Factors Affecting the Binding of Diltiazem to Rainbow Trout Plasma: Implications for the Risk Assessment of Pharmaceuticals in Aquatic Systems.

The accumulation of organic toxicants in fish plasma, and how they partition between the bound and unbound fraction once absorbed, are important metrics in models that seek to predict the risk of such contaminants in aquatic settings. Rapid equilibrium dialysis of diltiazem, an ionizable weak base and important human pharmaceutical contaminant of freshwaters, was conducted with rainbow trout (Oncorhynchus mykiss) plasma. The effect of fed state, fish sex, fish strain/size, and dialysis buffer pH on the binding of radiolabeled diltiazem (9 ng ml-1 ) was assessed. In fed fish, 24.6%-29.5% of diltiazem was free, unbound to plasma proteins. Although starvation of fish resulted in a decrease in plasma protein, the bound fraction of diltiazem remained relatively constant. Consequently, the protein-bound concentration of diltiazem increased with length of starvation. In general, rainbow trout strain was a significant factor affecting plasma binding, although the two strains tested also differed markedly in size. Dialysis buffer pH significantly influenced plasma binding, with a higher unbound diltiazem fraction at pH 6.8 than pH 8.0. These data indicate that empirical measures of plasma binding in fish are important for accurate risk assessment and that the physiological status of a fish is likely to impact its sensitivity to toxicants such as diltiazem. Environ Toxicol Chem 2022;41:3125-3133. © 2022 SETAC.

Molecular interactions of polyvinyl chloride microplastics and beta-blockers (Diltiazem and Bisoprolol) and their effects on marine meiofauna: Combined in vivo and modeling study.

The ecotoxicological effects of beta-blockers (i.e. Diltiazem and Bisoprolol) and their interactions with the microplastic polyvinyl chloride on marine meiofauna were tested in laboratory microcosms. An experimental factorial design was applied, using meiobenthic fauna collected from the Old Harbor of Bizerte (NE Tunisia), but with a main focus on the nematode communities. The meiobenthic invertebrates were exposed to two concentrations of Diltiazem and Bisoprolol, of 1.8 µg.L-1 and 1.8 mg.L-1, respectively, and one concentration of polyvinyl chloride (i.e. 20 mg.kg-1), separately and mixed. The overall meiofauna abundance was significantly reduced in all treatments, mainly that of polychaetes and amphipods. Moreover, the juveniles-gravid female ratios of the nematode communities were the lowest in the 1.8 µg.L-1 Bisoprolol treatment and for the 1.8 mg.L-1 mixture of Diltiazem and microplastics, suggesting that different dosages influence the maturity status of the examined species. The demographic results were also supported by in silico approach. The simulation of molecular interactions revealed acceptable binding affinities (up to -8.1 kcal/mol) and interactions with key residues in the germ line development protein 3 and sex-determining protein from Coenorhabditis elegans. Overall, the experimental outcome strongly indicates synergistic interactions among the beta-blockers Diltiazem and Bisoprolol and the microplastic polyvinyl chloride on marine nematode communities.

Interaction of the Olfactory System of Rainbow Trout (Oncorhynchus mykiss) with Diltiazem.

Diltiazem is ubiquitously prescribed and has been reported in many effluents and freshwater bodies. Being a calcium channel blocker, diltiazem could disrupt the function of the sensory and central nervous systems. In the present study, using electro-olfactography (EOG), we investigated the interaction of diltiazem with the olfactory sensory neurons (OSNs) of rainbow trout by looking into the detection threshold and effects of immediate (~5 min) and acute (24 h) exposure to diltiazem at 6.6, 66, and 660 µg/L. We also studied the accumulation of the drug in fish plasma and whole body. Brief exposure to diltiazem impaired the OSN response to a chemosensory stimulus in a concentration-dependent manner at 6.6 µg/L and higher, whereas OSNs exposed for 24 h only displayed an impairment at 660 µg/L. Chemical analysis showed that the accumulation of diltiazem in fish plasma and body correlated with the EOG response because it was 10 times higher in the group that displayed a significant impairment (660 µg/L) compared to the other 2 groups (6.6, 66 µg/L). This correlation suggests that the impact of diltiazem on OSNs might partially be through the accumulated molecules in the fish bloodstream. Fish did not detect diltiazem as a sensory stimulus even at concentrations as high as 660 µg/L; thus, fish could potentially swim toward or fail to escape harmful concentrations of diltiazem. Environ Toxicol Chem 2022;41:554-550. © 2020 SETAC.

Low dissolved oxygen increases uptake of a model calcium channel blocker and alters its effects on adult Pimephales promelas.

Human population growth accompanied with urbanization is urbanizing the water cycle in many regions. Urban watersheds, particularly with limited upstream dilution of effluent discharges, represent worst case scenarios for exposure to multiple environmental stressors, including down the drain chemicals (e.g., pharmaceuticals) and other stressors (e.g., dissolved oxygen (DO)). We recently identified the calcium channel blocker diltiazem (DZM) to accumulate in fish plasma exceeding human therapeutic doses (e.g., Cmin) in coastal estuaries impaired due to nonattainment of DO water quality standards. Thus, we examined whether DO influences DZM uptake by fish, and if changes in DO-dependent upatke alter fish physiological and biochemical responses. Low DO (3.0 mg DO/L) approximately doubled diltiazem uptake in adult fathead minnows relative to normoxic (8.2 mg DO/L) conditions and were associated with significant (p < 0.05) increases in fish ventilation rate at low DO levels. Decreased burst swim performance (Uburst) of adult fathead minnows were significantly (p < 0.05) altered by low versus normal DO levels. DO × DZM studies reduced Uburst by 13-31% from controls, though not significantly (p = 0.06). Physiological responses in fish exposed to DZM alone were minimal; however, in co-exposure with low DO, decreasing trends in Uburst appeared inversely related to plasma lactate levels. Such physiological responses to multiple stressors, when paired with internal tissue concentrations, identify the utility of employing biological read across approaches to identify adverse outcomes of heart medications and potentially other cardiotoxicants impacting fish cardiovascular function across DO gradients.

Food-Drug Interaction between the Adlay Bran Oil and Drugs in Rats.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) contains various phytonutrients for treating many diseases in Asia. To investigate whether orally administered adlay bran oil (ABO) can cause drug interactions, the effects of ABO on the pharmacokinetics of five cytochrome P450 (CYP) probe drugs were evaluated. Rats were given a single oral dose (2.5 mL/kg BW) of ABO 1 h before administration of a drug cocktail either orally or intravenously, and blood was collected at various time points. A single oral dose of ABO administration did not affect the pharmacokinetics of five probe drugs when given as a drug cocktail intravenously. However, ABO increased plasma theophylline (+28.4%), dextromethorphan (+48.7%), and diltiazem (+46.7%) when co-administered an oral drug cocktail. After 7 days of feeding with an ABO-containing diet, plasma concentrations of theophylline (+45.4%) and chlorzoxazone (+53.6%) were increased after the oral administration of the drug cocktail. The major CYP enzyme activities in the liver and intestinal tract were not affected by ABO treatment. Results from this study indicate that a single oral dose or short-term administration of ABO may increase plasma drug concentrations when ABO is given concomitantly with drugs. ABO is likely to enhance intestinal drug absorption. Therefore, caution is needed to avoid food-drug interactions between ABO and co-administered drugs.

The effects of diltiazem on growth, reproduction, energy reserves, and calcium-dependent physiology in Daphnia magna.

With the growth of both the pharmaceutical industry and the human population and longevity, more drugs are used and processed each day. Inevitably, these pharmaceuticals enter wastewater through human excretion and improper disposal of leftovers. One such medication, diltiazem, a calcium channel blocker, is of importance due to its widespread consumption, and prevalence in aquatic environments. To study the sub-lethal effects of diltiazem on aquatic animals, we investigated its impacts no feeding behaviour, heart rate, respiration, growth, and reproduction of a bioindicator species, Daphnia magna. When exposed to environmentally relevant concentrations, D. magna increased their heart rate by 12% and oxygen consumption by 48%. However, exposure did not have any effects on thoracic limb movement frequency or peristalsis (i.e. feeding behaviour). Individuals exposed to diltiazem for a longer duration (16 days) showed a 44% decrease in lipid reserves and produced between 17 and 28% fewer neonates which were 10-12% larger. Our study demonstrated that exposure to diltiazem creates an energy imbalance in D. magna which could, in the long run, influence their populations.

Diltiazem aggravates testicular function impairment induced by cyclosporine A or tacrolimus in unilateral nephrectomised rats.

The aim of this study was to elucidate the reproductive toxicity of the coadministration of diltiazem and cyclosporine A or tacrolimus. Testicular development, semen quality, sex hormones and testicular tissues were assessed in unilateral nephrectomised (UN) rats, including the control group, UN group, UN+CsA group, UN+FK506 group, UN+Rapa group, UN+CsA+Dil group and UN+FK506+Dil group. The testicular coefficient, the sperm number and the sperm motility were lower in the treatment groups (except UN+FK506) than in the control and UN groups (all p < 0.05). The lowest sperm number and motility were identified in the UN+CsA+Dil group, followed by the UN+CsA group. The proportion of abnormal sperm was higher in the UN+CsA and UN+CsA+Dil groups than in the control and UN groups, respectively (p < 0.05). The plasma concentrations of sex hormones were changed in the treatment groups. Dil can increase the blood concentrations of CsA and FK506 (◇p < 0.05, ◆p < 0.05). Therapeutic doses of these agents induced morphological changes in the testicular tissue and ultrastructural changes in the testosterone, mesenchymal cells and supporting cells. Our present study suggests that Dil can increase the testicular toxicity of CNIs (calcineurin inhibitors, including CsA and FK506) by enhancing the plasma concentrations of CNIs.

Conduction and contraction properties of human iPS cell-derived cardiomyocytes: analysis by motion field imaging compared with the guinea-pig isolated heart model.

We used motion field imaging to characterize the conduction and contraction of a sheet of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs). A hiPS-CMs sheet of 2.8 mm × 2.8 mm allowed us to simultaneously measure the conduction and the contraction properties in the same cells. Pharmacological responses in the hiPS-CMs of four typical cardiac functional modulators, Na+ channel blocker (lidocaine), Ca2+ channel blocker (diltiazem), gap-junction inhibitor (carbenoxolone), and ß-adrenergic stimulator (isoproterenol), were investigated, and the results were compared to those found using the isolated guinea-pig heart model perfused by the Langendorff method. The conduction speed of excitation waves in hiPS-CMs was decreased by lidocaine, diltiazem, and carbenoxolone, and increased by isoproterenol, and these results were in accordance with the changes in the conduction parameters of electrocardiogram (QRS duration, PR interval, and P duration) in the Langendorff guinea-pig heart model. The maximum speeds for contraction and relaxation, which respectively represent the contraction and relaxation kinetics of hiPS-CMs, were decreased by lidocaine and diltiazem, and increased by isoproterenol. These results also corresponded to alterations in the contractile and relaxation parameters found by measuring left ventricular pressure (LVdP/dtmax and LVdP/dtmin) in the Langendorff guinea-pig heart model. From these lines of evidence, it was suggested that hiPS-CMs enable us to evaluate the cardiac toxicities associated with conduction disturbance or contractile dysfunction, and thereby would be useful as an integrated assessment of cardiac function.

Influence of Diltiazem on Fathead Minnows Across Dissolved Oxygen Gradients.

Water resources in many arid to semi-arid regions are stressed by population growth and drought. Growing populations and climatic changes are influencing contaminant and water chemistry dynamics in urban inland waters, where flows can be dominated by, or even dependent on, wastewater effluent discharge. In these watersheds, interacting stressors such as dissolved oxygen and environmental contaminants (e.g., pharmaceuticals) have the potential to affect fish physiology and populations. Recent field observations from our group identified the calcium channel blocker (CCB) diltiazem in fish plasma exceeding human therapeutic doses (e.g., Cmin ) in aquatic systems impaired because of nonattainment of dissolved oxygen water quality standards. Therefore our study objectives examined: 1) standard acute and chronic effects of dissolved oxygen and diltiazem to fish, 2) influences of dissolved oxygen at criteria levels deemed protective of aquatic life on diltiazem toxicity to fish, and 3) whether sublethal effects occur at diltiazem water concentrations predicted to cause a human therapeutic level (therapeutic hazard value [THV]) in fish plasma. Dissolved oxygen × diltiazem co-exposures significantly decreased survival at typical stream, lake, and reservoir water quality standards of 5.0 and 3.0 mg dissolved oxygen/L. Dissolved oxygen and diltiazem growth effects were observed at 2 times and 10 times lower than median lethal concentration (LC50) values (1.7 and 28.2 mg/L, respectively). Larval fathead minnow (Pimephales promelas) swimming behavior following low dissolved oxygen and diltiazem exposure generally decreased and was significantly reduced in light-to-dark bursting distance traveled, number of movements, and duration at concentrations as low as the THV. Individual and population level consequences of such responses are not yet understood, particularly in older organisms or other species; however, these findings suggest that assessments with pharmaceuticals and other cardioactive contaminants may underestimate adverse outcomes in fish across dissolved oxygen levels considered protective of aquatic life. Environ Toxicol Chem 2018;37:2835-2850. © 2018 SETAC.

Lipid emulsion alleviates the vasodilation and mean blood pressure decrease induced by a toxic dose of verapamil in isolated rat aortae and an in vivo rat model.

This study aimed to examine the effects of lipid emulsion on the vasodilation and cardiovascular depression induced by toxic doses of calcium channel blockers. The effects of lipid emulsion on the vasodilation induced by bepridil, verapamil, nifedipine, and diltiazem were investigated in isolated endothelium-denuded rat aortae. The effect of lipid emulsion on the comparable hemodynamic depression induced by the continuous infusion of a toxic dose of either verapamil or diltiazem was examined in an in vivo rat model. The results showed the following decreasing order for the magnitude of lipid emulsion-mediated inhibition of vasodilation: bepridil, verapamil, nifedipine, and diltiazem. Lipid emulsion (0.5-2%) reversed the vasodilation induced by a toxic dose of verapamil, whereas only a higher concentration (2%) reversed the vasodilation induced by a toxic dose of diltiazem. Pretreatment with lipid emulsion alleviated the systolic and mean blood pressure decreases induced by a toxic dose of verapamil, whereas it had no effect on the decrease induced by diltiazem. Taken together, these results suggest that lipid emulsion alleviates the severe vasodilation and systolic blood pressure decrease induced by a toxic dose of verapamil, and this alleviation appears to be associated with the relatively high lipid solubility of verapamil.

The effect of metanephros transplantation on blood pressure in anephric rats with induced acute hypotension.

BACKGROUND: The kidney is an important organ for maintaining blood pressure. We have previously reported that transplanted metanephroi can reproduce some kidney functions. The aim of the present study was to determine the metabolic function of transplanted metanephroi with particular reference to maintaining blood pressure. METHODS: Male Wistar rats were transplanted with metanephroi (transplanted group, n = 28), following unilateral nephrectomy. For comparison, we performed unilateral nephrectomy without transplantation in 32 rats (non-transplanted group, n = 18; haeminephrectomy control group, n = 14). The remaining kidney was removed 2 weeks after the initial operation, while control rats had a sham operation. Hypotension was induced by intravenous infusion of diltiazem hydrochloride or rapid withdrawal of blood. Mean arterial blood pressure (MAP) was invasively monitored and plasma renin activity (PRA) was analysed at multiple time points. Renin expression by metanephroi was evaluated by real-time polymerase chain reaction and immunohistochemistry. RESULTS: Metanephroi in the transplanted group expressed renin messenger RNA. Metanephros transplantation significantly raised PRA and maintained MAP compared with the non-transplanted group. No significant differences between the transplanted and control groups were found with respect to PRA or MAP. PRA was positively correlated with metanephroi weight as well as MAP in the transplanted group. CONCLUSION: The present study shows that transplantation of metanephroi produces PRA and contributes to raising MAP in a rat model of acute hypotension.

Treatment of calcium channel blocker-induced cardiovascular toxicity with drug scavenging liposomes.

Calcium channel blocker (CCB) overdose is potentially lethal. Verapamil and diltiazem are particularly prone to acute toxicity due to their dual effect on cardiac and vascular tissues. Unfortunately, conventional decontamination measures are ineffective in accelerating blood clearance and, to date, few efforts have been made to develop antidotes. To address the issue, injectable long-circulating liposomes bearing a transmembrane pH-gradient are proposed as efficient detoxifying agents of CCB poisoning. By scavenging the drug in situ, these circulating nanocarriers can restrict its distribution in tissues and hinder its pharmacological effect. In vitro, we showed that liposomes stability in serum and their ability to sequester CCBs could be finely-tuned by modulating their internal pH, surface charge, and lipid bilayer structure. Subsequently, we verified their efficacy in reversing the cardiovascular effects of verapamil in rats implanted with telemetric pressure/biopotential transmitters. In animals orally intoxicated to verapamil, an intravenous injection of the liposomal antidote rapidly attenuated the reduction in blood pressure. Areas under diastolic, systolic, and mean pressures curves were significantly reduced by up to 60% and the time to hemodynamic recovery was shortened from 19 to only 11 h. These findings confirm the protective effect of pH-gradient liposomes against cardiovascular failure after CBB intoxication, and endorse their potential as efficient, versatile antidotes.

The in vitro hemolytic effect of diltiazem on erythrocytes exposed to varying osmolarity.

The hemolytic effect of diltiazem (a calcium channel blocker) on red blood cells (RBCs) exposed to varying osmolarity was investigated. Previous work on the in vitro hemolytic effect of verapamil shed some light on the potential for some drugs to induce hemolysis at moderate-to-high doses and in pre-existing osmotic stress. The current experimental approach used a modified red cell hemolysis assay with concentrations of diltiazem ranging from 50-1500 µM compared to drug free controls. The time-course of hemolytic effects was also investigated. In conditions representing decreasing osmolarity (dilution from 140 to 0 mM NaCl) there was a significant increase in erythrocyte hemolysis that was also dependent on diltiazem concentration (ANOVA, p<0.05). The red cells also showed a significantly increased rate of hemolysis over 5 h with increasing concentration of diltiazem (ANOVA, p<0.05). Overall the data suggested that diltiazem can cause hemolysis of RBCs in a predictable time- and concentration-dependent manner, and that diltiazem increases the fragility of the erythrocytes further during hypotonic osmotic stress. The mechanism of diltiazem-dependent hemolysis could involve various ion transport pathways (i.e. Ca pump, Ca Channels) and subsequent effects on cell volume control or membrane fragility.

In vivo and in vitro bradycardia induced by local anesthetics is potentiated by calcium channel blockers.

The present study examined the interactions of local anesthetics (LA) and calcium channel blockers (CCBs) on rhythmicity of heart using in vivo and in vitro experiments. ECG recordings were made from the anesthetized rats for in vivo preparations and spontaneously beating isolated rat right atrial potential for the in vitro experiments. The in vivo experiments with LA showed dose-dependent bradycardia with lignocaine (LIG, 100-500 microg/kg) and bupivacaine (BUP, 10-100 microg/kg). BUP was 4-5 times more potent than LIG. Verapamil (VML) and diltiazem (DTZ), CCBs also produced dose (10-100 microg/kg) -dependent bradycardia. However, none of them affected the PR/QT interval or QRS complex. Further, LA-induced bradycardia was potentiated by CCBs. In addition, flattening of P-wave in ECG was observed with doses (10-25 microg/kg) of LA in the presence of CCBs. Similarly, the in vitro experiments demonstrated a concentration-dependent decrease in atrial rate by BUP or VML. The BUP-induced decrease was potentiated in the presence of VML. Thus, the results suggest that CCBs potentiate the LA-induced bradycardia by involving pacemaker activity. Further, the flattening of P-wave in ECG serves as an early indicator of the cardiotoxicity produced by these drugs.

Verapamil toxicity dysregulates the phosphatidylinositol 3-kinase pathway.

OBJECTIVES: Recent animal research and clinical case reports suggest benefit from high-dose insulin therapy (HDIT) for the treatment of calcium channel blocker (CCB) toxicity. One molecular signaling pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, that contributes to CCB toxicity and the efficacy of HDIT, was examined for a role in this phenomenon. METHODS: A differentiated 3T3-L1 adipocyte model system was utilized to characterize metabolic and molecular signaling events dysregulated in response to acute CCB toxicity. Glucose uptake assays were performed in the presence of representatives of three classes of CCB drugs, and the ability of HDIT to reverse observed inhibition was assessed. Western blot analyses were utilized to probe which insulin-dependent signaling pathway was inhibited by CCB toxicity. RESULTS: Representative compounds from the dihydropyridine and phenylalkylamine classes of CCBs were more effective at inhibiting glucose uptake in differentiated 3T3-L1 adipocytes than a representative from the benzothiazepine class. Phosphorylation at serine 473 of the Akt protein (P-Akt), a protein representing a common pathway for insulin receptors (IR), insulinlike growth factor receptors (IGFR), and hybrid receptors formed by IR and IGFR subunits, was abolished in the presence of toxic doses of the phenylalkylamine CCB verapamil. Phosphorylation at serine 473 of Akt was rescued in the presence high concentrations of insulin. CONCLUSIONS: These data suggest that dysregulation of the insulin-dependent PI3K pathway is partially responsible for insulin resistance and the hyperglycemic state observed in response to acute CCB toxicity.

Aquatic toxicity of acetaminophen, carbamazepine, cimetidine, diltiazem and six major sulfonamides, and their potential ecological risks in Korea.

Pharmaceuticals are manufactured and used for specific biological functions in veterinary and human medicine. Their detection in the environment and their bioactivity have resulted in concern for potential adverse effects on non-target species. Notwithstanding recent attention for their occurrence in the environment, there are significant research gaps for existing pharmaceuticals with regard to their potential ecological consequences. In this study, the four most abundantly used pharmaceuticals in Korea, namely acetaminophen, carbamazepine, cimetidine, and diltiazem, and six sulfonamide related antibiotics, including sulfamethoxazole, sulfachlorpyridazine, sulfathiazole, sulfamethazine, sulfadimethoxine, and trimethoprim were examined for their acute aquatic toxicity employing a marine bacterium (Vibrio fischeri), a freshwater invertebrate (Daphnia magna), and the Japanese medaka fish (Oryzias latipes). In general, Daphnia was the most susceptible among the test organisms. The most acutely toxic among the chemicals tested in this study was diltiazem, with a median lethal concentration of 8.2 mg/L for D. magna. The resulting acute toxicity of these pharmaceuticals was reasonably predicted by physicochemical descriptors such as pH-dependent distribution coefficient and EHOMO-ELUMO gap. Predicted environmental concentrations (PECs) derived for the test pharmaceuticals in Korea ranged between 0.14 and 16.5 microg/L. Hazard quotients derived from PECs and predicted no effect concentrations (PNECs) for sulfamethoxazole and acetaminophen were 6.3 and 1.8, respectively, suggesting potential environmental concerns and a need for further investigation.

Release behaviour and biocompatibility of drug-loaded pH sensitive particles.

The purpose of this work was to investigate the physical properties of drug-loaded poly(methacrylic acid-g-ethylene glycol) {P(MAA-g-EG)} particles, their biocompatibility with the gastrointestinal tract of rats and also the effects of these particles on the tight junctions of the rat intestinal epithelium. Model drugs such as diltiazem HCl, diclofenac Na, ciprofloxacin HCl and isoniazid were used in this study. P(MAA-g-EG) particles were prepared by free radical solution polymerization of methacrylic acid (MAA) and poly(ethylene glycol) (PEG). The loading efficiency of the model drugs in the particles and in vitro release profiles were investigated in pH 7.4 phosphate buffer and in gradually pH changing buffers (pH 1.2, 5.8, 6.8 and 7.4). The stability of free particles and drug-loaded particles was established by Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). In conclusion, P(MAA-g-EG) particles controlled the release rate of small molecular weight model drugs according to the pH of the medium. Stability of those particles loaded with drugs did not change in accelerated stability conditions. Histopathological results indicated that loading drugs to the particles prevented cell and tissue damage after 20 h. Free particles showed no change of tight junctions after 2 and 10 h. The results of TEM showed that increasing the amount of P(MAA-g-EG) particles from 100 to 385 mg clearly opened the tight junction, but with serious epithelial cell disruption.

Photostability and phototoxicity studies on diltiazem.

The photostability of diltiazem was studied in aqueous solutions at different pH values. Firstly, the hydrolysis of the drug to desacetyldiltiazem in alkaline medium was evaluated and then the drug photodegradation under exposure to UVA-UVB radiation (solar simulator) was monitored by HPLC methods. The main photoproduct was isolated and characterized as diltiazem-S-oxide on the basis of the NMR and mass spectra. The HPLC method was also applied to the selective analysis of diltiazem in commercial formulations. Tests on mutagenicity and photomutagenicity of the drug were also carried out using Salmonella typhimurium TA 102 strain. In this testing the drug neither was mutagenic nor toxic.

Effects of verapamil on atrial fibrillation and its electrophysiological determinants in dogs.

BACKGROUND: Atrial tachycardia-induced remodeling promotes the occurrence and maintenance of atrial fibrillation (AF) and decreases L-type Ca(2+) current. There is also a clinical suggestion that acute L-type Ca(2) channel blockade can promote AF, consistent with an AF promoting effect of Ca(2+) channel inhibition. METHODS: To evaluate the potential mechanisms of AF promotion by Ca(2+) channel blockers, we administered verapamil to morphine-chloralose anesthetized dogs. Diltiazem was used as a comparison drug and autonomic blockade with atropine and nadolol was applied in some experiments. Epicardial mapping with 240 epicardial electrodes was used to evaluate activation during AF. RESULTS: Verapamil caused AF promotion in six dogs, increasing mean duration of AF induced by burst pacing, from 8+/-4 s (mean+/-S.E.) to 95+/-39 s (P<0.01 vs. control) at a loading dose of 0.1 mg/kg and 228+/-101 s (P<0.0005 vs. control) at a dose of 0.2 mg/kg. Underlying electrophysiological mechanisms were studied in detail in five additional dogs under control conditions and in the presence of the higher dose of verapamil. In these experiments, verapamil shortened mean effective refractory period (ERP) from 122+/-5 to 114+/-4 ms (P<0.02) at a cycle length of 300 ms, decreased ERP heterogeneity (from 15+/-1 to 10+/-1%, P<0.05), heterogeneously accelerated atrial conduction and decreased the cycle length of AF (94+/-4 to 84+/-3 ms, P<0.005). Diltiazem did not affect ERP, AF cycle length or AF duration, but produced conduction acceleration similar to that caused by verapamil (n=5). In the presence of autonomic blockade, verapamil failed to promote AF and increased, rather than decreasing, refractoriness. Neither verapamil nor diltiazem affected atrial conduction in the presence of autonomic blockade. Epicardial mapping suggested that verapamil promoted AF by increasing the number of simultaneous wavefronts reflected by separate zones of reactivation in each cycle. CONCLUSIONS: Verapamil promotes AF in normal dogs by promoting multiple circuit reentry, an effect dependent on intact autonomic tone and not shared by diltiazem.