Masaki Honda

Preclinical QT safety assessment: Cross-species comparisons and human translation from an industry consortium

INTRODUCTION In vivo models have been required to demonstrate relative cardiac safety, but model sensitivity has not been systematically investigated. Cross-species and human translation of repolarization delay, assessed as QT/QTc prolongation, has not been compared employing common methodologies across multiple species and sites. Therefore, the accurate translation of repolarization results within and between preclinical species, and to man, remains problematic. METHODS Six pharmaceutical companies entered into an informal consortium designed to collect high-resolution telemetered data in multiple species (dog; n=34, cynomolgus; n=37, minipig; n=12, marmoset; n=14, guinea pig; n=5, and man; n=57). All animals received vehicle and varying doses of moxifloxacin (3-100 mg/kg, p.o.) with telemetered ECGs (≥500 Hz) obtained for 20-24h post-dose. Individual probabilistic QT-RR relationships were derived for each subject. The rate-correction efficacies of the individual (QTca) and generic correction formulae (Bazett, Fridericia, and Van de Water) were objectively assessed as the mean squared slopes of the QTc-RR relationships. Normalized moxifloxacin QTca responses (Veh Δ%/μM) were derived for 1h centered on the moxifloxacin Tmax. RESULTS All QT-RR ranges demonstrated probabilistic uncertainty; slopes varied distinctly by species where dog and human exhibited the lowest QT rate-dependence, which was much steeper in the cynomolgus and guinea pig. Incorporating probabilistic uncertainty, the normalized QTca-moxifloxacin responses were similarly conserved across all species, including man. DISCUSSION The current results provide the first unambiguous evidence that all preclinical in vivo repolarization assays, when accurately modeled and evaluated, yield results that are consistent with the conservation of moxifloxacin-induced QT prolongation across all common preclinical species. Furthermore, these outcomes are directly transferable across all species including man. The consortium results indicate that the implementation of standardized QTc data presentation, QTc reference cycle lengths, and rate-correction coefficients can markedly improve the concordance of preclinical and clinical outcomes in most preclinical species.

Role of α2C-adrenoceptors in the reduction of skin blood flow induced by local cooling in mice

The reduction of skin blood flow induced by local cooling results from a reflex increase in sympathetic output and an enhanced vasoconstrictor activity of cutaneous vessels. The present study investigated the latter local response in vivo in tetrodotoxin‐treated mice, in which the sympathetic nerve tone was abolished.

Local regulation of skin blood flow during cooling involving presynaptic P2 purinoceptors in rats

1 This study investigated a local effect of cooling on the plantar skin blood flow (PSBF) of tetrodotoxin‐treated rats by laser‐Doppler flowmetry. 2 When the air temperature around the left foot was locally cooled from 25 to 10°C, the PSBF of the left foot decreased. 3 The response was inhibited by the α‐adrenoceptor antagonist phentolamine, the α1‐adrenoceptor antagonist bunazosin, the α2‐adrenoceptor antagonist RS79948, and bretylium and guanethidine that inhibit noradrenaline release from sympathetic nerves. Adrenalectomy of the rats did not affect the cooling‐induced response. 4 The P2 purinoceptor antagonists suramin and PPADS also significantly suppressed the cooling‐induced reduction of PSBF. However, the inhibitory effect of PPADS on the cooling‐induced response was abolished after the treatment with phentolamine. Intra‐arterial injections of ATPγS, a stable P2 purinoceptor agonist, at 25°C caused a transient decrease in PSBF in a dose‐dependent manner, which was significantly inhibited by phentolamine and guanethidine. 5 These results suggest a novel mechanism for local cooling‐induced reduction of skin blood flow in vivo; moderate cooling of the skin induces the release of ATP, which stimulates presynaptic P2 purinoceptors on sympathetic nerve terminals and facilitates the release of noradrenaline, thereby causing contractions of skin blood vessels via the activation of α1‐and α2‐adrenoceptors.

Sensitivity of common marmosets to detect drug-induced QT interval prolongation: Moxifloxacin case study

INTRODUCTION Moxifloxacin is the most widely used positive reference agent in clinical cardiac repolarization studies, but it has not been characterized in common marmosets which are uniquely suited to studies in early-stage development due to their small size and minimal test article requirements. The purpose of this study was to evaluate the sensitivity of the common marmoset to detect moxifloxacin-associated QT interval prolongation. METHODS Eight telemetered common marmosets were monitored for 24 h following oral administration of moxifloxacin by gavage at 0, 10, 30, and 100 mg/kg using a Latin square design. Concurrently, a pharmacokinetic evaluation in 8 non-telemetered animals was conducted. A rate-corrected QT (QTc) interval was derived using an individual probabilistic QT rate-correction. QTc (placebo-adjusted QTc change from the individual baseline) was calculated and the relationship between pharmacokinetics (PK) and pharmacodynamics (PD) was analyzed. RESULTS A slight, but not significant, increase in QTc was detected with 10 mg/kg of moxifloxacin. Moxifloxacin at 30 and 100 mg/kg elicited dose-dependent increases in QTc of 14.0+/-3.6 and 35.0+/-6.2 ms, respectively, with associated total moxifloxacin C(max) values of 6.5+/-0.5 and 16.5+/-1.6 microg/mL, respectively. From the PK/PD relationship, the plasma concentration which would attain QTc of 5 to 10 ms was estimated to be 1.67-3.73 microg/mL. The results were consistent with typical clinical trial results (QTc of 6.6-14.8 ms at 2.5-3.5 microg/mL). CONCLUSIONS The present study demonstrates that the common marmoset is highly sensitive to moxifloxacin-associated changes in cardiac repolarization, assessed as QTc. As such, this species is suitable for precise and reliable detection of small, but significant, drug-associated increases in QTc interval. Thus, the common marmoset should be regarded as a validated animal model for the detection of QT risk in early-stage drug development and represents an important addition to the current in vivo armamentarium.

Application of probabilistic analysis for precisely correcting the QT interval for heart rate in telemetered common marmosets.

INTRODUCTION QT intervals are strongly influenced by preceeding heart rate history and are also characterized by rate-independent variability, leading to difficulty in precise rate-correction of the raw QT interval. The present study elucidates a novel analytical method that effectively addresses this problematic phenomenon in telemetered common marmosets. METHODS ECGs were collected from telemetered common marmosets (male and female) and analyzed by computerized algorithms. Descriptive statistics were calculated from the mean of QT intervals for 5-ms increments of RR. The QT interval was corrected for the RR interval by applying Bazetts, Fridericias, and individual probabilistic QT rate-correction formulae. RESULTS The linear regression of log-transformed QT and RR intervals derived from a probabilistic approach yielded a well-correlated QT-RR fit. Assessed as the slope of the QTc-RR interval, application of individual probabilistic QT rate-corrections resulted in the most effective dissociation of the effects of rate from the raw QT interval, compared to generic rate-correction formulae. Using individual corrections, the QTc was stable while the interquartile range (IQR) of the QTc distribution was stable, spanning 5-10 ms for each subject over all physiological RR intervals. Heart rate variability distributions were centered about unity during both photoperiods and sinus arrhythmia was far less pronounced compared with measurements in dogs. DISCUSSION Probabilistic QT rate-correction eliminated the confounding effects of heart rate and provided a stable QTc baseline. These results indicate that application of this method of analysis in telemetered common marmosets results in a high degree of sensitivity for the consistent detection of small (5-10 ms) changes in the QTc interval.

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 β-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.