Temperature induced changes in cardiac electrophysiology in anaesthetised guinea pigs

Abstracts

Poster No: 7 Experience with different vehicle formulations in ECG studies with anaesthetized guinea pigs Rita Dost, Barbara Langen, Ramona Dieckmann, Helge Hartenhauer Biotie Therapies GmbH, Radebeul, Germany During the last years we were faced with an increasing number of compounds with low aqueous solubility in drug discovery. Due to the unfavorable physico-chemical properties we were forced to deal with identifying appropriate formulation for intravenous administration in early cardiovascular safety. We used common formulation approaches such as co-solvents, cyclodextrins or buffers in different compositions and compared these with saline in ECG studies in anaesthetized guinea pigs. The selection of vehicle was driven by their suitability for individual compounds. In the ECG studies, hemodynamic and electrophysiological parameters were continuously monitored 30 min prior to and up to 60 min post administration (Digital Power Lab system, AD instruments) of 12 different formulations (n = 3–7/group). During recording rectal body temperature, heart rate, systolic and diastolic arterial blood pressures and lead II ECG were measured. QT was corrected using Bazett formula. The different formulations were compared with saline treated animals by Two-Way-ANOVA provided that 6 animals/ group were tested. Among the different formulations investigated all co-solvents used (PEG, ethanol, DMSO, and Tween) influenced at least one parameter. In most cases arterial blood pressure was changed, e.g. PEG as frequently used co-solvent increased or reduced blood pressure depending on application route (iv. or ip.) even at low concentration. In addition a small effect on QT interval was noticed. The complexation of poorly water-soluble compounds with cyclodextrins represents a good alternative to saline. doi:10.1016/j.vascn.2011.03.014

Poster No: 8 Left ventricular pressure (LVP) measurements in anesthetized guinea-pigs: Method validation and comparison with the QA interval Pieter-Jan Guns a, Hanne Daems a, Jan Verrelst b, Johan Lissens a, Brigitte Loenders b, David Gallacher b a

Bio-Plus Safety Pharmacology, Vlasmeer 5/0003, B-2400 Mol, Belgium Center of Excellence for Cardiovascular Safety Research and Mechanistic Pharmacology, Johnson & Johnson, European Discovery Capability Organization, Division of Janssen Pharmaceutica N.V., B-2340, Beerse, Belgium

b

Introduction: Front-loading of cardiovascular safety pharmacology studies to the early stages of preclinical drug development has gained popularity. Thus far, the anesthetized guinea-pig represents a validated model for early in vivo screening of QT prolongation. This work aimed at evaluating the feasibility of adding indicators of contractility (left ventricular pressure (LVP) derived parameters dp/ dtmax and dp/dtmax/pd, and the QA interval) to the model. Methods: Female Dunkin Hartley guinea-pigs were anesthetized with sodium pentobarbital (60 mg/kg), placed on a heating plate and artificially ventilated. Sodium pentobarbital was continuously infused (6 mg/kg/h) into the left jugular vein and the test substance was administered via the right jugular vein. Blood pressure was measured in the left carotid artery. A tip-catheter (Millar, SPR-249) was introduced via the right carotid artery into the left ventricle. Four

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subcutaneous ECG-needles were placed to obtain a 6-lead ECG. All signals were analyzed using Notocord-Hem software. Results: Reproducible and stable LVP signals were obtained in anesthetized guinea-pigs. Meticulous positioning of the tip-catheter was crucial to avoid catheter-related arrhythmias. The clinical reference drugs dobutamine and verapamil respectively, increased and decreased dp/dtmax and dp/dtmax/pd. Furthermore, Bowditch staircase representation of the data showed true positive and negative inotropic effects of dobutamine and verapamil. In addition, the QA interval discriminated between vehicle, dobutamine (shortened QA) and verapamil (prolonged QA), and plotting of QA versus dp/dtmax values showed an inverted relationship between both parameters. Conclusions: Measurement of left ventricular contractility by dp/ dtmax or QA interval is feasible in anesthetized guinea-pigs. The addition of parameters of left ventricular contractility further complements the anesthetized guinea-pig model and makes it a more complete cardiovascular early in vivo screening platform. doi:10.1016/j.vascn.2011.03.015

Poster No: 9 IV infusion rate affects pharmacokinetics and pharmacodynamic (QTc) response in pentobarbital-anesthetized guinea pigs Masahiro Nishida, Pierre Morissette, Hiroyasu Miyazaki, Joseph J. Salata Safety and Exploratory Pharmacology, Safety Assessment, Merck Research Laboratories West Point, PA 19486, USA The anesthetized guinea pig may serve as a robust model to assess and mitigate cardiovascular (CV) risk, particularly for QTc prolongation during drug discovery and development. This study examines and compares 2 different IV infusion protocols in order to develop a more sensitive and predictive model for assessing QTc risk. Six reference agents that are well-known to induce QTc prolongation (Moxifloxacin, Haloperidol, Cisapride, Astemizole, Terfenadine, and MK-499) were administered IV to pentobarbital-anesthetized guinea pigs at rising doses, with protocol-1) five consecutive doses, each delivered over a 5min period vs. protocol-2) three consecutive doses, each delivered over a 20-min period. Plasma drug concentrations (PK) vs. QTc changes (PD) were determined at the end of each infusion period and the PK-PD relations were plotted. Dose- and concentration-dependent QTc prolongation was observed for all agents with both infusion protocols, but PK-PD relations with protocol-2 were generally positioned upward and to the left compared with protocol-1. Quantitatively, plasma drug concentrations inducing 5% or 10% QTc prolongation were significantly lower with the 20-min vs. 5-min protocol. Thus, the infusion rate in anesthetized guinea pigs significantly influenced the PK-PD relation for assessing QTc risk with positive control agents. In conclusion, careful selection of infusion protocols is important to optimize PK-PD relations and accurately determine CV risk in pre-clinical animal models and better predict clinical outcomes. doi:10.1016/j.vascn.2011.03.016

Poster No: 10 Temperature induced changes in cardiac electrophysiology in anaesthetised guinea pigs Jens Kagstrom, Eva-Lena Laumola, Niklas Poijes, Maria Johansson, Ann-Christin Ericson

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Abstracts

AstraZeneca R&D, Södertälje, Sweden

Poster No: 12

The anaesthetised guinea pig is used to evaluate drug effects on cardiac electrophysiology. As anesthesia compromises the ability to thermoregulate, the study aimed to evaluate the influence of changes in body temperature (BT) on electrophysiological parameters. Guinea pigs were vagotomised and β-blocked, an atrial pacing electrode attached, a MAP suction electrode placed on the left ventricular wall, ECG needle electrodes attached, and a rectal temp probe inserted. In control group animals BT was kept constant (37.9 °C); in a 2nd group animals were slowly warmed to 41.9 °C; and in a 3rd group animals were slowly cooled to 34.4 °C. Effects on repolarisation (MAPD90), AV conduction and ECG were recorded for up to 50 min during cardiac pacing. No timedependent changes were seen in the control group. In contrast, a linear correlation was found between changes in BT and MAPD90, AV conduction and QT interval. For each degree BT falls below 38 °C MAPD90 is prolonged by 6.1 ±0.3 ms (95% CI), and for each degree above 38 °C the MAPD90 is shortened by 3.9±0.3 ms. Corresponding effects on the QT interval is +12.7 ±1.0 ms and −10.7 ±0.8 ms, respectively, and for AV conduction time +9.5±0.3 ms −5.5 ±0.6 ms, respectively. In contrast, no effect on the QRS interval was seen. The data highlights the importance of maintaining body temperature when performing electrophysiological recordings in anaesthetised guinea pigs. Reduction by a single degree can prolong MAPD90 by 6 ms (approx 5%) and thus increase the risk for false positive identification of test compounds.

The FEAB dog-model: Now within reach for everyone! Henk J. van der Linde, Bruno Van Deuren, Yves Somers, Ard Teisman, David J. Gallacher

doi:10.1016/j.vascn.2011.03.017

Poster No: 11 Usefulness of a complete atrioventricular block dog model in the safety evaluation of drug-induced QRS prolongation Yoshiyuki Furukawa, Keiko Matsune, Yuki Takamatsu, Hirofumi Awatsuji, Keiji Yamamoto

Center of Excellence for Cardiovascular Safety Research and Mechanistic Pharmacology, Johnson & Johnson, European Discovery Capability Organization, Division of Janssen Pharmaceutica N.V., B-2340, Beerse, Belgium The fentanyl/etomidate-anaesthetised beagle (FEAB) dog model (JPTM, 60, 11–23) is used in cardiovascular safety studies and has minimal direct effects on the cardiovascular system, autonomic regulation and has proven value to detect seizure activity (JPTM, 2010). However, its use is still limited. To solve the limitation that lofentanil is not widely available, we adapted our anaesthetic regime by using fentanyl premedication in place of lofentanil. The anaesthetic regime now used is: induction by fentanyl (0.25 mg/kg), scopolamine (0.015 mg/kg) and succinylcholine (1 mg/kg), and maintenance by: etomidate (1.5 mg/kg/h) and fentanyl (0.01 mg/kg/h). Compared to the classic FEAB model no clear differences were noted in baseline values, e.g. heart rate (74 vs. 73 bpm.), systolic blood pressure (130 vs. 119 mm Hg), LV dp/dtmax (2783 vs. 2517 mm Hg/s), cardiac output (1.9 vs. 2.0 l/min), PQ (111 vs. 110 ms), QRS (41 vs. 46 ms), QT (241 vs. 249 ms) and narcotrend index (56 vs. 56). Also the stability of all parameters over 3 h was similar in both regimes. In addition, the baroreflex sensitivity during this new regime was completely comparable to the classic FEAB model: 13.7 ± 2.1 vs. 12.6 ± 3.1 ms/ mm Hg, respectively. Furthermore, dofetilide (0.01 mg/kg iv) induced comparable QTc Van de Water prolongations in both regimes (12% in the adapted and 15% in the classic regime). We suggest that these data can convince other groups to use the elegant and useful FEAB dog model in their cardiovascular safety evaluations. doi:10.1016/j.vascn.2011.03.019

Development Research Center, Takeda Pharmaceutical Company Limited, Osaka, Japan Drug-induced QRS prolongation is caused by many cardiac sodium channel blockers. The use-dependent effects on QRS prolongation and cardiac function in vivo have not been practically evaluated. The aim of this study was to investigate whether anesthetized dogs with an acute complete atrioventricular (AV) block, in which the heart rate is controlled by ventricular pacing, were useful for the safety evaluation of the use-dependent effect. After cumulative intravenous administration of flecainide (FL) or mexiletine (ME) (n = 5 each), QRS duration, LV dP/dt max and LVEDP were measured at the pacing rates of 60, 100, 150 and 200 bpm just before ending each 30-min infusion. FL showed slight and use-dependent QRS prolongation from 60 bpm at 1.5 mg/kg and induced marked and monotonically use-dependent QRS prolongation at 5 mg/kg. ME showed QRS prolongation not at 60 bpm but at 150 bpm at 5 mg/kg and induced steeply use-dependent QRS prolongation at higher pacing rates at 15 mg/kg. Pharmacokinetic analysis revealed that the plasma concentrations of FL and ME, causing QRS prolongation, were comparable to those in humans. At 60 bpm, both drugs decreased LV dP/dt max dose-dependently at the doses with QRS prolongation, but neither drug had effects on LVEDP at any dose. With higher pacing rates, FL decreased LV dP/dt max usedependently at 5 mg/kg and both drugs increased LVEDP dose- and use-dependently. These results suggest that this acute AV-block model is valuable for assessing the use-dependent characteristics of sodium channel blockers on QRS prolongation and cardiac function. doi:10.1016/j.vascn.2011.03.018

Poster No: 13 Differential effects of fenfluramine in regulating pulmonary and systemic circulation Brett R. Herzberg, Lee C. Preusser, Scott W. Mittelstadt, Bryan F. Cox, Eugene W. Shek Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL, USA Fenfluramine, an antiobesity agent, was withdrawn from therapeutic use due to the increased risk of pulmonary hypertension and cardiac valvular lesions. Fenfluramine has also been reported to produce peripheral cardiovascular actions such as hypertension, increased myocardial contractility and cardiac output. However, it remains to be determined whether fenfluramine exerts differential effects on pulmonary and systemic circulation. Therefore, the effects of fenfluramine on pulmonary and systemic circulation were examined during three 30-min escalating infusions (0.004, 0.038 and 0.114 mg/kg/min) in the pentobarbital anesthetized beagle dog (n = 6/group). Fifteen minutes into the mid-dose infusion, fenfluramine significantly increased pulmonary arterial pressure (35%), and pulmonary vascular resistance (48%) from baseline as compared with vehicle with minimal effects on mean arterial pressure, heart rate, myocardial contractility, cardiac output and systemic vascular resistance. Following the high-dose infusion of fenfluramine, both pulmonary arterial pressure and pulmonary vascular resistance were