Neuropharmacology and Analgesia
The novel small molecule α9α10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic

https://doi.org/10.1016/j.ejphar.2011.08.053Get rights and content

Abstract

Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the α9α10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the α9α10 subtype with an IC50 of 0.51 (0.35–0.72) nM. Antagonist activity at other nicotinic subtypes (α1β1δε, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3, α6/α3β4 and α7) was 10–1000-fold lower than at the α9α10 subtype. In competition binding assays, the ki of ZZ-204G at γ-aminobutyric acid(A), serotonin(3), γ-aminobutyric acid(B), κ- and μ-opioid receptors was 1000- to > 10,000-fold lower than at α9α10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for α9α10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain.

Introduction

Chronic pain afflicts approximately 20% of the adult population in developed nations (Brennan et al., 2007). Although numerous analgesic medications are available for treatment, these pharmacotherapies act via a limited number of molecular mechanisms. The primary classes of analgesics include opioids, non-steroidal anti-inflammatory drugs and various adjuvant agents including antidepressants, anticonvulsants, and α2-adrenergic agonists. Altogether, these medications inadequately manage pain, and a belief among many chronic pain patients is that no treatment will alleviate their suffering.

One promising new target is the neuronal nicotinic acetylcholine receptor, which is present in pain signaling pathways, including primary afferents, spinal cord excitatory and inhibitory interneurons, projecting neurons, brain nuclei with descending spinal projection, and dorsal root ganglia (D'Hoedt and Bertrand, 2009, Taly et al., 2009). Nicotinic receptors are pentameric ligand gated ion channels composed of α and/or β subunit. In mammals, nine α (α1–7, α9 and α10) and four β (β1–4) subunits have been described. Significant efforts have focused on agonists of central α4β2 ( denotes the possible presence of additional subunits) and α7 nicotinic acetylcholine receptors (Vincler, 2005). Activation of either α4β2 or α7 subtypes is associated with analgesic activity in preclinical pain models. Nevertheless, α4β2 and α7 nicotinic acetylcholine receptors are widespread in the CNS and thus, the potential for centrally-mediated side-effects make these agents less desirable analgesic drugs.

α-Conotoxins are small, disulfide rich peptides, isolated from carnivorous marine snails, which selectively target nicotinic receptors (Azam and McIntosh, 2009, Nicke et al., 2004). Recent studies employing α-conotoxins have suggested that the α9α10 nicotinic receptor subtype may be important as a potential target for novel analgesic agents. Interestingly in this case, antagonist rather than agonist activity is associated with analgesia. Transcripts for α9 and α10 subunits have been identified in dorsal root ganglion neurons and a variety of immune cells (Elgoyhen et al., 2001, Gomez-Casati et al., 2005, McIntosh et al., 2009, Weisstaub et al., 2002). Of note, the α9α10 subtype is absent in brain removing the potential for cognitive side effects from blockade of this receptor. To our knowledge, there are no reports of α9 expression in spinal cord.

Peripherally administered α9α10 selective α-conotoxin antagonists produce analgesia in rodent models of neuropathic and tonic inflammatory pain at doses that do not produce motor toxicity (McIntosh et al., 2009, Vincler and McIntosh, 2007, Vincler et al., 2006). In order to characterize non-peptide nicotinic antagonists, we have begun to examine tetrakis-azaaromatic quaternary ammonium salts. Such compounds have previously been shown to be potent inhibitors at α7 nicotinic acetylcholine receptors (Lopez-Hernandez et al., 2009) and to inhibit nicotinic receptor subtypes that mediate nicotine-evoked dopamine release (Zhang et al., 2008). Here, we report that one such analog that has high potency and selectivity for α9α10 nicotinic acetylcholine receptors and has analgesic properties in rat models of pain including chronic constriction nerve injury model of neuropathic pain and the formalin model of tonic inflammatory pain. The current results suggest the feasibility of designing orally active small molecules that exert their therapeutic action via the novel α9α10 target.

Section snippets

Synthesis of ZZ-204G

ZZ-204G [5,5′,5″,5‴-(1,2,4,5-benzenetetrayl)tetrakis-[1-(3-phenylpyridinium)-4-pentyne] tetrabromide] was synthesized utilizing the general procedure described by Zhang et al. (2008), by reacting 5,5′,5″,5‴-(1,2,4,5-benzenetetrayl)tetrakis-[1-bromo-4-pentyne] (0.46 mmol) with 3-phenylpyridine (2 mmol) at 60–70 °C for 18 hours. The resulting mixture was then treated with diethyl ether and the mixture dissolved in water (15 ml). The aqueous solution was extracted extensively with chloroform (5 × 30 ml)

Activity at nicotinic acetylcholine receptor subtypes and off target receptors

To assess the functional effects of ZZ-204G (structure illustrated in Fig. 1) the compound was applied to Xenopus oocytes heterologously expressing either neuronal or muscle nicotinic acetylcholine receptors. ZZ-204G potently blocked α9α10 nicotinic acetylcholine receptors, with 10 nM compound blocking 93.9 ± 1.4% inhibition of the response (n = 6 oocytes). Block was reversed upon washout of ZZ-204G. By comparison, ZZ-204G (100 nM) blocked only ~ 5% of the acetylcholine-induced currents from α4β2

Discussion

The current results show that the tetrakis-quaternary ammonium compound ZZ-204G is a potent antagonist of nicotinic acetylcholine receptors. To our knowledge this is the first report of a small molecule nicotinic ligand that shows selectivity for the α9α10 nicotinic acetylcholine receptor subtype. Extensive prior work on nicotine analogs and related compounds has focused on the development of analgesic agonists (Arneric et al., 2007, Buccafusco, 2004, D'Hoedt and Bertrand, 2009). Given the wide

Acknowledgments

This work was supported by U19 DA017548, NIH MH53631, GM48677 and a seed grant from the University of Utah Research Foundation to JMM. Radioligand binding studies were generously performed by the NIMH Psychoactive Drug Screening Program, contract # HHSN-271-2008-00025-C. The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA.

References (39)

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