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Experimental 3D structures of calcium channels with phenylalkylamines (PAAs) provide basis for further analysis of atomic mechanisms of these important cardiovascular drugs. In the crystal structure of the engineered calcium channel CavAb with Br-verapamil and in the cryo-EM structure of the Cav1.1 channel with verapamil, the ligands bind in the inner pore. However, there are significant differences between these structures. In the crystal structure the ligand ammonium group is much closer to the ion in the selectivity-filter region Site 3, which is most proximal to the inner pore, than in the cryo-EM structure. Here we used Monte Carlo energy minimizations to dock PAAs in calcium channels. Our computations suggest that in the crystal structure Site 3 is occupied by a water molecule rather than by a calcium ion. Analysis of the published electron density map does not rule out this possibility. In the cryo-EM structures the ammonium group of verapamil is shifted from the calcium ion in Site 3 either along the pore axis, towards the cytoplasm or away from the axis. Our unbiased docking reproduced these binding modes. However, in the cryo-EM structures detergent and lipid molecules interact with verapamil. When we removed these molecules, the nitrile group of verapamil bound to the calcium ion in Site 3. Models of Cav1.2 with different PAAs suggest similar binding modes and direct contacts of the ligands electronegative atoms with the calcium ion in Site 3. Such interactions explain paradoxes in structure-activity relationships of PAAs.

P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The role of N-methyl-D-aspartate receptor (NMDA-R) in heart is still unclear. For these ionotropic glutamate receptors is characteristic the necessity of both co-agonists, glutamate and glycine, for their activation, which primarily allows influx of calcium. The aim of the present study was to examine the effects of verapamil, as a calcium channel blocker, alone and its combination with glycine and/or glutamate on cardiac function, coronary flow, and oxidative stress in isolated rat heart or to examine the effects of potential activation of NMDA-R in isolated rat heart. The hearts of male Wistar albino rats were excised and perfused according to Langendorff technique, and cardiodynamic parameters and coronary flow were determined during the administration of verapamil and its combinations with glutamate and/or glycine. The oxidative stress biomarkers, including thiobarbituric acid-reactive substances, nitrites, superoxide anion radical, and hydrogen peroxide, were each determined spectrophotometrically from coronary venous effluent. The greatest decline in parameters of cardiac contractility and systolic pressure was in the group that was treated with verapamil only, while minimal changes were observed in group treated with all three tested substances. Also, the largest changes in coronary flow were in the group treated only with verapamil, and at least in the group that received all three tested substances, as well as the largest increase in oxidative stress parameters. Based on the obtained results, it can be concluded that NMDA-R activation allows sufficient influx of calcium to increase myocardial contractility and systolic pressure, as well as short-term increase of oxidative stress.

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

This study was performed to optimize the formulation of polymer-lipid hybrid nanoparticles (PLN) for the delivery of an ionic water-soluble drug, verapamil hydrochloride (VRP) and to investigate the roles of formulation factors. Modeling and optimization were conducted based on a spherical central composite design. Three formulation factors, i.e., weight ratio of drug to lipid (X1), and concentrations of Tween 80 (X2) and Pluronic F68 (X3), were chosen as independent variables. Drug loading efficiency (Y1) and mean particle size (Y2) of PLN were selected as dependent variables. The predictive performance of artificial neural networks (ANN) and the response surface methodology (RSM) were compared. As ANN was found to exhibit better recognition and generalization capability over RSM, multi-objective optimization of PLN was then conducted based upon the validated ANN models and continuous genetic algorithms (GA). The optimal PLN possess a high drug loading efficiency (92.4%, w/w) and a small mean particle size (∼100nm). The predicted response variables matched well with the observed results. The three formulation factors exhibited different effects on the properties of PLN. ANN in coordination with continuous GA represent an effective and efficient approach to optimize the PLN formulation of VRP with desired properties., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Verapamil and naproxen Parallel Artificial Membrane Permeability Assay (PAMPA) permeability was studied using lipids not yet reported for this model in order to facilitate the quantification of drug permeability. These lipids are 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an equimolar mixture of DMPC/DSPC, both in the absence and in the presence of 33.3 mol% of cholesterol. PAMPA drug permeability using the lipids mentioned above was compared with lecithin-PC. The results show that verapamil permeability depends on the kind of lipid used, in the order DMPC > DMPC/DSPC > DSPC. The permeability of the drugs was between 1.3 and 3.5-times larger than those obtained in lecithin-PC for all the concentrations of the drug used. Naproxen shows similar permeability than verapamil; however, the permeability increased with respect to lecithin-PC only when DMPC and DMPC/DSPC were used. This behavior could be explained by a difference between the drug net charge at pH 7.4. On the other hand, in the presence of cholesterol, verapamil permeability increases in all lipid systems; however, the relative verapamil permeability respect to lecithin-PC did not show any significant increase. This result is likely due to the promoting effect of cholesterol, which is not able to compensate for the large increase in verapamil permeability observed in lecithin-PC. With respect to naproxen, its permeability value and relative permeability respect lecithin-PC not always increased in the presence of cholesterol. This result is probably attributed to the negative charge of naproxen rather than its molecular weight. The lipid systems studied have an advantage in drug permeability quantification, which is mainly related to the charge of the molecule and not to its molecular weight or to cholesterol used as an absorption promoter.

Verapamil and its major metabolite norverapamil were identified to be both mechanism-based inhibitors and substrates of CYP3A and reported to have non-linear pharmacokinetics in clinic. Metabolic clearances of verapamil and norverapmil as well as their effects on CYP3A activity were firstly measured in pooled human liver microsomes. The results showed that S-isomers were more preferential to be metabolized than R-isomers for both verapamil and norverapamil, and their inhibitory effects on CYP3A activity were also stereoselective with S-isomers more potent than R-isomers. A semi-physiologically based pharmacokinetic model (semi-PBPK) characterizing mechanism-based auto-inhibition was developed to predict the stereoselective pharmacokinetic profiles of verapamil and norverapamil following single or multiple oral doses. Good simulation was obtained, which indicated that the developed semi-PBPK model can simultaneously predict pharmacokinetic profiles of S-verapamil, R-verapamil, S-norverapamil and R-norverapamil. Contributions of auto-inhibition to verapamil and norverapamil accumulation were also investigated following the 38th oral dose of verapamil sustained-release tablet (240mg once daily). The predicted accumulation ratio was about 1.3-1.5 fold, which was close to the observed data of 1.4-2.1-fold. Finally, the developed semi-PBPK model was further applied to predict drug-drug interactions (DDI) between verapamil and other three CYP3A substrates including midazolam, simvastatin, and cyclosporine A. Successful prediction was also obtained, which indicated that the developed semi-PBPK model incorporating auto-inhibition also showed great advantage on DDI prediction with CYP3A substrates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Accumulating evidence suggests that L-type calcium channel blockers (CCBs) attenuate the expression of opioid withdrawal and the dihydropyridine L-type CCB isradipine has been shown to block the behavioral effects of naloxone in opioid-maintained humans. This study determined whether two prototypic L-type CCBs with differing chemical structures, the benzothiazepine diltiazem and the phenylalkamine verapamil, attenuate the behavioral effects of naloxone in methadone-maintained humans trained to distinguish between low-dose naloxone (0.15 mg/70 kg, i.m.) and placebo under an instructed novel-response drug discrimination procedure. Once discrimination was acquired, diltiazem (0, 30, 60, 120 mg) and verapamil (0, 30, 60, 120 mg), alone and combined with the training dose of naloxone, were tested. Diltiazem alone produced 33-50% naloxone- and novel-appropriate responding at 30 and 60 mg and essentially placebo-appropriate responding at 120 mg. Verapamil alone produced 20-40% naloxone- and 0% novel-appropriate responding. Diltiazem at 60 mg decreased several ratings associated with positive mood and increased VAS ratings of "Bad Drug Effects" relative to placebo, whereas verapamil increased ratings associated with euphoria. When administered with naloxone, diltiazem produced 94-100% naloxone-appropriate-responding with 6% novel-appropriate responding at 60 mg (n=3). When administered with naloxone, verapamil produced 60-80% naloxone- and 0% novel-appropriate responding (n=5). Diltiazem decreased diastolic blood pressure and heart rate whereas verapamil decreased ratings of arousal relative to placebo. These results suggest that CCBs with different chemical structures can be differentiated behaviorally, and that diltiazem and verapamil do not attenuate the discriminative stimulus effects of naloxone in humans at the doses tested., (© 2013 Elsevier B.V. All rights reserved.)

The present study was undertaken to investigate the effect of hydrophilic, plastic and hydrophobic types of polymers and their content level on the release profile of drug from matrix systems. To improve therapeutic efficacy, systemic absorption and patient compliance a sustained release matrix tablets of Verapamil HCl (VHE) has been developed. VHE tablets were prepared by using various polymers like hydrophilic (HPMC K15M CR), plastic (Kollidon SR), hydrophobic (Eudragit RSPO) and combination of best two resulted polymers using direct compression. A 32 full factorial design was applied to study the effect of polymers on drug release. For the combination of polymers, selected factors HPMC K15 CR (X(1)) and Eudragit RSPO (X(2)) showed positive influence on drug release at 18 hrs and 20 hrs. The release profile of VHE formulation exhibits Higuchi model with anomalous diffusion release. Accelerated stability trials for 3 months proved reproducibility. A good correlation between the dissolution profiles and bioavailability indicated a linear relationship between in vitro - in vivo data. The current study attained the successful design, development and optimization of controlled release once-a-day formulation of VHE.

The present study aimed to produce verapamil hydrochloride-loaded solid lipid microparticles (SLM) by the w/o/w emulsion solvent evaporation technique, using diethyl ether as solvent phase, glyceryl monostearate as biodegradable polymer and Span 60 as surfactant. SLM of spherical shape were prepared by simple dilution of the emulsion with water. To increase the lipid load the process was conducted at 50 degrees C, and in order to reach sub-micron size, a high-shear homogenizer was used. The encapsulation efficiency of prepared SLM reached 74.29 +/- 0.76%. Particle size (98.55 +/- 1.42 microm), surface morphology (spherical) and drug loading efficiency (18.57 +/- 1.25% w/w) were investigated. And optimization of drug polymer ratio (3:1), nature and concentration of emulsion stabilizer in the external aqueous (0.1%), phase viscosity of external aqueous phase (0.5%), volume of external aqueous phase and stirring rate (1000 rpm) were detected. Analysis of microsphere content after processing showed that verapamil did not undergo any chemical modification within the micro-particles. The in-vitro release of verapamil from the microparticles was very low and an initial burst effect of 17% of the dose was observed. The slow release may help to avoid a high frequency of administration. The prepared solid lipid microparticles appear to have interesting perspectives as delivery systems for the oral administration of verapamil hydrochloride with improved half-life, improved bioavailability, and minimized local and systemic gastrointestinal disturbances of the drug.

Positron annihilation lifetime spectroscopy (PALS), a method well established for the study of polymers, is employed to characterize the temperature dependence of the free volume through T(g) in the amorphous pharmaceutical Verapamil hydrochloride. From the PALS spectra analyzed with the routine LifeTime9.0 the size (volume) distribution of local free volumes (subnanometre-size holes), its mean, v(h), and mean dispersion, sigma(h), were calculated. A comparison with the macroscopic volume from PVT-experiments delivered the hole density and the hole free volume fraction and in that way a complete characterization of the free volume microstructure. These data are used in correlation with structural (alpha-) relaxation data from broad-band dielectric spectroscopy in terms of the Cohen-Turnbull free volume model. An extension of this model, distinctions in the free volume behaviour of the glassy and supercooled-liquid state and different ways of extrapolating the equilibrium part of the free volume into the temperature range of the glass are discussed. The potential of the PALS method for the study of pharmaceuticals is briefly reviewed and some recently developed applications (analysis of density fluctuations) are illuminated.

Thermogravimetry (TG) and differential scanning calorimetry (DSC) are useful techniques that have been successfully applied in the pharmaceutical industry to reveal important information regarding the physicochemical properties of drug and excipient molecules such as polymorphism, stability, purity, formulation compatibility among others. Verapamil hydrochloride shows thermal stability up to 180 degrees C and melts at 146 degrees C, followed by total degradation. The drug is compatible with all the excipients evaluated. The drug showed degradation when subjected to oxidizing conditions, suggesting that the degradation product is 3,4-dimethoxybenzoic acid derived from alkyl side chain oxidation. Verapamil hydrochloride does not present the phenomenon of polymorphism under the conditions evaluated. Assessing the drug degradation kinetics, the drug had a shelf life (t90) of 56.7 years and a pharmaceutical formulation showed t90 of 6.8 years showing their high stability.

Verapamil hydrochloride (VH) is a very popular calcium channel blocker. Solubility of its crystalline form in the blood reaches only 10-20%. Thus, it seems to be very important to improve its bioavailability. In this article, we show that the preparation of the amorphous form of VH enhance its dissolution rate. In addition we performed dielectric measurements to describe molecular dynamics of this active pharmaceutical ingredient (API). Since examined sample is typical ionically conducting material, to gain information about structural relaxation we employed the dielectric modulus formalism. The temperature dependence of the structural relaxation time can be described over the entire measured range by a single Vogel-Fulcher-Tamman (VFT) equation. From the VFT fits the glass transition temperature was estimated as T(g) = 320.1 K. Below glass transition temperature one clearly visible secondary relaxation, with activation energy E(a) = 37.8 kJ/mol, was reported. Deviations of experimental data from KWW fits on high-frequency flank of alpha-peak indicate the presence of an excess wing in tested sample. Based on Kia Ngai's coupling model we identified the excess wing as true Johari-Goldstein process., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association.)

The potential effect of ethanol to accelerate drug release from sustained release (SR) oral formulations is a general concern. Marketed Verapamil is a calcium channel blocker, mainly used as antihypertensive and anti-anginal drug and available in various dose and dosage forms. One is Verapamil Meltrex, combining an innovative and unique SR formulation and technology that achieves a stable solid dispersion of drug by using melt extrusion technology. The aim of this investigation was to determine the influence of ethanol on the in vitro rate of release of marketed Verapamil (240 mg) Meltrex, in contrast to three compressed marketed Verapamil (240 mg) SR formulations. Dissolution was tested under standardized conditions, with mediums containing ethanol concentrations of 0, 5, 20, and 40%. The dissolution profiles for Verapamil Meltrex showed no differences between 5 and 40% ethanol versus 0% ethanol (P>0.05). The mean dissolution percentage (%) was identical at 1h (19%) in 0% versus 40% ethanol. In contrast, the three comparators showed significant increases in dissolution in 20 and 40% ethanol versus 0% ethanol (P<0.001). An initial rapid release (within 2h) was observed in 20 and 40% ethanol, with a mean dissolution of 99% (range 73-107%). Therefore, unlike the three SR Verapamil formulations tested, Verapamil Meltrex was found to be resistant to in vitro dose dumping when combined with readily accessible ethanol concentrations.

A novel gastro retentive controlled release drug delivery system of verapamil HCl was formulated in an effort to increase the gastric retention time of the dosage form and to control drug release. Hydroxypropylmethylcellulose (HPMC), carbopol, and xanthan gum were incorporated for gel-forming properties. Buoyancy was achieved by adding an effervescent mixture of sodium bicarbonate and anhydrous citric acid. In vitro drug release studies were performed, and drug release kinetics was evaluated using the linear regression method. The optimized intragastric floating tablet composed of 3:2 of HPMC K4M to xanthan gum exhibited 95.39% drug release in 24 h in vitro, while the buoyancy lag time was 36.2 s, and the intragastric floating tablet remained buoyant for >24 h. Zero-order and non-Fickian release transport was confirmed as the drug release mechanism from the optimized formulation (F7). X-ray studies showed that total buoyancy time was able to delay the gastric emptying of verapamil HCl intragastric floating tablet in mongrel dogs for more than 4 h. Optimized intragastric floating tablet showed no significant change in physical appearance, drug content, total buoyancy time, or in vitro dissolution pattern after storage at 40 degrees C/75% relative humidity for 3 months.

Excitation emission fluorescence matrices (EEMs) of Verapamil drug were obtained by direct and by derivatization fluorescence spectroscopy. The fluorescence excitation and emission wavelengths were displaced to longer wavelengths and the fluorescence intensity was enhanced upon derivation with respect to the native fluorescence of the drug. The complete EEM of the native fluorescence of the drug and of the derivatization product were rapidly acquired by using a charged-coupled device detector (CCD), which is advantageous in terms of speed in the analysis, with respect to the use of a conventional photomultiplier detector. The EEMs were analyzed by several second-order multivariate calibration methods exploiting the second order advantage. The three-dimensional decomposition methods used, based in different assumptions about the trilinearity of the three way data structure under analysis, were parallel factor analysis (PARAFAC), bilinear least squares (BLLS), parallel factor analysis 2 (PARAFAC2) and multivariate curve resolution-alternating least squares (MCR-ALS). The determination was performed by using the standard addition approach. The figures of merit of the PARAFAC and BLLS methods were calculated, obtaining a lower limit of detection with the derivatization procedure, when compared with the direct measurement of the fluorescence of the drug. In Verapamil drug the best estimations were found with the BLLS and the MCR-ALS models. In the quantification of Verapamil in a pharmaceutical formulation the best estimation, when compared with the result obtained by the US Pharmacopeia high performance liquid chromatography approach, was obtained by direct fluorescence spectroscopy with MCR-ALS and by derivatization fluorescence spectroscopy with the PARAFAC2 model.

Unlabelled: The multidrug efflux transporter P-glycoprotein (P-gp) is expressed in high concentrations at the blood-brain barrier (BBB) and is believed to be implicated in resistance to central nervous system drugs. We used small-animal PET and (R)-11C-verapamil together with tariquidar, a new-generation P-gp modulator, to study the functional activity of P-gp at the BBB of rats. To enable a comparison with human PET data, we performed kinetic modeling to estimate the rate constants of radiotracer transport across the rat BBB., Methods: A group of 7 Wistar Unilever rats underwent paired (R)-11C-verapamil PET scans at an interval of 3 h: 1 baseline scan and 1 scan after intravenous injection of tariquidar (15 mg/kg, n = 5) or vehicle (n = 2)., Results: After tariquidar administration, the distribution volume (DV) of (R)-11C-verapamil was 12-fold higher than baseline (3.68 +/- 0.81 vs. 0.30 +/- 0.08; P = 0.0007, paired t test), whereas the DVs were essentially the same when only vehicle was administered. The increase in DV could be attributed mainly to an increased influx rate constant (K1) of (R)-11C-verapamil into the brain, which was about 8-fold higher after tariquidar. A dose-response assessment with tariquidar provided an estimated half-maximum effect dose of 8.4 +/- 9.5 mg/kg., Conclusion: Our data demonstrate that (R)-11C-verapamil PET combined with tariquidar administration is a promising approach to measure P-gp function at the BBB.

In recent years pharmaceuticals and personal care products have been detected in increasing concentrations in hospital effluents, sewage treatment plants (STP) as well as in different environmental compartments such as surface water, groundwater and soil. Little is known about the elimination of these substances during sewage treatment or about the formation of potential metabolites in the environment caused by bacterial biotransformation. To assess the biodegradability of the popular cardiovascular drug verapamil and the possible formation of potential microbial degradation products, two tests from the OECD series were used in the present study: the widely used Closed Bottle test (OECD 301 D) and the modified Zahn-Wellens test (OECD 302 B). In the Closed Bottle test, a screening test that simulates the conditions of an environmental surface water compartment, no biological degradation was observed for verapamil at concentrations of 2.33mgl(-1). In the Zahn-Wellens test, a test for inherent biodegradability which allows evaluation of aerobic degradation at high bacterial density, only a partial biological degradation was found. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass spectrometry (HPLC-MSn) revealed 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methylamino)pentane nitrile, already known as D617 (Knoll nomenclature), a metabolite of mammalian metabolism, which is the major degradation product and dead-end transformation product of aerobic degradation of verapamil.

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.

To evaluate the effects of dietary salt on the stereoselective disposition of verapamil enantiomers in relation to the transporter ABCB1 2677GG/3435CC and 2677TT/3435TT haplotypes, ten healthy subjects were asked to take diets of three different salt levels for 7 days in a randomized, three-way crossover manner. The plasma concentrations of verapamil and norverapamil enantiomers were determined after a single oral dose of 240 mg verapamil on the last day of each phase. Pharmacokinetic parameters were calculated by non-compartmental analysis techniques and compared among the three different dietary salt phases. Compared with the medium salt diet, the high and low salt diets had no significant effect on the disposition of verapamil enantiomers. Moreover, the ABCB1 haplotypes did not alter the impact of dietary salt, although ABCB1 2677TT/3435TT subjects had slightly, but not significantly, higher C(max) and area under the curve (AUC) and lower T(max) for the verapamil enantiomers than did 2677GG/3435CC subjects in each salt phase.

Purpose: (R)-[(11)C]verapamil is a new PET tracer for P-glycoprotein-mediated transport at the blood-brain barrier. For kinetic analysis of (R)-[(11)C]verapamil PET data the measurement of a metabolite-corrected arterial input function is required. The aim of this study was to assess peripheral (R)-[(11)C]verapamil metabolism in patients with temporal lobe epilepsy and compare these data with previously reported data from healthy volunteers., Methods: Arterial blood samples were collected from eight patients undergoing (R)-[(11)C]verapamil PET and selected samples were analysed for radiolabelled metabolites of (R)-[(11)C]verapamil by using an assay that measures polar N-demethylation metabolites by solid-phase extraction and lipophilic N-dealkylation metabolites by HPLC., Results: Peripheral metabolism of (R)-[(11)C]verapamil was significantly faster in patients compared to healthy volunteers (AUC of (R)-[(11)C]verapamil fraction in plasma: 29.4 +/- 3.9 min for patients versus 40.8 +/- 5.0 min for healthy volunteers; p < 0.0005, Student's t-test), which resulted in lower (R)-[(11)C]verapamil plasma concentrations (AUC of (R)-[(11)C]verapamil concentration, normalised to injected dose per body weight: 25.5 +/- 2.1 min for patients and 30.5 +/- 5.9 min for healthy volunteers; p = 0.038). Faster metabolism appeared to be mainly due to increased N-demethylation as the polar [(11)C]metabolite fraction was up to two-fold greater in patients., Conclusions: Faster metabolism of (R)-[(11)C]verapamil in epilepsy patients may be caused by hepatic cytochrome P450 enzyme induction by antiepileptic drugs. Based on these data caution is warranted when using an averaged arterial input function derived from healthy volunteers for the analysis of patient data. Moreover, our data illustrate how antiepileptic drugs may decrease serum levels of concomitant medication, which may eventually lead to a loss of therapeutic efficacy.

The aim of this study was to synthesize stable copolymer (vinyl acetate-co-3-dimethyl[methacryloyloxyethyl] ammonium propane sulfinate) zwitterionic latex with different compositions for the first time by emulsifier-free emulsion copolymerization. Throughout the course of the study, a proposal was made for the explanation of the relationship between the "overshooting" phenomenon (a swelling kinetics with a maximum) and the specific self-association of the zwitterionic copolymers. The zwitterionic monomer unit mole fraction, pH, and ionic strength effects on this relationship, on the swelling kinetics of the zwitterionic copolymers, and on the sustained verapamil hydrochloride release from the model tablets were established by the study's authors.

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.

Stable co-polymer [vinyl acetate-co-3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate, p(VA-co-DMAPS)] latex of different compositions has been synthesized for the first time by emulsifier-free emulsion copolymerization. The unusual >>overshooting<< behavior of the co-polymer tablets has been explained by the formation of specific clusters from the opposite oriented dipoles-zwitterionic species. The change of their concentration with the DMAPS unit fraction (mDMAPS), pH and ionic strength has been considered responsible for the differences observed in the swelling kinetics. The results obtained prove that mDMAPS and ionic strength could be used to control the swelling degree of the p(VA-co-DMAPS) matrices and their sustained drug delivery. In this way, p(VA-co-DMAPS) matrices could be effectively used to control the sustained release of drugs with basic properties like verapamil hydrochloride from model tablets.

Any clinical intervention (e.g., coronary angioplasty, thrombolysis) used to reintroduce blood flow to an ischemic region of the myocardium is accompanied by a complex enzymatic cascade of reactions resulting in severe injury to the heart, termed myocardial ischemia/reperfusion (I/R) injury. In this study, we evaluated the ability of H-3010 (1-hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid (2-(3,4-dimethoxyphenyl)-5-([2-(3,4-dimethoxyphenyl)ethyl]-methylamino)-2-isopropylpentyl)-amide), a pyrroline modification of verapamil (2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethylmethyl-amino]-2-(1-methylethyl)pentanenitrile), to protect the heart against I/R-mediated injury. Isolated perfused rat hearts pretreated with verapamil and H-3010 were subjected to 30 min of global no-flow ischemia followed by 45 min of reperfusion. The recovery (expressed as a percentage of preischemic baseline) in contractile function (left ventricular developed pressure) of hearts subjected to I/R was significantly higher in hearts treated with H-3010 at 5 microM (51.0 +/- 6.4%) as well as at 50 microM (75.1 +/- 7.4%) as compared with verapamil at 5 microM (32.2 +/- 3.7%) or untreated control hearts (18.1 +/- 2.8%). Creatine kinase release was significantly attenuated in hearts treated with H-3010 (45.7 +/- 4.5 U/liter) as compared with untreated controls (131.5 +/- 6.4 U/liter). Similar trends were also observed for lactate dehydrogenase release as well. A marked reduction in percent area of infarction was observed in the H-3010 group (11.7 +/- 1.6%) compared with verapamil (25.1 +/- 2.9%) and control (41.3 +/- 1.9%) groups. Additional in vitro studies showed a marked decrease in reactive oxygen species generation with H-3010. In conclusion, our data clearly demonstrated that the verapamil derivative, H-3010, significantly decreased I/R-induced cardiac dysfunction. This can be attributed to the combined benefits of the pyrroline moiety (antioxidant) and the parent verapamil component (antiarrhythmic) in the protection of the heart from I/R-induced injury.

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Ca(v)1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Ca(v)1.2 (1). The shifted channel gating of these mutants (by 30-40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Ca(v)1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Ca(v)1.2 is able to accommodate a large cation such as (-)D888.

Purpose: Liposomes co-encapsulating doxorubicin (DOX) and verapamil (VER), and conjugated to transferrin (Tf-L-DOX/VER) were synthesized and evaluated in K562 leukemia cells. The design of this formulation was aimed at selective targeting of tumor cells, reducing cardiotoxicity of DOX and VER, as well as overcoming P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) phenotype., Methods: The liposomes were prepared by polycarbonate membrane extrusion, followed by pH-gradient driven remote loading and Tf conjugation. Kinetics of in vitro release of DOX and VER from liposomes was determined by measuring changes in the concentration of encapsulated drugs. Uptake of Tf-conjugated liposomes by K562 cells was evaluated by fluorescence microscopy and by fluorometry. Cytotoxicities of various formulations of DOX were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay., Results: Efficiencies for liposomal loading of DOX and VER were 95% and 70%, respectively. The mean particle diameter for the liposomes was approximately 110nm. Rates of release for DOX and VER were similar in singly-loaded and co-loaded liposomes. Tf-L-DOX/VER showed efficient uptake by the TfR+ K562 cells. In DOX-resistant K562 cells (K562/DOX), Tf-L-DOX/VER showed 5.2 and 2.8 times greater cytotoxicity (IC50 = 4.18 muM) than non-targeted liposomes (L-DOX/VER) (IC50 = 21.7 muM) and Tf-targeted liposomes loaded with DOX alone (Tf-L-DOX) (IC50 = 11.5 muM), respectively., Conclusions: The combination of TfR targeting and co-encapsulation of DOX and VER was highly effective in overcoming drug resistance in K562 leukemia cells.

Microcrystalline cellulose (MCC) and powdered cellulose (PC) are commonly used excipients for solid dosage forms e.g., pellets. The aim of this study was to compare the utility of the MCC and PC in the floating pellet cores comprising verapamil hydrochloride (VH) manufactured by extrusion and spheronization and influence on their physical properties like swelling, compressibility and VH release. It was found by scanning electron microscopy (SEM) investigation that porosity of surface of the pellets' cores increased with an increase of PC amount in composition. Differential scanning calorimetry (DSC) analysis indicated the lack of physicochemical interaction between PC and MCC either with VH or with any excipients in the pellet core. Formulation having the highest PC participation were characterized by the highest friability and compressibility and addition of MCC corresponded with a decrease of friability and compressibility. The results on pellets friability were not reflected by the results on the hardness test. It means that the PC contents growth contributes to the hardness growth. The swelling forces of physical mixture of powders containing PC and MCC was different and increased with increasing amount of PC in pellet's core. Pellets' cores were coated with Eudragit NE dispersion. It was found that VH release rate from coated pellets with higher amount of PC was considerably slower in comparison to the pellets containing highest MCC participation.

Verapamil and amlodipine are calcium ion influx inhibitors of wide clinical use. They are partially charged at neutral pH and exhibit amphiphilic properties. The noncharged species can easily cross the lipid membrane. We have measured with solid-state NMR the structural changes induced by verapamil upon incorporation into phospholipid bilayers and have compared them with earlier data on amlodipine and nimodipine. Verapamil and amlodipine produce a rotation of the phosphocholine headgroup away from the membrane surface and a disordering of the fatty acid chains. We have determined the thermodynamics of verapamil partitioning into neutral and negatively charged membranes with isothermal titration calorimetry. Verapamil undergoes a pK-shift of DeltapK(a) = 1.2 units in neutral lipid membranes and the percentage of the noncharged species increases from 5% to 45%. Verapamil partitioning is increased for negatively charged membranes and the binding isotherms are strongly affected by the salt concentration. The electrostatic screening can be explained with the Gouy-Chapman theory. Using a functional phosphate assay we have measured the affinity of verapamil, amlodipine, and nimodipine for P-glycoprotein, and have calculated the free energy of drug binding from the aqueous phase to the active center of P-glycoprotein in the lipid phase. By combining the latter results with the lipid partitioning data it was possible, for the first time, to determine the true affinity of the three drugs for the P-glycoprotein active center if the reaction takes place exclusively in the lipid matrix.

The pharmacokinetics of a new verapamil retard tablet formulation have been investigated in a randomized cross-over bioequivalence study on 12 healthy subjects. The drug was given orally at a single new or standard retard tablet dose of 240mg and at a single intravenous dose of 5mg. Plasma verapamil concentrations were determined by HPLC. New retard tablets produced peak plasma verapamil concentrations of 81.34+/-5.69microg/l, time to peak plasma concentrations of 4.91+/-0.89h and an AUC (0-24h) of 1291+/-103.4h x microg/l, with a terminal phase half-life of 55.1+/-14.9h. After intravenous administration verapamil exhibited biphasic elimination kinetics with a terminal plasma half-life of 2.36+/-0.42h and systemic clearance of 34.32+/-5.81 l/h. Bioavailability of the new peroral retard formulation ranged from 19.49+/-4.41% to 67.69+/-11.70%. Absorption rates and amounts were evaluated by means of the spline-convolutional method. Input rates for the new verapamil retard formulation ranged from 0.77+/-0.20mg/h to 5.57+/-1.58mg/h. The cumulative amount of verapamil input was 39.17+/-9.71% for the new retard tablets. All pharmacokinetic parameters for the new verapamil retard tablet formulation, were in reasonable agreement with the data obtained on already registered verapamil retard formulations, indicating their bioequivalence.

A cubic spline function for describing the verapamil concentration profile, resulting from the verapamil absorption input to be evaluated, has been used. With this method, the knots are taken to be the data points, which has the advantage of being computationally less complex. Because of its inherently low algorhythmic errors, the spline method is less distorted and more suitable for further data analysis than others. The method has been evaluated using simulated verapamil delayed release tablet concentration data containing various degrees of random noise. The accuracy of the method was determined by how well the estimates of input rate and extent represented the true values. It was found that the accuracy of the method was of the same order of magnitude as the noise level of the data. Spline functions in convolutional modeling of verapamil formulation bioavailability and bioequivalence, as shown in the numerical simulation investigation, are very powerful additional tools for assessing the quality of new verapamil formulations in order to ensure that they are of the same quality as already registered formulations of the drug. The development of such models provides the possibility to avoid additional or larger bioequivalence and/or clinical trials and to thus help shorten the investigation time and registration period.

A 52-year-old woman with a history of depression and personality disorders, hypertension, coronary disease and asthma was admitted to the Department of Clinical Toxicology after taking 60 tablets of Staveran (immediate release verapamil), and 4 tablets of acetaminophen. One and a half hours after ingestion her condition was critical. She required endotracheal intubation and artificial respiration. Her heart rate was 75 beats per minute, (bpm) and her blood pressure dropped from 70/50 to 50/00 mmHg. Additionally, drowsiness and headache were observed. On admission, R-(+)- and S-(-)-verapamil serum concentrations were 2252 ng/ml and 810 ng/ ml, respectively. Monitoring of the verapamil serum concentration was carried out over 74 h. Terminal elimination half-lives were 18.7 (21.3) and 17.0 (18.5) hours, respectively, for R-(+)- and S-(-)-verapamil. Monitoring of verapamil enantiomers concentrations in serum indicated a higher concentration of the less active form and slightly faster elimination of the more active enantiomer. The data-support a stereoselective difference between first pass clearance and later systemic clearance of verapamil, when taken in overdose.

Eudragit RL (ERL) and Eudragit RS (ERS) are biocompatible cationic copolymers, pH-independent and insoluble in aqueous environment. In this study drug delivery system consisting of a capsule filled with floating pellets with verapamil hydrochloride (VH) is proposed. The release of VH in the stomach results in better solubility in an acid gastric environment in vivo and may result in greater amount of the VH absorbed and its higher concentration in plasma. The scope of this study was to investigate the influence of ERL and ERS ratio on VH release in 0,1 M HCl from floating coating pellets. The stability of this film was also investigated. The ERL film is much more permeable than ERS, and an increase of ERL film thickness did not retard the release rate. The combination of ERL and ERS are forms of the sustained release film. It was a necessary to add the uncoated pellets, which constituted the initial dose. The start of flotation depends on permeability of polymeric film, and decreases with addition of ERS. There is no change in the start flotation time after 12 months under room condition (25 degrees C/60% RH). The drug delivery from uncoated pellets and pellets coated with ERL/ERS is stable after 12 months under room condition (25 degrees C/60% RH).

Ab initio Hartree-Fock calculations have been performed at the 6-31G level to study the pharmacophoric features of verapamil. Both the unprotonated and the protonated forms of verapamil have been studied. The study predicts that the drug enters the body in protonated form and is anchored to the receptor via H-bond formation involving protonated amine. Huge conformational change as well as deprotonation is required before the drug is capable of holding Ca(2+) ions. Folded form of drug is capable of holding Ca(2+) ion and the chiral center also seems to be involved to certain extent.

The purpose of this study was to work out a method of compression of floating pellets with verapamil hydrochloride (VH) in a dose of 40 mg. It was assumed that this form should reside in the stomach floating for several hours and gradually release the drug in a controlled way. Compression of pellets into tablets, being a modern technological process, is much more perfect than enclosing them in a hard gelatin capsule. Kollicoat SR 30 D was selected for coating. In experiments three plasticizers were examined-propylene glycol, triethyl citrate and dibuthyl sebecate (all at concentration of 10%). It was found that VH release from pellets coated by the films of the same thickness (70 microm), however, containing plasticizers is considerably different. Pellets were prepared by wet granulation of powder mixture, spheronization of the granulated mass and coating of the cores with a sustained release film. Two kinds of cellulose, microcrystalline and powdered, and sodium hydrocarbonate were the main components of pellet core. Proper pellet coating film thickness, ensuring obtaining desirable VH release profile and flotation effect, was defined. X compositions of tablets with pellets were examined in order to obtain formulation, from which VH release would mostly approximate pellets before compressing. The best formulation was evaluated taking into account the effect of compression force an tablet hardness and friability, and pellet agglomeration and flotation. Tablet cross-section photographs were taken confirming necessary coating film thickness preventing their deformation caused by compressing into tablets.

The genetic basis for polymorphic expression of CYP3A5 has been recently identified, but the significance of CYP3A5 expression is unclear. The purpose of this study is to quantify the capability of verapamil, a mechanism-based inhibitor of CYP3A, and its metabolites to inhibit the activities of CYP3A4 and CYP3A5, and to determine whether CYP3A5 expression in human liver microsomes alters the inhibitory potency of verapamil. Testosterone 6beta-hydroxylation or midazolam 1'-hydroxylation was used to quantify CYP3A activity. The possibility that verapamil and its metabolites form metabolic-intermediate complex (MIC) with CYP3A was assessed using dual beam spectrophotometry. Verapamil and N-desalkylverapamil (D617) were found to have little inhibitory effect on cDNA-expressed CYP3A5 activity and did not form a MIC with cDNA-expressed CYP3A5 as indicated by the appearance of the characteristic peak at 455 nm. At 50 microM, norverapamil showed time-dependent inhibition of CYP3A5 (30%), but to a much lesser extent compared with that of CYP3A4 (80%). The estimated values of the inactivation parameters k(inact) and K(I) of norverapamil were 4.53 microM and 0.07 min(-1) for cDNA-expressed CYP3A5, and 10.3 microM and 0.30 min(-1) for cDNA-expressed CYP3A4. Human liver microsomes that expressed CYP3A5 were less inhibited by both verapamil and norverapamil. The inactivation efficiency of verapamil and norverapamil was 30 times and 45 times lower, respectively, for CYP3A5-expressing microsomes compared with CYP3A5-non-expressing microsomes. These findings indicate that the presence of variable CYP3A5/CYP3A4 expression in the liver may contribute to the interindividual variability associated with verapamil-mediated drug interactions.

1 We have investigated the actions of the calcium entry blockers nifedipine, R-verapamil and S-verapamil in rat aorta, colon and vas deferens. 2 In aorta and colon, these agents produced concentration-dependent relaxations of KCl (80 mM)-induced contractions. In both tissues, the order of potency was nifedipine > S-verapamil > R-verapamil. However, nifedipine showed selectivity for aorta (potency ratio, colon/aorta: 4.36), S-verapamil showed no selectivity (0.62), but R-verapamil showed selectivity for colon (0.19). 3 In prostatic portions of rat vas deferens, nifedipine (10 microM) abolished the contraction to a single electrical stimulus, but R- and S-verapamil were without effect. In epididymal portions of rat vas deferens, R- and S-verapamil inhibited alpha1-adrenoceptor-mediated contractions to a single electrical stimulus at concentrations of 10 microM and above. 4 In conclusion, R-verapamil may prove useful as an intestinal selective calcium entry blocker in the treatment of intestinal disease with a hypermotility component, e.g. irritable bowel syndrome.

A stereoselective bioanalytical method for the simultaneous quantification of the enantiomers of verapamil and its active main metabolite norverapamil in human plasma has been developed and validated. The samples were analysed by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) in the Selected Reaction Monitoring (SRM) mode using a deuterated internal standard. The stationary phase used for the chiral separation was a Chiral-AGP. The enantiomers of verapamil were selectively detected from those of norverapamil by the mass spectrometer due to different molecular masses, although there was a chromatographic co-elution. Thus, time-consuming procedures like achiral preseparation or chemical derivatisation could be avoided. Higher detection sensitivity than earlier published methods based on fluorescence detection was obtained, although a mobile phase of high water-content and high flow-rate was introduced into the electrospray interface (85% aqueous ammonium acetate pH 7.4 +15% acetonitrile at 0.6 ml/min). The enantiomers of verapamil and norverapamil could be quantified at levels down to 50 pg and 60 pg/500 microl plasma sample, respectively, with R.S.D. in the range of 3.6-7.8%. The presented method was successfully applied to an in vivo intestinal absorption and bioavailability study in humans, using the Loc-I-Gut method.

Objective: Release of cellular cholesterol and phospholipid mediated by helical apolipoprotein and ATP-binding cassette transporter (ABC) A1 is a major source of plasma HDL. We investigated the effect of calcium channel blockers on this reaction., Methods and Results: Expression of ABCA1, apoA-I-mediated cellular lipid release, and HDL production were enhanced in cAMP analogue-treated RAW264 cells by verapamil, and similar effects were also observed with other calcium channel blockers. The verapamil treatment resulted in rapid increase in ABCA1 protein and its mRNA, but not the ABCG1 mRNA, another target gene product of the nuclear receptor liver X receptor (LXR). By using the cells transfected with a mouse ABCA1 promoter-luciferase construct (-1238 to +57bp), verapamil was shown to enhance the transcriptional activity. However, it did not increase transcription of LXR response element-driven luciferase vector., Conclusions: The data demonstrated that verapamil increases ABCA1 expression through LXR-independent mechanism and thereby increases apoA-I-mediated cellular lipid release and production of HDL.

In vitro studies of enantioselective metabolism of R-(+)- and S-(-)verapamil (VER) were conducted using human cDNA-expressed CYP3A isoforms, CYP3A4, CYP3A5, and CYP3A7. N-dealkylated products nor-VER [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and D617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] were the major metabolites for all CYP3A isoforms regardless of enantiomer. Enantioselectivity of CYP3A4 and CYP3A7 was most similar among the three isoforms. This coincides with the degree of homology of amino acids at the active sites and in the total amino acid sequences of the enzymes. Biphasic substrate inhibition was observed for the formation of nor-VER and D617, whereas simple biphasic kinetics were observed for the formation of O-demethylated products for both enantiomers with CYP3A4. The biphasic substrate inhibition was observed only for nor-VER, and simple biphasic kinetics were observed for D617 and O-demethylated products for both enantiomers with CYP3A5. However, with CYP3A7, D617 and O-demethylated products showed typical Michaelis-Menten kinetics, and only nor-VER displayed substrate (monophasic) inhibition. When metabolic rates of VER were determined in the presence of three different effectors, midazolam, testosterone, and nifedipine, activation, inhibition, or activation and inhibition of VER metabolism was observed depending on the enantiomers, metabolites, effectors, and cytochrome P450 isoforms. Addition of anti-CYP3A4 antibody inhibited formation of all metabolites for both CYP3A4 and CYP3A5. The atypical phenomena (biphasic substrate inhibition, activation, and inhibition depending on product formation) of VER kinetics could be adequately explained by introducing the concept of steric interaction into a two binding-site model.

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as beta-glucuronidase (beta-gluc). Regulation of beta-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of beta-gluc activity by the calcium channel blocker verapamil. To further explore this phenomenon, we investigated the effect of verapamil on beta-gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2. Treatment of HepG2 cells with verapamil revealed down-regulation of beta-gluc activity, protein, and mRNA level down to 50% of the control with EC(50) values of 25 microM. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human beta-glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by beta-glucuronidase.

Patients with cancer often develop major electrolyte disorders, which are aggravated by radiation therapy and chemotherapy and by the concomitant impairment of the renal function and the development of drug resistance. In addition, tumour cells have membranes with more negative charges than normal eukaryotic cells. This study was designed to test the hypothesis that the ability of the Ca(2+) blocker verapamil to mediate the reversal of multidrug resistance (MDR) by interacting with the membrane phospholipids may be correlated with the ionic strength and membrane surface potential in resistant tumours. The permeation properties of verapamil, which is the best-known MDR-modulator, were therefore studied by quantifying its ability to induce the leakage of carboxyfluorescein through unilamellar liposomes containing various mole fractions of phosphatidic acid (x(EPA)=0, 0.1 and 0.3), at four different ionic strengths (I=0.052, 0.124, 0.204 and 0.318 M). The dye leakage induced by verapamil varied greatly with I, depending on x(EPA). The permeation process was a co-operative one (1.3

A series of novel neuronal voltage-dependent calcium channel (VDCC) blockers, with inhibitory activity at low micromolar and moderate solubility in water, was discovered by constructing and screening a focused library based on emopamil (1) left hand (ELH) as a bioactive template.

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents.

ATP-driven pumping of a variety of drugs out of cells by the human P-glycoprotein poses a serious problem to medical therapy. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Membrane permeability of transported drugs and consequent lack of an experimentally defined drug position have made resolution of the transport mechanism difficult by classical techniques. To overcome these obstacles we devised a novel EPR spin-labeled verapamil for use as a transport substrate. Spin-labeled verapamil was an excellent transport substrate with apparent turnover number, K(m) and K(i) values of 5.8 s(-1), 4 microm, and 210 microm, respectively, at pH 7.4 and 37 degrees C. The apparent affinities were approximately 10-fold higher than for unlabeled verapamil. Spin-labeled verapamil stimulated ATPase activity approximately 5-fold, was relatively hydrophilic, and had a very low flip-flop rate, making it an ideal transport substrate. The K(m) for MgATP activation of transport was 0.8 mm. By measuring the mobility of spin-labeled verapamil during transport experiments, we were able to resolve the location of the drug in proteoliposome suspensions. Steady state gradients of spin-labeled verapamil within the range of K(i)/K(m) ratios were observed.

A simple CE assay for the rapid determination of the in vitro metabolic stability of verapamil in human liver microsomes has been developed and validated. Verapamil was used as the prototype drug since it is extensively metabolized in human liver microsomes. The assay showed good intra- (CV < or = 10%) and interday (CV < or = 8%) reproducibility. The recovery of verapamil after incubation at 37 degrees C for 60 min with human liver microsomes was low (15 +/- 1%) and two metabolites were detected. The method is currently in use for assessing the metabolic stability of new drug candidates at an early stage of lead optimization at Cardiome Pharma Corp. (Vancouver, BC, Canada).

Polymeric sodium alginate interpenetrating network membranes containing verapamil hydrochloride were fabricated for transdermal application. The membranes were evaluated for their physical properties, weight and thickness uniformity, water vapor transmission, as well as drug content uniformity. All the thin patches were transparent, smooth, and flexible. The drug-loaded membranes were analyzed by X-ray diffraction to understand the drug polymorphism inside the membrane. The transdermal patches were permeable to water vapor, indicating the permeability characteristics of the polymers. The in vitro drug release was performed in distilled water using a Keshary-Chien diffusion cell. The release data were analyzed to understand the mechanism of drug release.