Rate-limiting biotransformation of triamterene is mediated by CYP1A2.

OBJECTIVE: Triamterene (TA), a potassium-sparing diuretic, is extensively metabolized by hydroxylation in 4'-position and subsequent conjugation by cytosolic sulfotransferases. To identify the cytochrome P450 enzyme(s) catalyzing hydroxylation of triamterene (the rate-limiting step in the formation of the sulfate ester (STA)), in vitro incubation studies were performed with human liver microsomes. METHODS: Initial rates of TA hydroxylation (0 - 300 microM) were determined during a ten-minute-incubation period with liver microsomes of two donors. The role of individual CYP enzymes was determined by pre-incubation with selective inhibitors/alternative substrates. Vice versa, the effect of TA (0 - 500 microM) on 3-demethylation of caffeine (0 - 1,000 microM) was assessed. Metabolite concentrations were estimated by reversed-phase HPLC methods. RESULTS: TA Km values without inhibitors were 60 and 142 microM, Vmax was 177 and 220 pmol/min/mg protein, respectively. Mean inhibitor induced changes of 4'-hydroxy-TA formation were as follows: Furafylline 25 microM (CYP1A2), complete inhibition (-100%); omeprazole 250 microM (CYP1A2 inhibitor/CYP2C 19 substrate), -30%; coumarin 25 microM (CYP2A6), -11%; quinidine 25 microM (CYP2D6), -9%; ketoconazole 25 microM (CYP3A), -18%; and erythromycin 250 microM (CYP3A), -8%. In the reverse inhibition studies, TA competitively inhibited caffeine 3-demethylation with Ki values of 65 and 111 microM, respectively. CONCLUSION: 4'-hydroxylation of TA in humans appears to be mediated exclusively by CYP1A2. Inhibition or induction of CYP1A2 will change the time course of both TA and its active phase-II metabolite. The net pharmacodynamic effect of such changes is difficult to predict and needs to be evaluated in clinical studies.

Absorption pattern of trospium chloride along the human gastrointestinal tract assessed using local enteral administration.

BACKGROUND AND OBJECTIVES: The antimuscarinic drug trospium chloride is hydrophilic and therefore does not enter the CNS when used for the treatment of overactive bladder disturbances. However, the same property is the main reason for low and variable oral bioavailability. The present study was performed to assess the influence of intestinal site on absorption of the drug as the basis for the development of modified release preparations. METHODS: In a change-over pilot study, 8 healthy male volunteers received single 20 mg doses oftrospium chloride orally as a tablet (reference), as Eudragit-coated tablets dissolving at pH 6.0 (local administration into the small intestine), and rectally via a mini enema (corresponding to local administration into the large intestine). Plasma concentrations of trospium chloride were determined up to 36 hours after administration using GC/MS. RESULTS: Extent and rate of trospium chloride absorption declined rapidly upon administration into more distal regions of the gastrointestinal tract. C(max) (median: 6.42 ng/ml) and AUC(0.tlast) (42.28 ng/ml x h) were highest and t(max) (3.5 h) was shortest after administration of the reference tablet. AUC(0-tlast) reached 78% (90% CI 43 - 139%) after small intestine administration and 2% (90% CI 1 - 9%) following rectal administration, respectively, relative to the values for the oral tablet. CONCLUSION: Trospium chloride is absorbed primarily in the upper gastrointestinal tract. Development of modified release preparations must balance prolonged apparent absorption rates of the drug against a decrease in bioavailability.

Screening for inhibitory effects of antineoplastic agents on CYP3A4 in human liver microsomes.

BACKGROUND: The human cytochrome P450 enzyme CYP3A4 is involved in the metabolism of many anticancer drugs. Since these drugs are usually administered in a polychemotherapy regimen, the objective of this study was to examine their inhibitory potency on CYP3A4 with regard to possible mutual drug interactions. METHOD: CYP3A4 activities in human liver microsomes from 2 donors were determined using the oxidation of the dihydropyridine denitronifedipine, a specific CYP3A4 substrate, at a concentration of 50 microM (= KM). Formation of the pyridine metabolite was measured using HPLC. Inhibitor concentrations used were 0.5, 5 and 50 microg/ml, except for cyclophosphamide and ifosfamide (0.5, 2.5 and 5 mg/ml) and for paclitaxel (0.05, 0.15, 0.5, 1.5 and 5 microg/ml). RESULTS: The following substances showed an inhibitory effect on CYP3A4 (IC50 values for the 2 microsome samples are parenthesized): cyclophosphamide (12.3/9.2 mmol/l), mafosfamide generated 4-OH-cyclophosphamide (152/163 [micromol/l), ifosfamide (3.6/2.5 mmol/l), vinblastine sulfate (20/44 micromol/l), vincristine sulfate (67/176 micromol/l), daunorubicin hydrochloride (206/200 micromol/l), doxorubicin hydrochloride (160/215 micromol/l), teniposide (64/84 micromol/l) and docetaxel (6.4/12.7 micromol/l). No inhibitory effect on CYP3A4 was observed with epirubicin, etoposide, paclitaxel, cytarabine, 5-FU, 6-mercaptopurine, methotrexate, cisplatin, carboplatin, bleomycin, busulfan, chlorambucil and mitomycin. CONCLUSION: Comparing IC50 values with plasma concentrations present during antineoplastic therapy, the agents cyclophosphamide, ifosfamide, vinblastine, teniposide and docetaxel could possibly cause clinical drug interactions by inhibition of CYP3A4. Some recently described clinical interactions with antineoplastic agents may be explained by these results.

Variation of CYP1A2-dependent caffeine metabolism during menstrual cycle in healthy women.

BACKGROUND AND OBJECTIVES: The activity of the human cytochrome P450 CYP1A2 is decreased by female sex hormones during pregnancy or treatment with oral contraceptives. However, the influence of menstrual cycle on CYP 1A2 activity is not clear. METHODS: CYP1A2 activity was monitored in 15 women (13 with confirmed ovulatory cycles, 2 smokers, age (mean +/- SD) 27.8 +/- 3.8 years, body mass index 23.8 +/- 3.8 kg x m-2) using the specific substrate caffeine (mean doses 149 mg). After a run-in period started one week prior to expected onset of menses, daily saliva samples were taken 7.3 +/- 0.7 hours after caffeine intake throughout the cycle, and caffeine clearance was estimated from the paraxanthine to caffeine ratio therein. Ovulation was confirmed by progesterone serum concentration above 3 ng/ml in the second half of the cycle. RESULTS: Initial (day 2) caffeine clearance (n = 15, geometric mean) was 1.37 ml/min/kg body weight (coefficient of variation (CV) 48%). The ratio of caffeine clearance for the luteal (day -9 to -4 prior to onset of the next menses) to the follicular phase (days 5-10) was (n = 13, point estimate) 1.03 (90% CI 0.95-1.12), indicating that there was no difference in CYP1A2 activity between these cycle phases. The median intraindividual CV in ovulatory cycles (n = 13) was 23% (range 11% to 39%). As an additional finding, there was evidence for long-term fluctuations of CYP1A2 activity in most individuals. CONCLUSIONS: A dose adaptation according to the phase of menstrual cycle based on pharmacokinetics is not required for CYP1A2 substrates.

Problems and perspectives of phenotyping for drug-metabolizing enzymes in man.

Pronounced interindividual differences in drug disposition are mainly caused by differences in the activity of liver drug-metabolizing enzymes. These depend on known and unknown covariates, including genetic as well as environmental factors. Phenotyping, i.e. assessment of enzyme activities in vivo after administration of a test dose, seems to be a promising tool for determining actual metabolic capacities. Although it is a well-established experimental approach, phenotyping has not yet found its way into clinical practice. Main reasons for this are lack of validation for many probes and assays used, complicated procedures, invasiveness, semi-quantitative test results, non-compliance on behalf of the subjects tested, high costs, and lack of prospective clinical studies to assess the benefit of phenotyping for patients. Problems and perspectives of phenotyping are exemplified for the cytochrome P-450 enzymes CYP1A2 and CYP3A4, two major human drug-metabolizing enzymes.

Omeprazole weakly inhibits CYP1A2 activity in man.

BACKGROUND AND OBJECTIVES: Omeprazole is an inducer of human cytochrome P450 1A (CYP1A) enzymes, but shows inhibitory effects on CYP2C19 and CYP3A4. In this study, a potential inhibitory effect of omeprazole on caffeine metabolism, a validated CYP1A2 marker, was examined. METHODS: A randomized, balanced crossover single-dose study was conducted in 16 healthy volunteers comprising 12 extensive (EM) and 4 poor metabolizers (PM) for CYP2C19. All volunteers received a 40 mg omeprazole dose or placebo 0.5 h prior to caffeine 3 mg/kg body weight. Six EMs were re-tested with 80 mg of omeprazole. In vitro, effects of omeprazole on caffeine N3-demethylation were determined in human liver microsomes. RESULTS: In vivo, non-parametric point estimates (90% confidence intervals) for the ratios of caffeine pharmacokinetics with/without co-administration of the 40 mg omeprazole dose were: AUC 1.08 (1.04 - 1.13), MRT 1.09 (0.99 - 1.19), and plasma ratio of paraxanthine/caffeine 6 h post-dose 0.91 (0.80 - 1.00). Inhibition of caffeine N3-demethylation by omeprazole was slightly more pronounced in PM than in EM of CYP2C19. Estimates for the 80 mg omeprazole dose were: AUC 1.12 (1.05 -1.18), MRT 1.18 (1.07 - 1.30), and paraxanthine/caffeine ratio 0.83 (0.74 -0.94). In vitro, omeprazole was mainly a competitive CYP1A2 inhibitor with K(i) values of around 150 microM. CONCLUSIONS: Omeprazole exerts a concentration-dependent inhibition of CYP1A2 activity in man. However, even after single oral doses up to 80 mg, this effect is weak and without clinical relevance.

Estimation of cytochrome P-450 CYP1A2 activity in 863 healthy Caucasians using a saliva-based caffeine test.

A pronounced variability limits the usefulness of CYP1A2 phenotyping for drug therapy, for evaluating liver function, and for assessing the role of this enzyme in carcinogenesis. To identify and quantify sources of this variation, we estimated CYP1A2 activity in 863 healthy Caucasians using caffeine clearance derived from saliva concentrations before and 5-7 h after a caffeine test dose. Data from 786 individuals were eligible for evaluation (mean age 39 years, 415 women including 94 taking oral contraceptives, 401 non-smokers). Overall geometric mean (geometric SD) caffeine clearance was 1.34 ml min(-1) kg b.w.(-1) (1.65). The effect of the following covariates was evaluated by analysis of covariance: age, sex, oral contraceptives, body height, body weight, body mass index, number of cigarettes smoked, tar exposure from smoking, several indices of dietary caffeine consumption, intake of sauerkraut, and country of residence (Germany, Bulgaria or Slovakia). Estimated changes relative to arbitrarily defined basal caffeine clearance (male, non-smoking, German resident) exerted by significant (P < 0.05) covariates were: coffee, 1.45-fold per litre of coffee drunk daily; body mass index, 0.99-fold per kg m(-2); smoking, 1.22-fold, 1.47-fold, 1.66-fold, and 1.72-fold for 1-5, 6-10, 11-20, and > 20 cigarettes smoked per day, respectively; oral contraceptives, 0.72-fold; country of residence, 0.81-fold and 0.74-fold for Bulgaria and Slovakia, respectively; female, 0.90-fold. These covariates explained 37% of overall variation. The 95% confidence interval of individual clearance was 0.46-2.20 times the predicted value. No relevant polymorphism was found for CYP1A2 activity when adjusted for covariate effects.

Interaction of pefloxacin and enoxacin with the human cytochrome P450 enzyme CYP1A2.

BACKGROUND AND OBJECTIVES: Pefloxacin is reported to cause clinically relevant inhibition of theophylline metabolism in vivo, but in vitro pefloxacin was only a weak inhibitor of the cytochrome P450 CYP1A2, mediating main theophylline biotransformation. We therefore further characterized the interaction between pefloxacin and CYP1A2. METHODS: A randomized 3-period change-over study was conducted in 12 healthy young volunteers on the steady-state interactions between pefloxacin or enoxacin (400 mg twice a day) with caffeine (183 mg once daily), a validated marker of CYP1A2. Caffeine pharmacokinetics were estimated after its fifth dose. Studies in human liver microsomes were carried out to measure the effect of pefloxacin and norfloxacin on caffeine 3-demethylation, an in vitro CYP1A2 probe, and to identify the enzyme(s) that mediate pefloxacin N-4'-demethylation with selective inhibitors. RESULTS: For the in vivo study, ANOVA-based point estimates (90% confidence intervals [CI]) for the ratios of caffeine pharmacokinetics with and without pefloxacin coadministration were 1.11 for maximal steadystate plasma concentrations (Cmax,ss; 90% CI, 0.99 to 1.26), 0.53 for total clearance (CLt,ss; 90% CI, 0.49 to 0.58), and 1.04 for the beta-phase distribution volume (Vdbeta; 90% CI, 0.96 to 1.13). The values for enoxacin were 1.99 for Cmax,ss (90% CI, 1.77 to 2.23), 0.17 for CLt,ss (90% CI, 0.16 to 0.19), and 1.01 for Vdbeta (90% CI, 0.90 to 1.13). Thus pefloxacin caused a 2-fold decrease in caffeine clearance, and enoxacin caused a 6-fold decrease in caffeine clearance. In vitro, norfloxacin and pefloxacin competitively inhibited CYP1A2, with inhibition constant (Ki) values of 0.1 and 1 mmol/L, respectively, and CYP1A2 was the only enzyme with a relevant contribution (approximately 50%) to pefloxacin N-4'-demethylation. CONCLUSIONS: Enoxacin and to a lesser extent pefloxacin may cause clinically relevant interactions with further CYP1A2 substrates. The data suggest that the pefloxacin interaction is partly mediated by its major metabolite norfloxacin.

Grapefruit juice increases oral nimodipine bioavailability.

The bioavailability of dihydropyridine calcium channel blockers following oral administration was shown to be increased by concomitant intake of grapefruit juice for all drugs of this class tested up to now. Here we report a randomized crossover interaction study on the effects of grapefruit juice on the pharmacokinetics of nimodipine and its metabolites. Eight healthy young men (4 smokers/4 nonsmokers) were included. Nimodipine was given as a single 30 mg tablet (Nimotop) with either 250 ml of water or 250 ml of grapefruit juice (751 mg naringin/l). Drug concentrations in plasma withdrawn up to 24 hours postdose were measured by GC-ECD, and model-independent pharmacokinetic parameters were estimated. The study was handled as an equivalence problem. Point estimators and ANOVA based 90% confidence intervals (CI) were calculated for the test (= grapefruit juice period) to reference (= water period) ratios using dose-normalized concentrations. The absence of a relevant interaction was assumed if the CIs were within the 0.67-1.50 range. Cmax for nimodipine reached 124% of the reference period (90% CI 0.76-2.01), AUC was increased to 151% (90% CI 114%-200%), respectively. The null hypothesis "relevant interaction" thus could not be rejected for the primary pharmacokinetic parameters AUC and Cmax. The ratios of metabolite AUC to parent drug AUC were slightly reduced with grapefruit juice intake. Additionally, there was evidence for a more pronounced hemodynamic response in the grapefruit juice period. To avoid the interaction, nimodipine should not be taken with grapefruit juice.

[Bioavailability of a new theophylline chewable pill]. / Bioverfügbarkeit einer neuen Theophyllin-Kautablette.

Oral administration of theophylline as a chewable tablet is an alternative to the conventional parenteral route used in the treatment of acute dyspnoea in asthma bronchiale and other obstructive pulmonary diseases. To investigate the bioavailability of this preparation, a randomised 2-period cross-over study on the pharmacokinetics of a 100 mg theophylline single dose was conducted comparing oral administration as a chewable tablet (test medication) or as solution (reference) in 14 healthy male volunteers (age 21-31 years, body weight 60-90 kg). Bioequivalence of the tested formulations could be confirmed basing on the primary pharmacokinetic parameters (AUC, Cmax) for the extent and rate of theophylline absorption. The criterion of bioequivalence was also met for the secondary parameters, including terminal elimination half-life, mean residence time, time point of maximal plasma concentration and the ratio Cmax/AUC. Hence, the chewable theophylline tablet is bioequivalent to theophylline given as a solution. With regard to therapeutic efficacy, equivalence to medication by droplets can be expected.