Biomedical subjects

Jeffrey C Stevens

Publications and source records attributed to Jeffrey C Stevens.

8 recordsLinked to original sources

PubMed2006

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.

Adolescent↗

PubMed2006

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.

Age Factors↗

PubMed2003

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.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

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.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

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.

Acetylene↗

PubMed2003

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.

Education, Graduate↗

PubMed2003

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.

Animals↗

PubMed2002

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.

Alamethicin↗