Abstract

The discovery of (±)-epibatidine, a naturally occurring neuronal nicotinic acetylcholine receptor (nAChR) agonist with antinociceptive activity 200-fold more potent than that of morphine, has renewed interest in the potential role of nAChRs in pain processing. However, (±)-epibatidine has significant side-effect liabilities associated with potent activity at the ganglionic and neuromuscular junction nAChR subtypes which limit its potential as a clinical entity. ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine] is a novel, potent cholinergic nAChR ligand with analgesic properties (see accompanying paper by Bannon et al., 1998b) that shows preferential selectivity for neuronal nAChRs and a consequently improved in vivo side-effect profile compared with (±)-epibatidine. ABT-594 is a potent inhibitor of the binding of [³H](−)-cytisine to α4β2 neuronal nAChRs (K_i = 37 pM, rat brain;K_i = 55 pM, transfected human receptor). At the α1β1δγ neuromuscular nAChR labeled by [¹²⁵I]α-bungarotoxin (α-Btx), ABT-594 has aK_i value of 10,000 nM resulting in a greater than 180,000-fold selectivity of the compound for the neuronal α4β2 nAChR. In contrast, (±)-epibatidine has K_ivalues of 70 pM and 2.7 nM at the α4β2 and α1β1δγ nAChRs, respectively, giving a selectivity of only 38-fold. TheS-enantiomer of ABT-594, A-98593 has activity at the neuronal α4β2 nAChR identical with ABT-594 (K_i = 34–39 pM), which demonstrates a lack of stereospecific binding similar to that reported previously for (±)-epibatidine. A similar lack of stereoselectivity is seen at the human α7 receptor. However, A-98593 is 3-fold more potent at the neuromuscular nAChR (K_i = 3420 nM) and the brain α-Btx-sensitive nAChR (K_i = 4620 nM) than ABT-594. ABT-594 has weak affinity in binding assays for adrenoreceptor subtypes alpha-1B (K_i = 890 nM), alpha-2B (K_i = 597 nM) and alpha-2C (K_i = 342 nM), and it has negligible affinity (K_i > 1000 nM) for approximately 70 other receptors, enzyme and transporter binding sites. Functionally, ABT-594 is an agonist. At the transfected human α4β2 neuronal nAChR (K177 cells), with increased ⁸⁶Rb⁺ efflux as a measure of cation efflux, ABT-594 had an EC₅₀ value of 140 nM with an intrinsic activity (IA) compared with (−)-nicotine of 130%; at the nAChR subtype expressed in IMR-32 cells (sympathetic ganglion-like), an EC₅₀ of 340 nM (IA = 126%); at the F11 dorsal root ganglion cell line (sensory ganglion-like), an EC₅₀ of 1220 nM (IA = 71%); and viadirect measurement of ion currents, an EC₅₀ value of 56,000 nM (IA = 83%) at the human α7 homo-oligimeric nAChR produced in oocytes. A-98593 is 2- to 3-fold more potent and displays approximately 50% greater intrinsic activity than ABT-594 in all four functional assays. In terms of potency, ABT-594 is 8- to 64-fold less active than (±)-epibatidine and also has less IA in these functional assays. ABT-594 (30 μM) inhibits the release of calcitonin gene-related peptide from C-fibers terminating in the dorsal horn of the spinal cord, an effect mediated via nAChRs. Pharmacologically, ABT-594 has an in vitro profile distinct from that of the prototypic nicotinic analgesic (±)-epibatidine, with the potential for substantially reduced side-effect liability and, as such, represents a potentially novel therapeutic approach to pain management.