Azithromycin/Chloroquine Combination Does Not Increase Cardiac Instability despite an Increase in Monophasic Action Potential Duration in the Anesthetized Guinea Pig

Anthony A. Fossa 0 1 Todd Wisialowski 0 1 J. Neil Duncan 0 1 Shibing Deng 0 1 Michael Dunne 0 1 0 search and Development , Eastern Point Rd, MS 8260-2630, Groton, CT 06340 1 Pfizer Global Research and Development, Departments of Translational and Molecular Medicine, Safety Pharmacology, Drug Metabolism and Biostatistics , Groton, Connecticut Prolongation of the electrocardiogram QT interval by some, but not all drugs, has been associated with increased incidence of sudden cardiac death. Current preclinical regulatory assays cannot discriminate the arrhythmia liability of these drugs. Consequently, many new medications that prolong the QT interval are not developed despite their potential therapeutic benefit. Alternans (action potential duration alternations) is a measure of cardiac instability in humans and animals associated with the onset of ventricular fibrillation. Due to potential arrhythmia risk from observed QT prolongation, alternans was assessed in the anesthetized guinea pig after azithromycin or chloroquine alone and after combination treatment at clinically relevant concentrations proposed for the management of malaria. Chloroquine alone, but not azithromycin, caused a profound increase in action potential duration but with only minimal effects on alternans (10 ms). Azithromycin alone and in combination with chloroquine showed no increase in alternans beyond vehicle baseline responses indicating no additional arrhythmia liability. - Since 1997, International Conference of Harmonization (ICH) regulatory guidelines1,2 have been established to assess properties of new drugs that cause QT prolongation because of the association with the increased occurrence of a rare but sometimes fatal ventricular arrhythmia, torsades de pointes. Many anti-bacterials such as erythromycin and moxifloxacin are known to prolong the QT interval but are associated with different likelihoods of torsades de pointes or cardiac risk.3 The current ICH regulatory required assays36 used in pharmaceutical development cannot accurately discriminate arrhythmia susceptibility below approximately 30-fold the intended therapeutic indices.7 Moxifloxacin and erythromycin, for example, are associated with a 1- to 8-fold therapeutic safety margin based on these assays and most likely would not be developed in todays regulatory environment. There is no doubt that erythromycin and other antibacterial and antiparasitic agents have improved our lives by reducing the threat from infections, particularly malaria. Many of these highly valued agents may need to be replaced in the future with new generations of medications as organism resistance develops. Therefore, new methods to assess the arrhythmia potential are needed. To improve the confidence in their predictive value, these new methods can be benchmarked against drugs with clinical histories that are known/recognized by most physicians. Azithromycin (Zithromax) is being considered for use in combination with chloroquine in patients with chloroquineresistant malaria because synergy has been reported in vitro.8 Preliminary studies indicated an increase in clinical success from approximately 30% with each drug used alone to 97% with combination therapy.9 Azithromycin3 and chloroquine10,11 have well-established safety profiles. However, each drug is known to delay cardiac repolarization through inhibition of rapidly activating delayed rectifier potassium current known as hERG,10,12 which in the case of chloroquine, can result in QT prolongation.13 Beat-to-beat alternations of the cardiac action potential duration (APD) known as alternans are thought to be a precursor/substrate to the development of ventricular arrhythmias.14,15 This occurs when a series of cardiac cycles oscillate between short-long duration and generates a reciprocating short-long sequence of APD as heart rate accelerates (see example in Figure 1). When the APD oscillations become large enough, they act as waves that break and may cause conduction block, reentry, and synchronous ventricular tachycardia that can transition to fibrillation.16 Our laboratory has developed a rapid pacing model (30 sequential cycles at increasing rates of stimulation after intermittent periods of rest) in the anesthetized guinea pig and shown it be predictive of the concentration-dependent onset of torsades de pointes type ventricular arrhythmias with a wide variety of both cardiovascular and non-cardiovascular commercial compounds17,18 including several anti-bacterials.19 To determine whether an arrhythmogenic effect could be detected with either azithromycin or chloroquine alone and in combination, studies were conducted to examine the generation of alternans in this model at concentrations within or exceeding the clinically relevant range for management of malaria.20 MATERIALS AND METHODS Monophasic action potential duration (MAPD) alternans measurements in the anesthetized guinea pig. Surgical procedure. All studies were conducted in compliance with an Animal Care and Usage Protocol approved by the Institutional Animal Care and Use Committee (IACUC). Male Hartley guinea pigs weighing 400700 grams were anesthetized with pentobarbital 40 mg/kg, intraperitoneally. Supplemental anesthetic was given if necessary and the guinea pigs were placed on a heating pad for the duration of the surgery and experiment. A tracheotomy was performed and the guinea pigs were ventilated with room air using a Harvard Apparatus Rodent Ventilator Model 683 (South Natick, MA). The right jugular vein and left carotid artery were cannulated with PE50 tubing for drug administration and blood pressure monitoring, respectively. A sternal (or medial) thoracotomy was performed allowing the heart to be suspended in a pericardial cradle. The heart was frequently moistened with lactated Ringers solution. Two Teflon-coated silver wires (0.013 diameter; A-M Systems, Inc., Carlsborg, WA) were sutured to the left ventricular apex for pacing with a Bloom Technologies DTU 215 Programmable Stimulator (Fischer Imaging Corporation, Denver, CO). Monophasic action potential (MAP) signals were recorded from the left ventricle with an EP Technologies EPT 200 Probe (San Jose, CA) that was held in position with a modified stereotaxic stand. Once the MAP signal was stable, the probe remained fixed in the same position throughout the entire study. Lead II ECG signals were recorded with 22-gauge needle electrodes placed subcutaneously around the thoracotomy. The MAP, pacing stimulus, ECG, and blood pressure waveforms were sampled at 1000 Hz and saved to disk using a PoNeMah data acquisition and analysis system (Transoma, St. Paul, MN). Dosing protocol. Guinea pigs received one of the six infusion protocols outlined in Figure 2 and Table 1. These protocols were designed to achieve free plasma concentrations of azithromycin and chloroquine that were near or exceeded the clinical use concentrations of each drug without causing excessive hemodynamic (...truncated)