 42.6 microM (K(m)) and 3.56 nmol/min/nmol of enzyme (V(max)). Human FMO3 was observed to catalyze the S-oxidation of sulindac sulfide, with respective K(m) and V(max) values of 69.3 microM and 35.4 nmol/min/nmol of enzyme. dFMO3 also catalyzed sulindac sulfide S-oxidation with 6.8 nmol/min/nmol of enzyme being the highest velocity observed. Finally, Western blot analysis indicated protein expression levels of dFMO3 in pooled dog liver and lung microsomes to be 27 and 9 pmol/mg, respectively. In summary, dFMO3 appears to be a functional enzyme expressed at appreciable levels in liver, but one with some kinetic properties that are substantially different from its human homolog hFMO3.

Human hepatic CYP2B6 developmental expression: the impact of age and genotype.

Although CYP2B6 is known to metabolize numerous pharmaceuticals and toxicants in adults, little is known regarding CYP2B6 ontogeny or its possible role in pediatric drug/toxicant metabolism. To address this knowledge gap, hepatic CYP2B6 protein levels were characterized in microsomal protein preparations isolated from a pediatric liver bank (N=217). Donor ages ranged from 10 weeks gestation to 17 years of age with a median age of 1.9 months. CYP2B6 levels were measured by semi-quantitative western blotting. Overall, CYP2B6 expression was detected in 75% of samples. However, the percentage of samples with detectable CYP2B6 protein increased with age from 64% in fetal samples to 95% in samples from donors >10 years of age. There was a significant, but only 2-fold increase in median CYP2B6 expression after the neonatal period (birth to 30 days postnatal) although protein levels varied over 25-fold in both age groups. The median CYP2B6 level in samples over 30 postnatal days to 17 years of age (1.3 pmol/mg microsomal protein) was lower than previously reported adult levels (2.2-22 pmol/mg microsomal protein), however, this likely relates to the median age of these samples, i.e., 10.3 months. CYP2B6 expression did not vary significantly by gender. Furthermore, CYP2B6 levels did not correlate with CYP3A4, CYP3A5.1 or CYP3A7 activity, consistent with different mechanisms controlling the ontogeny and constitutive expression of these enzymes and the lack of significant induction in the pediatric samples.

Developmental changes in human liver CYP2D6 expression.

Within the human cytochrome P450 family, specific forms show developmental expression patterns that can affect drug clearance, efficacy, and safety. The objective of this study was to use dextromethorphan O-demethylase activity and quantitative Western blotting to identify CYP2D6 developmental expression patterns in a large (n = 222) and developmentally diverse set of pediatric liver samples. Immunodetectable levels of CYP2D6 protein determined for selected samples across all age categories showed a significant correlation with the corresponding dextromethorphan O-demethylase activity. Of gender, ethnicity, postmortem interval, and genotype, only increasing gestational age was associated with CYP2D6 activity and protein content in prenatal samples. In contrast, both age and genotype were associated with CYP2D6 expression in postnatal samples. CYP2D6 expression in liver samples from neonates less than 7 days of age was higher than that observed in first and second trimester samples, but not significantly higher than third trimester fetal samples. In contrast, expression in postnatal samples greater than 7 days of age was substantially higher than that for any earlier age category. Higher CYP2D6 expression also was observed in liver samples from Caucasians versus African Americans. Finally, using phenotype categories inferred from genotype, CYP2D6 activity was higher in postnatal samples predicted to be extensive or intermediate metabolizers versus poor metabolizers. These results suggest that age and genetic determinants of CYP2D6 expression constitute significant determinants of interindividual variability in CYP2D6-dependent metabolism during ontogeny.

Epirubicin glucuronidation and UGT2B7 developmental expression.

The usefulness of epirubicin in the treatment of adult and childhood malignant diseases is related in part to the potential reduction in cardiac toxicity compared with that of other anthracyclines given at equivalent doses. An important pathway for epirubicin detoxification is UGT2B7-dependent glucuronidation. This study was implemented to provide a preclinical evaluation of the metabolism of epirubicin with respect to age-related changes in epirubicin glucuronidation in pediatric liver microsomes. Rates of epirubicin glucuronidation and levels of UGT2B7 were determined for liver microsomes from four pediatric age categories (n = 32) and one adult age category (n = 8). Both sets of data showed an increase in UGT2B7 activity and content with increasing age. Epirubicin glucuronidation activity in the adult group was statistically higher compared with all pediatric age groups (p < or = 0.01). UGT2B7 expression also was statistically higher in adults compared with children below 11 years of age, with evidence of significant differences in protein levels among the pediatric age categories. A positive correlation (r = 0.68) between UGT2B7 levels and postnatal age was observed, suggesting a progressive increase in UGT2B7 protein expression with increasing age. However, allometric scaling using the (3/4) power rule suggested no difference in activity between any of the pediatric age categories and the adult, although only a single neonatal sample was included in the analysis. In summary, these in vitro data show differences in epirubicin glucuronidation and UGT2B7 content within pediatric age groups and support the use of epirubicin in pediatric patients at least 6 months of age.

New perspectives on the impact of cytochrome P450 3A expression for pediatric pharmacology.

Advances in the basic and clinical sciences of drug actions and safety have been applied almost exclusively to the largest demographic patient group--adults. Metabolism-dependent drug clearance is not only a primary determinant for obtaining efficacious drug exposure, but could also demonstrate clear age-dependence. These concepts are exemplified by the three major human cytochrome P450 (CYP) 3A enzymes: CYP3A4, CYP3A5 and CYP3A7. Recent preclinical and clinical studies show CYP3A7 is the most abundant CYP3A enzyme in fetal liver, with a gradual shift towards CYP3A4 expression throughout childhood. However, the polymorphic nature and regulatory intricacies of CYP3A5 and CYP3A7 expression could cause an underappreciated contribution to interindividual variability in drug response and safety.

Developmental expression of human hepatic CYP2C9 and CYP2C19.

The CYP2C subfamily is responsible for metabolizing many important drugs and accounts for about 20% of the cytochrome p450 in adult liver. To determine developmental expression patterns, liver microsomal CYP2C9 and -2C19 were measured (n = 237; ages, 8 weeks gestation-18 years) by Western blotting and with diclofenac or mephenytoin, respectively, as probe substrates. CYP2C9-specific content and catalytic activity were consistent with expression at 1 to 2% of mature values (i.e., specific content, 18.3 pmol/mg protein and n = 79; specific activity, 549.5 pmol/mg/min and n = 72) during the first trimester, with progressive increases during the second and third trimesters to levels approximately 30% of mature values. From birth to 5 months, CYP2C9 protein values varied 35-fold and were significantly higher than those observed during the late fetal period, with 51% of samples exhibiting values commensurate with mature levels. Less variable CYP2C9 protein and activity values were observed between 5 months and 18 years. CYP2C19 protein and catalytic activities that were 12 to 15% of mature values (i.e., specific content, 14.6 pmol/mg and n = 20; specific activity, 18.5 pmol/mg/min and n = 19) were observed as early as 8 weeks of gestation and were similar throughout the prenatal period. CYP2C19 expression did not change at birth, increased linearly over the first 5 postnatal months, and varied 21-fold from 5 months to 10 years. Adult CYP2C19 protein and activity values were observed in samples older than 10 years. The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0- to 5-months postnatal samples, implying different developmental regulatory mechanisms.

Developmental expression of the major human hepatic CYP3A enzymes.

The human cytochrome P4503A forms show expression patterns subject to developmental influence. CYP3A7 and CYP3A4 are generally classified as the major fetal and adult liver forms, respectively. However, characterization of CYP3A4, -3A5, and -3A7 developmental expression has historically been confounded by the lack of CYP3A isoform-specific antibodies or marker enzyme activities. Therefore, the objective of this study was to characterize the developmental expression of hepatic CYP3A forms from early gestation to 18 years of age using up to 212 fetal and pediatric liver samples. Based on immunoquantitation, CYP3A5 protein expression was found to be highly variable, generally independent of age, and more frequently observed for African-American individuals. For differentiation of CYP3A4 and -3A7 levels, dehydroepiandrosterone metabolite patterns for expressed CYP3A forms were characterized and used for simultaneous quantitation of protein levels within liver microsome samples. The major metabolite formed by CYP3A4, 7beta-hydroxy-dehydroepiandrosterone, was identified based on cochromatography and mass spectra matching with the authentic standard. Kinetic analysis showed a 34-fold greater intrinsic clearance of 7beta-hydroxy-dehydroepiandrosterone by CYP3A4 versus -3A7, whereas CYP3A7 showed the highest 16alpha-hydroxy-dehydroepiandrosterone intrinsic clearance. Metabolite profiles for the expressed enzymes were fit to a multiple response model and CYP3A4 and -3A7 levels in fetal and pediatric liver microsome samples were calculated. Fetal liver microsomes showed extremely high CYP3A7 levels (311-158 pmol/mg protein) and significant expression through 6 months postnatal age. Low CYP3A4 expression was noted for fetal liver (< or =10 pmol/mg), with mean levels increasing with postnatal age.

The changing environment of graduate and postdoctoral training in drug metabolism: viewpoints from academia, industry, and government.

This article is an invited report of a symposium sponsored by the Drug Metabolism Division of the American Society for Pharmacology and Experimental Therapeutics held at Experimental Biology 2002 in New Orleans. The impetus for the symposium was a perceived shortage in the supply of graduate students qualified for drug metabolism research positions in industry, academia, and government. For industry, recent hiring stems largely from the expansion of drug metabolism departments in an effort to keep pace with the demands of drug discovery and new technologies. In turn, regulatory scientists are needed to review and verify the results of the increased number and volume of studies required for drug development and approval. Thus the initial source of training, academia, has been forced to recognize these external hiring pressures while trying to attract and retain the faculty, postdoctoral scientists, and students necessary for active teaching and research programs. The trend of the expansion of the interdisciplinary nature of traditional drug metabolism to include emerging technologies such as pharmacogenetics, transporters, and proteomics and the implications for future needs in training and funding were acknowledged. There was also consensus on the value of partnerships between academia and industry for increasing student interest and providing training in disciplines directly applicable to industrial drug metabolism research. Factors affecting the sources of these trainees, such as federal funding, the number of trainees per institution, and recent issues with immigration restrictions that have limited the flow of scientists were also discussed.

Expression and characterization of functional dog flavin-containing monooxygenase 1.

A full-length dog (beagle) flavin-containing monooxygenase 1 (FMO1) cDNA (dFMO1) was obtained from liver by reverse transcription-polymerase chain reaction. The amino acid sequence of dFMO1 was 89% homologous to human FMO1. Using a baculovirus expression system in Sf-9 insect cells, dFMO1 was expressed to protein levels of 0.4 nmol/mg, as determined by immunoquantitation. The flavin content of the expressed enzyme was consistent with immunodetectable dFMO1 protein levels. Expressed dFMO1 catalyzed NADPH-dependent methyl p-tolyl sulfide oxidation, with K(m) and V(max) values of 98.6 microM and 63.8 nmol of S-oxide formed/min/mg of protein, respectively. By comparison, human FMO1 showed similar values of 87.1 microM (K(m)) and 51.0 nmol/min/mg (V(max)). Activity for dFMO1 showed characteristic pH dependence, with a 4.5-fold increase in S-oxidase activity as the incubation pH increased from 7.6 to 9.0. Human FMO1 also showed an increase in reaction rate with pH but a somewhat lower optimum of 8.0 to 8.4. dFMO1 also catalyzed imipramine N-oxidation, with a K(m) of 4.7 microM and a V(max) of 82.1 nmol/min/mg of protein. This enzyme displayed other characteristics of FMO enzymes, with rapid depletion of enzyme activity upon heating in the absence of NADPH. Protein levels of 74 pmol of dFMO1/mg of microsomal protein were determined for a pooled liver microsome sample, suggesting that this enzyme is a major canine hepatic monooxygenase. In conclusion, the expression and characterization of catalytically active dFMO1 will allow the role of this enzyme in the metabolism of xenobiotics to be determined.

Mechanism-based inactivation of cytochrome P450 2B6 by a novel terminal acetylene inhibitor.

N-(3,5-Dichloro-4-pyridyl)-3-(cyclopentyloxy)-4-methoxybenzamide (DCMB) is a known marker substrate for cytochrome p450 2B6. Based on the chemical template of DCMB, a novel terminal acetylene compound, N-(3,5-dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy)benzamide (TA) was synthesized and evaluated as a mechanism-based inactivator of p450 2B6. The pseudo first-order inactivation of expressed p450 2B6 by TA was both substrate and time-dependent. The kinetics of inhibition resulted in a maximal rate constant (k(inactivation)) of 0.09 min(-1) and an apparent K(I) of 5.1 microM. Incubation of expressed p450 2B6 with TA and NADPH resulted in a 68% loss in enzyme activity and a concurrent 62% loss in the formation of a reduced carbon monoxide complex, suggesting that heme destruction is the primary mode of enzyme inactivation. Enzyme inactivation of p450 2B6 was not reduced by the presence of 10 mM glutathione and was protected by incubation of excess DCMB with TA. The production of the carboxylic acid metabolite, N-(3,5-Dichloro-4-pyridyl)-3-(2-carboxyethoxy)-4-methoxybenzamide (TA-COOH), during the incubation of TA with 2B6 suggests that inactivation proceeds through a ketene intermediate. For 2B6 inactivation, the partition ratio was approximately 1.5 nmol TA-COOH formed/nmol P450 inactivated. Finally, TA was evaluated for mechanism-based inactivation of p450 3A4, 2C9, 2C19, 2D6, and 2E1 using human liver microsomes. In addition to 2B6, p450 2C forms were also found to be sensitive to TA-mediated inactivation, suggesting that subtle changes in the O-alkyl chain of the parent may be critical for the selectivity of enzyme inactivation.

Characterization of raloxifene glucuronidation in vitro: contribution of intestinal metabolism to presystemic clearance.

Raloxifene, a selective estrogen receptor modulator used for the treatment of osteoporosis, undergoes extensive conjugation to the 6-beta- and 4'-beta-glucuronides in vivo. This paper investigated raloxifene glucuronidation by human liver and intestinal microsomes and identified the responsible UDP-glucuronosyltransferases (UGTs). UGT1A1 and 1A8 were found to catalyze the formation of both the 6-beta- and 4'-beta-glucuronides, whereas UGT1A10 formed only the 4'-beta-glucuronide. Expressed UGT1A8 catalyzed 6-beta-glucuronidation with an apparent K(m) of 7.9 microM and a V(max) of 0.61 nmol/min/mg of protein and 4'-beta-glucuronidation with an apparent K(m) of 59 microM and a V(max) of 2.0 nmol/min/mg. Kinetic parameters for raloxifene glucuronidation by expressed UGT1A1 could not be determined due to limited substrate solubility. Based on rates of raloxifene glucuronidation and known extrahepatic expression, UGT1A8 and 1A10 appear to be primary contributors to raloxifene glucuronidation in human jejunum microsomes. For human liver microsomes, the variability of 6-beta- and 4'-beta-glucuronide formation was 3- and 4-fold, respectively. Correlation analyses revealed that UGT1A1 was responsible for 6-beta- but not 4'-beta-glucuronidation in liver. Treatment of expressed UGTs with alamethicin resulted in minor increases in enzyme activity, whereas in human intestinal microsomes, maximal increases of 8-fold for the 6-glucuronide and 9-fold for the 4'-glucuronide were observed. Intrinsic clearance values in intestinal microsomes were 17 microl/min/mg for the 6-glucuronide and 95 microl/min/mg for the 4'-isomer. The corresponding values for liver microsomes were significantly lower, indicating that intestinal glucuronidation may be a significant contributor to the presystemic clearance of raloxifene in vivo.