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J Steven Leeder

Publications and source records attributed to J Steven Leeder.

31 records · Page 2Linked to original sources
PubMed2003

Genome-wide prediction, display and refinement of binding sites with information theory-based models.

BACKGROUND: We present Delila-genome, a software system for identification, visualization and analysis of protein binding sites in complete genome sequences. Binding sites are predicted by scanning genomic sequences with information theory-based (or user-defined) weight matrices. Matrices are refined by adding experimentally-defined binding sites to published binding sites. Delila-Genome was used to examine the accuracy of individual information contents of binding sites detected with refined matrices as a measure of the strengths of the corresponding protein-nucleic acid interactions. The software can then be used to predict novel sites by rescanning the genome with the refined matrices. RESULTS: Parameters for genome scans are entered using a Java-based GUI interface and backend scripts in Perl. Multi-processor CPU load-sharing minimized the average response time for scans of different chromosomes. Scans of human genome assemblies required 4-6 hours for transcription factor binding sites and 10-19 hours for splice sites, respectively, on 24- and 3-node Mosix and Beowulf clusters. Individual binding sites are displayed either as high-resolution sequence walkers or in low-resolution custom tracks in the UCSC genome browser. For large datasets, we applied a data reduction strategy that limited displays of binding sites exceeding a threshold information content to specific chromosomal regions within or adjacent to genes. An HTML document is produced listing binding sites ranked by binding site strength or chromosomal location hyperlinked to the UCSC custom track, other annotation databases and binding site sequences. Post-genome scan tools parse binding site annotations of selected chromosome intervals and compare the results of genome scans using different weight matrices. Comparisons of multiple genome scans can display binding sites that are unique to each scan and identify sites with significantly altered binding strengths. CONCLUSIONS: Delila-Genome was used to scan the human genome sequence with information weight matrices of transcription factor binding sites, including PXR/RXRalpha, AHR and NF-kappaB p50/p65, and matrices for RNA binding sites including splice donor, acceptor, and SC35 recognition sites. Comparisons of genome scans with the original and refined PXR/RXRalpha information weight matrices indicate that the refined model more accurately predicts the strengths of known binding sites and is more sensitive for detection of novel binding sites.

Binding Sites↗
PubMed2003

Combined phenotypic assessment of cytochrome p450 1A2, 2C9, 2C19, 2D6, and 3A, N-acetyltransferase-2, and xanthine oxidase activities with the "Cooperstown 5+1 cocktail".

Previously, we have validated a 4-drug phenotyping cocktail, the "Cooperstown cocktail," using caffeine (cytochrome p450 [CYP] 1A2, N-acetyltransferase-2 [NAT2], and xanthine oxidase [XO]), dextromethorphan (CYP2D6), omeprazole (CYP2C19), and intravenous midazolam (hepatic CYP3A). Data suggest that warfarin can be used as a safe and accurate biomarker for CYP2C9, and if warfarin is administered with vitamin K, the pharmacodynamic effect is ablated. Twelve subjects received the Cooperstown cocktail, warfarin plus vitamin K, and both sets of biomarkers (Cooperstown 5+1 cocktail) in a randomized crossover fashion. On the basis of log-transformed data and a paired t test, no significant difference was seen for S-warfarin area under the serum concentration-time curve from time 0 to infinity (P =.09), omeprazole metabolic ratio (P =.374), caffeine metabolic ratio (P =.169 for CYP1A2 activity), midazolam plasma clearance (P =.573), or dextromethorphan metabolic ratio (P =.747) with the Cooperstown cocktail, warfarin plus vitamin K alone, or the Cooperstown 5+1 cocktail. During drug administration, the only side effect was mild and short-lived sedation after intravenous midazolam administration. Phenotypic measurements were in concordance with the subject's CYP2C9, CYP2C19, and CYP2D6 genotypes. The Cooperstown 5+1 cocktail may be used to simultaneously assess the activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A, NAT2, and XO.

Acetyltransferases↗
PubMed2003

Bioactivation of clozapine by murine cardiac tissue in vivo and in vitro.

Clozapine, an atypical neuroleptic, undergoes bioactivation to a chemically reactive nitrenium ion. This has been implicated in the pathogenesis of clozapine-induced agranulocytosis. Clozapine also causes myocarditis and cardiomyopathy, the mechanisms of which are unknown. To investigate this, we have evaluated whether clozapine undergoes bioactivation by murine cardiac tissue, in comparison to hepatic tissue. Mice were administered clozapine (5 and 50 mg/kg i.p.), and the extent of covalent binding was assessed by Western blotting. There was an increase in irreversible binding of clozapine to several proteins, ranging in mass from 30 to 250 kDa in both hepatic and cardiac tissue. Bioactivation by hepatic and cardiac microsomes was assessed by LC/MS using glutathione to trap the intermediate. Metabolism of radiolabeled clozapine to a glutathionyl conjugate by liver and cardiac microsomes was 30.5 +/- 3.3 and 3.6 +/- 0.3% of the initial incubation concentration, respectively. Ketoconazole (20 muM), a P450 inhibitor, significantly reduced binding in both hepatic and cardiac microsomes to 6.2 +/- 0.2 and 0.5 +/- 0.06%, respectively. These data indicate that clozapine undergoes bioactivation in the heart to a chemically reactive nitrenium metabolite that may be important in the pathogenesis of myocarditis and cardiomyopathy observed in man.

Animals↗
PubMed2003

Cisapride: a potential model substrate to assess cytochrome P4503A4 activity in vivo.

OBJECTIVES: Cisapride was compared with midazolam in vivo to determine its potential applicability as a cytochrome P450 (CYP) 3A4 "probe." As well, we evaluated whether cisapride was transported by P-glycoprotein. METHODS: Bidirectional transport assays were conducted in LLC-PK1 cells and the derivative cell line L-MDR1 to determine whether cisapride was a substrate for P-glycoprotein. A pharmacokinetic study was also conducted in 17 healthy adults (n = 8 women) who received intravenous midazolam (0.025 mg/kg), oral midazolam (0.15 mg/kg), and oral cisapride (0.07 mg/kg) in a randomized crossover design. Plasma concentrations were quantitated from repeated after-dosing blood samples by HPLC with ultraviolet detection for midazolam and HPLC with tandem mass spectrometry detection for cisapride and norcisapride. Pharmacokinetic parameters were determined by noncompartmental methods. Both linear and nonlinear regression analyses were used to examine the association between the apparent plasma clearance of midazolam and cisapride and the cisapride/norcisapride plasma concentration ratios. RESULTS: Although not a substrate for P-glycoprotein, cisapride inhibited P-glycoprotein with an apparent inhibition constant (K(i)) of 16.1 micromol/L. Linear correlations between cisapride clearance and both intravenous and oral midazolam clearance (P =.01, r(2) = 0.43 and P =.001, r(2) = 0.46, respectively) were found. Cisapride/norcisapride plasma concentration ratios at 8 hours (P =.001, r(2) = 0.90) and 12 hours (P =.001, r(2) = 0.96), as well as cisapride plasma concentrations at these time points, were shown to accurately predict the area under the plasma concentration versus time curve for cisapride. CONCLUSIONS: CYP3A4 activity reflected by the total body clearance after oral administration of cisapride should be independent of transport by P-glycoprotein. Concordance between the pharmacokinetics for cisapride and midazolam support the applicability of oral cisapride as a pharmacologic substrate to assess total CYP3A4 activity in vivo. Cisapride plasma concentration ratios at 8 or 12 hours after a single oral cisapride dose may prove useful as a single-point determination to reflect the area under the plasma concentration versus time curve and the plasma clearance of cisapride and, as well, total CYP3A4 activity in vivo.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Information theory-based analysis of CYP2C19, CYP2D6 and CYP3A5 splicing mutations.

Several mutations are known or suspected to affect mRNA splicing of CYP2C19, CYP2D6 and CYP3A5 genes; however, little experimental evidence exists to support these conclusions. The present study applies mathematical models that measure changes in information content of splice sites in these genes to demonstrate the relationship between the predicted phenotypes of these variants to the corresponding genotypes. Based on information analysis, the CYP2C19*2 variant activates a new cryptic site 40 nucleotides downstream of the natural splice site. CYP2C19*7 abolishes splicing at the exon 5 donor site. The CYP2D6*4 allele similarly inactivates splicing at the acceptor site of exon 4 and activates a new cryptic site one nucleotide downstream of the natural acceptor. CYP2D6*11 inactivates the acceptor site of exon 2. The CYP3A5*3 allele activates a new cryptic site 236 nucleotides upstream of the exon 4 natural acceptor site. CYP3A5*5 inactivates the exon 5 donor site and CYP3A5*6 strengthens a site upstream of the natural donor site, resulting in skipping of exon 7. Other previously described missense and nonsense mutations at terminal codons of exons in these genes affected splicing. CYP2D6*8 and CYP2D6*14 both decrease the strength of the exon 3 donor site, producing transcripts lacking this exon. The results of information analysis are consistent with the poor metabolizer phenotypes observed in patients with these mutations, and illustrate the potential value of these mathematical models to quantitatively evaluate the functional consequences of new mutations suspected of altering mRNA splicing.

Amino Acid Substitution↗
PubMed2003

Developmental and pediatric pharmacogenomics.

Children, as well as adults, should benefit from the discoveries of the genomic era. Many diseases with complex etiologies originate during childhood (e.g., asthma, autism, attention deficit/hyperactivity disorder, epilepsy and juvenile rheumatoid arthritis) and persist into adulthood. Attempts to better understand the genetic basis of age-specific disease processes requires an appreciation that the period of human development encompasses the prenatal period through adolescence, and is a rapidly changing, dynamic process. As a result, pharmacologic modulation of developing gene networks may have unintended and unanticipated consequences that do not become apparent or relevant until later in life. Thus, there is considerable potential for large-scale pharmacogenomic technologies to impact the development and utilization of new therapeutic strategies in children.

Child↗
PubMed2002

Unique CYP2D6 activity distribution and genotype-phenotype discordance in black Americans.

BACKGROUND: Although CYP2D6 has been studied extensively in different population groups, relatively little is known for black Americans. METHODS: CYP2D6 activity was assessed with dextromethorphan in 283 black American subjects and correlated with their genotype (2D6*2 to *12, 2D6*14, 2D6*15, 2D6*17, 2D6*18, and 2D6*29 and gene duplications). Volunteers provided information about ethnicity and concurrent medication, and they participated in either phenotyping (n = 225), genotyping (n = 251), or both (n = 193). RESULTS: The median urinary dextromethorphan/dextrorphan metabolic ratio (MR) indicated significantly lower CYP2D6 activity in the black American group (0.016) than in a white control population (0.0044; P =.0001) studied previously. The reduced function allele 2D6*17 was more common (frequency [f] = 0.395) among intermediate metabolizers (0.03 < or = MR < or= 0.3) than extensive metabolizers (MR < or = 0.03; f = 0.148; P =.0001). Consistent with reduced function toward dextromethorphan of COS cell-expressed 2D6.29 protein, 2D6*29 also was more frequent in intermediate metabolizers (f = 0.114) than in extensive metabolizers (f = 0.057; P = NS). Frequencies for 2D6*17 and 2D6*29 were f = 0.213 and 0.072, respectively. Of the 193 genotyped and phenotyped subjects, 14 were determined to be poor metabolizers, with dextromethorphan/dextrorphan ratios >0.3 (7.25%), but only 2 subjects (1.04%) carried 2 nonfunctional alleles (2D6*3/*4x2 and 2D6*4/*4). A new allelic variant, 2D6*40, was subsequently found in 2 discordant subjects (2D6*4/*40 and 2D6*6/*40), implying that the 18-base pair (bp) insertion found in 2D6*40 renders it nonfunctional. The frequency of 2D6*40 was 0.006. For genotypes that contain 2D6*2, median MR values were consistently higher in black Americans than in white subjects, indicating that other unidentified factors also contribute to lower CYP2D6 activity in black Americans. CONCLUSIONS: The lower CYP2D6 activity observed in a black American population is in part attributable to the presence of variant alleles that occur at a higher frequency in this population than in white subjects. Additional studies are required to ascertain the pharmacokinetic and pharmacodynamic consequences of these pharmacogenetic data in black Americans.

Adolescent↗
PubMed2002

Pathways of carbamazepine bioactivation in vitro I. Characterization of human cytochromes P450 responsible for the formation of 2- and 3-hydroxylated metabolites.

In vitro studies were conducted to identify the cytochromes P450 (P450s) involved in the formation of 2- and 3-hydroxycarbamazepine, metabolites that may serve as precursors in the formation of protein-reactive metabolites. Human liver microsomes (HLMs) converted carbamazepine (30-300 microM) to 3-hydroxycarbamazepine at rates >25 times those of 2-hydroxycarbamazepine. Both the 2- and 3-hydroxylation of carbamazepine appeared to conform to monophasic Michaelis-Menten kinetics in HLMs (apparent K(m) values, approximately 1640 and approximately 217 microM; apparent V(max) values, approximately 5.71 and approximately 46.9 pmol/mg of protein/min, respectively). Rates of carbamazepine 2- and 3-hydroxylation correlated strongly with CYP2B6 activity (r >or= 0.757) in a panel of HLMs (n = 8). Carbamazepine 3-hydroxylation also correlated significantly with CYP2C8 activity at a carbamazepine concentration of 30 microM. Formation of 2- and 3-hydroxycarbamazepine did not correlate significantly with any other P450 activities. The chemical inhibitors ketoconazole (CYP3A) and 7-EFC (CYP2B6) inhibited both 2- and 3-hydroxycarbamazepine formation whereas 4-methylpyrazole (CYP2E1) markedly decreased 2-hydroxycarbamazepine formation. Several recombinant P450s catalyzed carbamazepine 2- and 3-hydroxylation, but after adjustment for relative hepatic abundance, CYP3A4 and CYP2B6 appeared to be the major catalysts of carbamazepine 3-hydroxylase activity, and at least five P450s were significant contributors to 2-hydroxycarbamazepine formation; CYP2E1 made the greatest contribution to the Cl(int) of carbamazepine 2-hydroxylation (approximately 30%), but P450s CYP1A2, 2A6, 2B6, and 3A4 also made significant contributions (approximately 13-18%). These results suggest that CYP2B6 and CYP3A4 are largely responsible for the formation of 3-hyrdoxycarbamazepine, whereas multiple P450s (CYP1A2, 2A6, 2B6, 2E1, and 3A4) contributed to 2-hydroxycarbamazepine formation.

Anticonvulsants↗
PubMed2002

Characterization of cytochrome P450 2D6.1 (CYP2D6.1), CYP2D6.2, and CYP2D6.17 activities toward model CYP2D6 substrates dextromethorphan, bufuralol, and debrisoquine.

Over 50 allelic variants of cytochrome P450 2D6 (CYP2D6) encoding fully functional, reduced-activity, or nonfunctional proteins have been described. Compared with Caucasians, studies in black populations demonstrate a tendency toward slower CYP2D6 activity, attributed in part to the presence of a variant allele associated with reduced activity, the CYP2D6*17 allele. To investigate the kinetic characteristics of this variant protein, expression constructs coding for CYP2D6.1, CYP2D6.2, and CYP2D6.17 gene products were prepared and transfected into mammalian COS-7 and insect (Trichoplusia ni) cells for expression. Microsomal fractions containing the expressed proteins were used to determine the kinetic parameters K(m), V(max), and intrinsic clearance (Cl(int)) for the model substrates dextromethorphan, bufuralol, and debrisoquine. Relative to the wild-type CYP2D6.1 protein expressed in COS-7 cells, CYP2D6.17 exhibited a 2-fold higher K(m) and a 50% reduction in V(max) using dextromethorphan as the substrate. In contrast, no appreciable change in bufuralol K(m) was observed with CYP2D6.17 whereas V(max) was decreased by 50%. When expressed in the baculovirus expression system, CYP2D6.17 exhibited a 6-fold increase in K(m) but no change in V(max) with dextromethorphan as the substrate, a 2-fold higher K(m) and 50% reduction in V(max) with bufuralol, and a 3-fold increase in K(m) and no change in V(max) with debrisoquine relative to CYP2D6.1. These data indicate that CYP2D6.17 exhibits reduced metabolic activity toward all three commonly used CYP2D6 substrates, although specific effects on substrate affinity and turnover demonstrate some substrate dependence.

Animals↗