Interaction of azimilide with neurohumoral and channel receptors Robert R. Brooks a, *, Schwe Fang Pong a , Nicholas J. Izzo a , Thomas J. Moorehead a , Murali Gopalakrishnan b , David J. Triggle c a Procter & Gamble Pharmaceuticals, 11810 E. Miami River Rd., Cincinnati, OH 45252, USA b Abbott Laboratories, Abbott Park, IL 60064, USA c State University of New York, Buffalo, NY 14260, USA Received 23 August 2000; accepted 31 January 2001 Abstract Binding of the class III antiarrhythmic agent azimilide to brain, heart, and other organ receptors was assessed by standard radioligand binding techniques. In a survey of 60 receptors, azimilide at 10 M inhibited binding by more than 50% at serotonin uptake ( K i : 0.6 M), muscarinic ( K i : 0.9 to -3.0 M), Na + channel site 2 ( K i : 4.3 M), and central sigma ( K i : 6.2 M) sites. Lesser (20 – 40%) inhibition was seen at adrenergic, histamine, serotonin, purinergic, angiotensin II, dopamine uptake, and norepinephrine sites and at a voltage-sensitive K + channel. In rat ventricle, azimilide inhibited binding to  1 - and -adrenergic and muscarinic receptors ( K i : 5 M) and to the L-type Ca 2+ channel ( K i : 37.3 M). In rat brain, azimilide blocked ligand binding to these same receptors and to a serotonin receptor, and the breadth and potency of its interaction pattern differentiated it from ten other class III antiarrhythmics. Azimilide displayed agonist and antagonist action at five muscarinic receptor subtypes in transfected NIH 3T3 cells producing receptor-sensitive mitogenesis and -galac- tosidase activity. Agonist action predominated at M 2 and M 4 subtypes, and antagonist action predominated at M 1 ,M 3 , and M 5 subtypes. The azimilide concentration for 50% maximum stimulation (EC 50 ) in M 2 -expressing cells was 1.97 M (vs 0.14 M for carbachol). Azimilide’s receptor interactions occur at concentrations from one to forty times those required to block cardiac delayed-rectifier channels but could contribute to the efficacy and safety of the drug. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Azimilide; Class III antiarrhythmic; Radioligand binding; Adrenergic receptors; Muscarinic receptors 1. Introduction Azimilide dihydrochloride (NE-10064, (E)-1-[[[5-(4- chlorophenyl)-2-furanyl]methylene]amino]-3-[4-(4-methyl- 1-piperazinyl)butyl]-2,4-imidazolidinedione dihydrochlo- ride) prolongs cardiac refractoriness by blocking the fast ( I Kr ) and slow ( I Ks ) components of the delayed-rectifier potassium current [1–3]. These in vitro electrophysiologic properties could account for its efficacy in rodent and dog models of ischemia-induced ventricular arrhythmia [4,5], termination of atrial arrhythmias in dogs [6,7], suppression of fibrillation in a dog model of sudden cardiac death [8], and clinical efficacy [9,10]. Azimilide blocks delayed-rec- tifier currents at 0.2 to 3 M (50% inhibition in vitro in guinea pig) and also interacts with other ion channels, blocking cardiac Na + channels at 12 M (guinea pig), an L-type Ca 2+ channel at 18 M (dog), and the inward rec- tifier channel at 50 M (dog) [11–13]. Drugs targeted primarily at one biological receptor can exhibit some interaction with other receptors, where effects as either agonist or antagonist may contribute to efficacy or side-effects. Azimilide’s block of ligand binding at 60 re- ceptors, including neurotransmitters, ion channels, second messengers, steroids, brain and gut peptides, growth factors, and peptides was evaluated by standard protocols at the Oceanix Biosciences Corp. These receptors cover many body systems with possible targets for the drug from an efficacy or safety viewpoint. The study was designed, in particular, to identify possible targets for the compound from a side-effect perspective. A survey approach, involv- ing 60 receptors found at many sites, was considered ap- propriate for identifying potentially important interactions * Corresponding author. Tel.: +1-513-627-0313; fax: +1-513-627- 0929. E-mail address: brooks.rr@pg.com (R.R. Brooks). Abbreviations: I Kr , rapidly activating component of the delayed-recti- fier current; I Ks , slowly activating component of the delayed-rectifier current; EC 50 , concentration giving 50% of the maximum stimulation; K i , inhibition constant; and HT, hydroxytryptamine (serotonin). Biochemical Pharmacology 62 (2001) 883– 892 0006-2952/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0006-2952(01)00737-7