In Vitro Drug Metabolism Using Liver Microsomes.

Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping) allows prediction, at least at the qualitative level, of factors (including metabolic drug-drug interactions) likely to alter the clearance of both new chemical entities (NCEs) and established drugs. Microsomes are typically used as the enzyme source for the measurement of metabolic stability and for reaction phenotyping because they express the major drug-metabolizing enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), along with others that contribute to drug metabolism.

A multi-endpoint evaluation of cytochrome P450 1A2, 2B6 and 3A4 induction response in human hepatocyte cultures after treatment with β-naphthoflavone, phenobarbital and rifampicin.

U.S. FDA and EMEA guidance recommend that the preferred in vitro model for cytochrome P450 induction testing is human hepatocytes coupled with acceptable inducers as controls.

Differential time- and NADPH-dependent inhibition of CYP2C19 by enantiomers of fluoxetine.

Fluoxetine [+/--N-methyl-3-phenyl-3-[(alpha, alpha, (-trifluoro-p-tolyl)oxy]-propylamine)] a selective serotonin reuptake inhibitor, is widely used in treating depression and other serotonin-dependent disease conditions. Racemic, (R)- and (S)-fluoxetine are potent reversible inhibitors of CYP2D6, and the racemate has been shown to be a mechanism-based inhibitor of CYP3A4. Racemic fluoxetine also demonstrates time- and concentration-dependent inhibition of CYP2C19 catalytic activity in vitro.

A novel design of artificial membrane for improving the PAMPA model.

Purpose: Since the first demonstration of PAMPA, the artificial membrane has been traditionally prepared by impregnating a porous filter with a solution of lipid mixture. While the lipid solution-based method is simple and seems to provide good predictability for many compounds, it is challenged by several shortcomings including reproducibility, stability, mass retention and the incorrect prediction of a group of highly permeable compounds including caffeine and antipyrine. Here we present the validation of a novel artificial membrane formed by constructing a lipid/oil/lipid tri-layer in the porous filter.

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.

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.

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.

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.

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.

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.

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.

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.

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.

High-Performance Liquid Chromatography Analysis of CYP2C8-Catalyzed Paclitaxel 6α-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 6α-hydroxylation reaction. A reverse-phase, high-performance liquid chromatographic assay is described for the analysis of paclitaxel 6α-hydroxylation catalyzed by human liver microsomes or cDNA-expressed P450 enzyme CYP2C8.

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.

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.

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.

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.

Thiourea toxicity in mouse C3H/10T1/2 cells expressing human flavin-dependent monooxygenase 3.

Human flavin-dependent monooxygenase (FMO) isoforms 1 and 3 were expressed by retroviral gene transfer in mouse C3H/10T1/2 cells. FMO function was determined by the sulfoxidation of p-tolylmethylsulfide (TMS). Enzyme activity ranged from 4 to 30 nmol p-tolylmethylsulfoxide (TMSO)/30 min/mg cell protein for FMO 3 clones; for FMO 1 clones, the range was 1-6 nmol TMSO/30 min/mg.

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.

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.