Effect of intralipid administration on calcium therapy in verapamil toxicity.

AIM: Lipid emulsions are promising with regard to the treatment of toxicity by agents of high lipophilic nature. Our objective is to investigate the efficacy of intralipid 20% and calcium administration at different times when symptoms of cardiac toxicity occur during verapamil infusion. METHOD: 24 adult male Spraque-Dawley rats were randomly divided into 4 different groups, the control group, calcium group, calcium following 20% intralipid group and concomitant 20% intralipid and calcium group. Following monitoring under ketamine anesthesia, all groups were administered 37.5 mg kg-1 h-1 verapamil infusion until a 50% decrease occurred in MAPb. At the end of the infusion, verapamil infusion was decreased down to 15 mg kg-1h-1 and the treatment agents predetermined for the groups were administered concomitantly. RESULTS: There is no statistically significant difference between the administration of 20% intralipid synchronized with calcium or as a pretreatment, but both groups provided a higher survival rate when compared to the other groups. CONCLUSIONS: The administration of calcium alone in verapamil toxicity is not sufficient; when calcium and 20% intralipid are administered together, there is no difference between the administration of lipid and calcium concomitantly and the administration of lipid prior to calcium (Tab. 1, Fig. 2, Ref. 23).

Alteration in DNA structure, molecular responses and Na+ -K+ -ATPase activities in the gill of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) in response to sub-lethal verapamil.

The ecotoxicological consequences of residues from pharmaceutical drugs on aquatic biota have necessitated the development of sensitive and reliable techniques to assess the impact of these xenobiotics on aquatic organisms. This study investigated the alteration in DNA structure, molecular responses and the activities of Na+ -K+ -ATPase and antioxidant enzymes in the gill of Nile tilapia, Oreochromis niloticus, exposed to long-term effects at the concentrations (0.14, 0.28 and 0.57mgL-1) of verapamil in static renewal system for 15, 30, 45 and 60 days. Evaluation of DNA structure, using single cell gel electrophoresis, revealed certain degree of DNA damages in the gill in a time and concentration-dependent relationship. Transcription of mRNA of superoxide dismutase (sod), catalase (cat) and heat shock protein (hsp70) genes in the gill of the fish showed the genes were up-regulated. Na+-K+-ATPase activity was inhibited in a concentration and time dependent manner. The indices of oxidative stress biomarkers (lipid peroxidation and carbonyl protein) as well as superoxide dismutase, glutathione peroxidase, glutathione-S-transferase were elevated in the treated fish in comparison to the control. Further, the level of reduced glutathione and catalase activity were inhibited at 0.28mgL-1 after day 30. Long-term exposure to sub-lethal concentration of verapamil can cause DNA damages, molecular effects and oxidative stress in O. niloticus. The biomarkers analysed can be used as early warning signals in environmental biomonitoring and assessment of drug contamination in aquatic ecosystem.

Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment.

Verapamil is a Ca2+ channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca2+ -permeable N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca2+ entry through L-type Ca2+ channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca2+ channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca2+ release suggesting that ALCAR acts via effectors downstream of Ca2+ . In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca2+ during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Assessment of mutagenic, hematological and oxidative stress biomarkers in liver of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) in response to sublethal verapamil exposure.

The influx of pharmaceutical drugs and their metabolites have been reported to cause negative impact on aquatic biota. In this study, effects of long-term exposure of verapamil on mutagenic, hematological parameters and activities of the oxidative enzymes of Nile tilapia, Oreochromis niloticus were investigated for 60 days exposure at the concentrations of 0.29, 0.58 and 1.15 mg L-1 in the fish liver. The exposure resulted in significantly high (p < 0.05) micronuclei induction of peripheral blood cells at the peak on day 30 at 1.15 mg L-1. Compared with the control, there was significant increase (p < 0.05) in white blood cell counts and red blood cell distribution width (RDW), with a reduction in hemoglobin (Hb), red blood cell counts (RBCs), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) level as the concentration of the drug increased. The indices of oxidative stress biomarkers (lipid peroxidation and carbonyl protein) showed elevated level, depicting a positive correlation with both time and concentration. More so, the activity of energy-related parameter (Na+ -K+- ATPase) in the tissue was significantly inhibited (p < 0.05) at the end of 60 days exposure period. Further, the activity of catalase (CAT) was inhibited while reduced glutathione (GSH) level was decreased in the liver tissue. There was increase in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) after 30 days at 0.29 mg L-1. The study demonstrated that prolonged exposure to verapamil at sublethal concentration can result in mutagenic effects and oxidative dysfunctions in O. niloticus.

Comparison of Effects of Separate and Combined Sugammadex and Lipid Emulsion Administration on Hemodynamic Parameters and Survival in a Rat Model of Verapamil Toxicity.

BACKGROUND: Toxicity of calcium channel blockers leads to high patient mortality and there is no effective antidote. The benefit of using 20% lipid emulsion and sugammadex has been reported. The present study measured the effect of sugammadex and 20% lipid emulsion on hemodynamics and survival in a rat model of verapamil toxicity. MATERIAL/METHODS: In this single-blinded randomized control study, rats were separated into 4 groups of 7 rats each: Sugammadex (S), Sugammadex plus 20% lipid emulsion (SL), 20% lipid emulsion (L), and control (C). Heart rates and mean arterial pressures were monitored and noted each minute until death. RESULTS: Average time to death was 21.0±9.57 minutes for group C, 35.57±10.61 minutes for group S, 37.14±16.6 minutes for group L and 49.86±27.56 minutes for group SL. Time to death was significantly longer in other groups than in the control group (p<0.05). CONCLUSIONS: Verapamil overdose is has a comparatively high mortality rate and there is no effective antidote. Treatment generally involves gastric decontamination and symptomatic treatment to counteract the drug's negative effects. In animal studies sugammadex and lipid emulsion had a positive effect on survival in patients with calcium channel blocker toxicity. Sugammadex and intralipid increased survival in a rat model of verapamil toxicity. The combination of both drugs may decrease cardiotoxicity. Sugammadex alone or combined with 20% lipid emulsion reduce the need for inotropic agents. The mechanism requires clarification with larger studies.

Inhibition of MDR3 Activity in Human Hepatocytes by Drugs Associated with Liver Injury.

MDR3 dysfunction is associated with liver diseases. We report here a novel MDR3 activity assay involving in situ biosynthesis in primary hepatocytes of deuterium (d9)-labeled PC and LC-MS/MS determination of transported extracellular PC-d9. Several drugs associated with DILI such as chlorpromazine, imipramine, itraconazole, haloperidol, ketoconazole, sequinavir, clotrimazole, ritonavir, and troglitazone inhibit MDR3 activity. MDR3 inhibition may play an important role in drug-induced cholestasis and vanishing bile duct syndrome. Several lines of evidence demonstrate that the reported assay is physiologically relevant and can be used to assess the potential of chemical entities and their metabolites to modulate MDR3 activity and/or PC biosynthesis in hepatocytes.

Uptake of Pharmaceuticals Influences Plant Development and Affects Nutrient and Hormone Homeostases.

The detection of a range of active pharmaceutical ingredients (APIs) in the soil environment has led to a number of publications demonstrating uptake by crops, however very few studies have explored the potential for impacts on plant development as a result of API uptake. This study investigated the effect of carbamazepine and verapamil (0.005-10 mg/kg) on a range of plant responses in zucchini (Cucurbita pepo). Uptake increased in a dose-dependent manner, with maximum leaf concentrations of 821.9 and 2.2 mg/kg for carbamazepine and verapamil, respectively. Increased carbamazepine uptake by zucchini resulted in a decrease in above (<60%) and below (<30%) ground biomass compared to the controls (p < 0.05). At soil concentrations >4 mg/kg the mature leaves suffered from burnt edges and white spots as well as a reduction in photosynthetic pigments but no such effects were seen for verapamil. For both APIs, further investigations revealed significant differences in the concentrations of selected plant hormones (auxins, cytokinins, abscisic acid and jasmonates), and in the nutrient composition of the leaves in comparison to the controls (p < 0.05). This is some of the first research to demonstrate that the exposure of plants to APIs is likely to cause impacts on plant development with unknown implications.

Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.

BACKGROUND: Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. METHODS AND RESULTS: Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations. CONCLUSIONS: We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.

Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish.

Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.

Dual agent loaded PLGA nanoparticles enhanced antitumor activity in a multidrug-resistant breast tumor eenograft model.

Multidrug-resistant breast cancers have limited and ineffective clinical treatment options. This study aimed to develop PLGA nanoparticles containing a synergistic combination of vincristine and verapamil to achieve less toxicity and enhanced efficacy on multidrug-resistant breast cancers. The 1:250 molar ratio of VCR/VRP showed strong synergism with the reversal index of approximately 130 in the multidrug-resistant MCF-7/ADR cells compared to drug-sensitive MCF-7 cells. The lyophilized nanoparticles could get dispersed quickly with the similar size distribution, zeta potential and encapsulation efficiency to the pre-lyophilized nanoparticles suspension, and maintain the synergistic in vitro release ratio of drugs. The co-encapsulated nanoparticle formulation had lower toxicity than free vincristine/verapamil combinations according to the acute-toxicity test. Furthermore, the most effective tumor growth inhibition in the MCF-7/ADR human breast tumor xenograft was observed in the co-delivery nanoparticle formulation group in comparison with saline control, free vincristine, free vincristine/verapamil combinations and single-drug nanoparticle combinations. All the data demonstrated that PLGANPs simultaneously loaded with chemotherapeutic drug and chemosensitizer might be one of the most potential formulations in the treatment of multidrug-resistant breast cancer in clinic.

Synergism of ochratoxin B and calcium-channel antagonist verapamil caused mitochondrial dysfunction.

We examined the mechanism by which the ochratoxin B induced interaction with calcium-channel antagonist verapamil and mitochondrial dysfunction of the rat trachea in vitro experiment. The tracheas were cut into 2-3 mm wide rings and suspended in a tissue bath. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Verapamil (1 × 10(-6) M) produced a concentration-dependent contraction response in rat's tracheal rings pre-contracted by acetylcholine. Incubation of rat's tracheal rings with the ochratoxin B significantly potentiated the contraction responses of verapamil. Verapamil and OTB accelerate the overloading of Ca(2+) in tracheal smooth muscle contributes the tissue toxicity as shown in electron microscopy and mitochondrial enzymes, through a mechanism that could involve perturbations of Ca(2+) homeostasis. These results proved that ochratoxin B is a potential vasoconstrictor mycotoxin with the presence of calcium-channel antagonist. In conclusion, disturbance of Ca(2+) homeostasis caused by OTA and plays a significant role in produces toxicity through mitochondrial enzyme inhibition.

Dorsal hippocampus is necessary for novel learning but sufficient for subsequent similar learning.

Our current understanding of brain mechanisms involved in learning and memory has been derived largely from studies using experimentally naïve animals. However, it is becoming increasingly clear that not all identified mechanisms may generalize to subsequent learning. For example, N-methyl-D-aspartate glutamate (NMDA) receptors in the dorsal hippocampus are required for contextual fear conditioning in naïve animals but not in animals previously trained in a similar task. Here we investigated how animals learn contextual fear conditioning for a second time-a response which is not due to habituation or generalization. We found that dorsal hippocampus infusions of voltage-dependent calcium channel blockers or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) agonist impaired the first, not the second contextual learning. Only manipulations of the entire hippocampus led to an impairment in second learning. Specifically, inactivation of either the dorsal or ventral hippocampus caused the remaining portion of the hippocampus to acquire and consolidate the second learning. Thus, dorsal hippocampus seems necessary for initial contextual fear conditioning, but either the dorsal or ventral hippocampus is sufficient for subsequent conditioning in a different context. Together, these findings suggest that prior training experiences can change how the hippocampus processes subsequent similar learning.

Transmural dispersion of repolarization as a preclinical marker of drug-induced proarrhythmia.

Torsade de Pointes (TdP) proarrhythmia is a major complication of therapeutic drugs that block the delayed rectifier current. QT interval prolongation, the principal marker used to screen drugs for proarrhythmia, is both insensitive and nonspecific. Consequently, better screening methods are needed. Drug-induced transmural dispersion of repolarization (TDR) is mechanistically linked to TdP. Therefore, we hypothesized that drug-induced enhancement of TDR is more predictive of proarrhythmia than QT interval. High-resolution transmural optical action potential mapping was performed in canine wedge preparations (n = 19) at baseline and after perfusion with 4 different QT prolonging drugs at clinically relevant concentrations. Two proarrhythmic drugs in patients (bepridil and E4031) were compared with 2 nonproarrhythmic drugs (risperidone and verapamil). Both groups prolonged the QT (all P < 0.02), least with the proarrhythmic drug bepridil, reaffirming that QT is a poor predictor of TdP. In contrast, TDR was enhanced only by proarrhythmic drugs (P < 0.03). Increased TDR was due to a preferential prolongation of midmyocardial cell, relative to epicardial cell, APD, whereas nonproarrhythmic drugs similarly prolonged both cell types. In contrast to QT prolongation, augmentation of TDR was induced by proarrhythmic but not nonproarrhythmic drugs, suggesting TDR is a superior preclinical marker of proarrhythmic risk during drug development.

Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh-7 and HCC-T cells with the highest potential to study drug metabolism.

Primary human hepatocytes (hHeps) are still gold standard to perform human drug metabolism studies, but their availability is limited by donor organ scarcity. Therefore, hepatoma cell lines are widely used as alternatives, although their phases I and II drug-metabolizing activities are substantially lower compared with hHeps. The major advantage of these cell lines is immediate availability, standardized culture conditions and unlimited life span. Therefore, the aim of this study was to investigate the drug-metabolizing profile of five human hepatoma cell lines (HepG2, Hep3B, HCC-T, HCC-M and Huh-7) over a culture period of 10 passages. Fluorescent-based assays for seven different cytochrome P450 (CYP) isoforms and seven different phase II enzymes were performed and compared with enzymatic activities of hHeps. CYP activities were much lower in the cell lines (5-15% of hHeps), whereas phase II enzyme activities that are involved in buffering oxidative stress (e.g., Glutathione-S-transferase) reached levels comparable with hHeps. Furthermore, phases I and II enzyme activities in hepatoma cell lines vary strongly during culture time. Interestingly, the most constant results were obtained with Huh-7 cells. Huh-7 cells as well as HCC-T cells exhibited a drug-metabolizing profile closest to hHeps between passages two and four. Toxicity studies with Diclofenac, Paracetamol and Verapamil in both cell lines show dose-response characteristics and EC(50) values similar to hHeps. Therefore, we propose that due to the more consistent results throughout the passages, Huh-7 could be an alternative system to the limitedly available hHeps and frequently used HepG2 cell line in the study of drug metabolism.

Verapamil does not modify catalytic activity of CYP450 in rainbow trout after long-term exposure.

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 µg l(-1)) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Rescue extracorporeal life support for acute verapamil and propranolol toxicity in a neonate.

Extracorporeal life support (ECLS) to manage acute antiarrhythmic drugs toxicity in neonates has never been reported. Here presented is a case of venoarterial extracorporeal membrane oxygenation support in a newborn with refractory low cardiac output as a result of acute Ca-channel and ß-receptor antagonist toxicity for treatment of paroxysmal supraventricular tachycardia (SVT). Shortly after onset of ECLS, the baby recovered sinus rhythm and subsequent bouts of SVT were controlled by amiodarone infusion and repeated DC shocks. Weaning was possible on the 5th day after implant, once recovery of the left ventricular function and optimization of the antiarrhythmic medication were achieved. In neonates with severe but potentially reversible cardiac dysfunction caused by drug toxicity, ECLS can maintain cardiac output and vital organ perfusion while allowing time for drug redistribution, metabolism, and clearance.

Toxicity evaluation of verapamil and tramadol based on toxicity assay and expression patterns of Dhb, Vtg, Arnt, CYP4, and CYP314 in Daphnia magna.

In this study, the toxicities of two pharmaceuticals, verapamil and tramadol were evaluated in Daphnia magna using the conventional toxicity tests (acute and chronic test) and the expression patterns of five stress responsive genes. In the chronic toxicity test, several parameters, such as the survival percentage, the body length of D. magna, the time of first reproduction, and the number of offspring per female, were adversely affected during the exposure to 4.2 mg L(-1) verapamil and 34 mg L(-1) tramadol. During the 24-h short-term exposure, verapamil particularly caused a downregulated expression of the CYP4 and CYP314 genes, whereas tramadol upregulated the expression of the CYP314 gene. Neither pharmaceutical affected the expression of Dhb, Arnt, and Vtg. However, during the 21-day long-term exposure, both verapamil and tramadol significantly reduced the expression level of the Vtg gene, a biomarker of the reproduction ability in an oviparous animal, whereas neither affected the other genes.

[Verapamil-induced linear IgA disease mimicking toxic epidermal necrolysis]. / Dermatose à IgA linéaire induite par le vérapamil : présentation inhabituelle à type de nécrolyse épidermique toxique.

BACKGROUND: Linear IgA bullous dermatosis is a rare auto-immune bullous dermatitis characterized by linear IgA deposits in the basal membrane zone. Clinical diagnosis may be difficult due to the various clinical presentations mimicking bullous pemphigoid, pemphigus, erythema multiforme or toxic epidermal necrolysis. Linear IgA may be idiopathic or due to drugs, particularly vancomycin. PATIENTS AND METHODS: We describe a 91-year-old woman treated with verapamil for coronary disease who developed an eruption presenting as a toxic epidermal necrolysis, although the diagnosis was amended after direct immunofluorescence revealed IgA deposits in the basal membrane zone. DISCUSSION: Ours appears to be the first reported case of verapamil-induced linear IgA bullous dermatosis.

Torsadogenic Action of Cisapride, dl-Sotalol, Bepridil, and Verapamil Analyzed by the Chronic Atrioventricular Block Cynomolgus Monkeys: Comparison With That Reported in the CiPA In Silico Mechanistic Model.

In order to bridge the gap of information between the in silico model and human subjects, we evaluated torsadogenic risk of cisapride, dl-sotalol, bepridil and verapamil selected from 12 training compounds in the comprehensive in vitro proarrhythmia assay using the chronic atrioventricular block monkeys. Cisapride (0, 1, and 5 mg/kg, n = 5 for each dose), dl-sotalol (0, 1, 3, and 10 mg/kg, n = 5 for each dose), bepridil (0, 10, and 100 mg/kg, n = 4 for each dose), verapamil (0, 1.5, 15, and 75 mg/kg, n = 4 for each dose) were orally administered to the monkeys in conscious state. Five mg/kg of cisapride, 1, 3, and 10 mg/kg of dl-sotalol and 100 mg/kg of bepridil prolonged ΔΔQTcF, which was not observed by verapamil. Torsade de pointes was induced by 5 mg/kg of cisapride in 2 out of 5 animals, by 10 mg/kg of dl-sotalol in 5 out of 5 and by 100 mg/kg of bepridil in 2 out of 4, which was not induced by verapamil. These torsadogenic doses were normalized by their maximum clinical daily ones to estimate torsadogenic risk. The order of risk was dl-sotalol >bepridil ≥cisapride >verapamil in our study. Since the order was bepridil ≥dl-sotalol >cisapride >verapamil in comprehensive in vitro proarrhythmia assay (CiPA) in silico mechanistic model validation, sympathetic regulation on the heart may play a pivotal role in the onset of torsade de pointes in vivo.

K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport.

BACKGROUND: Lung epithelial Na+ channels (ENaC) are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport. METHODS: Verapamil-induced depression of heterologously expressed human alphabetagamma ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM. RESULTS: The rate of in vivo AFC was reduced significantly (40.6+/-6.3% of control, P<0.05, n=12) in mice intratracheally administrated verapamil. KCa3.1 (1-EBIO) and KATP (minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in alphabetagamma ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na), K Ca3.1 (1-EBIO), and KATP (minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways. CONCLUSIONS: Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal.