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Charles L Crespi

Publications and source records attributed to Charles L Crespi.

16 recordsLinked to original sources
PubMed2006

Human cytochrome p450 induction and inhibition potential of clevidipine and its primary metabolite h152/81.

Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 microM. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 microM and 100 microM concentrations of clevidipine tested. Induction response for CYP3A4 to 100 microM clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC(50) values below 10 microM, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC(50) values >70 microM). The K(i) values for CYP2C9 and CYP2C19 were 1.7 and 3.3 microM, respectively, and those for CYP3A4 were 8.3 and 2.9 microM, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC(50) values >or= 69 microM). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans.

Calcium Channel Blockers↗
PubMed2006

Determination of CYP2C9-catalyzed diclofenac 4'-hydroxylation by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatography method is described for quantification of diclofenac 4'-hydroxylation catalyzed by human liver microsomes or cDNA-expressed CYP2C9. Analytical separation is achieved using a C18 column developed with a gradient of 30% acetonitrile and 2 mM perchloric acid in water to 100% methanol, with detection at 280 nm. This method is applicable to enzymatic studies for determination of CYP2C9-catalyzed diclofenac 4'-hydroxylation activity.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

CYP2C19-mediated (S)-mephenytoin 4'-hydroxylation assayed by high-performance liquid chromatography with radiometric detection.

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 4'-hydroxymephenytoin formed enzymatically from 14C-labeled (S)-mephenytoin following incubation with cDNA-expressed CYP2C19 or human liver microsomes. Analytical separation is achieved using a C18 column developed with a gradient from 10 to 100% methanol, with detection using a scintillation detector. This method is applicable to enzymatic studies for determination of CYP2C19-catalyzed (S)-mephenytoin 4'-hydroxylation activity.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

CYP2D6-dependent bufuralol 1'-hydroxylation assayed by reverse-phase ion-pair high-performance liquid chromatography with fluorescence detection.

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 1'-hydroxybufuralol formed enzymatically by the incubation of bufuralol with cDNA-expressed CYP2D6 or human liver microsomes. Analytical separation is achieved using a C18 column and a mobile phase consisting of 30% acetonitrile and 2 mM perchloric acid, with detection by fluorescence using an excitation wavelength of 252 nm and an emission wavelength of 302 nm. This method is applicable to enzymatic studies for determination of CYP2D6-catalyzed bufuralol 1'-hydroxylation activity.

Chromatography, High Pressure Liquid↗
PubMed2006

Spectrophotometric analysis of human CYP2E1-catalyzed p-nitrophenol hydroxylation.

The cytochrome P450 enzyme CYP2E1 catalyzes the oxidative metabolism of many solvents and other small organic molecules. A spectrophotometric method is described for determination of CYP2E1 activity by monitoring the formation of p-nitrocatechol from p-nitrophenol by cDNA-expressed CYP2E1 or isolated liver microsomes. The enzymatic product, p-nitrocatechol, is assayed at 535 nm after acidification of the reaction mixture with trichloroacetic acid followed by neutralization using 2 M NaOH. This method is applicable to enzymatic studies for determination of P450-catalyzed p-nitrophenol hydroxylation activity.

Catalysis↗
PubMed2006

Determination of CYP4A11-catalyzed lauric acid 12-hydroxylation by high-performance liquid chromatography with radiometric detection.

Lauric acid serves as an endogenous substrate for the cytochrome P450 enzyme CYP4A11. A reverse-phase, high-performance liquid chromatography method is described for the quantification of 12-hydroxylauric acid formed enzymatically by incubation of 14C-labeled lauric acid with cDNA-expressed CYP4A11 or human liver microsomes. Analytical separation is achieved using a C18 column and a gradient of 30% acetonitrile and 2 mM perchloric acid to 100% methanol, using a detection scintillation counter. This method is applicable to enzymatic studies for determination of lauric acid 12-hydroxylation activity.

Catalysis↗
PubMed2006

Determination of CYP2B6 component of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity in human liver microsomes.

The cytochrome P450 enzyme CYP2B6 plays an important role in the metabolism of structurally diverse drugs, including the anticancer drug cyclophosphamide, and may be an important determinant of clinical responses to these agents. A spectrofluorometric method is described for the determination of CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity in human liver microsomes. The specificity of this method for CYP2B6 is increased by the use of inhibitory antibodies to CYP1A2, CYP2C, and CYP2E1, which block the contributions of these higher-K(m) enzymes to human liver microsomal metabolism of 7-ethoxy-4-trifluoromethylcoumarin. The enzymatic product, 7-hydroxy-4-trifluoromethylcoumarin, is monitored by fluorescence using an excitation wavelength of 410 nm and an emission wavelength of 510 nm. This approach can be modified to assay the catalytic activity of cDNA-expressed CYP2B6.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

High-performance liquid chromatography analysis of CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation.

Paclitaxel (Taxol) is a naturally occurring member of the taxane family of antitumor drugs, which act by stabilizing microtubules. Paclitaxel is inactivated in human liver by a cytochrome P450 (P450)-catalyzed 6alpha-hydroxylation reaction. A reverse-phase, high-performance liquid chromatographic assay is described for the analysis of paclitaxel 6alpha-hydroxylation catalyzed by human liver microsomes or cDNA-expressed P450 enzyme CYP2C8. Analytical separations are achieved using a C18 column with a linear gradient of 10-100% methanol, with detection at 230 nm. This method is applicable to enzymatic studies for determination of CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation activity.

Aryl Hydrocarbon Hydroxylases↗
PubMed2004

Human-cell mutagens in respirable airborne particles in the northeastern United States. 1. Mutagenicity of fractionated samples.

Few studies have characterized the regional scale (300-500 km) variability of the mutagenicity of respirable airborne particles (PM2.5). We previously collected 24-h PM2.5 samples for 1 year from background, suburban, and urban sites in Massachusetts (MA) and rural and urban sites in upstate New York (NY) (n = 53-60 samples per site). Bimonthly composites of these samples were mutagenic to human cells. The present report describes our effort to identify chemical classes responsible for the mutagenicity of the samples, to quantify spatial differences in mutagenicity, and to compare the mutagenicity of samples composited in different ways. Organic extracts and HPLC fractions (two nonpolar, one semipolar, and one polar) of annual composites were tested for mutagenicity in the h1A1v2 cells, a line of human B-lymphoblastoid cells that express cytochrome P450 CYP1A1 cDNA. The mutagenic potency (induced mutant fraction per microg organic carbon) of the semipolarfractions was the highest at all five sites, accounting for 35-82% of total mutagenic potency of the samples, vs the nonpolar (4-38%) and polar (14-32%) fractions. These results are consistent with previous studies. While unfractionated extracts exhibited no spatial variations, the mutagenicity of semipolar fractions at the NY sites was approximately 2-fold higher than at the MA sites. This suggests there may be significant regional differences in the sources and/ or transport and transformation of mutagenic compounds in PM2.5. In addition, mutagenic potency was sensitive to whether samples were fractionated and how they were composited: unfractionated annual composite samples at the NY sites were significantly less mutagenic than their semipolar fractions and the annual average of bimonthly composites; spatial differences in the mutagenic potency of bimonthly composites and the semipolar fractions were not apparent in the annual composites.

Air Movements↗
PubMed2004

Highly selective inhibition of human CYP3Aa in vitro by azamulin and evidence that inhibition is irreversible.

Azamulin [14-O-(5-(2-amino-1,3,4-triazolyl)thioacetyl)-dihydromutilin] is an azole derivative of the pleuromutilin class of antiinfectives. We tested the inhibition potency of azamulin toward 18 cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. In a competitive inhibition model, IC(50) values for CYP3A (0.03-0.24 microM) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 ( approximately 50-fold lower). The IC(50) value with heterologously expressed CYP3A4 was 15-fold and 13-fold less than those of CYP3A5 and CYP3A7, respectively. The reference inhibitor ketoconazole was less selective and exhibited potent inhibition (IC(50) values <10 microM) for CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP4F2, and CYP4F12. Inhibition of CYP3A by azamulin appeared sigmoidal and well behaved with the substrates 7-benzyloxy-4-trifluoromethylcoumarin, testosterone, and midazolam. Preincubation of 4.8 microM azamulin in the presence of NADPH for 10 min inhibited approximately 95% of testosterone 6beta-hydroxylase activity compared with preincubation in the absence of NADPH. Catalytic activities of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 were unaffected by similar experiments. Incubation of azamulin with heterologously expressed CYP3A4 yielded a type I binding spectrum with a spectral dissociation constant of 3.5 microM, whereas no interaction was found with CYP2D6. Azamulin exhibited good chemical stability when stored in acetonitrile for up to 12 days. Aqueous solubility was found to be >300 microM. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.

Anti-Infective Agents↗
PubMed2002

CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6alpha-hydroxylase activity in human liver microsomes.

Published cDNA sequences suggest the existence of non-synonymous single nucleotide polymorphisms in the cytochrome P450 CYP2C8. To determine whether these polymorphisms could be confirmed in a Caucasian population and to investigate whether additional polymorphisms occur in the coding and upstream regions of this gene, we screened for previously described and for novel polymorphisms using PCR-RFLP and SSCP analysis. We confirmed the existence of two of the previously detected polymorphisms which give rise to the amino acid substitutions I264M and K399R, respectively, but failed to detect three others in our population. We also confirmed that a recently identified polymorphism (R139K) is linked to K399R (CYP2C8*3) in our study population. The allele frequencies for the I264M (CYP2C8*4 allele) and the CYP2C8*3 allele were 0.075 and 0.15, respectively. Three novel polymorphisms (T-370G, C-271A and T1196C/L390S) were also detected with the upstream polymorphisms showing allele frequencies of 0.061 and 0.196, respectively, but the L390S polymorphism detected only in a single subject. An additional single subject was heterozygous for a polymorphism recently described in African-Americans (A805T; CYP2C8*2 allele). The functional significance of the two upstream polymorphisms and the CYP2C8*3 and CYP2C8*4 alleles was investigated in human liver microsomes. Samples heterozygous for CYP2C8*3 showed significantly lower paclitaxel 6alpha-hydroxylase activity compared with wild-type samples. Median activity associated with CYP2C8*4 also appeared lower than the wild-type but the difference was not significant. There was no evidence that either upstream polymorphism gave rise to altered CYP2C8 expression.

Amino Acid Sequence↗
PubMed2002

Assessing pregnancy risks of azole antifungals using a high throughput aromatase inhibition assay.

UNLABELLED: Human aromatase (CYP19) converts C19 androgens to aromatic C18 estrogenic steroids. Its activity is critical for early and mid pregnancy maintenance and in regulating parturition in late pregnancy. Past studies have utilized placental microsome tritiated water release assay to assess drug-hormone interactions with estrogen synthesis. We compared data from human placental assays with BD Gentest's high throughput recombinant CYP19 enzyme assay using the fluorometric substrate dibenzylfluorescein. We tested a panel of azole antifungal agents that are commonly administered to women of childbearing potential, for their potential to inhibit aromatase. Potency varied by several orders of magnitude. Plasma and tissue levels of some azole drugs following oral or topical administration are at or above these IC50 values. These include the oral agents fluconazole and ketoconazole, and the topical agents econazole, bifonazole, clotrimazole, miconazole, and sulconazole. CONCLUSIONS: 1. Recombinant enzyme assay data are comparable to the human placental assay data in both SAR rank order and potency. 2. Plasma and tissue levels of some azole drugs following oral or topical administration are at or above these IC50 values. Therefore, some azole drugs may disrupt estrogen production in pregnancy, affecting pregnancy outcome. 3. Recombinant CYP19 assay using the fluorometric substrate dibenzylfluorescein, demonstrates rapid screening potential for chemicals that may affect pregnancy outcome as a result of CYP19 inhibition.

Animals↗
PubMed2002

Cytochrome P450 fluorometric substrates: identification of isoform-selective probes for rat CYP2D2 and human CYP3A4.

We have tested a panel of 29 cDNA-expressed rat and human enzymes with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products. The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and 7-benzyloxy-4-trifluoromethylcoumarin (BFC). For most substrates, multiple cDNA-expressed cytochrome P450 isoforms were found to catalyze the formation of the fluorescent product. However, among the combinations tested, rat CYP2D2 displayed high selectivity for AMMC demethylation (a substrate selective for CYP2D6 in human liver microsomes). AMMC demethylation activity was 15-fold lower in microsomes isolated from female Dark Agouti rats, a model known to have a low abundance of CYP2D2, and apparent K(M) values were similar for cDNA-expressed CYP2D2 and male Sprague-Dawley liver microsomes. BFC dealkylation and BQ dealkylation were selective but not exclusive for human CYP3A4. A small role for CYP1A2 could be demonstrated. The CYP3A4 selectivity in hepatic microsomes was supported by studies using chemical and antibody inhibitors and a correlation analysis within a panel of liver microsomes from individual donors. BQ demonstrated a higher degree of selectivity for and higher rates of metabolism by CYP3A than BFC. However, per unit enzyme the fluorescent signal is lower for BQ than BFC. AMMC, BQ, and BFC should find uses as enzyme-selective probe substrates.

Animals↗
PubMedDate not supplied

Comparison of species differences of P-glycoproteins in beagle dog, rhesus monkey, and human using Atpase activity assays.

P-glycoprotein (P-gp) is a transmembrane efflux transporter which possesses many important functions in drug absorption, disposition, metabolism, and toxicity. The ultimate goal of investigating drug interactions between P-gp and drug molecules in early drug discovery is to understand the contribution of P-gp to the pharmacokinetic and pharmacodynamic properties of drug candidates and to project drug-drug interaction (DDI) potentials in humans. Understanding species differences in P-gp activities further helps the prediction of P-gp-mediated drug disposition and DDI in humans from preclinical pharmacokinetics data. The objective of the present study is to investigate the species difference in P-gp activities, via P-gp ATPase assays, using rhesus monkey Mdr1, beagle dog Mdr1, and human MDR1 expressed insect cell membranes. Twenty-one compounds with diverse chemical structures and different P-gp binding sites were chosen for the ATPase assays. P-gp ATPase binding affinities (alphaKa) and fold increases in P-gp ATPase activities (beta) of P-gp substrates were determined. Consistent with the gene and amino acid similarity, the binding affinities of test compounds to rhesus monkey P-gp were much closer to those of human P-gp than beagle dog P-gp. This is the first study which investigates the ligand affinities of P-gp from three different species. The result of this study provides an example of how to use membrane P-gp ATPase assays to evaluate interspecies P-gp differences.

ATP Binding Cassette Transporter, Subfamily B↗