Impact of inhalational exposure to ethanol fuel on the pharmacokinetics of verapamil, ibuprofen and fluoxetine as in vivo probe drugs for CYP3A, CYP2C and CYP2D in rats.

Occupational toxicology and clinical pharmacology integration will be useful to understand potential exposure-drug interaction and to shape risk assessment strategies in order to improve occupational health. The aim of the present study was to evaluate the effect of exposure to ethanol fuel on in vivo activities of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2C and CYP2D by the oral administration of the probe drugs verapamil, ibuprofen and fluoxetine. Male Wistar rats exposed to filtered air or to 2000 ppm ethanol in a nose-only inhalation chamber during (6 h/day, 5 days/week, 6 weeks) received single oral doses of 10 mg/kg verapamil or 25 mg/kg ibuprofen or 10 mg/kg fluoxetine. The enantiomers of verapamil, norverapamil, ibuprofen and fluoxetine in plasma were analyzed by LC-MS/MS. The area under the curve plasma concentration versus time extrapolated to infinity (AUC(0-∞)) was calculated using the Gauss-Laguerre quadrature. Inhalation exposure to ethanol reduces the AUC of both verapamil (approximately 2.7 fold) and norverapamil enantiomers (>2.5 fold), reduces the AUC(0-∞) of (+)-(S)-IBU (approximately 2 fold) and inhibits preferentially the metabolism of (-)-(R)-FLU. In conclusion, inhalation exposure of ethanol at a concentration of 2 TLV-STEL (6 h/day for 6 weeks) induces CYP3A and CYP2C but inhibits CYP2D in rats.

Verapamil effect on phenytoin pharmacokinetics in rats.

Efflux transporter and enzyme overexpression can be induced by certain antiepileptic drugs. Phenytoin (PHT) is at the same time substrate and inducer of CYP2C isoenzymes and efflux carriers. Its inductive effect has been postulated to be concentration and time-dependent. Since verapamil (VPM) is a well known substrate and inhibitor of P-glycoprotein, its administration could modify PHT systemic exposure. The objective of this work was to determine if single doses (40mg/kg) of VPM might change PHT body fate in the same way when given at the beginning or several days after 100mg/kg of PHT daily doses were started. Both drugs were administered intraperitoneally to female Sprague Dawley rats. VPM increased plasma PHT concentrations after one day of treatment, while a decrease in PHT plasma exposure was observed when VPM was added at the fifth day of the antiepileptic treatment. These results suggested that VPM would have different impact on PHT pharmacokinetics, depending on the level of expression of both efflux transporters and enzymes. Before the hepatic cells could acquire a high content of enzymes due to the inductive effect of PHT dosing, VPM decreased the predominant intestinal clearance of PHT. But, once the enzymatic machinery at the hepatocyte became more important than that at the intestine, although ineffective because of the high hepatobiliary efflux transporter overexpression, VPM blockade from the liver resulted in an increased total PHT clearance.

Reduction of enantioselectivity in the kinetic disposition and metabolism of verapamil in rats exposed to toluene.

Toluene and verapamil are subject to extensive oxidative metabolism mediated by CYP enzymes, and their interaction can be stereoselective. In the present study we investigated the influence of toluene inhalation on the enantioselective kinetic disposition of verapamil and its metabolite, norverapamil, in rats. Male Wistar rats (n = 6 per group) received a single dose of racemic verapamil (10 mg/kg) orally at the fifth day of nose-only toluene or air (control group) inhalation for 6 h/day (25, 50, and 100 ppm). Serial blood samples were collected from the tail up to 6 h after verapamil administration. The plasma concentrations of verapamil and norverapamil enantiomers were analyzed by LC-MS/MS by using a Chiralpak AD column. Toluene inhalation did not influence the kinetic disposition of verapamil or norverapamil enantiomers (p > 0.05, Kruskal-Wallis test) in rats. The pharmacokinetics of verapamil was enantioselective in the control group, with a higher plasma proportion of the S-verapamil (AUC 250.8 versus 120.4 ng x h x mL(-1); p < or = 0.05, Wilcoxon test) and S-norverapamil (AUC 72.3 versus 52.3 ng x h x mL(-1); p < or = 0.05, Wilcoxon test). Nose-only exposure to toluene at 25, 50, or 100 ppm resulted in a lack of enantioselectivity for both verapamil and norverapamil. The study demonstrates the importance of the application of enantioselective methods in studies on the interaction between solvents and chiral drugs.

Simultaneous analysis of the enantiomers of verapamil and norverapamil in rat plasma by liquid chromatography-tandem mass spectrometry.

An enantioselective micromethod for the simultaneous analysis of verapamil (VER) and norverapamil (NOR) in plasma was developed, validated and applied to the study of the kinetic disposition of VER and NOR after the administration of a single oral dose of racemic-VER to rats. VER, NOR and the internal standard (paroxetine) were extracted from only 100-microL plasma samples using n-hexane and the enantiomers were resolved on a Chiralpak AD column using n-hexane:isopropanol:ethanol:diethylamine (88:6:6:0.1) as the mobile phase. The analyses were performed in the selected reaction monitoring mode. Transitions 456>166 for VER enantiomers, 441>166 for NOR enantiomers and 330>193 for the internal standard were monitored and the method had a total chromatographic run time of 12 min. The method allows the determination of VER and NOR enantiomers at plasma levels as low as 1.0 ng/mL. Racemic VER hydrochloride (10mg/kg) was given to male Wistar rats by gavage and blood samples were collected from 0 to 6.0 h (n=6 at each time point). The concentration of (-)-(S)-VER was three folds higher than (+)-(R)-VER, with an AUC ratio (-)/(+) of 2.66. Oral clearance values were 12.17 and 28.77 L/h/kg for (-)-(S)-VER and (+)-(R)-VER, respectively. The pharmacokinetic parameters of NOR were not shown to be enantioselective.

Verapamil quantification in human plasma by liquid chromatography coupled to tandem mass spectrometry. An application for bioequivalence study.

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of Verapamil in human plasma using Metoprolol as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction and chromatographed on a C(8) analytical column. The mobile phase consisted of methanol-water (70:30; v/v)+12 mM formic acid. The method had a chromatographic total run time of 3.5 min and was linear within the range 1.00-500 ng/mL. Detection was carried out on a Micromass Quattro Ultima tandem mass spectrometer by multiple reaction monitoring (MRM). The intra-run imprecision was less than 5.1% calculated from the quality control (QC) samples, and 16.3% from the limit of quantification (LOQ). The accuracy determined from QC samples were between 92.9 and 103.1%, and 95.2 and 115.3% from LOQ. Concerning the inter-batch analysis, the imprecision was less than 5.8% and 17.3% from QC samples and LOQ, respectively. The accuracy varied between 98.2 and 100.8% from QC and it was 103.1% from LOQ. The protocol herein described was employed in a bioequivalence study of two tablet formulations of Verapamil.

Measurement of plasma verapamil levels by high-performance liquid chromatography.

1. We have developed an alternative procedure for the measurement of verapamil levels in human plasma by reverse-phase high performance liquid chromatography with fluorimetric detection. 2. Prior to assay, plasma is submitted to a double extraction procedure, using first n-heptane in alkaline medium and then an acid phosphate buffer. Flecainide, a compound not related to verapamil, is used as internal standard. Mean recoveries of 70 and 63% were obtained for verapamil and flecainide, respectively. 3. The sensitivity (5 ng/ml), reproducibility (inter-assay %CV = 1.7-8.7; intra-assay %CV = 2-4) and high recovery during sample clean-up make this method useful for the quantitation of verapamil in therapeutic monitoring and pharmacokinetic studies. 4. The method is illustrated with the pharmacokinetic results obtained for 14 healthy male volunteers who received a single 240 mg dose of the commercially available tablets of Dilacoron Retard 240 mg. The mean values for the area under the curve from 0 to 24 h (AUC[0-24]), maximum achieved concentration (Cmax) and time to achieve the maximum concentration (Tmax) were 863 ng h-1 ml-1, 112 ng/ml and 4 h, respectively.

Measurement of plasma verapamil levels by high-performance liquid chromatography

1. We have developed an alternative procedure for the measurement of verapamil levels in human plasma by reverse-phase high performance liquid chromatography with fluorimetric detection. 2. Prior to assay, plasma is submitted to a double extraction procedure, using first n-heptane in alkaline medium and then an acid phosphate buffer. Flecainide, a compound not related to verapamil, is used as internal standard. Mean recoveries of 70 and 63 per cent were obtained for verapamil and flecainide, respectively. 3. The sensitivity (5 ng/ml), reproducibility (inter-assay per cent CV = 1.7-8.7; intra-assay per cent CV = 2-4) and high recovery during sample clean-up make this method useful for the quantitation of verapamil in therapeutic monitoring and pharmacokinetic studies. 4. The method is illustrated with the pharmacokinetic results obtained for 14 healthy male volunteers who received a single 240 mg dose of the commercially available tablets of Dilacoron Retard 240 mg. The mean values for the area under the curve from 0 to 24 h (AUC[0-24]), maximum achieved concentration (Cmax) and time to achieve the maximum concentration (Tmax) were 863 ng h-1 ml-1, 112 ng/ml and 4 h, respectively

Relationships between verapamil plasma concentrations and its antihypertensive action.

Twenty-seven hypertensive outpatients were studied to evaluate the efficacy of verapamil after a single oral dose as well as following a short-term treatment and also in combination with oxprenolol. Blood pressure was significantly reduced (p 0.01) after verapamil monotherapy and the combined treatment. PR interval was lengthened from 30 min to 4 h during acute testing, and also after short-term treatment. QT was only prolonged after the verapamil monotherapy. Systolic time intervals (STIs) were not modified, except left ventricular ejection time (LVETc). Direct correlations were found among verapamil plasma concentrations and changes provoked on blood pressure and PR interval. The mean side effects found were disturbance of atrioventricular conduction in two patients without ulterior complications. The results suggest that verapamil monotherapy or in combination with oxprenolol could be useful in the treatment of essential hypertension.