To construct a non-clinical database for drug-induced QT interval prolongation, the electrophysiological effects of 11 positive and 10 negative compounds on action potentials (AP) in guinea-pig papillary muscles were investigated in a multi-site study according to a standard protocol. Compounds with a selective inhibitory effect on the rapidly activated delayed rectifier potassium current (IKr) prolonged action potential duration at 90% repolarization (APD90) in a concentration-dependent manner, those showing Ca2+ current (ICa) inhibition shortened APD30, and those showing Na+ current (INa) inhibition decreased action potential amplitude (APA) and Vmax. Some of the mixed ion-channel blockers showed a bell-shaped concentration-response curve for APD90, probably due to their blockade of INa and/or ICa, sometimes leading to a false-negative result in the assay. In contrast, all positive compounds except for terfenadine and all negative compounds with IKr-blocking activity prolonged APD30–90 regardless of their INa- and/or ICa-blocking activities, suggesting that APD30–90 is a useful parameter for evaluating the IKr-blocking activity of test compounds. Furthermore, the assay is highly informative regarding the modulation of cardiac ion channels by test compounds. Therefore, when APD90 and APD30–90 are both measured, the action potential assay can be considered a useful method for assessing the risk of QT interval prolongation in humans in non-clinical safety pharmacology studies. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-A1
Sixteen pharmaceutical companies and 6 contract research organizations in QT PRODACT acquired data on the action potentials in isolated guinea-pig papillary muscles using a standard protocol established by the QT PRODACT. Inter- and intra-facility variability for each of the parameters in the pre-application values and Δ% change after vehicle (0.1% DMSO) or dl-sotalol (30 μmol/L) treatment were examined using a nested model. Inter-facility variability of each of the parameters in the pre-application values were Vmax > APDs = APD30–90 > APA = RMP (descending order). The inter-facility variability of all of the parameters was almost the same or was less as compared with the intra-facility variability. Inter-facility variability for the Δ% change for each parameter after dl-sotalol treatment showed a tendency similar to the results for the pre-application values. Comparing the inter- and the intra-facility variability, the inter-facility variation did not exceed the intra-facility variation. All facilities found that dl-sotalol prolonged APD. Therefore, it is suggested that the test system using this standard protocol is useful as a non-clinical evaluation system for QT prolongation. Moreover, the results are considered to be directly comparable between multiple facilities. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-B5.
Certain compounds that prolong QT interval in humans have little or no effect on action-potential (AP) duration used traditionally, but they inhibit rapidly-activated-delayed-rectifier potassium currents (IKr) and/or human ether-a-go-go-related gene (hERG) currents. In this study using isolated guinea-pig papillary muscles, we investigated whether new parameters in AP assays can detect the inhibitory effects of various compounds on IKr and/or hERG currents with high sensitivity. The difference in AP duration between 60% and 30% repolarization, 90% and 60% repolarization, and 90% and 30% repolarization (APD30–60, APD60–90, and APD30–90, respectively) were calculated as the new parameters. All the 15 IKr and/or hERG current inhibitors that have been reported (9 compounds) or not reported (6 compounds) to inhibit calcium currents prolonged APD30–60, APD60–90, and/or APD30–90; and 8 of the 15 inhibitors prolonged APD30–60, APD60–90, and/or APD30–90 more potently than APD90. The APD30–60, APD60–90, and APD30–90 measurements revealed no difference in sensitivity when evaluating the effects of the IKr and/or hERG current inhibitors on the three parameters. On the other hand, compounds with little or no effect on hERG currents had no effect on APD30–60, APD60–90, or APD30–90. Therefore, it is concluded that in AP assays using isolated guinea-pig papillary muscles, APD30–60, APD60–90, and APD30–90 are useful indexes for evaluating the inhibitory effects of compounds including mixed ion-channel blockers on IKr and/or hERG currents. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-C8
The goal of the present study was to examine the utility of the conscious dog model by assessing the QT-interval-prolonging potential of ten positive compounds that have been reported to induce QT interval prolongation in clinical use and seven negative compounds considered not to have such an effect. Three doses of test compounds or vehicle were administered orally to male beagle dogs (n = 4), and telemetry signals were recorded for 24 h after administration. All positive compounds (astemizole, bepridil, cisapride, E-4031, haloperidol, MK-499, pimozide, quinidine, terfenadine, and thioridazine) caused a significant increase in the corrected QT (QTc) interval, with a greater than 10% increase achieved at high doses. In contrast, administration of negative compounds (amoxicillin, captopril, ciprofloxacin, diphenhydramine, nifedipine, propranolol, and verapamil) did not produce any significant change in the QTc interval, with the exception of nifedipine that may have produced an overcorrection of the QTc interval due to increased heart rate. The estimated plasma concentrations of the positive compounds that caused a 10% increase in the QTc interval were in good agreement with the plasma/serum concentrations achieved in humans who developed prolonged QT interval or torsade de pointes (TdP). Although careful consideration should be given to the interpretation of QT data with marked heart rate change, these data suggest that an in vivo QT assay using the conscious dog is a useful model for the assessment of QT interval prolongation by human pharmaceuticals. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-A2
The purpose of this study was to assess the utility of the isoflurane-anesthetized dog model for detecting the potential for QT interval prolongation by human pharmaceuticals. The effects of 10 positive compounds with torsadogenic potential, 8 negative compounds with little torsadogenic potential, and dl-sotalol as a common positive compound were evaluated in 5 facilities in accordance with the common protocol approved by QT PRODACT. Each test compound was cumulatively infused into male beagle dogs anesthetized with isoflurane. Surface lead II ECG, blood pressure, and plasma concentrations for the positive compounds were measured. Repeated administration of the vehicle examined in each facility before the start of the experiments resulted in a slight, but not significant, change in corrected QT (QTc) interval, indicating that this model only shows slight experimental variation. Although an inter-facility variability in the extent of dl-sotalol-induced QT interval prolongation was observed, dl-sotalol significantly prolonged QTc interval in all facilities. All positive compounds significantly prolonged QTc interval at plasma levels up to 10 times those in patients who developed prolonged QTc interval or TdP, whereas no negative compounds did so. These data suggest that the in vivo QT assay using the anesthetized dog is a useful model for detecting the potential for QT interval prolongation by human pharmaceuticals. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-A3
In safety pharmacology studies, the effects on the QT interval of electrocardiograms are routinely assessed using a telemetry system in cynomolgus monkeys. However, there is a lack of integrated databases concerning in vivo QT assays in conscious monkeys. As part of QT Interval Prolongation: Project for Database Construction (QT PRODACT), the present study examined 10 positive compounds with the potential to prolong the QT interval and 6 negative compounds considered to have no such effect on humans. The experiments were conducted at 7 facilities in accordance with a standard protocol established by QT PRODACT. The vehicle or 3 doses of each test compound were administered orally to male cynomolgus monkeys (n = 3 – 4), and telemetry signals were recorded for 24 h. None of the negative compounds prolonged the corrected QT using Bazett’s formula (QTcB) interval. On the other hand, almost all of the positive compounds prolonged the QTcB interval, but haloperidol, terfenadine, and thioridazine did not. The failure to detect the QTcB interval prolongation appeared to be attributable for the differences in metabolism between species and/or disagreement with Bazett’s formula for tachycardia. In the cynomolgus monkeys, astemizole induced Torsade de Pointes and cisapride caused tachyarrhythmia at lower plasma concentrations than those observed in humans and dogs. These results suggest that in vivo QT assays in conscious monkeys represent a useful model for assessing the risks of drug-induced QT interval prolongation. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-A4
To investigate whether miniature pigs are useful for evaluating the potential of drugs for drug-induced prolongation of the QT interval, we performed an in vivo QT assay using conscious and unrestricted miniature pigs. Compared with the vehicle average baseline values, haloperidol at 3 and 10 mg/kg, p.o. prolonged the QTcF interval (Fridericia’s formula) by 8% – 16%. The plasma concentration of haloperidol at which QT interval was prolonged (Cmax = 42.9 ng/mL) was almost equal to that in humans. dl-Propranolol at 3, 10, and 30 mg/kg, p.o. caused no alterations in QT interval. dl-Propranolol at 3, 10, and 30 mg/kg, at which plasma concentrations were lower than in humans treated with dl-propranolol at the therapeutic dose level, shortened QTcF interval by 7% – 12%. dl-Sotalol at 10 mg/kg, p.o. prolonged QTcF interval by 7%. From the above results, we considered that the miniature pig can be used for prediction of drug-induced prolongation of QT interval in humans, and thus, it is one of the useful animal species for assessing electrocardiograms in safety pharmacology studies.
In safety pharmacology studies, the effect of a test compound on the electrocardiogram is routinely examined by using conscious dogs. However, the results may be widely variable. The monkey, on the other hand, has scarcely been used for such studies; and as yet, there have not been reported studies on monkeys conducted at several facilities with a standard protocol. In the present study, we examined inter-facility variabilities in electrocardiographic and hemodynamic parameters as described below. We analyzed the data from 8 facilities (9 test groups) on dogs and 5 facilities (7 test groups) on monkeys. This data was obtained from the studies employing the following standard protocol: dl-Sotalol or a vehicle (0.5 w/v% methylcellulose solution) was given to animals; and the PR interval, QRS duration, QT interval, heart rate, and mean blood pressure were determined time-sequentially before and after administration of the vehicle or dl-sotalol in each test group. dl-Sotalol produced a prolongation of the maximum mean QTcF interval in conscious dogs and QTcB interval in conscious monkeys by more than 10% in every test group. No difference in the corrected QT interval among the test groups was observed in dogs, but a difference was observed in monkeys. Supplementary material (Appendix): available only at http://dx.doi.org/10.1254/jphs.QT-B6
The purpose of this investigation was to define the sensitivity and specificity of the canine telemetry assay for detecting drug-induced QT interval prolongation. Data from twelve studies generated in the QT PRODACT project were used in this investigation. The study design was a 4 × 4 Latin square cross-over design and included the following drugs: MK-499, E-4031, terfenadine, haloperidol, cisapride, bepridil, propranolol, diphenhydramine, captopril, verapamil, amoxicillin, and ciprofloxacin. The estimated root squared error of the model, which estimated the slope of the QT-RR relationships for each animal, for all dogs during the pre-dosing period was 5.45%. Using the QT-RR model, the sensitivity and specificity in each cutoff value that judges QT prolongation were estimated based on the experiment errors and measurement errors in the 12 studies. When the cutoff value was 5%, the sensitivity in 10% prolongation was 0.978 and the specificity in 0% was 0.996. In conclusion, it was judged that a 5% cutoff value for changes in heart rate corrected QT interval using the canine telemetry assay is practical, and the sensitivity and specificity of the telemetry assay are very high when using the analytical method presented here. Based upon this information, the canine telemetry assay using the individual subject heart rate correction model is recommended as a sensitive test system for the in vivo assessment of risk for QT interval prolongation.
Drug concentrations that would prolong repolarization parameters by 10%, including action potential duration (APD90, APD30 – 90), in in vitro assays using guinea-pig papillary muscle and QTc intervals in in vivo assays using conscious dogs, conscious monkeys, and anesthetized dogs were compared. Although, both the in vitro and in vivo assays showed concentration-dependent responses for compounds that have been classified as torsadogenic in humans, only a weak correlation in EC10 values was observed between the in vitro and in vivo assays. Among the in vivo QT assays, the EC10 values obtained from conscious dogs, conscious monkeys, and anesthetized dogs correlated well with each other, but the EC10 values in monkeys were somewhat lower in comparison to those in dogs. When in vivo QT assay EC10 values were compared to the respective human effective therapeutic plasma concentration (ETPC), the ratios of EC10 values to ETPCs were less than 20 for most torsadogenic compounds. In conclusion, the relationships between the extent of QTc interval prolongation and the concentration of drugs was highly consistent among the three in vivo models, suggesting that the ratios of EC10 values in in vivo QT assays are useful for estimating the safety margin of drugs that prolong the QTc interval.