Lactobacillus rhamnosus induces CYP3A and changes the pharmacokinetics of verapamil in rats.

Verapamil, a calcium channel blocker, has been approved as the first-line drug for treatment of angina pectoris, hypertension and supraventricular tachycardia. Lactobacillus rhamnosus, one of the normal strains in human intestinal tract, is very popular in the probiotic market for conferring a health benefit on the host. This report investigated the potential of gut microbiota-drug interactions between lactobacillus rhamnosus and verapamil via using wild type (WT) and Cyp3a1/2 knockout (KO) rats. In WT rats, administration of Lactobacillus rhamnosus for 14 days decreased systemic exposure of verapamil and increased its metabolite norverapamil in vivo, and resulted in gut microbiota-drug interactions. In Cyp3a1/2 KO rats, however, this interaction disappeared. Further studies found that Lactobacillus rhamnosus induced CYP3A activity and expression, and changed the composition of gut microbiota, thus changing the pharmacokinetics of verapamil. These results demonstrated the interaction between lactobacillus rhamnosus and verapamil, and indicated that the effect of gut microbiota on metabolic enzymes cannot be ignored.

Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application.

A novel, fast and sensitive enantioselective HPLC assay with a new core-shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(-)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1-450 ng/mL (r2 ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(-)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(-)-VER established higher Cmax and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core-shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).

Successful Prediction of Human Steady-State Unbound Brain-to-Plasma Concentration Ratio of P-gp Substrates Using the Proteomics-Informed Relative Expression Factor Approach.

In order to optimize central nervous system (CNS) drug development, accurate prediction of the drug's human steady-state unbound brain interstitial fluid-to-plasma concentration ratio (Kp,uu,brain ) is critical, especially for drugs that are effluxed by the multiple drug resistance transporters (e.g., P-glycoprotein, P-gp). Due to lack of good in vitro human blood-brain barrier models, we and others have advocated the use of a proteomics-informed relative expressive factor (REF) approach to predict Kp,uu,brain . Therefore, we tested the success of this approach in humans, with a focus on P-gp substrates, using brain positron emission tomography imaging data for verification. To do so, the efflux ratio (ER) of verapamil, N-desmethyl loperamide, and metoclopramide was determined in human P-gp-transfected MDCKII cells using the Transwell assay. Then, using the ER estimate, Kp,uu,brain of the drug was predicted using REF (ER approach). Alternatively, in vitro passive and P-gp-mediated intrinsic clearances (CLs) of these drugs, estimated using a five-compartmental model, were extrapolated to in vivo using REF (active CL) and brain microvascular endothelial cells protein content (passive CL). The ER approach successfully predicted Kp,uu,brain of all three drugs within twofold of observed data and within 95% confidence interval of the observed data for verapamil and N-desmethyl loperamide. Using the in vitro-to-in vivo extrapolated clearance approach, Kp,uu,brain was reasonably well predicted but not the brain unbound interstitial fluid drug concentration-time profile. Therefore, we propose that the ER approach be used to predict Kp,uu,brain of CNS candidate drugs to enhance their success in development.

Determination of Verapamil Hydrochloride and Norverapamil Hydrochloride in Rat Plasma by Capillary Electrophoresis With End-Column Electrochemiluminescence Detection and Their Pharmacokinetics Study.

In this study, we developed a new method for simultaneous determination of verapamil hydrochloride (VerHCl) and its metabolite norverapamil hydrochloride (NorHCl) by using the capillary electrophoresis-electrochemiluminescence. Under optimized experimental conditions, the linear ranges of the VerHCl and NorHCl concentrations were 0.015-10.0 and 0.060-10.0 µg/mL, respectively. The linearity relations were determined using the respective regression equations y = 581.2x + 19.94 and y = 339.4x + 29.16. The respective limits of detection (S/N = 3) were 0.006 and 0.024 µg/mL. The proposed method was used to study the pharmacokinetics of both agents in rat plasma. The maximum concentration (Cmax), half-life time (T1/2) and time to peak (Tmax) were 683.21 ± 74.81 ng/mL, 0.52 ± 0.21 h and 2.49 ± 0.32 h for VerHCl and 698.42 ± 71.45 ng/mL, 1.14 ± 0.26 h and 2.83 ± 0.23 h for NorHCl, respectively, following oral administration of 10 mg/kg VerHCl.

Drug-drug interaction of rivaroxaban and calcium channel blockers in patients aged 80 years and older with nonvalvular atrial fibrillation.

Objectives For revealing the peculiarities of the drug-drug interaction of rivaroxaban (substrate CYP3A4 and P-gp) and calcium channel blockers (CCBs) (verapamil - inhibitor CYP3A4 and P-gp and amlodipine - substrate CYP3A4) in patients 80 years and older with nonvalvular atrial fibrillation (NAF) we studied 128 patients. Methods All patients were divided into groups depending on the therapy taken: the 1st - rivaroxaban + amlodipine (n=51), the 2nd - rivaroxaban + verapamil (n=30), the control group - rivaroxaban without CCBs (n=47). A trough steady-state plasma concentration (C min,ss) of rivaroxaban, prothrombin time (PT) in the blood plasma and the event of clinically relevant non-major (CRNM) bleeding were assessed for each patient. Results Patient in group 2 had higher C min,ss of rivaroxaban, PT and CRNM than subjects in the control group (Me 73.8 [50.6-108.8] ng/mL vs. 40.5 [25.6-74.3] ng/mL; Me 14.8 [13.4-17.3] s vs. 13.8 [12.6-14.4] s; 34% vs. 13%, respectively, p<0.05 for all). When compared, the PT and complication rate in group 1 with the control group C min,ss of rivaroxaban were practically the same (p>0.05 for all). Conclusions In patients ≥80 years with NAF, the use of rivaroxaban in combination with verapamil may not be safe and can lead to CRNM bleeding.

Direct analysis in real time-mass spectrometry for rapid quantification of five anti-arrhythmic drugs in human serum: application to therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART-MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART-MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART-MS/MS method yielded satisfactory linearity (R2 ≥ 0.9906), accuracy (86.1-109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART-MS/MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.

Predicted values for human total clearance of a variety of typical compounds with differently humanized-liver mouse plasma data.

Prediction of human pharmacokinetics is important in the preclinical stage. Values for total clearance of compounds from plasma should be one of the most important pharmacokinetic parameters for predictions. Although several physiological and empirical methods including single-species allometry for prediction of values for human clearance of compounds using humanized-liver mice have been reported, further improvement of prediction accuracies would be still expected. To optimize these approaches, we proposed methods for unbound intrinsic clearance in virtually 100% humanized-liver mouse by incorporating unbound plasma fractions of compounds in differently humanized-liver mice. Comparisons of prediction accuracies of values for human clearance of 15 model compounds were performed among our current physiological and previously reported models and single-species allometry using humanized-liver mice. Incorporation of the actual unbound plasma fractions of compounds and correction of residual mice hepatocyte in humanized-liver mice showed comparable prediction accuracy to that by single-species allometry. After exclusion of 3 compounds with large species differences in values of clearance and unbound plasma fractions between mice and humans out of 15 compounds, prediction accuracies were improved in the methods investigated. The previously and present reported physiological methods could show the good prediction accuracy of values for clearance of drugs from plasma.

Peculiarities of Pharmacokinetics and Bioavailability of Some Cardiovascular Drugs under Conditions of Antiorthostatic Hypokinesia.

We studied pharmacokinetics and bioavailability of verapamil, propranolol, and ethacizine in healthy volunteers after single oral administration under normal conditions and on the second day of simulated antiorthostatic hypokinesia modeling some effects of microgravity. Under conditions of antiorthostatic hypokinesia, a tendency to a decrease in half-elimination period, mean retention time, and volume of distribution and an increase in the rate of absorption, ratio of maximum concentrations, and relative rate of absorption of verapamil and propranolol were revealed. For ethacizine, a statistically significant increase in the time of attaining maximum concentration and volume of distribution and a decrease in the maximum concentration, rate of absorption, ratio of maximum concentrations, and relative rate of absorption under conditions of antiorthostatic hypokinesia were found.

Influence of verapamil on the pharmacokinetics of oridonin in rats.

Context: Oridonin has been traditionally used in Chinese treatment of various cancers, but its poor bioavailability limits its therapeutic uses. Verapamil can enhance the absorption of some drugs with poor oral bioavailability. Whether verapamil can enhance the bioavailability of oridonin is still unclear.Objective: This study investigated the effect of verapamil on the pharmacokinetics of oridonin in rats and clarified its main mechanism.Materials and methods: The pharmacokinetic profiles of oral administration of oridonin (20 mg/kg) in Sprague-Dawley rats with two groups of six animals each, with or without pre-treatment of verapamil (10 mg/kg/day for 7 days) were investigated. The effects of verapamil on the transport and metabolic stability of oridonin were also investigated using Caco-2 cell transwell model and rat liver microsomes.Results: The results showed that verapamil could significantly increase the peak plasma concentration (from 146.9 ± 10.17 to 193.97 ± 10.53 ng/mL), and decrease the oral clearance (from 14.69 ± 4.42 to 8.09 ± 3.03 L/h/kg) of oridonin. The Caco-2 cell transwell experiments indicated that verapamil could decrease the efflux ratio of oridonin from 1.67 to 1.15, and the intrinsic clearance rate of oridonin was decreased by the pre-treatment with verapamil (40.06 ± 2.5 vs. 36.09 ± 3.7 µL/min/mg protein).Discussion and conclusions: These results indicated that verapamil could significantly change the pharmacokinetic profile of oridonin in rats, and it might exert these effects through increasing the absorption of oridonin by inhibiting the activity of P-gp, or through inhibiting the metabolism of oridonin in rat liver. In addition, the potential drug-drug interaction should be given special attention when verapamil is used with oridonin. Also, the dose of oridonin should be carefully selected in the clinic.

The Effects of Special Patient Population Plasma on Pharmacokinetic Quantifications Using LC-MS/MS.

BACKGROUND: Clinical development of lesinurad, a selective uric acid reabsorption inhibitor, required analysis of lesinurad in plasma from special patient populations. METHODS: EMA and FDA bioanalytical method validation guidance have recommended studying matrix effects on quantitation if samples from special patient populations are to be analyzed. In addition to lesinurad (plasma protein binding 98.2%), the matrix effects from special population plasma on the quantitation of verapamil (PPB 89.6%), allopurinol and oxypurinol (PPB negligible) were also investigated. RESULTS: The plasma from special population patients had no matrix effects on the three quantification methods with stable isotope labeled internal standard, protein precipitation extraction, and LC-MS/MS detection. The validated lesinurad plasma quantification method was successfully applied for the pharmacokinetic evaluations to support the clinical studies in renal impaired patients. CONCLUSION: Special population plasma did not affect quantitation of drugs with a wide range of plasma protein binding levels in human plasma. With the confirmation that there is no impact on quantification from the matrix, the bioanalytical method can be used to support the pharmacokinetic evaluations for clinical studies in special populations.

High-throughput quantification of ten antiarrhythmic drugs in human plasma using UPLC-MS/MS.

In this paper we present an FDA validated method to analyze ten antiarrhythmic drugs (atenolol, bisoprolol, carvedilol, diltiazem, flecainide, lidocaine, metoprolol, propranolol, sotalol and verapamil). A simple and fast sample preparation protocol with protein precipitation followed by ultra performance liquid chromatography (UPLC) for chromatographic separation and mass spectrometric detection applying electrospray ionization (ESI+) and selected reaction monitoring mode (MS/MS) was used. Only 50 µl plasma sample is needed for the simultaneous quantification of all compounds within a 5 min run-to-run analysis time. Sotalol-D6, carvedilol-D5 and verapamil-D6 were used as internal standards. The method was validated according to the FDA guidelines. Correlation coefficients were higher than 0.998 for all compounds. Intra- and interday accuracies were within 15 CV(%) for all analytes. The method is currently successfully applied for routine analysis in our hospital.

Telemetered common marmosets is useful for the assessment of electrocardiogram parameters changes induced by multiple cardiac ion channel inhibitors.

The objective of this study is to assess the response of telemetered common marmosets to multiple cardiac ion channel inhibitors and to clarify the usefulness of this animal model in evaluating the effects of drug candidates on electrocardiogram (ECG). Six multiple cardiac ion channel inhibitors (sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine) were orally administered to telemetered common marmosets and changes in QTc, PR interval and QRS duration were evaluated. Drugs plasma levels were determined to compare the sensitivity in common marmosets to that in humans. QTc prolongation was observed in the marmosets dosed with sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine. PR prolongation was noted after flecainide and verapamil administration, and QRS widening occurred following treatment with flecainide and quinidine. Drugs plasma levels associated with ECG changes in marmosets were similar to those in humans, except for verapamil-induced QTc prolongation. Verapamil-induced change is suggested due to body temperature decrease. These results indicate that telemetered common marmoset is a useful animal for evaluation of the ECG effects of multiple cardiac ion channel inhibitors and the influence of body temperature change should be considered in the assessment.

Quantitative Analysis of Tozadenant Using Liquid Chromatography-Mass Spectrometric Method in Rat Plasma and Its Human Pharmacokinetics Prediction Using Physiologically Based Pharmacokinetic Modeling.

Tozadenant is one of the selective adenosine A2a receptor antagonists with a potential to be a new Parkinson's disease (PD) therapeutic drug. In this study, a liquid chromatography-mass spectrometry based bioanalytical method was qualified and applied for the quantitative analysis of tozadenant in rat plasma. A good calibration curve was observed in the range from 1.01 to 2200 ng/mL for tozadenant using a quadratic regression. In vitro and preclinical in vivo pharmacokinetic (PK) properties of tozadenant were studied through the developed bioanalytical methods, and human PK profiles were predicted using physiologically based pharmacokinetic (PBPK) modeling based on these values. The PBPK model was initially optimized using in vitro and in vivo PK data obtained by intravenous administration at a dose of 1 mg/kg in rats. Other in vivo PK data in rats were used to validate the PBPK model. The human PK of tozadenant after oral administration at a dose of 240 mg was simulated by using an optimized and validated PBPK model. The predicted human PK parameters and profiles were similar to the observed clinical data. As a result, optimized PBPK model could reasonably predict the PK in human.

Impaired Rivaroxaban Clearance in Mild Renal Insufficiency With Verapamil Coadministration: Potential Implications for Bleeding Risk and Dose Selection.

Pharmacokinetics and antithrombotic effects of the Factor Xa inhibitor rivaroxaban were studied in subjects with mild renal insufficiency concurrently taking the P-glycoprotein and moderate CYP3A inhibitor verapamil, a drug commonly administered to patients with hypertension, ischemic heart disease, or atrial fibrillation. Age-matched controls with normal renal function were studied concurrently. Subjects' overall mean age was 59 years. Mean creatinine clearance values in the 2 groups were 105 and 71 mL/min. After single 20-mg oral doses, rivaroxaban area under the curve (AUC) was increased by a factor of 1.11 (ratio of geometric means [RGM]) in mild renal insufficiency compared to controls. Verapamil coadministration independently increased AUC to the same extent in both the mild renal insufficiency and control groups (RGM, 1.39 and 1.43). Concurrent mild renal insufficiency and verapamil produced additive inhibition compared to controls without verapamil (RGM, 1.58). Prothrombin time (PT) prolongation and Factor Xa inhibition tracked plasma rivaroxaban, and were enhanced by verapamil. Concentration-response relationships for PT (linear) and Factor Xa inhibition (hyperbolic) were unaffected by renal function or verapamil. The absolute and relative increases in rivaroxaban AUC caused by verapamil in mild renal insufficiency subjects are potentially associated with an increased bleeding risk. Modification of recommended dosage may be required in this combination of circumstances to reduce risk to patients.

Dispersive micro-solid-phase extraction using carbon-based adsorbents for the sensitive determination of verapamil in plasma samples coupled with capillary electrophoresis.

A dispersive micro-solid-phase extraction procedure coupled with capillary electrophoresis ultraviolet detection was developed for determination of verapamil in plasma samples. Graphene oxide/polydopamin was synthesized by a one-step polymerization method, and graphene oxide/Fe3 O4 (magnetic graphene oxide) nanocomposite was prepared by coprecipitation method. Moreover, they were fully characterized. The use of hazardous and water-immiscible solvents was scaled down, and only 500 µL of acetone was required as the desorption solvent. The detector response concentration plots were linear in the range of 5-500 ng/mL, and the proposed method was validated according to guidelines. The precision and accuracy were less than 15%. Dispersive micro-solid-phase extraction method provides a rapid, environmentally friendly, and sensitive analysis for the verapamil in patient plasma samples, which is adequate for therapeutic drug monitoring and pharmacokinetic studies.

Development and Application of Zirconia Coated Paper Substrate for High Sensitivity Analysis of Therapeutic Drugs in Dried Blood Spots.

Paper spray mass spectrometry has been demonstrated to be promising for direct analysis of therapeutic drugs in dried blood spots (DBS); however, the strong hydrogen bond and van de Waals interactions between paper substrate and analytes containing polar functional groups (e.g., therapeutic drugs) affect greatly the elution behavior and analysis sensitivity of compounds of interest during paper spray. Herein, we developed a one-sided ZrO2 coated paper substrate through a facile vacuum filtration approach using commercial ZrO2 particles as coating material and soluble starch as adhesive agent. Owing to the unique surface properties, as-prepared ZrO2 paper substrate has been shown to have excellent performance for analysis of therapeutic drugs in DBS during paper spray mass spectrometry. In contrast to original cellulose paper substrates, improvements of 43-189-fold in lower limit of quantitation (LLOQ) were obtained for the tested drugs using ZrO2 coated paper for paper spray. In comparing with the previously reported grade SG81 paper and one-sided silica coated paper, the LLOQs of the tested drugs with as-prepared ZrO2 paper decreased 1.5-16.5-fold relative to those from the above two, revealing that ZrO2 coated paper is a good candidate for paper spray in high sensitivity analysis of therapeutic drugs in DBS.

Comparison of Measured and Predicted Bioconcentration Estimates of Pharmaceuticals in Fish Plasma and Prediction of Chronic Risk.

Evaluation of the environmental risk of human pharmaceuticals is now a mandatory component in all new drug applications submitted for approval in EU. With >3000 drugs currently in use, it is not feasible to test each active ingredient, so prioritization is key. A recent review has listed nine prioritization approaches including the fish plasma model (FPM). The present paper focuses on comparison of measured and predicted fish plasma bioconcentration factors (BCFs) of four common over-the-counter/prescribed pharmaceuticals: norethindrone (NET), ibuprofen (IBU), verapamil (VER) and clozapine (CLZ). The measured data were obtained from the earlier published fish BCF studies. The measured BCF estimates of NET, IBU, VER and CLZ were 13.4, 1.4, 0.7 and 31.2, while the corresponding predicted BCFs (based log Kow at pH 7) were 19, 1.0, 7.6 and 30, respectively. These results indicate that the predicted BCFs matched well the measured values. The BCF estimates were used to calculate the human: fish plasma concentration ratios of each drug to predict potential risk to fish. The plasma ratio results show the following order of risk potential for fish: NET > CLZ > VER > IBU. The FPM has value in prioritizing pharmaceutical products for ecotoxicological assessments.

In Vitro Studies Indicate Intravenous Lipid Emulsion Acts as Lipid Sink in Verapamil Poisoning.

Intravenous lipid emulsion (ILE), a component of parenteral nutrition, consists of a fat emulsion of soy bean oil, egg phospholipids, and glycerin. Case reports suggest that ILE may reverse hypotension caused by acute poisoning with lipophilic drugs such as verapamil, but the mechanism remains unclear. The methods used are the following: (1) measurement of ILE concentration in serum samples from a patient with verapamil poisoning treated with ILE, (2) measurement of free verapamil concentrations in human serum mixed in vitro with increasing concentrations of ILE, and (3) measurement of murine ventricular cardiomyocyte L-type Ca(2+) currents, intracellular Ca(2+), and contractility in response to verapamil and/or ILE. Maximum patient serum ILE concentration after infusion of 1 L ILE over 1 h was approximately 1.6 vol%. In vitro GC/MS verapamil assays showed that addition of ILE (0.03-5.0 vol%) dose-dependently decreased the free verapamil concentration in human serum. In voltage-clamped myocytes, adding ILE to Tyrode's solution containing 5 µM verapamil recovered L-type Ca(2+) currents (ICa). Recovery was concentration dependent, with significant ICa recovery at ILE concentrations as low as 0.03 vol%. ILE had no effect on ICa in the absence of verapamil. In field-stimulated intact ventricular myocytes exposed to verapamil, adding ILE (0.5 %) resulted in a rapid and nearly complete recovery of myocyte contractility and intracellular Ca(2+). Our in vitro studies indicate that ILE acts as a lipid sink that rapidly reverses impaired cardiomyocyte contractility in the continued presence of verapamil.

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.

PBPK modeling to unravel nonlinear pharmacokinetics of verapamil to estimate the fractional clearance for verapamil N-demethylation in the recirculating rat liver preparation.

We applied physiologically based pharmacokinetic (PBPK) modeling to study the dose-dependent metabolism and excretion of verapamil and its preformed metabolite, norverapamil, to unravel the kinetics of norverapamil formation via N-demethylation. Various initial verapamil (1, 50, and 100 µM) and preformed norverapamil (1.5 and 5 µM) concentrations, perfused at 12 ml/min, were investigated in the perfused rat liver preparation. Perfusate and bile were collected over 90 minutes, and livers were harvested at the end of perfusion for high-performance liquid chromatography analysis. After correction for the adsorption of 10%-25% dose verapamil and norverapamil onto Tygon tubing and binding to albumin and red blood cell, fitting of verapamil and formed and preformed norverapamil data with ADAPT5 revealed nonlinearity for protein binding, N-demethylation (V(max,met1)(VER --> NOR) = 96.6 ± 33.4 nmol/min; K(m,met1)(VER --> NOR) = 10.4 ± 4.1 µM), formation of other metabolites (V(max,met2(VER -->others) 288 ± 51 nmol/min; K(m.met2)(VER -->others )= 14.1 ± 4.9 µM), as well as biliary excretion (V(max,sec)(VER)= 0.911 ± 0.505 nmol/min; K(m,sec)(VER) = 4.75 ± 2.29 µM). The hepatic clearance of verapamil (CL(L)(VER) decreased with the dose (8.16-10.2 ml/min), with values remaining high relative to perfusate blood flow rate among the doses. The hepatic clearance of preformed norverapamil (11 ml/min) remained unchanged for the concentrations studied and approximated perfusate blood flow rate, suggesting a high norverapamil extraction ratio. The fractional formation of norverapamil and biliary excretion of verapamil based on fitted constants were 31.1% and 0.64% of CL(L)(VER), respectively. Enantiomeric disposition and auto-inhibition of verapamil failed to perturb these estimaties according to PBPK modeling, due to the low values of the Michaelis-Menten constant, Km, and inhibition parameter, kI.