Validation of Risk-Stratification Method for the Chronic Atrioventricular Block Cynomolgus Monkey Model and Its Mechanistic Interpretation Using 6 Drugs With Pharmacologically Distinct Profile.

Validation of risk-stratification method for the chronic atrioventricular block cynomolgus monkey model and its mechanistic interpretation was performed using 6 pharmacologically distinct drugs. The following drugs were orally administered in conscious state, astemizole: 1, 5, and 10 mg/kg (n = 6); haloperidol: 1, 10, and 30 mg/kg (n = 5); amiodarone: 30 mg/kg (n = 4); famotidine: 10 mg/kg (n = 4); levofloxacin: 100 mg/kg (n = 4); and tolterodine: 0.2, 1, and 4.5 mg/kg (n = 4). Astemizole of 5 and 10 mg/kg significantly prolonged ΔΔQTcF, whereas no significant change was observed by the others. Torsade de pointes (TdP) was induced by astemizole of 5 and 10 mg/kg in 3/6 and 6/6, and by haloperidol of 10 and 30 mg/kg in 1/5 and 1/5, respectively, which was not observed in the others. Torsadogenic risk of the drugs was quantified using the criteria for the monkey model specified in our previous study. Namely, high-risk drugs induced TdP at ≤ 3 times of their maximum clinical daily dose. Intermediate-risk drugs did not induce TdP at this dose range, but induced it at higher doses. Low/no-risk drugs never induced TdP at any dose tested. The magnitude of risk was intermediate for astemizole and haloperidol, and low/no risk for the others. The prespecified, risk-stratification method for the monkey model may solve the issue existing between nonclinical models and patients with labile repolarization, which can reinforce the regulatory decision-making and labeling at time of marketing application of nondouble-negative drug candidate (hERG assay positive and/or in vivo QT study positive).

Usefulness of cardiotoxicity assessment using calcium transient in human induced pluripotent stem cell-derived cardiomyocytes.

Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be an attractive strategy for assessing compounds on cardiac contractility. In addition, as arrhythmogenic compounds are known to induce characteristic waveform changes in hiPSC-CMs, it is expected that calcium transient would allow evaluation of not only compound-induced effects on cardiac contractility, but also compound arrhythmogenic potential. Using a combination of calcium transient in hiPSC-CMs and a fast kinetic fluorescence imaging detection system, we examined in this study changes in calcium transient waveforms induced by a series of 17 compounds that include positive/negative inotropic agents as well as cardiac ion channel activators/inhibitors. We found that all positive inotropic compounds induced an increase in peak frequency and/or peak amplitude. The effects of a negative inotropic compound could clearly be detected in the presence of a ß-adrenergic receptor agonist. Furthermore, most arrhythmogenic compounds raised the ratio of peak decay time to peak rise time (D/R ratio) in calcium transient waveforms. Compound concentrations at which these parameters exceeded cutoff values correlated well with systemic exposure levels at which arrhythmias were reported to be evoked. In conclusion, we believe that peak analysis of calcium transient and determination of D/R ratio are reliable methods for assessing compounds' cardiac contractility and arrhythmogenic potential, respectively. Using these approaches would allow selection of compounds with low cardiotoxic potential at the early stage of drug discovery.

Visualizing voltage dynamics in zebrafish heart.

The zebrafish heart provides a useful vertebrate cardiovascular model with outstanding advantages, including genetic manipulatability, optical accessibility and rapid development. In addition, an emerging topic in cardiotoxicity assay and drug discovery is its use in phenotype-based chemical screening. Here, we report a technique that allows non-invasive voltage mapping in beating heart using a genetically encoded probe for transmembrane potential. Application of the anti-allergy compound astemizole resulted in aberrant propagation of excitation, which accounted for a lack of ventricular contraction. This optical method will provide new opportunities in broad areas of physiological, developmental and pharmacological cardiovascular research.

Simultaneous gene expression signature of heart and peripheral blood mononuclear cells in astemizole-treated rats.

We investigated the effects of astemizole, a second-generation antihistamine, on the heart and peripheral blood mononuclear cells (PBMCs) and identified the early markers of its cardiotoxicity using gene expression profiling. Astemizole causes torsades de pointes, which is a type of ventricular tachycardia. We administered astemizole (dosage: 20, 60 mg/kg) to male Sprague-Dawley rats, using an oral gavage. Cardiac tissue and PBMCs were collected from the rats 4 h after treatment. Gene expression profiles were obtained using an Affymetrix GeneChip. The most deregulated genes were associated with energy metabolism pathways and calcium ion homeostasis in the heart of astemizole-treated rats. The most altered genes in the PBMCs were those involved in developmental processes and cardiotoxicity. Genes related to the response to oxidative stress, reactive oxygen species, heat shock proteins, hypoxia, immunity, and inflammation were also deregulated in the heart and PBMCs. These data provide further insight into the genetic pathways affected by astemizole. In addition, the simultaneously deregulated genes identified herein may be further studied. It will be interesting to find out whether single genes or certain sets of these genes could finally serve as biomarkers for cardiotoxicity of astemizole or other similar antihistamine drugs.

Improved methodology for identifying the teratogenic potential in early drug development of hERG channel blocking drugs.

Drugs blocking the potassium current IKr of the heart (via hERG channel-inhibition) have the potential to cause hypoxia-related teratogenic effects. However, this activity may be missed in conventional teratology studies because repeat dosing may cause resorptions. The aim of the present study was to investigate an alternative protocol to reveal the teratogenic potential of IKr-blocking drugs. The IKr blocker astemizole, given as a single dose (80 mg/kg) on gestation day (GD) 13 to pregnant rats caused digital defects. In whole rat embryo culture (2h) on GD 13, astemizole caused a decrease in embryonic heart rate at 20 nM, and arrhythmias at 200-400 nM. Cetirizine, without IKr-blocking properties, did not affect the rat embryonic heart in vitro. The present study shows that single dose testing on sensitive days of development, together with whole embryo culture, can be a useful methodology to better characterize the teratogenic potential of IKr-blocking drugs.

Temporal variability of QT interval and changes in T wave morphology in dogs as markers of the clinical risk of drug-induced proarrhythmia.

INTRODUCTION: The aim of this experiment was to establish the usefulness of assessing temporal variability of the QT interval and changes in the T wave morphology in dogs as markers of pro-arrhythmic risk in humans. For this purpose, the electrocardiographic effects of astemizole and cisapride, two well known pro-arrhythmic drugs in humans, were assessed in dogs. METHODS: Astemizole was administered intravenously at single doses of 1 and 3 mg/kg whilst cisapride was administered orally at doses of 1.5 and 6 mg/kg. Electrocardiograms (ECG) were recorded before and after treatment. From each ECG tracing, QT intervals were recorded over 100 beats for calculation of the mean and standard deviation (SD) of QT and mean corrected QT (QTc). The coefficient of variation of QT (CV=SD/mean) was calculated as an indicator of QT temporal variability. The changes in T wave morphology were assessed in precordial lead CV5RL. RESULTS: Astemizole increased both the QTc interval and the CV of QT. Increases in these parameters also occurred after cisapride, but were less marked than after astemizole. In addition, both compounds produced a notching of the T wave, consisting of the presence of two peaks. DISCUSSION: The effects of astemizole and cisapride on the CV of QT and their propensity to induce of T wave notching are consistent with the blocking of I(Kr) channels and indicate, respectively, an increase in temporal variability of cardiac repolarization and an increased heterogeneity of the repolarization of cardiac cells across the myocardium. These changes are key triggers of arrhythmic events and are thus consistent with the pro-arrhythmic properties of these drugs. This study therefore indicates that the evaluation of CV of QT and T wave morphology in dogs may help in predicting drug-induced pro-arrhythmic risk in humans.

Embryonic cardiac arrhythmia and generation of reactive oxygen species: common teratogenic mechanism for IKr blocking drugs.

In the adult organism, it is well established that hypoxia followed by reperfusion may be fatal and result in generation of reactive oxygen species (ROS) and subsequent tissue damage. There is also considerable evidence that temporary decrease or interruption in oxygen supply to the embryo and ROS generation during reperfusion result in tissue damage in embryonic tissues. A wide spectrum of different malformations by transient embryonic hypoxia could be produced, depending on the duration, extent, and timing of the hypoxic event. It is the contention of this paper that drugs that block the potassium channel IKr, either as an intended pharmacologic effect or as an unwanted side-effect, are potentially teratogenic by a common ROS related mechanism. Drugs blocking the IKr channel, such as almokalant, dofetilide, phenytoin, cisapride and astemizole, do all produce a similar pattern of hypoxia-related malformations. Mechanistic studies show that the malformations are preceded by embryonic cardiac arrhythmia and periods of hypoxia/reoxygenation in embryonic tissues. Pretreatment or simultaneous treatment with radical scavengers with capacity to capture ROS, markedly decrease the teratogenicity of different IKr blocking drugs. A second aim of this review is to demonstrate that the conventional design of teratology studies is not optimal to detect malformations caused by IKr blocking drugs. Repeated high doses result in high incidences of embryonic death due embryonic cardiac arrhythmia, thus masking their teratogenic potential. Instead, single dosing on specific days is proposed to be a better way to characterize the teratogenic potential of Ikr blocking drugs.

QT prolongation in guinea pigs for preliminary screening of torsadogenicity of drugs and drug-candidates. II.

Experimental approaches on anaesthetised guinea pigs have been shown recently to be satisfactorily predictive of the torsadogenic risk of drugs. This work aimed at obtaining additional data, for a further understanding of the reliability and/or the limits of this model. Clonidine (non-torsadogenic in humans) induced a lengthening of the ECG parameter of RR in anaesthetised guinea pigs, without any corresponding increase of QT (corrected by the algorithms of Bazett and Fridericia). Thus, 'QT correct' prolonging effects produced by drugs torsadogenic in humans, on the guinea pig model are primarily due to inhibition of cardiac repolarisation. The corresponding RR prolongation is a consequence (not the cause) of this primary effect. Astemizole, haloperidol and terfenadine, torsadogenic in humans, produced in Langendorff perfused guinea pig hearts a prolongation of the QT interval. Chlorprotixene (non-torsadogenic) did not produce any significant effect on QT. These results are fully consistent with previous observations in anaesthetised guinea pigs. In Langendorff perfused hearts, pentobarbital does not affect cardiac repolarisation and does not potentiate the QT-prolonging effect of astemizole. Together with the findings reported by many authors, these data suggest that ECG recording in anaesthetised guinea pigs is a reliable model for cardiac safety studies evaluating the influence of drugs on the repolarisation process.

Development of telemetry system in the common marmoset--cardiovascular effects of astemizole and nicardipine.

The purpose of this study was to evaluate a telemetry system for examining the cardiovascular system in the conscious common marmoset. Parameters obtained were blood pressure, heart rate, respiratory rate, ECG, body temperature and locomotor activity, and these were continuously recorded on a data recorder via the telemetry system and then processed by a computerized system. Diurnal rhythms of blood pressure, heart rate, body temperature and locomotor activity were observed in this system. We studied the effects of astemizole (antihistamine) and nicardipine (Ca2+ channel blocker) on cardiovascular parameters. Astemizole at 30 mg/kg (p.o.) and at 1 to 3 mg/kg (i.v.), prolonged QT interval and induced ventricular extrasystole. Torsades de pointes occurred in one of three cases at 3 mg/kg (i.v.) and 30 mg/kg (p.o.), while it did not affect the blood pressure, respiratory rate and body temperature. Nicardipine at 30 mg/kg (p.o.) caused sustained hypotension and tachycardia. These results demonstrate the usefulness of the telemetry system using the common marmoset for evaluating the cardiovascular effects of drugs under physiological conditions.

Comparative effects of maternal prenatal and postnatal exposures to astemizole on reproductive parameters of rats.

The prenatal and postnatal effects of administration of a nonsedative antihistamine H1, astemizole (ATZ), were compared. ATZ (10 mg/kg) was injected daily into female Wistar rats throughout pregnancy (prenatal treatment) or during the first 6 days of lactation (postnatal treatment). Neither treatment modified dam body weight. Prenatal exposure reduced offspring body weight and lead to a latter expression of the vaginal opening of female offspring. In addition, a long-term disruption of male reproductive behavior was observed, while female sexual behavior was not modified. The sexual activity index and the intromission frequency were increased in prenatally treated animals. Testes wet weight was reduced, but no modifications were detected in vas deferens or seminal vesicles. Postnatal treatment did not alter offspring body weight, open-field activity, sexual behavior and organ weight as well as did not delay testes descent but reduced the time until vaginal opening. Hypothalamic serotonin (5-HT), dopamine (DA) and noradrenaline (NA) as well as DA and NA metabolites were not modified by both prenatal and postnatal treatments. Increased striatal 3,4-dihydroxyphenylacetic acid (DOPAC) levels were observed after prenatal and postnatal treatments, while only postnatal 5-HT levels were increased. We propose that the present results indicate that prenatal ATZ exposure can have long-lasting organizational effects on reproductive behavior of male rats, while postnatal exposure to this drug did not alter mating behavior. In relation to female rats, prenatal and postnatal exposures to ATZ accelerated puberty but did not alter sexual behavior. Neurochemical data show that both treatments increased striatal dopaminergic system activity, suggesting a central ATZ effect after perinatal exposure.

Electrophysiological effects of cetirizine, astemizole and D-sotalol in a canine model of long QT syndrome.

Observations of torsades de pointes during therapy with terfenadine and astemizole has raised concern about the cardiac safety of non-sedating H1-antagonist agents. We compared cetirizine, another compound of that class, to D-sotalol and to astemizole in a model of acquired long QT syndrome. Open-chest surgery was performed in adult beagle dogs anaesthetized with halothane and thiopental. Bradycardia was produced with beta-adrenergic blockade and sinus node crush. Four left ventricular intramyocardial unipolar monophasic action potentials (MAP) were recorded during atrial pacing at basic cycle lengths (BCL) 400-1500 msec, before and during three successive 1-h drug infusions (0.14, 0.45 and 1.4 mg/kg/h for astemizole and cetirizine and 1.1, 2.2 and 4.5 mg/kg/h for D-sotalol). Dose- and bradycardia-dependent prolongations of MAP duration (MAPD) were produced by D-sotalol (P < 0.001) and astemizole (P < 0.001) but not by cetirizine. At BCL 1500 ms, the three infusions of astemizole prolonged endocardial MAPD from 323 +/- 8 msec (mean +/- SE) at baseline to 343 +/- 10, 379 +/- 13 and 468 +/- 26 msec, respectively (n = 9). Sotalol prolonged that MAPD from 339 +/- 6 msec to 377 +/- 7, 444 +/- 15 and 485 +/- 24 msec (n = 7). In contrast, cetirizine did not prolong MAPD: 341 +/- 8 msec at baseline Vs 330 +/- 8, 324 +/- 9 and 323 +/- 11 msec (n = 9). Drug-induced increase in transmural dispersion reached +79 +/- 19 msec after astemizole, +59 +/- 21 msec after D-sotalol and only +7 +/- 11 msec after cetirizine. Runs of ventricular tachycardias and torsades de pointes occurred during dose three of astemizole (5/9 dogs) and D-sotalol (4/7 dogs) but never during cetirizine. In the present model, astemizole and D-sotalol but not cetirizine prolonged MAPD and transmural dispersions of repolarization and produced torsades de pointes. These results suggest that the halothane-anaesthetized bradycardic dog could be a valuable model to discriminate drugs for their class III effects and proarrhythmic potencies.

Effects of nonsedating antihistamine, astemizole, on the in situ canine heart assessed by cardiohemodynamic and monophasic action potential monitoring.

The possible mechanisms of cardiac adverse effects of astemizole were studied using a halothane-anesthetized in vivo canine model under the cardiohemodynamic and monophasic action potential monitoring. A dose of 0.3 mg/kg of iv astemizole (n = 7), which is close to the recommended dose for clinical use, showed a bradycardic effect and a reversed use-dependent lengthening of repolarization. The increase in the repolarization was greater than in the effective refractory period. These effects persisted even when the plasma drug concentration became undetectable. Additional administration of 3.0 mg/kg of iv astemizole (n = 7) decreased the mean blood pressure, suppressed the cardiac contraction and conduction, and induced early after depolarization-like potential in addition to the qualitatively similar effects compared to those observed by the lower dose. The decrease of the plasma concentration of astemizole followed the pattern predicted by the two-compartment theory of pharmacokinetics, but the drug concentration in the cardiac muscle was estimated to be more than 100 times greater than that in plasma. Our study emphasizes that each cardiac consequence of astemizole overdose may be related to proarrhythmic effects and the monitoring of plasma drug concentration will be less helpful in predicting the cardiac adverse effects of astemizole. The results provide some insights into the clinical cardiotoxicity of astemizole. Drugs or interventions inducing positive chronotropic, inotropic, and dromotropic effects can become good candidates for the treatment of astemizole intoxication, which may attenuate the cardiac effects of astemizole including the lengthening of repolarization.

Carcinogenicity studies of astemizole in mice and rats.

The histamine H1 antagonist astemizole (Hismanal) was tested for carcinogenicity in Swiss mice and Wistar rats. Astemizole was administered with the food to mice for 18 and to rats for 24 consecutive months. The doses given--approximately 5, 20, and 80 mg/kg body weight.day--were equivalent to 25, 100, and 400 times, respectively, the recommended human dose of 10 mg/day. Survival of both mice and rats was comparable between groups. Peto's age-adjusted, dose-related trend analysis for the tumor-bearing rats did not reveal a statistically significant difference for males or females. There was no evidence that astemizole led to an increased incidence of spontaneously or unusually occurring neoplastic lesions in either mice or rats. Special attention was given to the effect of astemizole on the progression of spontaneously occurring mammary gland adenomas and fibroadenomas. Peto's analysis applied to the number of female rats bearing these benign mammary gland tumors disclosed no statistically significant dose-related trend. There was no positive trend for the onset of this tumor type, and the median size of the tumor over time per rat was also not statistically significantly different in a comparison of the control group with each of the dosed groups. The findings from these carcinogenicity studies suggest that astemizole is not tumorigenic and that it does not promote tumor growth.

Electrophysiological and arrhythmogenic effects of the histamine type 1-receptor antagonist astemizole on rabbit Purkinje fibers: clinical relevance.

Astemizole is a potent histamine H1-antagonist that has been associated with cases of life-threatening cardiac arrhythmias, including torsade de pointes and atrioventricular (AV) block. However, its effects on cardiac action potential (AP) has not been described. We examined the electrophysiological effects of astemizole on rabbit Purkinje fibers using conventional glass microelectrodes in parallel with the effects of the widely used histamine H2-antagonist cimetidine, selected because it has no known cardiac arrhythmic toxicity. Astemizole (0.01-3 microM) exerted a concentration-dependent prolonging effect on final repolarization that did not reach steady state after 3 h of exposure. This effect was more pronounced at low stimulation frequency and was less marked at high stimulation frequency. In addition, early afterdepolarizations (EADs) occurred in one third of the fibers. Increasing extracellular concentration of KCl (2.7-5.4 mM) or MgCl2 (1-5 mM) suppressed EADs and reversed the prolonging effect that was conversely exaggerated by decreasing KCl (4-2.7 mM) or MgCl2 (1-0.5 mM) concentration. At higher concentrations (3-30 microM), astemizole induced an increasing depressant effect on the maximal rate of depolarization (Vmax) that became more pronounced with high stimulation frequency. All parameters were strongly depressed at 10 microM astemizole, leading to cellular inexcitability in 5 of 12 fibers when exposed to 30 microM astemizole. In comparison, cimetidine induced minor changes on AP characteristics, i.e., a prolongation in plateau duration at high (30-100 microM) concentrations. These results provide evidence that astemizole exerts quinidine-like effects on cardiac APs that are compatible with the occurrence of the clinically observed arrhythmias.

Enhanced cancer growth in mice administered daily human-equivalent doses of some H1-antihistamines: predictive in vitro correlates.

BACKGROUND: Present studies of drug-induced tumor growth promotion have evolved from earlier investigations into the mechanism of action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy[ethanamine.HCl, a tamoxifen derivative which potently inhibits lymphocyte mitogenesis in vitro and stimulates tumor growth in vivo. It is thought that potency to bind to intracellular histamine receptors (HIC), some of which are on cytochromes P450, may correlate with tumor growth-promoting activity. PURPOSE: We assessed the effectiveness of five in vitro assays in predicting in vivo tumor growth stimulation by the H1-antihistamines loratadine, astemizole, cetirizine, hydroxyzine, and doxylamine. METHODS: Potency of each agent was ranked 1-5 in each of the following in vitro assays: 1) inhibition of [3H]histamine binding to microsomal HIC, 2) inhibition of histamine binding to microsomal P450, 3) inhibition of the P450-catalyzed demethylation of aminopyrine, 4) inhibition of lymphocyte mitogenesis, and 5) stimulation of tumor colony formation. An overall rank score was assigned to each drug and correlated with tumor growth stimulation in vivo. Two laboratories conducted in vivo studies in a blinded fashion. Female C57BL and C3H mice were given a subcutaneous injection on day 1 of syngeneic B16F10 melanoma cells (5 x 10(5)) or C-3 fibrosarcoma cells (1 x 10(5)), respectively. Mice were randomly assigned to treatment groups, then received a single, daily intraperitoneal injection of an estimated human-equivalent dose (or range of doses) of antihistamine or vehicle control for 18-21 days before being killed. Tumors were surgically removed and wet weights compared statistically among groups. RESULTS: The cumulative potency of each drug in affecting tumor growth or growth mechanisms in the five in vitro assays ranked as follows: Loratidine and astemizole ranked highest and were equally potent, followed in decreasing order by hydroxyzine, doxylamine, and cetirizine. A significant correlation (r = .97; P < .02) was observed between the rank order of potency of the antihistamines in all five in vitro assays and the rank order to enhance tumor growth in vivo: Loratidine and astemizole significantly (P < .001) promoted the growth of both melanoma and fibrosarcoma, hydroxyzine significantly (P < .001) promoted the growth of melanoma, while doxylamine and cetirizine did not promote the growth of either tumor. CONCLUSION: Data demonstrate that the in vitro assays predicted the propensity of each H1-antihistamine to stimulate cancer growth in vivo. IMPLICATION: These in vitro tests may prove valuable to screen potential tumor growth promoters.