Mitchell A. Hamman

The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and clarithromycin

To assess the relative contribution of intestinal and hepatic CYP3A inhibition to the interaction between the prototypic CYP3A substrates midazolam and clarithromycin.

The effects of St John's wort (Hypericum perforatum) on human cytochrome P450 activity

St Johns wort(Hypericum perforatum) is a popular over‐the‐counter dietary supplement and herbal remedy that has been implicated in drug interactions with substrates of several cytochrome P450 (CYP) isozymes. The effect of St Johns wort on CYP activity in vivo was examined with a probe drug cocktail.

The Effect of Echinacea (Echinacea purpurea Root) on Cytochrome P450 Activity in Vivo

Echinacea is a widely available over‐the‐counter herbal remedy. Tinctures of echinacea have been shown to inhibit cytochrome P450 (CYP) in vitro. The effect of echinacea (Echinacea purpurea root) on CYP activity in vivo was assessed by use of the CYP probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A).

The interaction between St John's wort and an oral contraceptive

The popular herbal remedy St Johns wort is an inducer of cytochrome P450 (CYP) 3A enzymes and may reduce the efficacy of oral contraceptives. Therefore we evaluated the effect of St Johns wort on thedisposition and efficacy of Ortho‐Novum 1/35 (Ortho‐McNeil Pharmaceutical, Inc, Raritan, NJ), a popular combination oral contraceptive pill containing ethinyl estradiol (INN, ethinylestradiol) and norethindrone (INN, norethisterone).

Regioselective and stereoselective metabolism of ibuprofen by human cytochrome P450 2C

The cytochrome P450s responsible for the regio- and stereoselectivity in the 2- and 3-hydroxylation of the chiral non-steroidal antiinflammatory drug ibuprofen were characterized in human liver microsomes. The rates of formation of both the 2- and 3-hydroxy metabolites exhibited monophasic (N = 2; N is the number of microsomal preparations) and biphasic (N = 2) substrate concentration dependence for both enantiomers of ibuprofen. The high affinity enzyme class parameters for S-ibuprofen (N = 4) were: 2-hydroxylation, Vmax = 566 +/- 213 pmol/min/mg, Km = 38 +/- 13 microM; 3-hydroxylation, Vmax = 892 +/- 630 pmol/min/mg, Km = 21 +/- 6 microM. For R-ibuprofen, the corresponding parameters were: 2-hydroxylation, Vmax = 510 +/- 117 pmol/min/mg, Km = 47 +/- 20 microM; 3-hydroxylation, Vmax = 593 +/- 113 pmol/min/mg, Km = 29 +/- 8 microM. cDNA-expressed CYP2C9 (Arg 144 and Cys 144) favored S-2- and S-3-hydroxyibuprofen formation, but CYP2C8 favored R-2-hydroxyibuprofen formation. Sulfaphenazole, retinol, and arachidonic acid competitively inhibited the rate of formation of all hydroxyibuprofens; Ki values (N = 3) for sulfaphenazole on the 2- and 3-hydroxylations of S-ibuprofen were 0.12 +/- 0.05 and 0.07 +/- 0.04 and of R-ibuprofen were 0.11 +/- 0.07 and 0.06 +/- 0.03 microM, respectively. Sulfaphenazole also competitively inhibited ibuprofen hydroxylation by cDNA-expressed CYP2C9 (Arg 144 and Cys 144) with Ki values in the range of 0.05 to 0.18 microM and CYP2C8 in the range of 0.36 to 0.55 microM. In a bank of 14 human liver microsome samples, significant correlations (r = 0.72 to 0.90; P < 0.01) were observed between the rates of formation of all four hydroxyibuprofens, and for each hydroxyibuprofen and prototypical CYP2C8/9 biotransformations. The regio- and stereoselectivities observed in vitro were consistent with those noted in vivo. The relative levels of both CYP2C8 and CYP2C9 and the expression of the corresponding variants may influence the disposition of ibuprofen in vivo.

The effect of rifampin administration on the disposition of fexofenadine

Our objective was to assess the effect of rifampin (INN, rifampicin) on the pharmacokinetics of fexofenadine and to assess the influence of advanced age and sex.

Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes.

The stereoselective sulfoxidation of the pharmacologically active metabolite of sulindac, sulindac sulfide, was characterized in human liver, kidney, and cDNA-expressed enzymes. Kinetic parameter estimates (pH = 7.4) for sulindac sulfoxide formation in human liver microsomes (N = 4) for R- and S-sulindac sulfoxide were V(max) = 1.5 +/- 0.50 nmol/min/mg, K(m) = 15 +/- 5.1 microM; and V(max) = 1.1 +/- 0.36 nmol/min/mg, K(m) = 16 +/- 6.1 microM, respectively. Kidney microsomes (N = 3) produced parameter estimates (pH = 7.4) of V(max) = 0.9 +/- 0.29 nmol/min/mg, K(m) = 15 +/- 2.9 microM; V(max) = 0.5 +/- 0.21 nmol/min/mg, K(m) = 22 +/- 1.9 microM for R- and S-sulindac sulfoxide, respectively. In human liver and flavin-containing monooxygenase 3 (FMO3) the V(max) for R-sulindac sulfoxide increased 60-70% at pH = 8.5, but for S-sulindac sulfoxide was unchanged. In fourteen liver microsomal preparations, significant correlations occurred between R-sulindac sulfoxide formation and either immunoquantified FMO or nicotine N-oxidation (r = 0.88 and 0.83; P < 0.01). The R- and S-sulindac sulfoxide formation rate also correlated significantly (r = 0.85 and 0.75; P < 0.01) with immunoquantified FMO in thirteen kidney microsomal samples. Mild heat deactivation of microsomes reduced activity by 30-60%, and a loss in stereoselectivity was observed. Methimazole was a potent and nonstereoselective inhibitor of sulfoxidation in liver and kidney microsomes. n-Octylamine and membrane solubilization with lubrol were potent and selective inhibitors of S-sulindac sulfoxide formation. cDNA-expressed CYPs failed to appreciably sulfoxidate sulindac sulfide, and CYP inhibitors were ineffective in suppressing catalytic activity. Purified mini-pig liver FMO1, rabbit lung FMO2, and human cDNA-expressed FMO3 efficiently oxidized sulindac sulfide with a high degree of stereoselectivity towards the R-isomer, but FMO5 lacked catalytic activity. The biotransformation of the sulfide to the sulfoxide is catalyzed predominately by FMOs and may prove to be useful in characterizing FMO activity.

The effect of short- and long-term administration of verapamil on the disposition of cytochrome P450 3A and P-glycoprotein substrates

Verapamil has the capability to inhibit and induce cytochrome P450 (CYP) 3A and P‐glycoprotein (P‐gp), but the relative extent and time course of these events in vivo are unclear. The effect of verapamil on CYP3A and P‐gp activity was determined by examining its effect on its own disposition and on the disposition of fexofenadine, respectively.

Quantification of dextromethorphan and metabolites : a dual phenotypic marker for cytochrome P450 3A4/5 and 2D6 activity

A sensitive and selective liquid chromatographic procedure using fluorimetric detection was developed to quantify dextromethorphan (DTM), 3-methoxymorphinan (3MM), dextrorphan (DT), 3-hydroxymorphinan (3OH) and two internal standards, codeine (COD) and ethylmorphine (ETM), in urine. Precision and accuracy of the assay were determined over a concentration range of 5-3200 ng/ml urine for DTM, 5-400 ng/ml urine for 3MM, 400-40 000 ng/ml urine for DT and 200-16 000 ng/ml urine for 3OH, by assaying freshly prepared calibration standards and replicates of six quality control (QC) samples on separate days. All of the inter-day and intra-day coefficients of variation (C.V.s) were less than 20% except for a low QC for 3MM. The inter-day and intra-day accuracies were less than 20% for the low QCs, less than 15% for the medium QCs and less than 12% for the high QCs, for all compounds. The limit of quantification (LOQ) was 2 ng/ml urine for DTM and 3MM, 250 ng/ml urine for DT, and 100 ng/ml urine for 3OH. Absolute recovery was 76% for DTM, 74% for 3MM, 77% for DT, 46% for 3OH, 73% for ETM, and 57% for COD. The frequency distribution of the CYP2D6 metabolic ratio (DTM/DT) illustrated a bimodal distribution whereas, the CYP3A metabolic ratio (DTM/3MM) exhibited a unimodal distribution in overnight urine samples of volunteers who ingested 30 mg dextromethorphan hydrobromide. The CYP2D6 metabolic ratio significantly correlated with 3MM/3OH (r=0.82) and DTM/3OH (r=0.95) but did not correlate with the CYP3A metabolic ratio (r=0.27).

Inhibition of human intestinal wall metabolism by macrolide antibiotics: effect of clarithromycin on cytochrome P450 3A4/5 activity and expression.

Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450 (CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent of intestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition.