α4β2 neuronal nicotinic receptor positive allosteric modulation: An approach for improving the therapeutic index of α4β2 nAChR agonists in pain
Graphical abstract
Positive allosteric modulator alone (PAM) does not have intrinsic activity at the receptor, but can amplify the physiological effects of the endogenous transmitter Ach or agonists (ABT-594, nicotine) in a spatial and temporally restricted manner.
Introduction
Pain is one of the most prominent reasons for patient visits to physicians. Current treatments for pain are either refinements of opiates or nonsteroidal anti-inflammatory drugs (NSAIDs). Although these treatment approaches are available for more than a decade, several types of pain, particularly associated with nerve damage, are still poorly managed by these medications (for review [2]). Neuropathic pain is predominately treated by use of antidepressants and antiepileptics agents such as duloxetine, gabapentin and pregabalin. However, neuropathic pain is still poorly managed by these agents either due to limited efficacy across the patient population or due to the dose limited side effect/tolerability profiles. Given the need for novel analgesic agents with improved efficacy, interest continues in the identification and validation of novel molecular targets and approaches for pain.
Neuronal nicotinic receptors (nAChRs) are ligand-gated ion channels, each composed of five subunits surrounding a cation pore. Various neuronal nAChR subunits (α2–α10 and β2–β4) are differentially expressed throughout the nervous system and combine to form diverse subtypes with a wide range of physiological and pharmacological profiles. The two most abundant nAChRs in the CNS can be differentiated by their relative affinities for nicotine and α-bungarotoxin. nAChRs with high affinity for nicotine but low affinity for α-bungarotoxin largely contain combinations of α4 and β2 subunits, whereas nAChRs with low affinity for nicotine but high affinity for α-bungarotoxin are predominantly α7-containing. nAChRs derived from α3β4 subunits are in much higher abundance in the autonomic nervous system than in the CNS and are generally regarded to be responsible for many of the adverse effects of nicotine.
α4β2 nAChR agonists have the potential as broad-spectrum analgesics based on preclinical studies demonstrating their efficacy in diverse pain states including multiple forms of acute, chronic, inflammatory and neuropathic pain [2]. These ligands act at multiple locations throughout the pain pathway to relieve pain. For example, α4β2 nAChRs are found on primary sensory neurons (periphery) where nociceptive information is initiated, in the cell body regions of these neurons (i.e. the dorsal root ganglion or DRG), the dorsal spinal cord where the first pain synapse is located, in the brainstem cell body regions that control descending innervation, as well as in the higher brain regions that integrate and perceive sensory information such as the thalamus and the cortex. In addition to studies with nAChR agonists, evidence in support for a role in α4β2 nAChRs in pain has also emerged from antisense and knockout studies. Antisense knockdown of the α4 subunit has been found to decrease the analgesic effect of agonists [2]. Reduced antinociceptive responses to nicotine also are seen in α4 and β2 knockout animals [3], [4]. Antinociceptive effects through α4β2 nAChRs have been attributed to enhancing descending inhibition, particularly in the raphe [5]. However, α4β2 nAChR stimulation of GABAergic and glycinergic inhibitory transmission in the spinal cord also may contribute [6], [7].
ABT-594, the first nAChR agonist demonstrated clinical efficacy in the treatment of painful diabetic neuropathy [8]. With ABT-594, however the efficacious doses were associated with an undesirable side effect profile, particularly with respect to nausea and emesis. Although targeting the α4β2 subtype with nAChR agonists with enhanced subtype selectivity remains a viable approach, robust efficacy in pain may be limited by the range of side effects (nausea, emesis, etc.) arising from possible interactions with the ganglionic nAChR (α3β4*) at efficacious doses. In light of the significance of chronic pain and the limitations in current therapeutic agents, it would be beneficial to further exploit the nAChR platform toward developing new approaches for treating such disorders, particularly those aimed at reducing adverse effects.
One approach to selectively enhance activity at the α4β2 nAChR is via positive allosteric modulation. As reported previously, positive allosteric modulators (PAMs) can enhance the efficacy and potency of agonist(s) so as to selectively amplify effects at the α4β2 nAChR [9], [10]. A positive allosteric modulator alone, in principle, does not exhibit intrinsic activity at the receptor, but can amplify the effects of agonists [11]. Because PAMs do not interact with agonist-binding site, they could, in principle, display a higher degree of selectivity at the target α4β2 subtype vs. other nAChR subtypes. Studies reported herein reveal that the analgesic efficacy of nAChR agonists such as ABT-594 in pain can be enhanced in combination with PAMs. Under these conditions, the overall preclinical side effect profiles (gastrointestinal, cardiovascular, etc.) are not affected. A portion of this study has been previously presented in an abstract form [12].
Section snippets
FLIPR calcium imaging assay
Experiments were carried out according to the methods described previously [13], [14]. HEK-293 cell lines stably expressing human α4β2, α3β4, or α4β4 nAChRs were established and maintained using standard procedures [13], [14]. Agonist-evoked Ca2+ increases were measured using Fluo-4/AM to detect intracellular Ca2+ in conjunction with a fluorescence imaging plate reader (FLIPR) equipped with an argon laser and a CCD camera (Molecular Devices/Danaher Corp., Sunnyvale, CA). Black-walled 96-well
In-vitro pharmacology
As previously reported, NS9283 (also known as A-969933, Table 1) was found to increase agonist-evoked response amplitude of α4β2 nAChRs in Ca2+-imaging and electrophysiology paradigms but did not itself produce receptor activation [9], [10]. In this study, NS9283 was initially tested for in vitro activity as positive allosteric modulator in HEK-293 cells expressing the human α4β2 nAChRs using ABT-594 as the agonist. As shown in Fig. 1A, NS9283 left-shifted the concentration response profile of
Discussion
Discovery of highly selective and positive allosteric modulators of nicotinic receptor provides an unprecedented opportunity for further interrogation of the physiological roles of receptor subtypes, as in this study, dissecting pain efficacy vs. adverse effects of nAChR ligands using an α4β2 selective PAM. α4β2 nAChR agonism is a clinically validated approach for treatment of pain as revealed with ABT-594, the first nAChR agonist demonstrating efficacy (phase 2) in the treatment of diabetic
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