Atsuhiko T. Naito

Effects of moxifloxacin on the proarrhythmic surrogate markers in healthy Filipino subjects: Exposure-response modeling using ECG data of thorough QT/QTc study

Effects of moxifloxacin on QTc as well as proarrhythmic surrogate markers including J-Tpeakc, Tpeak-Tend and short-term variability (STV) of repolarization were examined by using both standard E14 time-based evaluation and exposure-response modeling. The study was conducted with a single-blind, randomized, single-dose, placebo-controlled and two-period cross-over design in healthy Filipino subjects. QT interval was corrected by Fridericias formula (QTcF). In the E14 time-based evaluation of ECG data, the largest ΔΔQTcF with 90% confidence interval was 14.1xa0ms (11.2-16.9) with Cmax of 3.39xa0μg/mL at 3xa0h post-dose (nxa0=xa069; male: 35, female: 34), indicating a positive effect on the QTcF. Moxifloxacin significantly increased the ΔΔJ-Tpeakc and ΔΔTpeak-Tend, whereas the ΔΔSTV was not altered. Meanwhile in the exposure-response modeling of the same ECG data, the slope of moxifloxacin plasma concentration-ΔΔQTcF relationship was 4.84xa0ms per μg/mL and the predicted ΔΔQTcF with 90% confidence interval was 13.8xa0ms (13.1-15.1) at Cmax, also indicating a positive effect on the QTcF. Importantly, results in each proarrhythmic surrogate marker obtained by the exposure-response modeling also showed high similarity to those obtained by the E14 statistical evaluation. Thus, these results of moxifloxacin may become a guide to estimate proarrhythmic potential of new chemical entities.

Analysis of proarrhythmic potential of an atypical antipsychotic drug paliperidone in the halothane-anesthetized dogs

Fatal cases with the use of atypical antipsychotic drug paliperidone have been reported; however, there was no clinical report describing paliperidone-induced torsade de pointes. In this study we assessed its electropharmacological effects together with its proarrhythmic potential in intravenous doses of 0.03, 0.3 and 3xa0mg/kg using the halothane-anesthetized dogs (nxa0=xa05), which could provide approximately 2, 20 and 200 times higher peak plasma drug concentrations than its therapeutic level, respectively. Paliperidone exerted potent vasodilator effect resulting in hypotension, which may be largely explained by its α1-adrenoceptor blocking action. Inxa0vivo electrophysiological results suggest that paliperidone may inhibit human ether-à-go-go-related gene K+ channel in a dose-related manner and modestly suppress Na+ channel in the in situ heart. The high dose of paliperidone may have some potential to induce early afterdepolarization that can trigger lethal ventricular arrhythmias, whereas the low and middle doses lack such proarrhythmic possibility, indicating that at least 20 times higher plasma concentration may be considered to be safe.

Analysis of torsadogenic and pharmacokinetic profile of E-4031 in dogs bridging the gap of information between in vitro proarrhythmia assay and clinical observation in human subjects

We analyzed torsadogenic and pharmacokinetic profile of E-4031 using chronic atrioventricular block dogs. E-4031 in intravenous doses of 0.03, 0.1 and 0.3xa0mg/kg over 10xa0min prolonged QT/QTc, and increased short-term variability of QT in a dose-related manner (nxa0=xa04), resulting in onset of torsade de pointes in 1 animal after the middle dose and 4 animals after the high dose, while it attained peak plasma concentrations of 16.5, 60.5 and 182.5xa0ng/mL at 10xa0min after their start of administration, respectively (nxa0=xa02). These results bridge the gap of information between inxa0vitro proarrhythmia assay and clinical observation in human subjects.

Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD) of hiPSC-CMs have been corrected by clinically used Fridericias and/or Bazetts formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDcxa0=xa0FPD/RR0.22 with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action.

Sensitivity and Reliability of Halothane-Anaesthetized Microminipigs to Assess Risk of Drug-induced Long QT Syndrome

Using moxifloxacin and terfenadine, which are known to induce benign and malignant QT interval prolongation, respectively, we analysed whether halothane‐anaesthetized microminipigs are an appropriate model for assessing the risk of drug‐induced long QT syndrome. Moxifloxacin (0.03, 0.3 and 3 mg/kg) and terfenadine (0.03, 0.3 and 3 mg/kg) were intravenously infused over 10 min. with a pause of 20 min. to the halothane‐anaesthetized microminipigs (n = 4 for each drug). Moxifloxacin decreased the heart rate, whereas it increased the blood pressure in a dose‐related manner. It also prolonged the PR interval and QT/QTc in a dose‐related manner without altering the QRS width. Terfenadine decreased the heart rate and blood pressure, whereas it prolonged the PR interval, QRS width and QT/QTc in a dose‐related manner. Terfenadine significantly prolonged the beat‐to‐beat variability of QT interval reflecting its pro‐arrhythmic potential, which was not observed with moxifloxacin. The peak plasma concentrations of moxifloxacin and terfenadine after doses of 3 mg/kg were 4.81 and 10.15 μg/mL, respectively, which were both 1.5 times less in microminipigs than those previously reported in dogs. These results indicate that halothane‐anaesthetized microminipigs would be useful for detecting drug‐induced cardiovascular responses as well as differentiating benign from malignant QT interval prolongation like dogs, although there may be some differences in pharmacokinetic profile between these animals.

PTGER2-β-Catenin Axis Links High Salt Environments to Autoimmunity by Balancing IFNγ and IL-10 in FoxP3+ Regulatory T cells

Foxp3+ regulatory T cells (Tregs) are the central component of peripheral immune tolerance. While dysregulation of the Treg cytokine signature has been observed in autoimmune diseases such as multiple sclerosis (MS) and type 1 diabetes, the regulatory mechanisms balancing pro- and anti-inflammatory cytokine production are not known. Here, we identify imbalance between IFNγ and IL-10 as a shared Treg signature, present in patients with MS and under high salt conditions. By performing RNA-seq analysis on human Treg subpopulations, we identify β-catenin as a key regulator that controls the expression of IFNγ and IL-10. The activated β-catenin signature is enriched specifically in IFNγ+Tregs in humans, and this was confirmed in vivo with Treg-specific β-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional, IFNγ-producing, Treg phenotype. Moreover, we identify PTGER2 as a major factor balancing IFNγ and IL-10 production in the context of a high salt environment, with skewed activation of the β-catenin/SGK1/Foxo axis in IFNγ+Tregs. These findings identify a novel molecular mechanism underlying inflammatory Tregs in human autoimmune disease and reveal a new role for a PTGER2-β-catenin loop in Tregs linking environmental high salt conditions to autoimmunity.

Electropharmacological characterization of microminipigs as a laboratory animal using anti-influenza virus drug oseltamivir

We analyzed electropharmacological characteristics of microminipigs under halothane-anesthesia using anti-influenza virus drug oseltamivir, which has been known to possess multi-channel blocking properties, including Na+, Ca2+ and K+ channels (n = 4). Oseltamivir in doses of 0.3, 3 and 30 mg/kg was intravenously infused over 10 min with an interval of 20 min, which provided peak plasma concentrations 1.4, 7.4 and 125.5 µg/mL, respectively. The low dose did not alter any of the cardiovascular variables. The middle dose decreased the heart rate at 30 min after the start of the infusion. The high dose transiently returned the heart rate toward the baseline for 10-15 min, but decreased it for 20-60 min; decreased the mean blood pressure for 5-60 min; prolonged the PR interval for 10-60 min, and the QRS width for 10-20 min; but shortened the QT interval for 10-30 min, and the QTc for 5-60 min. Thus, oseltamivir can suppress the sinus automaticity, and atrioventricular nodal and intraventricular conduction; and decrease the mean blood pressure, extents of which were greater in microminipigs than in beagle dogs in our previous observation in spite of similar plasma concentrations, reflecting higher sensitivity of microminipigs for Na+ and Ca2+ channel inhibition than that of beagle dogs. In contrast to beagle dogs, oseltamivir shortened the repolarization period in microminipigs, indicating that oseltamivir can more potently inhibit the inward currents than the outward ones in the hearts of microminipigs. This information may help improve utilizatione of microminipigs as a laboratory animal.

Comparison of electropharmacological effects between terfenadine and its active derivative fexofenadine using a cross-over study in halothane-anesthetized dogs to analyze variability of pharmacodynamic and pharmacokinetic profiles of terfenadine and torsadogenic risk of fexofenadine

In order to better understand the variability of pharmacodynamic and pharmacokinetic profiles of terfenadine between the previous studies as well as to qualitatively and quantitatively examine the proarrhythmic potential of its major active metabolite fexofenadine in comparison with that of terfenadine, we directly compared their electropharmacological effects with halothane-anesthetized dogs (n = 3). For this purpose, we adopted a cross-over design, which can directly compare the effects of terfenadine and fexofenadine under the identical metabolic condition. Terfenadine in doses of 0.03 and 0.3 mg/kg increased the mean blood pressure, but that of 3 mg/kg decreased it. Terfenadine also increased the heart rate and ventricular contractility in a dose-related manner; but delayed the atrioventricular nodal and intraventricular conductions as well as repolarization suggesting its proarrhythmic potential. Meanwhile, fexofenadine in the same dose increased the mean blood pressure in a dose-related manner without affecting any of the electrophysiological variables in the same animals that proarrhythmic risk of terfenadine was confirmed, indicating its lack of proarrhythmic risk. Peak plasma concentrations for fexofenadine were 3.7, 8.1 and 11.2 times greater than for terfenadine at each matching dose, indicating terfenadine may be metabolized much faster than fexofenadine. Taken together, after the low and middle doses of terfenadine, vasopressor effect of a metabolite fexofenadine could be greater than the depressor effect of parent compound terfenadine, but its reverse would be correct after the high dose. Thus, the cross-over analysis can be an effective way to better understand drug-induced cardiovascular responses.

Application of human induced pluripotent stem cell-derived cardiomyocytes sheets with microelectrode array system to estimate antiarrhythmic properties of multi-ion channel blockers

We examined electrophysiological indices of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) sheets in order to quantitatively estimate Na+, K+ and Ca2+ channel blocking actions of bepridil and amiodarone using microelectrode array system in comparison with that of E-4031. We analyzed the field potential duration, effective refractory period, current threshold and conduction property using a programmed electrical stimulation protocol to obtain the post repolarization refractoriness and coefficient a of the relationship between the pacing cycle length and field potential duration. Electropharmacological profile of each drug was successfully characterized; namely, 1) the changes in the current threshold and conduction property provided basic information of Na+ channel blocking kinetics, 2) the relationship between pacing cycle length and field potential duration reflected drug-induced inhibition of human ether-à-go-go-related gene (hERG) K+ channel, 3) the post repolarization refractoriness indicated the relative contribution of these drugs to Na+ and K+ channel blockade, and 4) L-type Ca2+ channel blocking action was more obvious in the field potential waveform of the hiPSC-CMs sheets than that expected in the electrocardiogram in humans. Thus, this information may help to better utilize the hiPSC-CMs sheets for grasping the properties and net effects of drug-induced Na+, Ca2+ and K+ channel blockade.

Changes of electrocardiogram and hemodynamics in response to dipyridamole: In vivo comparative analyses using anesthetized beagle dogs and microminipigs

Microminipigs are expected as a novel animal model for cardiovascular pharmacological experiments. Since inherent vulnerability of coronary circulation of microminipigs has not been characterized, we performed dipyridamole-stress test to both microminipigs and beagle dogs, and compared the results. Dipyridamole in doses of 0.056 and 0.56xa0mg/kg were intravenously infused over 10xa0min (nxa0=xa04 for each animal). Dipyridamole decreased the systolic/diastolic blood pressures and double product in dogs as well as in microminipigs; but it did not significantly alter the heart rate or the global balance between the myocardial oxygen demand and supply in either animal. While organic coronary arterial stenosis was not detected in either animal, dogs have well-developed epicardial intracoronary networks unlike microminipigs. Like in humans, dipyridamole did not affect the ST segment of microminipigs, whereas it substantially depressed that in dogs. The results indicate the onset of subendocardial ischemia by dipyridamole in dogs may be partly associated with their well-developed native coronary collateral channels. Microminipigs would be more useful to evaluate the drugs which may affect the coronary circulation in the pre-clinical study than dogs.