Improved seizure liability detection by combining rat hippocampal brain slice electrophysiology with in vivo behavior observation following intracerebroventricular drug administration.

An adverse effect of drug candidates, seizure is a serious issue in drug development. Improving evaluation systems for seizure liability is crucial for selecting good candidates. Firstly, in vitro electrophysiological measurement by a multielectrode array system in rat hippocampal brain slices was employed to confirm an increase in electrically evoked population spike (PS) area, the occurrence of multiple population spikes (MPSs), and thereby the seizure liability of five positive control chemicals: picrotoxin, 4-aminopyridine, pentylenetetrazole, penicillin G, and chlorpromazine. Aspirin, a negative control, did not affect PS area or generate MPSs. Furthermore, baclofen, an anticonvulsant drug, decreased PS area and inhibited the increase in PS area or occurrence of MPSs induced by picrotoxin. A comparative study of seizure liability among carbapenem antibiotics revealed that tienam > carbenin > omegacin and finibax. Despite leading to a strong decrease in PS area, physostigmine, cisplatin, and paroxetine still produced MPSs. Therefore, the increase in PS area or the occurrence of the MPS are considered significant evaluation parameters for seizure liability. In contrast, the in vitro electrophysiological measurement could not detect the seizure liability of diphenhydramine or fluvoxamine. A follow-up study of in vivo mouse behavioral change induced by intracerebroventricular administration of these drugs clearly detected convulsions. The in vitro electrophysiological study using hippocampal brain slices combined with in vivo behavior observation study of drug candidates administered by intracerebroventricular injection can implement to assess the seizure liability of even small amounts, especially in the early stages of drug development.

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

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

The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT4 receptor agonist, DSP-6952.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine4 (5-HT4) receptor agonists. DSP-6952 had a strong affinity of Ki = 51.9 nM for 5-HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 µM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT4 receptor antagonist, and another 5-HT4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 µM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Significance of integrated in silico transmural ventricular wedge preparation models of human non-failing and failing hearts for safety evaluation of drug candidates.

INTRODUCTION: To evaluate the usefulness of in silico assay in predicting drug-induced QTc prolongation and ventricular proarrhythmia, we describe in this study 2-dimensional transmural ventricular wedge preparation model (2D model) of non-failing (non-FH) and failing hearts (FH) based on O'Hara-Rudy dynamic model of human ventricular myocytes. METHODS: Using the prepared 2D model, we simulated ventricular action potential and recorded electrocardiogram for the non-FH and FH. The FH model was constructed based on differences in mRNA, protein, and/or current levels of ion channels between non-diseased heart and failing heart. To simulate the effects of selected drugs, we incorporated changes in ion channel conductance depending on the IC50 value and Hill coefficient at unbound drug blood concentrations. RESULTS: Dofetilide concentration-dependently induced QTc prolongation at therapeutic concentration in the 2D model of both non-FH and FH. The QTc prolongation in FH was longer than that in non-FH. These findings are consistent with previously reported clinical data. At supratherapeutic concentration 20nM, dofetilide induced Torsade de Pointes-like arrhythmia in the 2D non-FH model. In contrast, the single ventricular myocyte model did not quantitatively reproduce experimental data due to lack of electrotonic interaction. The simulated QTc change induced by six drugs examined in the IQ-CSRC prospective study was almost equivalent to that recorded in drug-treated healthy volunteers. DISCUSSION: Our 2D model with or without heart failure faithfully reproduced drug-induced QT prolongation and ventricular arrhythmias, suggesting that the in silico approach is a powerful tool for predicting cardiac safety of drug candidates at preclinical stage.

Inhibition of late sodium current attenuates ionic arrhythmia mechanism in ventricular myocytes expressing LaminA-N195K mutation.

BACKGROUND: Lamin A and C are nuclear filament proteins encoded by the LMNA gene. Mutations in the LMNA gene cause many congenital diseases known as laminopathies, including Emery-Dreifuss muscular dystrophy, Hutchinson-Gilford progeria syndrome, and familial dilated cardiomyopathy (DCM) with conduction disease. A missense mutation (N195K) in the A-type lamins results in familial DCM and sudden arrhythmic death. OBJECTIVE: The purpose of this study was to investigate the ion current mechanism of arrhythmia and DCM caused by the LaminA-N195K variant. METHODS: A homozygous mouse line expressing the Lmna-N195K mutation (LmnaN195K/N195K) that exhibited arrhythmia, DCM, and sudden death was used. Using whole cell patch-clamp technique, we measured action potential duration (APD), Na+ currents (INa) in ventricular myocytes isolated from LmnaN195K/N195K, and wild-type mice. RESULTS: Both peak and late INa were significantly (P <.05) increased in LmnaN195K/N195K ventricular myocytes. Similarly, LmnaN195K/N195K ventricular myocytes exhibited significant (P <.005) prolongation of APD (time to 50% [APD50] and 90% [APD90] repolarization) and triggered activity. Acute application of ranolazine inhibited late INa, shortened APD, and abolished triggered activity in LmnaN195K/N195K ventricular myocytes. CONCLUSION: Inhibition of late INa may be an effective therapy in preventing arrhythmia in patients with LmnaN195K mutation-related DCM.

Swallowing disorder and inhibition of cough reflex induced by atropine sulfate in conscious dogs.

In this study, the effects of atropine sulfate (atropine) on swallowing and cough reflex were evaluated in the two experimental models in conscious dogs. To evaluate the effects of atropine on swallowing, 1 mL of marker (contrast medium) was injected into the pharynx under X-ray exposure to induce swallowing. Baclofen, used as a positive control, caused marker congestion in the upper esophagus. In our experimental model, atropine (0.02 and 0.1 mg/kg, i.v.) dose-dependently increased not only the number of marker congestions but also that of the swallows. In addition, atropine significantly shortened the onset of first swallowing. In the evaluation of atropine effects on electrically evoked cough reflex induced by two electrodes implanted into the trachea, atropine strongly inhibited the number of coughs at 0.01 or 0.05 mg/kg accompanied with 0.01 or 0.05 mg/kg per hour (i.v.), respectively. These findings indicate that atropine has the potential of causing aspiration pneumonia through induction of swallowing disorder and inhibition of the cough reflex.

Stimulatory action of itopride hydrochloride on colonic motor activity in vitro and in vivo.

We investigated the effects of itopride hydrochloride (itopride, N-[4-[2-(dimethylamino)ethoxy]benzyl]-3,4-dimethoxybenzamide hydrochloride), a gastroprokinetic agent, on the colonic motor activity in vitro and in vivo, in comparison with benzamides, cisapride hydrate (cisapride), and mosapride citrate (mosapride). Itopride stimulated both peristaltic and segmental motility induced by applying intraluminal pressure to the isolated guinea pig colon. Although cisapride and mosapride enhanced the segmental motility, they markedly reduced the peristaltic motility. In conscious dogs with implanted strain gauge force transducers, itopride stimulated contractile activity in the gastrointestinal tract from the stomach to the colon. Cisapride stimulated contractile activity in the gastric antrum, ileum, and ascending colon. Mosapride stimulated contractile activity only in the gastric antrum and ileum. In guinea pigs and rats, itopride accelerated colonic luminal transit. On the other hand, cisapride and mosapride failed to enhance colonic transit. These results demonstrate that itopride has a stimulatory action on colonic peristalsis, propelling colonic luminal contents, different from that of cisapride and mosapride. Therefore, itopride may be a useful drug for the treatment of functional bowel disorders such as functional constipation.