Kelvin W. Gee

Chronic neuropathic pain may be caused, in part, by loss of inhibition in spinal pain processing pathways due to attenuation of local GABAergic tone. Nociception and nocifensive behaviors are reduced after enhancement of tonically activated extrasynaptic GABA_AR-mediated currents by agonist ligands for δ subunit–containing GABA_ARs. However, typical ligands that target δ subunit–containing GABA_ARs are limited due to sedative effects at higher doses. We used the spinal nerve ligation (SNL) and gp120 models of experimental neuropathic pain to evaluate compound 2-261, a nonbenzodiazepine site positive allosteric modulator of α₄β₃δ GABA_ARs optimized to be nonsedative by selective activation of β_2/3-subunit–containing GABA_ARs over receptor subtypes incorporating β₁subunits. Similar levels of 2-261 were detected in the brain and plasma after intraperitoneal administration. Although systemic 2-261 did not alter sensory thresholds in sham-operated animals, it significantly reversed SNL-induced thermal and tactile hypersensitivity in a GABA_AR-dependent fashion. Intrathecal 2-261 produced conditioned place preference and elevated dopamine levels in the nucleus accumbens of nerve-injured, but not sham-operated, rats. In addition, systemic pretreatment with 2-261 blocked conditioned place preference from spinal clonidine in SNL rats. Moreover, 2-261 reversed thermal hyperalgesia and partially reversed tactile allodynia in the gp120 model of HIV-related neuropathic pain. The effects of 2-261 likely required interaction with the α₄β₃δ GABA_AR because 2-301, a close structural analog of 2-261 with limited extrasynaptic receptor efficacy, was not active. Thus, 2-261 may produce pain relief with diminished side effects through selective modulation of β_2/3-subunit–containing extrasynaptic GABA_ARs.

Type I positive allosteric modulators (PAMs) of the alpha7-nicotinic receptor enhance its cholinergic activation while preserving the spatiotemporal features of synaptic transmission and the receptor’s characteristic rapid desensitization kinetics. Alpha7-nicotinic receptor agonists have shown promise for improving cognition in schizophrenia, but longer-term trials have been disappointing. Therefore, the type I PAM AVL-3288 was evaluated for safety and preliminary evidence of neurocognitive effect in healthy human subjects. Single-dose oral administration in ascending doses was conducted in a double-blind, placebo-controlled Phase I trial in non-smokers. The trial found indication of positive but non-significant effects on neurocognition at 10 and 30 mg, two doses that produced overlapping peak levels. There was also some evidence for effects on inhibition of the P50 auditory evoked potential to repeated stimuli, a biomarker that responds to alpha7-nicotinic receptor activation. The pharmacokinetic characteristics were consistent between subjects, and there were no safety concerns. The effects and safety profile were also assessed at 3 mg in a cohort of smokers, in whom concurrent nicotine administration did not alter either effects or safety. The trial demonstrates that a type I PAM can be safely administered to humans and that it has potential positive neurocognitive effects in central nervous system (CNS) disorders.

In order to improve upon the pharmacological properties of the neuroactive steroid ganaxolone, it was used as the starting point in the design of novel neurosteroids that replace the 17β-acetyl side chain with an isoxazole bioisostere. UCI-50027 (3-[3α-hydroxy-3β-methyl-5α-androstan-17β-yl]-5-(hydroxymethyl)isoxazole) was designed as an orally active neuroactive steroid specifically targeted at the gamma-aminobutyric acid(A) receptor (GABA_AR). UCI-50027 was tested in vitro in Xenopus oocytes expressing human GABA_ARs and in vivo as an anticonvulsant, for ataxic effects and for anxiolytic activity. In vitro, UCI-50027 dose-dependently enhanced the activity of GABA at human α₁β₂γ_2L, α₂β₁γ_2L, and α₄β₃δ GABA_ARs. Consistent with its action as a positive allosteric modulator (PAM), it had no direct activity in the absence of GABA. UCI-50027 protected against acute pentylenetetrazol (PTZ)-induced convulsions with an ED₅₀ of 6 mg/kg p.o. In the rotarod (RR) paradigm in mice, the AD₅₀ (the ataxic dose where half of the animals fail the RR test) was found to be 38 mg/kg p.o., giving a therapeutic index (TI = RR AD₅₀/PTZ ED₅₀)∼6 versus 2.8 for ganaxolone. In the mouse-elevated plus maze (EPM) model for anxiety, UCI-50027 showed a minimum effective dose (MED) ≤0.3 mg/kg p.o. Thus, the TI (TI = RR AD₅₀/EPM MED) for the compound as an anxiolytic is ≥127 versus 3.3 for ganaxolone. UCI-50027 is an orally active neuroactive steroid with pharmacological activity consistent with a GABA_AR PAM that has an improved separation between anticonvulsant/anxiolytic and rotarod effects, potent activity as an anticonvulsant and anxiolytic when compared to ganaxolone.

GABAergic anxiolytics have well-documented centrally mediated side effects including sedation, potentiation of ethanol, tolerance, abuse liability and memory impairment. Most research directed towards identifying an anxioselective GABAergic therapeutic has been based upon the theory that these side effects could be mitigated by avoiding α1/5-subunit GABA_A receptors while specifically targeting those with the α2/3-subunit. Unfortunately, there are prominent exceptions to this theory and it has yet to be translated into clinical success. We previously demonstrated that β2/3-subunit-selective GABA_A receptor-positive allosteric modulators act as anxiolytics with reduced sedation and ethanol potentiation regardless of their activity at α1-subunit GABA_A receptors. The prototypical β2/3-subunit-selective positive allosteric modulator, 2-261, is further characterized here for additional side effects commonly associated with central GABA_A receptor activation. In mice, 10 times the anxiolytic dose (10 mg/kg) of 2-261 does not induce behavioral tolerance in the elevated plus maze following a 2 week subchronic treatment. In rats, an anxiolytic dose (10 mg/kg) of 2-261 is inactive in conditioned place preference, suggesting a reduced abuse liability. In rats, 10 times the anxiolytic dose (100 mg/kg) of 2-261 does not have a significant amnestic effect in the radial arm maze, suggesting a greater therapeutic index for memory impairment. These results suggest that β2/3-subunit subtype-selective GABA_A receptor-positive allosteric modulators not only have reduced sedative liability, but also a reduction in other central side effects commonly associated with broader GABA_A receptor activation. β2/3-subunit-selective compounds may represent a novel design template for anxiolytics with benzodiazepine-like efficacy and mitigated side effects.

GABA_A receptor (R) positive allosteric modulators that selectively modulate GABA_ARs containing β₂- and/or β₃- over β₁-subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,“β_2/3-selective” GABA_AR positive allosteric modulators are nonbenzodiazepines (nonBZs), do not show α-subunit isoform selectivity, yet have anxiolytic efficacy with reduced ataxic/sedative effects in animal models and humans. Here, we report on an enantiomeric pair of nonBZ GABA_AR positive allosteric modulators that demonstrate differential β-subunit isoform selectivity. We have tested this enantiomeric pair along with a series of other β_2/3-subunit selective, α-subunit isoform-selective, BZ and nonBZ GABA_A positive allosteric modulators using electrophysiological, pharmacokinetic, and behavioral assays to test the hypothesis that ataxia may be correlated with the extent of modulation at β₁-subunit-containing GABA_ARs. Our findings provide an alternative strategy for designing anxioselective allosteric modulators of the GABA_AR with BZ-like anxiolytic efficacy by reducing or eliminating activity at β₁-subunit-containing GABA_ARs.

Drugs that antagonize nicotinic acetylcholine receptors (nAChRs) can be used to inhibit nicotine-induced behavior in both humans and animals. The aim of our experiments is to establish a proof-of-principle that antagonism of nAChRs by negative allosteric modulation can alter behavior in a relevant animal model of addiction, nicotine self-administration. We have identified a novel, negative allosteric modulator of nAChRs, UCI-30002 [N-(1,2,3,4-tetrahydro-1-naphthyl)-4-nitroaniline], with selectivity for the major neuronal nAChR subtypes over muscle-type nAChRs. After systemic administration, UCI-30002 significantly reduces nicotine self-administration in rats on both fixed ratio and progressive ratio schedules of reinforcement. The minimum effective dose that significantly alters nicotine self-administration corresponds to brain concentrations of UCI-30002 that produce at least 30% inhibition of the major neuronal nAChR subtypes measured in vitro. UCI-30002 has no effect on responding for food reinforcement in rats on either type of schedule, indicating that there is no effect on general responding or natural reward. UCI-30002 represents validation of the concept that negative allosteric modulators may have significant benefits as a strategy for treating nicotine addiction and encourages the development of subtype-selective modulators.

Activation of brain α7 nicotinic acetylcholine receptors (α7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of α7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective α7 nAChR-positive allosteric modulator (PAM) from a library of GABA _A receptor PAMs. Compound 6 ( N -(4-chlorophenyl)-α-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at α7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of α7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.