Differential analysis of transient increases of serum cTnI in response to handling in rats.

Serum cardiac troponins are the key biomarkers of myocardial necrosis in humans and in preclinical species. The use of ultrasensitive assays for serum cardiac troponin I (cTnI) as a biomarker in safety studies is hampered by interindividual differences. In this study, we investigated the effect of handling procedures on serum cTnI and explored modeling and simulation approaches to mitigate the impact of these interindividual differences. Femoral-catheterized male Crl:WI(Han) rats (n = 16/group) were left undisturbed in their cages with no handling; subjected to 5 min of isoflurane/O2 anesthesia (A); or placed into a rodent restrainer followed by simulated tail vein injection (RR). Serum cTnI concentrations were assessed over a 24-h period using an ultrasensitive assay, and the study was repeated for confirmation. The mean serum cTnI concentration pre-procedure was 4.2 pg/mL, and remained stable throughout the duration of the study in the rats submitted to the A procedure. Serum cTnI concentrations increased transiently after the RR procedure with a median time to maximum concentration (T max), of 1 and 2 h and a mean maximum value concentration (C max), of 53.0 and 7.2 pg/mL in the initial and repeat studies, respectively. A population pharmacodynamic model identified interindividual, procedure- and study-specific effects on serum cTnI concentrations in rats. It is concluded that a modeling and simulation approach more appropriately describes and statistically analyzes the data obtained with this ultrasensitive assays.

Myocardial mononuclear cell infiltrates are not associated with increased serum cardiac troponin I in cynomolgus monkeys.

Myocardial mononuclear cell infiltrate is a spontaneous cardiac finding commonly identified in laboratory cynomolgus monkeys. The infiltrates are predominantly composed of macrophages with lesser lymphocytes and are not typically associated with histologically detectable cardiomyocyte degeneration. These infiltrates are of concern because they confound interpretation of test article-related histopathology findings in nonclinical safety toxicology studies. The interpretation of safety studies would be simplified by a biomarker that could identify myocardial infiltrates prior to animal placement on study. We hypothesized that monkeys with myocardial mononuclear cell infiltrates could be identified before necropsy using an ultrasensitive immunoassay for cardiac troponin I (cTnI). Serum cTnI concentrations in monkeys with myocardial infiltrates were not higher than those in monkeys without infiltrates at any of the sampling times before and on the day of necropsy. Increased serum cTnI levels are not suitable for screening monkeys with myocardial mononuclear cell infiltrates before placement in the study.

The complete pharmacokinetic profile of serum cardiac troponin I in the rat and the dog.

Recent improvements in assays have allowed serum cardiac troponin I (cTnI) to be measured at previously undetectable concentrations, which may have implications for cardiotoxicity studies. We characterized the pharmacokinetics of cTnI after a single iv administration of purified cTnI in rats at doses of 0.005, 0.05, and 0.5 µg/kg and in beagle dogs at doses of 0.05, 0.1, and 0.2 µg/kg. Serum cTnI concentration-time profiles were well described by a two-compartment pharmacokinetic model with first-order elimination in both species. The estimated mean (SD) values of total serum clearance, volume of distribution of the central compartment, and terminal half-life were 318 ml/h/kg, 52.9 ml/kg, and 0.8 h in rats and 481 (135) ml/h/kg, 230 (70) ml/kg, and 1.85 (0.5) h in dogs, respectively. In both species, a fast distribution phase was followed by a relatively slow elimination phase. These data indicate that the current practice in cardiotoxicity studies of unguided blood sampling should be revised. A targeted case-by-case approach is required whereby samples are collected not only relative to the kinetics of the test article but also in relation to the kinetics of the biomarker in the test species and the type and severity of anticipated cardiovascular perturbation. This approach is essential for the identification of subtle increases of serum cTnI concentrations in the low dynamic range.

Primary endothelial damage is the mechanism of cardiotoxicity of tubulin-binding drugs.

The use of tubulin binders (TBs) in the treatment of cancer often is associated with cardiotoxicity, the mechanism of which has not been elucidated. To test the hypothesis that interstitial cells of the myocardium are the primary target of TBs, we evaluated the acute effects of a single iv administration of three reference TBs: colchicine (0.2 and 2 mg/kg), vinblastine (0.5 and 3 mg/kg), and vincristine (0.1 and 1 mg/kg) 6 and 24 h after dosing. Mitotic arrest was identified at 24 h in all high-dose groups based on an increase in the number of mitotic figures in the interstitium coupled with a decrease in the number of Ki67-positive interstitial cells. Analysis of the myocardial transcriptomic data further supported G2/M cell cycle arrest 6 h after dosing with the high-dose groups of all three compounds. Apoptotic figures and an increase in the number of cleaved caspase 3-positive cells were identified at 6 and 24 h at the highest dose of each compound predominantly in interstitial cells, whereas a few cardiomyocytes were affected as well. Transcriptomic profiling of the myocardium further suggested that some of the affected interstitial cells were endothelial cells based on the upregulation of genes typically associated with vascular damage and downregulation of endothelial cell-specific molecule 1 and apelin. Taken together, these data identify endothelial cells of the myocardium as the primary target of the cardiotoxicity of TBs and identify cell cycle arrest as the mechanism of this toxicity.

Assessment of the toxicity of hydralazine in the rat using an ultrasensitive flow-based cardiac troponin I immunoassay.

The purpose of this study was to correlate the histologic changes in the heart to serum cardiac troponin I (cTnI) concentrations assayed with the Erenna Immunoassay System in Wistar rats (Crl:Wi[Han]) using the hydralazine model of cardiotoxicity. A single dose of hydralazine caused an increase of cTnI concentrations at six hours post-dose, followed by a sharp decrease at twenty-four hours and a return to baseline at forty-eight hours. The second dose of hydralazine caused a smaller magnitude increase in cTnI concentrations at six hours as compared to the first dose. Also, cTnI concentrations returned to baseline at twenty-four hours after the second dose. The increased cTnI concentrations coincided with acute myocardial necrosis at histology. However, increased cTnI concentrations in the absence of microscopic lesions were identified in several rats. As cTnI concentrations decreased, microscopic changes in the heart matured to cardiomyophagy. In conclusion, the increases in cTnI concentrations six hours after the administration of hydralazine were indicative of a myocardial damage that did not consistently have a microscopic correlate. However, the window of increased cTnI concentrations was short, and only microscopic evaluation of the heart detected the damage at twenty-four to forty-eight hours after the episode of acute myocardial necrosis.

Temporal gene expression profiling indicates early up-regulation of interleukin-6 in isoproterenol-induced myocardial necrosis in rat.

Gene expression was evaluated in the myocardium of male Wistar rats after a single subcutaneous administration of 0.5 mg of isoproterenol, a beta-adrenergic agonist that causes acute tachycardia with subsequent myocardial necrosis. Histology of the heart, clinical chemistry, and hematology were evaluated at 9 time points (0.5 hours to 14 days postinjection). Myocardial gene expression was evaluated at 4 time points (1 hour to 3 days). Contraction bands and loss of cross-striation were identified on phosphotungstic acid-hematoxylin-stained sections 0.5 hours postdosing. Plasma troponin I elevation was detected at 0.5 hours, peaked at 3 hours, and returned to baseline values at 3 days postdosing. Interleukin 6 (Il6) expression spiked at 1 to 3 hours and was followed by a short-lived, time-dependent dysregulation of its downstream targets. Concurrently and consistent with the kinetics of the histologic findings, many pathways indicative of necrosis/apoptosis (p38 mitogen-activated protein kinase [MAPK] signaling, NF-kappaB signaling) and adaptation to hypertension (PPAR signaling) were overrepresented at 3 hours. The 1-day and 3-day time points indicated an adaptive response, with down-regulation of the fatty acid metabolism pathway, up-regulation of the fetal gene program, and superimposed inflammation and repair at 3 days. These results suggest early involvement of Il6 in isoproterenol-induced myocardial necrosis and emphasize the value of early time points in transcriptomic studies.