Imatinib induces diastolic dysfunction and ventricular early-repolarization delay in the halothane-anesthetized dogs: Class effects of tyrosine kinase inhibitors.

Imatinib has been reported to induce heart failure and/or QTc prolongation. To better understand their underlying mechanisms, we assessed its effects on cardiohemodynamic, electrocardiographic and echocardiographic variables along with biomarkers of myocardial damage. Imatinib mesylate in doses of 1 and 10 mg/kg was intravenously administered to the halothane-anesthetized beagle dogs (n = 4). Effects of imatinib on each phase of isovolumetric contraction, ejection, isovolumetric relaxation and filling were studied, whereas its electrophysiological effects on early and late repolarization were analyzed by measuring J-Tpeak and Tpeak-Tend, respectively. The low and high doses of imatinib provided peak plasma concentrations of 3.23 and 17.39 µg/mL, reflecting clinically-relevant and supratherapeutic concentrations, respectively. Neither lethal ventricular tachyarrhythmia nor cardiohemodynamic collapse was observed. Imatinib decreased amplitude of peak -dP/dt, indicating suppression of isovolumetric relaxation, whereas no significant change was detected in the other phases. Imatinib prolonged QTc and J-Tpeakc without altering Tpeak-Tend, indicating increase of net inward current, which leads to intracellular Ca2+ overload. Thus, imatinib suppressed ventricular active relaxation and early repolarization, which may suggest the association of mitochondrial dysfunction-associated inhibition of ATP production. Since those findings were also reported for dasatinib, sunitinib and lapatinib, they could be common cardiac phenotype of tyrosine kinase inhibitors in vivo.

Utilization of the chronic atrioventricular block cynomolgus monkey as an in vivo model to evaluate drug interaction-associated torsade de pointes.

It has been difficult to experimentally reproduce synergistic effects of ketoconazole on terfenadine-induced torsade de pointes. We assessed proarrhythmic effects of terfenadine (30 mg/kg, p.o.) with/without ketoconazole (100 mg/kg, p.o.) pretreatment using the chronic atrioventricular block cynomolgus monkeys with repeated-measured design (n = 4). Terfenadine with ketoconazole pretreatment repeatedly induced non-sustained torsade de pointes in each animal, although terfenadine alone did not induce it at all. Thus, the chronic atrioventricular block cynomolgus monkeys can be used for studying drug interaction-associated torsade de pointes, providing a non-clinical strategy to circumvent untoward drug interactions in patients specially under polypharmacy.

Dasatinib can Impair Left Ventricular Mechanical Function But May Lack Proarrhythmic Effect: A Proposal of Non-clinical Guidance for Predicting Clinical Cardiovascular Adverse Events of Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors are known to clinically induce various types of cardiovascular adverse events; however, it is still difficult to predict them at preclinical stage. In order to explore how to better predict such drug-induced cardiovascular adverse events, we tried to develop a new protocol by assessing acute electrophysiological, cardiohemodynamic, and cytotoxic effects of dasatinib in vivo and in vitro. Dasatinib at 0.03 and 0.3 mg/kg was intravenously administered to the halothane-anesthetized dogs for 10 min with an interval of 20 min between the dosing (n = 4). Meanwhile, that at 0.1, 0.3, and 1 µM was cumulatively applied to the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (n = 7). In the dogs, the low and high doses provided peak plasma concentrations of 40 ± 5 (0.08) and 615 ± 38 ng/mL (1.26 µM), respectively. The low dose decreased the heart rate, impaired the left ventricular mechanical function, and prolonged the ventricular effective refractory period. The high dose prolonged the repolarization period, induced hemorrhagic tendency, and increased plasma cardiac troponin I level in addition to enhancement of the changes observed after the low dose, whereas it neither affected the cardiac conduction nor induced ventricular arrhythmias. In the hiPSC-CMs, dasatinib prolonged the repolarization and refractory periods like in dogs, while it did not induce apoptotic or necrotic process, but that it increased the conduction speed. Clinically observed major cardiovascular adverse events of dasatinib were observed qualitatively by currently proposed assay protocol, which may become a useful guide for predicting the cardiotoxicity of new tyrosine kinase inhibitors.

Sunitinib does not acutely alter left ventricular systolic function, but induces diastolic dysfunction.

PURPOSE: Cancer chemotherapies have improved the prognosis of cancer patients in recent years; however, their side effects on the cardiovascular systems have emerged as a major concern in the field of both cardiology and oncology. In particular, multi-targeted tyrosine kinase inhibitors are known to induce various types of cardiovascular adverse events including hypertension, QT-interval prolongation and heart failure, but their underlying mechanisms remain elusive. To explore how to better predict such drug-induced cardiovascular adverse events, we assessed the electropharmacological effects of sunitinib using the halothane-anesthetized dogs (n = 5), while plasma concentrations of cardiac enzymes including aspartate aminotransferase, lactate dehydrogenase, creatinine kinase and cardiac troponin I  were measured. METHODS: Sunitinib was intravenously administered at 0.01 and 0.1 mg/kg for 10 min with 20 min interval. RESULTS: Sunitinib decreased the amplitude of maximum downstroke velocity of the left ventricular pressure, prolonged the isovolumic relaxation time and increased the left ventricular end-diastolic pressure in a dose-related manner without affecting the other cardiohemodynamic and electrophysiological variables. More importantly, sunitinib significantly elevated cardiac troponin I level for 30-60 min after the high dose without altering the other biomarkers. CONCLUSIONS: Monitoring of the cardiac diastolic function together with cardiac troponin I after the start of sunitinib administration may become a reliable measure to predict the onset of sunitinib-induced cardiovascular adverse events.

Effects of Red Wine Vinegar Beverage on the Colonic Tissue of Rodents: Biochemical, Functional and Pharmacological Analyses.

A beverage made of red wine vinegar and grape juice (Yamanashi-no-megumi™) was developed as a supplemental fluid containing polyphenols, which has been clinically shown to enhance the colonic transit. In this study, we assessed the mechanism of its prokinetic action by analyzing the effects on both the colonic phosphodiesterase activity of rats (n=4) and the isolated colonic strip preparation of guinea pigs (n=4). The 7% (v/v) solution of the beverage significantly decreased the phosphodiesterase activity by 9% (n=4). The beverage in concentrations of 0.7, 2.1 and 7% (v/v) relaxed the colonic strips pre-contracted by 1 µmol/L of carbachol in a concentration-related manner with 50, 58 and 79%, each response of which was diminished to 11, 19 and 46%, respectively in the presence of 100 µmol/L of L-nitro-arginine methyl ester. These results obtained by biochemical, functional and pharmacological analyses suggest that the beverage could relax the colon through both cAMP-associated and nitric oxide-dependent pathways, which may partly explain clinically observed prokinetic effect of the beverage.

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 3 mg/kg using the halothane-anesthetized dogs (n = 5), 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. In vivo 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.

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.

Possibility as an anti-cancer drug of astemizole: Evaluation of arrhythmogenicity by the chronic atrioventricular block canine model.

Since astemizole in an oral dose of 50 mg/kg/day was recently reported to exert anti-cancer effect in mice, we evaluated its proarrhythmic potential using the atrioventricular block dogs in order to clarify its cardiac safety profile. An oral dose of 3 mg/kg prolonged the QT interval without affecting the QTc (n = 4), whereas that of 30 mg/kg increased the short-term variability of repolarization and induced premature ventricular contractions in each animal, resulting in the onset of torsade de pointes in 1 animal (n = 4). Thus, proarrhythmic dose of astemizole would be lower than anti-cancer one, limiting its re-profiling as an anti-cancer drug.

Comparison of direct effects of clinically available vasodilators; nitroglycerin, nifedipine, cilnidipine and diltiazem, on human skeletonized internal mammary harvested with ultrasonic scalpel.

Direct vasodilator effects of nitroglycerin, nifedipine, cilnidipine and diltiazem on human skeletonized internal mammary artery graft harvested with ultrasonic scalpel were assessed in the presence of 0.1 or 0.2 µM of noradrenaline. Ring preparations were made of distal end section of the bypass grafts, and those dilated by acetylcholine were used for assessment. Each drug dilated the artery in a concentration-related manner (0.01-10 µM, n = 6 for each drug) with a potency of nitroglycerin > nifedipine = cilnidipine > diltiazem. These results indicate that nitroglycerin can be useful for treating internal mammary artery spasm, that clinical utility of diltiazem may not depend on its vasodilator effect on the bypass graft, and that cilnidipine as well as nifedipine will have anti-spastic action which is in the middle between those of nitroglycerine and diltiazem.

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses.

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of ß-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Disruption of Stard10 gene alters the PPARα-mediated bile acid homeostasis.

STARD10, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein family, is highly expressed in the liver and has been shown to transfer phosphatidylcholine. Therefore it has been assumed that STARD10 may function in the secretion of phospholipids into the bile. To help elucidate the physiological role of STARD10, we produced Stard10 knockout mice (Stard10(-/-)) and studied their phenotype. Neither liver content nor biliary secretion of phosphatidylcholine was altered in Stard10(-/-) mice. Unexpectedly, the biliary secretion of bile acids from the liver and the level of taurine-conjugated bile acids in the bile were significantly higher in Stard10(-/-) mice than wild type (WT) mice. In contrast, the levels of the secondary bile acids were lower in the liver of Stard10(-/-) mice, suggesting that the enterohepatic cycling is impaired. STARD10 was also expressed in the gallbladder and small intestine where the expression level of apical sodium dependent bile acid transporter (ASBT) turned out to be markedly lower in Stard10(-/-) mice than in WT mice when measured under fed condition. Consistent with the above results, the fecal excretion of bile acids was significantly increased in Stard10(-/-) mice. Interestingly, PPARα-dependent genes responsible for the regulation of bile acid metabolism were down-regulated in the liver of Stard10(-/-) mice. The loss of STARD10 impaired the PPARα activity and the expression of a PPARα-target gene such as Cyp8b1 in mouse hepatoma cells. These results indicate that STARD10 is involved in regulating bile acid metabolism through the modulation of PPARα-mediated mechanism.