Urinary CYP eicosanoid excretion correlates with glomerular filtration in African-Americans with chronic kidney disease.

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid (AA), i.e., 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), play an important role in the regulation of renal tubular and vascular function. The present study for the first time profiled HETEs and epoxygenase derived dihydroxyeicosatetraenoic acid diHETEs levels in spot urines and plasma in 262 African American patients from the University of Mississippi Chronic Kidney Disease Clinic and 31 African American controls. Significant correlations in eGFR and urinary 20-HETE/creatinine and 19-HETE/creatinine levels were observed. The eGFR increased by 17.47 [p=0.001] and 60.68 [(p=0.005]ml/min/for each ng/mg increase in 20-HETE and 19-HETE levels, respectively. Similar significant positive associations were found between the other urinary eicosanoids and eGFR and also with 19-HETE/urine creatinine concentration and proteinuria. We found that approximately 80% of plasma HETEs and 30% diHETEs were glucuronidated and the fractional excretion of 20-HETE was less than 1%. These results suggest that there is a significant hepatic source of urinary 20-HETE glucuronide and EETs with extensive renal biotransformation to metabolites which may play a role in the pathogenesis of CKD.

Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin-induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-ß/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-ß1-induced renal tubular epithelial-mesenchymal transition in vitro. These findings highlight the beneficial roles of the CYP epoxygenase-EET system in the pathogenesis of diabetic nephropathy.

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats.

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.

Detection of aminophenylnorharman, a possible endogenous mutagenic and carcinogenic compound, in human urine samples.

Mutagenic/carcinogenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is formed from norharman and aniline in the presence of cytochrome P450 3A4/1A2. Because both precursors are widely distributed in the environment, human exposure is unavoidable. To clarify APNH formation in the human body, amounts of the compound in 24-h human urine collected from smokers and nonsmokers, eating a normal diet, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry. In addition, norharman and aniline were also analyzed by high-performance liquid chromatography and gas chromatography, respectively. APNH could be detected in all urine samples at levels 49 to 449 pg for smokers and 21 to 594 pg for nonsmokers per 24-h urine, respectively. The amounts of norharman and aniline were 46 to 185 ng and 0.70 to 8.10 microg for smokers and 52 to 447 ng and 0.49 to 5.72 microg for nonsmokers, respectively, per 24-h urine (none of the levels differing significantly between smokers and nonsmokers). To exclude exogenous exposure to norharman and aniline, we analyzed the levels of APNH, norharman, and aniline in urine samples collected from inpatients receiving parenteral alimentation. Similar to the healthy volunteers, all urine samples contained 12 to 338 pg of APNH, 6 to 75 ng of norharman, and 0.33 to 1.86 microg of aniline per 24-h urine. These results suggest that APNH should be considered as a novel endogenous mutagen/carcinogen; thus, it is very important to determine the biological significance of this carcinogen for human cancer development.

Study of urinary 6 beta-hydroxycortisol/cortisol ratio in spot urine sample as a biomarker of 3A4 enzyme activity in healthy and epileptic subjects of Egyptian population.

The ratio of urinary 6 beta-hydroxycortisol/cortisol (6 beta-OHC/FC) in morning spot urine samples collected from 8:00 a.m. to 12:00 p.m. was studied using ELIZA kits (Stabiligen) in a group of healthy adult Egyptians (control group) of both sex (n=65, age range: 16-48 years). The frequency distribution of urinary 6 beta-OHC/FC ratio was widely distributed among subjects with higher values in males in comparison to females. No bimodality in either sex was observed. Another group of adult epileptic patients (n=16) was studied for the influence of chronic carbamazepine antiepileptic drug administration on urinary 6 beta-OHC/FC ratio in spot urine samples. The induction property of carbamazepine on CYP3A4 was observed through significant increase (p=0.01) in 6 beta-OHC/FC ratio among epileptic patients in comparison with control subjects. In conclusion, the frequency distribution of urinary 6 beta-OHC/FC ratio among Egyptians shows sexual dimorphism. Also, measurement of urinary 6 beta-OHC/FC ratio provides a simple non-invasive method to monitor CYP3A4 enzyme induction during administration of carbamazepine antiepileptic drug.

Frequency distribution of dextromethorphan O-demethylation in a Greek population.

OBJECTIVE: To determine the CYP2D6 phenotype in a Greek population by using dextromethorphan (DM) as a probe drug. METHODS: DM (30 mg) was given orally to 102 unrelated Greek subjects and 8-hour urine samples were collected. Concentrations of DM and its metabolite dextrorphan (DX) were determined using a validated HPLC assay. Metabolic molar ratio (MR) of DM to free DX in log form was used as an in vivo index of metabolic status. RESULTS: The frequency distribution histogram of MR was bimodal. An antimode of 0.25 for the mean log MR was determined using probit analysis. Seven of 102 subjects (6.9%) were poor metabolizers (PMs). CONCLUSION: The PM frequency of CYP2D6 in Greek subjects was similar to other Caucasian populations.

Measurement of hepatic and intestinal CYP3A4 and PGP activity by combined po + iv [14C]erythromycin breath and urine test.

The aim of the present study was to develop a test for measuring hepatic and intestinal removal of cytochrome p-450 3A4 (CYP3A4)- and P-glycoprotein (PGP)-dependent xenobiotics that would be applicable for clinical use in humans. Orally and intravenously administered [N-methyl-14C]erythromycin was used for evaluation of 14C-labeled excretion dynamics in breath and urine. Simultaneous breath and urine test measurements were performed in 32 healthy volunteers and in 23 renal transplant recipients. Mathematical analysis of the excretion rate of labeled CO2 in breath and labeled carbon in urine resulted in 1). separation of both CYP3A4 and PGP activity in the liver and the intestinal mucosa and 2). numerical calculation of the dynamics of the different processes. The test was sufficiently sensitive to detect theoretically predicted process-specific pharmacological modulations by different drugs in healthy volunteers and after recent renal transplantation. It is concluded that the combined oral and intravenous erythromycin breath and urine test is a reliable and noninvasive test to measure phenotypic intestinal and hepatic CYP3A4 and PGP activity and may be a promising tool for prediction of drug interactions and dose adjustment of many pharmacotherapeutics in clinical practice, e.g., immunosuppressive agents after renal transplantation.

Implication of P450-metabolite complex formation in the nonlinear pharmacokinetics and metabolic fate of (+/-)-(1'R*,3R*)-3-phenyl-1-[(1',2',3',4'-tetrahydro-5',6'-methylene-dioxy-1'-naphthalenyl) methyl] pyrrolidine methanesulfonate (ABT-200) in dogs.

Following a single oral or intravenous administration of the R,R(+) and S,S(-) (14)C-pseudoracemate of (+/-)-(1'R*,3R*)-3-phenyl-1-[(1',2',3',4'-tetrahydro-5',6'-methylene-dioxy-1'-naphthalenyl) methyl] pyrrolidine methanesulfonate (ABT-200/I) to dogs, a total of six (R,R[+]) and eight (S,S[-]) metabolites were identified by high-pressure liquid chromatography/mass spectral techniques. Greater than 99% of the dose was eliminated as metabolites indicating that the clearance of I was predominantly metabolic. The catechol was the major excreted metabolite (fecal), whereas the urine and bile predominantly contained metabolites resulting from secondary biotransformation of the catechol via O-methylation, glucuronidation, and sulfation. After a single 12 mg/kg oral dose of racemic I to dogs, the mean area under the plasma curve (AUC(0-24h)) averaged 4.55 micro g. h/ml, with an apparent plasma clearance value of 2.70 l/h. kg. After 14 daily doses, the apparent plasma clearance was 3.5-fold lower (0.78 l/h. kg) and the AUC(0-24h) about 4-fold higher (18.58 micro g. h/ml). Isolation of liver microsomes from these animals indicated that a cytochrome p450 (p450)-metabolite complex (MI complex) was formed in the liver after both single and multiple dosing. The mean concentration of the MI complex 24 h after a single dose averaged 31 pmol/mg of microsomal protein, whereas the amount in the animals given multiple doses of I averaged 163 pmol/mg. There was a positive correlation (R(2) = 0.993) between the plasma AUC for I and the amount of the MI complex found in the liver of each animal. Formation of the MI complex was demonstrated in vitro in control dog liver microsomes, was NADPH-dependent, and was dissociated from p450 with 2-methylbenzimidazole. In vitro preincubation studies indicated that I was a mechanism-based inhibitor and that formation of the complex inhibited catechol formation. These results demonstrate that the liver p450s that metabolize I form an inhibitory MI complex after in vivo administration in dogs. Formation of the complex increases during multiple dosing and inhibits the enzymes from further metabolism of I resulting in nonlinear pharmacokinetics.

Epidemiological study of urinary 6beta-hydroxycortisol to cortisol ratios and breast cancer risk.

The ratio of urinary 6beta-hydroxycortisol:cortisol is a measure of the activity of cytochrome p450 3A4 (CYP3A4). CYP3A4 catalyzes the formation of the genotoxic estrogen, 16alpha-hydroxyestrone. It is also involved in the activation of many other mammary carcinogens, such as the polycyclic aromatic hydrocarbons and heterocyclic amines. We evaluated the association between urinary cortisol ratios and breast cancer risk in a subgroup of women who participated in a population-based case-control study in Shanghai. Overnight urine samples from 246 case-control pairs were assayed for 6beta-hydroxycortisol (6beta-OHC) to cortisol. The urine samples from all of the breast cancer patients were collected before any chemotherapy or radiotherapy. In-person interviews were conducted to obtain comprehensive information on dietary habits, reproductive history, and other lifestyle factors. The median levels of 6beta-OHC:cortisol ratios were 2.61 in cases and 2.16 in controls, a 20.8% difference (P < 0.001). The case-control difference was larger in women over 45 years of age (31.3% difference; P < 0.001) than younger women (6.0%; P = 0.45). After adjusting for confounding variables, the risks of breast cancer were increased from 1.0 (reference) to 1.6 [95% confidence interval (CI), 0.9-3.1], 2.2 (95% CI, 1.1-4.2), and 3.7 (95% CI, 1.9-7.4; P for trend, <0.001) with increasing levels of 6beta-OHC:cortisol ratios. The positive association was more pronounced among older women (>45 years) than among younger women (< or = 45 years). The adjusted odds ratios associated with the highest cortisol ratio were 6.0 (95%CI, 2.2-16.1) among older women and 2.2 (95%CI, 0.8-6.1) among younger women. The association of the 6beta-OHC:cortisol ratio was stronger among older women who had a high body mass index, late age at menopause, and early age at menarche (factors related to high endogenous estrogen exposure) than those who did not have these factors. These findings are consistent with the role of CYP3A4 in estrogen and carcinogen metabolism and suggest that high CYP3A4 activity may be a risk factor for breast cancer risk.

Fluoxetine pharmacokinetics and effect on CYP2C19 in young and elderly volunteers.

The objective of this study was to assess in both young and elderly volunteers the pharmacokinetics of fluoxetine and norfluoxetine and effects on cytochrome P450 (CYP) 2C19. Male volunteers aged 18 to 40 years (N = 14) or older than 65 years (N = 16) received fluoxetine 20 mg/day for 6 weeks and fluoxetine 40 mg/day for an additional 6 weeks. Blood was drawn over a 24-hour period after the initial dose and after 6 weeks and 12 weeks to determine AUC0-24, Cmax, and tmax; weekly to evaluate predose levels (C0); and over a 3-week period after discontinuation to evaluate washout (t1/2). Mephenytoin was used to assess CYP2C19 activity before and after 6 weeks and 12 weeks of fluoxetine. Fluoxetine AUC0-24, C0, and Cmax did not differ in young and elderly subjects. The norfluoxetine C0 was 22% lower in elderly subjects (p < .05), with comparable decreases in AUC0-24 and Cmax. In the elderly volunteers, the t1/2 for fluoxetine was 25% longer (5.0 vs. 4.0 days) and for norfluoxetine was 33% longer (20 vs. 15 days), although variability and sample size precluded statistical significance. Fluoxetine dosing inhibited CYP2C19 activity in both age groups, increasing the (S)- to (R)-mephenytoin ratio 3- to 4-fold (p < .01). The half-lives of fluoxetine and norfluoxetine at 40 mg/day were longer than commonly reported in the literature and may be longer in elderly subjects. Fluoxetine substantially inhibited the metabolism of the CYP2C19 substrate (S)-mephenytoin.

[Energy metabolism of free-radical and microsomal oxidation in workers engaged into oil-processing industry]. / Energeticheskii obmen cvobodnoradikal'nogo i mikrosomal'nogo okisleniia v organizme rabotnikov neftgepererabatyvaiushchei promyshlennosti.

The authors present data on disorders of energy metabolism in erythrocytes, of microsomal monooxygenases and of free-radical oxidation in blood and urine of workers engaged into oil-processing industry. Studies revealed considerable changes in RBC adenyl system parameters, in chemiluminescence of blood and urine, in monooxygenase system. Nonspecific therapy in sanatorium appears to better these aspects.

Metabolism of 3-butene-1,2-diol in B6C3F1 mice. Evidence for involvement of alcohol dehydrogenase and cytochrome p450.

3-Butene-1,2-diol (BDD), a metabolite of 1,3-butadiene, is rapidly metabolized by B6C3F1 mice at doses ranging from 10 to 250 mg/kg. Calculation of plasma clearance suggested that the kinetics of BDD metabolism were dose-dependent. Clearance varied 5-fold in this dose range. Urinary excretion of BDD was also dose-dependent but did not exceed 5% of the administered dose. A small fraction of the dose (<1%) was excreted as glucuronide or sulfate conjugates. Benzylimidazole, a cytochrome P450 inhibitor, decreased the clearance of BDD (25 mg/kg) by 44%, whereas 4-methylpyrazole, an alcohol dehydrogenase and cytochrome P450 inhibitor, decreased BDD clearance by 82%. BDD administration (250 mg/kg) resulted in depletion of hepatic and renal nonprotein thiols, by 48 and 22%, respectively. Pretreatment of mice with 4-methylpyrazole provided partial protection against depletion of nonprotein thiols, whereas pretreatment with benzylimidazole was ineffective. Incubation of BDD with NADPH and mouse liver microsomes resulted in time-dependent inactivation of p-nitrophenol hydroxylase (PNPH) activity, a marker for cytochrome P450. Inclusion of glutathione, with or without glutathione peroxidase, did not attenuate the inactivation of PNPH, whereas deferoxamine, superoxide dismutase, catalase, and mannitol provided modest protection. These results are consistent with suicide inhibition of PNPH by BDD, with a minor role for reactive oxygen species in the loss of PNPH. Treatment of mice with BDD (250 mg/kg) inactivated hepatic microsomal PNPH activity by 50% after 60 min. These results suggest that BDD is extensively and rapidly metabolized in mice, and they provide evidence for the formation of reactive intermediates that could play a role in the toxicity of 1, 3-butadiene.

Determination of S/R ratio of mephenytoin in human urine by chiral HPLC and ultraviolet detection and its comparison with gas chromatography.

AIM: To improve HPLC method for rapid determination of urinary S/R-ratio of mephenytoin, a widely used metabolic index for cytochrome P-450 2C19 (CYP2C19) activity. METHODS: Aliquots of 0-8-h urine sample after dosing racemic mephenytoin 100 mg underwent one-step extraction with dichloromethane. Analysis was performed on a chiral column (250 mm x 4 mm, 5 microns) at lambda = 207 nm. The eluent was a mixture of acetronitrile and water containing both 0.1% glacial acetic acid and 0.2% triethylamine (14:86, vol/vol) at a flow-rate of 0.9 mL.min-1. RESULTS: The enatiomers of mephenytoin in urine were well separated within 9 min. A linear correlation was observed between 50-5000 micrograms.L-1 with the detection limit of 12.5 micrograms.L-1 for both enantiomers of mephenytoin. This HPLC analysis was comparable to gas chromatography in accuracy and sensitivity, but with much shorter retention time and better resolution. CONCLUSION: The present HPLC method is good for rapid determination of the ability of subjects to hydroxylate S-mephenytoin after oral administration of the racemic drug.

Lack of effect of chloroquine on the debrisoquine (CYP2D6 and S-mephenytoin (CYP2C19) hydroxylation phenotypes.

The effects of chloroquine (CHQ) on debrisoquine hydroxylase (CYP2D6) and S-mephenytoin hydroxylase (CYP2C19) were assessed in 11 black Zimbabwean and 12 white Swedish healthy volunteers. The activity of CYP2D6 was measured as the urinary debrisoquine to 4-hydroxydebrisoquine metabolic ratio and that of CYP2C19 as the urinary S- to R-mephenytoin enantiomer ratio (S/R). There were no statistically significant differences in either metabolic ratio as a result of prophylactic or loading doses of CHQ. This indicates that CHQ does not inhibit CYP2D6 or CYP2C19 in vivo and is unlikely to compromise the metabolism of substrates for these two enzymes. It is, therefore, also unlikely that residual CHQ in populations under study will interfere with phenotyping of either CYP2D6 or CYP2C19.

Effects of omeprazole and cimetidine on the urinary metabolic ratio of proguanil in healthy volunteers.

OBJECTIVE: To examine the effects of omeprazole and cimetidine on the urinary recovery and metabolic ratio of proguanil in healthy subjects. METHODS: A metabolic interaction study was conducted in 12 young, healthy extensive metabolisers of proguanil, a CYP2C19 substrate, following a single 100 mg oral dose by analysis of urine collected for 8 hours. RESULTS: Concomitant administration of omeprazole (20 mg), a CYP2C19 substrate, had no significant effect on the urinary recovery of proguanil or cycloguanil, or the ratio of cycloguanil to proguanil [mean 0.76 (95% CI: 0.53 to 0.98) proguanil alone; mean 0.65 (95% CI: 0.40 to 0.89) proguanil plus omeprazole]. In contrast, cimetidine (400 mg), a general CYP inhibitor and renal organic cation secretion inhibitor, decreased the urinary recovery of cycloguanil and reduced the metabolic ratio from a mean of 0.76 to 0.54 (P < 0.01). In 3 poor metabolisers of proguanil, cimetidine had no effect on the proguanil metabolic ratio. CONCLUSION: The concomitant administration of omeprazole or cimetidine will not result in phenocopying extensive metabolisers of proguanil, although cimetidine inhibits the formation of cycloguanil in extensive metabolisers.

Non invasive in vivo study of the maturation of CYP IIIA in neonates and infants.

OBJECTIVE: Cytochrome P450 III (CYP III) has previously been demonstrated in vitro in fetal liver. METHODS: A study was performed in 7 premature, 13 term neonates and 30 infants to assess whether or not maturation had any influence on CYP IIIA activity in children from birth to 1 year of age. A simple non invasive procedure was used (6 beta OHF/FF concentration ratio in a morning spot urine sample). RESULTS: The 6 beta OHF/FF ratio was 7.2, 7.9 and 5.0 in premature and term neonates and infants, respectively. CYP IIIA activity is present at birth, with a 6 beta OHF/FF ratio comparable to adults. Its activity seemed lower in infants. This might be due to the decrease in CYP IIIA7 activity during maturation after birth, which might be more rapid than the increase in CYP IIIA3/4 activity.

CYP1A2 activity as a risk factor for bladder cancer.

CYP1A2, CYP2D6 and N-acetyltransferase activities were estimated in 100 patients with bladder cancer and 84 control subjects from measurements of theophylline, metoprolol and isoniazid and their metabolites in urine, respectively. The frequency of occurrence of slow acetylators of isoniazid and poor metabolizers of metoprolol were 16.7% and 1.2% in the control group and 16.3% and 2.0% in the cancer patient group. These differences were not significant. The recovery ratio of 1-methyluric acid(1-MU) from theophylline was significantly higher in patients with bladder cancer than in control subjects(0.340 +/- 0.016 versus 0.260 +/- 0.020, p < 0.05). The 1-MU recovery ratio was a significant, independent risk factor among the metabolic capacities tested as shown by logistic regression analysis, controlling for N-acetylation of isoniazid, hydroxylation of metoprolol, age, sex, and smoking. We concluded that the capacity for 3-demethylation of theophylline, as a reflection of CYP1A2 activity, is significantly associated with increased risk of nonoccupational urinary bladder cancer.

Metabolic interactions of methoxsalen and coumarin in humans and mice.

Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro. To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin. Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM). Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx. 30% of the activity in mouse liver microsomes). Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes. NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate. In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody. Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5. Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6. Methoxsalen and coumarin have the potential of strong metabolic interactions in man.

Gas-uptake pharmacokinetics of 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123).

The in vivo metabolic rate constants for the metabolism of the chlorofluorocarbon replacement 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) were determined for both male and female rats with a physiologically based pharmacokinetic model. Uptake studies with 500-5,000 ppm HCFC-123 indicated that a single saturable component was involved in both sexes, and no significant differences were observed in in vivo metabolic rate constants between male and female rats. The in vivo metabolic rate constants obtained from computer simulation studies were: for male rats--KM = 1.2 mg liter-1 (7.85 mumol liter-1) and Vmaxc = 7.20 +/- 0.28 mg kg-1 hr-1 (47.1 +/- 1.83 mumol kg-1 hr-1); for female rats--KM = 1.2 mg liter-1 (7.85 mumol liter-1) and Vmaxc = 7.97 +/- 0.30 mg kg-1 hr-1 (52.1 +/- 1.96 mumol kg-1 hr-1). The physiologically based pharmacokinetic model failed to simulate the reduction in HCFC-123 uptake in female rats at 2,000-5,000 ppm. The production and excretion of trifluoroacetic acid, the major urinary metabolite of HCFC-123, was also predicted by the physiologically based pharmacokinetic model with in vivo metabolic rate constants obtained in the gas-uptake simulation studies. Diallyl sulfide, a selective, mechanism-based inhibitor of cytochrome P450 2E1, inhibited the metabolism of HCFC-123, as indicated by a decreased uptake of HCFC-123 and by a lowered urinary excretion of trifluoroacetic acid in diallyl sulfide-treated rats.