Characterization of an anesthetized dog model of transient cardiac ischemia and rapid pacing: a pilot study for preclinical assessment of the potential for proarrhythmic risk of novel drug candidates.

INTRODUCTION: Preclinical proarrhythmic risk assessment of drug candidates is focused predominantly on arrhythmias arising from repolarization abnormalities. However, drug-induced cardiac conduction slowing is associated with significant risk of life-threatening ventricular arrhythmias, particularly in a setting of cardiac ischemia. Therefore, we optimized and characterized an anesthetized dog model to evaluate the potential proarrhythmic risk of drug candidates in ischemic heart disease patients. METHODS: Anesthetized dogs were instrumented with atrial and ventricular epicardial electrodes for pacing and measurement of conduction times, and a balloon occluder and flow probe placed around the left anterior descending coronary artery (LAD) distal to the first branch. Conduction times, ECG intervals and incidence of arrhythmias were assessed serially at the end of each dose infusion (flecainide: 0.32, 0.63, 1.25, 2.5 and 5mg/kg, i.v.; dofetilide:1.25, 2.5, 5, 10 and 20 µg/kg, i.v.; or vehicle; n=6/group) both during normal flow (with and without rapid pacing) and during 5-min LAD occlusion (with and without rapid pacing). Compound X, a development candidate with mild conduction slowing activity, was also evaluated. RESULTS: Flecainide produced pronounced, dose-dependent slowing of conduction that was exacerbated during ischemia and rapid pacing. In addition, ventricular tachycardia (VT) and fibrillation (VF) occurred in 4 of 6 dogs (3 VF @ 0.63 mg/kg; 1VT @ 2.5mg/kg). In contrast, no animals in the vehicle group developed arrhythmias. Dofetilide, a potent IKr blocker that does not slow conduction, prolonged QT interval but did not cause further conduction slowing during ischemia with or without pacing and there were no arrhythmias. Compound X, like flecainide, produced marked conduction slowing and arrhythmias (VT, VF) during ischemia and pacing. DISCUSSION: This model may be useful to more accurately define shifts in safety margins in a setting of ischemia and increased cardiac demand for drugs that slow conduction.

The fentanyl/etomidate-anaesthetised beagle (FEAB) dog: a versatile in vivo model in cardiovascular safety research.

The purpose of conducting cardiovascular safety pharmacology studies is to investigate the pharmacological profiles of new molecular entities (NMEs) and provide data that can be used for optimization of a possible new drug, and help make a selection of NMEs for clinical development. An anaesthetised dog preparation has been used for more than two decades by our department to measure multiple cardiovascular and respiratory parameters and to evaluate different scientific models, leading to more in-depth evaluation of drug-induced cardiovascular effects. An anaesthetic regime developed in house (induction with lofentanil, scopolamine and succinylcholine, and maintenance with fentanyl and etomidate) gives us a preparation free of pain and stress, with minimal effects on the cardiovascular system. This anaesthetic regime had minimal influences on circulating catecholamine levels, on the baroreflex sensitivity, and on all measured basal parameters compared to conscious dogs. All parameters were stable for at least 3 h, with acceptable tolerance intervals, evaluated over 99 safety studies with 3 vehicle treatments (saline, 10% and 20% hydroxypropyl-beta-cyclodextrin). This translates into a highly sensitive model for detecting possible drug-induced effects of NMEs with different mechanisms of action such as: Ca-, Na-, I(Kr)-, I(Ks)-channel blockers, K- and Ca-channel activators, alpha1- and beta-agonists, and muscarinic antagonists. Fentanyl in combination with etomidate is a successful anaesthetic regime in humans [Stockham, R.J., Stanley, T.H., Pace, N.L., King, K., Groen, F. & Gillmor, S.T. (1987). Induction of anaesthesia with fentanyl or fentanyl plus etomidate in high-risk patients. Journal of Cardiothoracic Anesthesia. 1(1), 19-23.]. In the anaesthetised dog, QT correction factors (Van de Water correction and body temperature correction) and risk factors (total, short-term and long-term instability) have been evaluated, using this regime [Van de Water, A., Verheyen, J., Xhonneux, R. & Reneman, R. (1989). An improved method to correct the QT interval of the electrocardiogram for changes in heart rate. Journal of Pharmacological Methods, 22, 207-217.; van der Linde, H.J., Van Deuren, B., Teisman, A., Towart, R. & Gallacher, D.J. (2008). The effect of changes in core body temperature on the QT interval in beagle dogs: A previously ignored phenomenon, with a method for correction. British Journal of Pharmacology, 154, 1474-1481.; van der Linde, H.J., Van de Water, A., Loots, W., Van Deuren, B., Lu, H.R., Van Ammel, K., et al. (2005) A new method to calculate the beat-to-beat instability of QT duration in drug-induced long QT in anaesthetised dogs. Journal of Pharmacological and Toxicological Methods, 52, 168-177.]. Furthermore, this anaesthetic protocol has been used to create different scientific models (long QT, short QT) with different specific end-points (ventricular fibrillation, adrenergic- or pause-dependent TdP) and also their specific precursors: e.g. aftercontractions, phase 2 EADs, phase 3 EADs, DADs, T-wave morphology changes, T-wave alternans, R-on-T, transmural and interventricular dispersion [Gallacher, D.J., Van de Water, A., van der Linde, H.J., Hermans, A.N., Lu, H.R., Towart, R., et al. (2007). In vivo mechanisms precipitating torsade de pointes in canine model of drug-induced long QT1 syndrome. Cardiovascular Research, 76-2, 247-256.]. This paper gives a brief overview of the stability, reproducibility, sensitivity and utility of a well-validated anaesthetised dog model.

Ultrasound agents may open the blood-brain barrier in rats and aggravate pathologic consequences of experimental head trauma.

Unilateral intracarotid injection of contrast agents may considerably destabilize the blood-brain barrier in rats. This leads to vasogenic edema in the ipsilateral hemisphere. Mortality and extravasation increased significantly when administration of these ultrasound contrast agents was followed by mild traumatic brain injury. Direct administration to the cerebral circulation is, therefore, indicative for edema-related pathology and may amplify the consequences of experimental neurotrauma.

Comparison of intracranial pressure measured in the cerebral cortex and the cerebellum of the rat.

In this study, we evaluated the accuracy of intracranial pressure (ICP) measurement in rats by insertion of a miniature ICP probe in the parenchyma of the cerebellum. A comparison was made between the ICP values measured simultaneously in the parenchyma of the cerebral cortex and the cerebellum. In order to obtain a wide range of ICP, animals were subjected to a severe closed head injury (CHI), a moderate CHI or to a sham operation. ICP values ranged from 0.8 to 43.9 mmHg. After 15 min stabilisation the first measurement was taken and followed by a second measurement 25 min after onset to allow comparison of ICP changes at the two implantation sites. Linear regression analysis showed a highly significant correlation at 15 min: Y = 0.919X + 0.655 (R(2) = 0.977), and at 25 min: Y = 0.931X + 0.698 (R(2) = 0.976). The differences in ICP measurement between cerebellar and cerebral site were not significantly different from zero at both time points. Altman-Bland plots showed that the difference in ICP readings between the two locations could differ maximally by 5.3 mmHg. The largest differences were detected when high ICP values were recorded. We conclude that in rats the ICP measurement in the cerebellum is comparable to the ICP measurement in the cerebral cortex. The cerebellar ICP can be used as a valuable alternative during experimental procedures.