Timothy B. Johnstone

Nootropic α7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators

Activation of brain α7 nicotinic acetylcholine receptors (α7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimers 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 GABAA 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.

Reversible lipidization for the oral delivery of leu-enkephalin.

The endogenous opioid peptide leu-enkephalin (ENK) was chemically modified by a method known as reversible aqueous lipidization (REAL) with a novel amine-reacting lipophilic dimethylmaleic anhydride analog, 3,4-bis(decylthiomethyl)-2,5-furandione. The binding affinity of the product, REAL-ENK, to opioid receptors was greatly reduced. This prodrug was stable in neutral and basic phosphate buffers but underwent rapid hydrolysis under acidic conditions in the presence of 50% acetonitrile. It also showed increased stability toward enzymatic degradations in various tissue preparations. The half-lives of REAL-ENK in mouse small intestinal mucosal homogenate and liver homogenate were 12 and 80 min, representing a 12- and 32-fold increase over those of ENK itself. In contrast to ENK (t1/2 6.7 min), REAL-ENK was stable in mouse plasma. More importantly, REAL-ENK produced significant and sustained antinociception mediated by peripheral opioid receptors in a rodent inflammatory pain model. Pharmacokinetic studies employing a radioimmunoassay (RIA) demonstrated that significantly higher and sustained plasma peptide levels were detected up to 24 h following the oral administration of REAL-ENK in normal mice. The peak concentration and area under the curve of oral REAL-ENK were 4.4 and 21 times higher than that of oral ENK. Our results indicate that like its disulfide-based counterpart, amine-based REAL may be an enabling technology which can be applied to enhance metabolic stability, increase oral absorption, and preserve and possibly prolong the pharmacological activity of peptide drugs.

Limiting Activity at β1-Subunit-Containing GABAA Receptor Subtypes Reduces Ataxia

GABAA receptor (R) positive allosteric modulators that selectively modulate GABAARs containing β2- and/or β3- over β1-subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,“β2/3-selective” GABAAR 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 GABAAR positive allosteric modulators that demonstrate differential β-subunit isoform selectivity. We have tested this enantiomeric pair along with a series of other β2/3-subunit selective, α-subunit isoform-selective, BZ and nonBZ GABAA 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 GABAARs. Our findings provide an alternative strategy for designing anxioselective allosteric modulators of the GABAAR with BZ-like anxiolytic efficacy by reducing or eliminating activity at β1-subunit-containing GABAARs.

First in human trial of a type I positive allosteric modulator of alpha7-nicotinic acetylcholine receptors: Pharmacokinetics, safety, and evidence for neurocognitive effect of AVL-3288:

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.

Allosteric modulation of related ligand-gated ion channels synergistically induces long term potentiation in the hippocampus and enhances cognition.

α5 Subunit-containing GABAA receptors (GABAARs) and α7 neuronal nicotinic-acetylcholine receptors (nAChRs) are members of the Cys-loop family of ligand-gated ion channels (LGICs) that mediate cognitive and attentional processes in the hippocampus. α5 GABAARs alter network activity by tonic inhibition of CA1/CA3 pyramidal cells of the hippocampus. Postsynaptic α7 nAChRs in the hippocampus regulate inhibitory GABAergic interneuron activity required for synchronization of pyramidal neurons in the CA1, whereas presynaptic α7 nAChRs regulate glutamate release. Can simultaneous allosteric modulation of these LGICs produce synergistic effects on cognition? We show that combined transient application of two allosteric modulators that individually 1) inhibit α5 GABAARs and 2) enhance α7 nAChRs causes long-term potentiation (LTP) of mossy fiber stimulation-induced excitatory postsynaptic currents (EPSC) from CA1 pyramidal neurons of rat hippocampal slices. The LTP effect evoked by two compounds is replicated by 3-(2,5-difluorophenyl)-6-(N-ethylindol-5-yl)-1,2,4-triazolo[4,3-b]pyridazine (522-054), a compound we designed to simultaneously inhibit α5 GABAARs and enhance α7 nAChRs. Selective antagonists for either receptor block sustained EPSC potentiation produced by 522-054. In vivo, 522-054 enhances performance in the radial arm maze and facilitates attentional states in the five-choice serial reaction time trial with similar receptor antagonist sensitivity. These observations may translate into therapeutic utility of dual action compounds in diseases of hippocampal-based cognitive impairment.

Design, Synthesis, and Activity of a Series of Arylpyrid-3-ylmethanones as Type I Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors

A series of novel arylpyrid-3-ylmethanones (7a-aa) were designed as modulators of α7 nicotinic acetylcholine receptors (nAChRs). The methanones were found to be type I positive allosteric modulators (PAMs) of human α7 nAChRs expressed in Xenopus ooctyes. Structure-activity relationship (SAR) studies resulted in the identification of compound 7v as a potent and efficacious type I PAM with maximum modulation of a nicotine EC5 response of 1200% and EC50 = 0.18 μM. Compound 7z was active in reversing the effect of scopolamine in the novel object recognition (NOR) paradigm with a minimum effective ip dose of 1.0 mg/kg (2.7 μmol/kg). This effect was blocked by the selective α7 nAChR antagonist methyllycaconitine (MLA). These compounds are potent type I positive allosteric modulators of α7 nAChRs that may have therapeutic value in restoring impaired sensory gating and cognitive deficits in schizophrenia and Alzheimers disease.

Limited central side effects of a β-subunit subtype-selective GABAA receptor allosteric modulator

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 GABAA 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 GABAA receptor-positive allosteric modulators act as anxiolytics with reduced sedation and ethanol potentiation regardless of their activity at α1-subunit GABAA receptors. The prototypical β2/3-subunit-selective positive allosteric modulator, 2-261, is further characterized here for additional side effects commonly associated with central GABAA 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 GABAA receptor-positive allosteric modulators not only have reduced sedative liability, but also a reduction in other central side effects commonly associated with broader GABAA receptor activation. β2/3-subunit-selective compounds may represent a novel design template for anxiolytics with benzodiazepine-like efficacy and mitigated side effects.

Role of β 2/3 -specific GABA-A receptor isoforms in the development of hippocampus kindling epileptogenesis

OBJECTIVE Subunit-specific positive allosteric modulators (PAMs) of gamma-aminobutyric acid-A (GABA-A) receptors are commonly used to uncover the role of GABA-A receptor isoforms in brain function. Recently, we have designed novel PAMs selective for β2/3-subunit containing GABA-A receptors (β2/3-selective PAMs) that are nonbenzodiazepine site-mediated and do not show an α-subunit isoform selectivity, yet exhibit anxiolytic efficacy with reduced potential for sedation, cognitive impairment, and tolerance. In this study, we used three novel β2/3-selective PAMs (2-261, 2-262, and 10029) with differential β2/3-subunit potency to identify the role of β2/3-selective receptor isoforms in limbic epileptogenesis. METHODS Experimental epileptogenesis was induced in mice by daily hippocampus stimulations until each mouse showed generalized (stage 5) seizures. Patch-clamp electrophysiology was used to record GABA-gated currents. Brain levels of β2/3-selective PAMs were determined for mechanistic correlations. RESULTS Treatment with the β2/3-selective PAMs 2-261 (30mg/kg), 2-262 (10mg/kg), and 10029 (30mg/kg), 30min prior to stimulations, significantly suppressed the rate of development of kindled seizure activity without affecting the afterdischarge (AD) signal, indicating their disease-modifying activity. The β2/3-selective agents suppressed chemical epileptogenesis in the pentylenetetrazol model. Test doses of these agents were devoid of acute antiseizure activity in the kindling model. CONCLUSION These findings demonstrate that β2/3-selective PAMs can moderately retard experimental epileptogenesis, indicating the protective role of β2/3-subunit GABA-A receptor isoforms in the development of epilepsy.

Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model

&NA; Autism spectrum disorder (ASD) is associated with two core symptoms (social communication deficits and stereotyped repetitive behaviors) in addition to a number of comorbidities. There are no FDA‐approved drugs for the core symptoms and the changes that underlie these behaviors are not fully understood. One hypothesis is an imbalance of the excitation (E)/inhibition (I) ratio with excessive E and diminished I occurring in specific neuronal circuits. Data suggests that both gamma‐aminobutyric acidA (GABAA) and &agr;7 nicotinic acetylcholine receptors (nAChRs) significantly impact E/I. BTBR T+ tf/J (BTBR) mice are a model that display an autism‐like phenotype with impaired social interaction and stereotyped behavior. A &bgr;2/3‐subunit containing GABAA receptor (GABAAR) subtype selective positive allosteric modulator (PAM), 2–261, and an &agr;7 nAChR subtype selective PAM, AVL‐3288, were tested in social approach and repetitive self‐grooming paradigms. 2–261 was active in the social approach but not the self‐grooming paradigm, whereas AVL‐3288 was active in both. Neither compound impaired locomotor activity. Modulating &agr;7 nAChRs alone may be sufficient to correct these behavioral and cognitive deficits. GABAergic and nicotinic compounds are already in various stages of clinical testing for treatment of the core symptoms and comorbidities associated with ASD. Our findings and those of others suggest that compounds that have selective activities at GABAAR subtypes and the &agr;7 nAChR may address not only the core symptoms, but many of the associated comorbidities as well and warrant further investigation in other models of ASD. HighlightsGABAAR subtype selective PAMs ameliorate aberrant social but not repetitive behaviors.&agr;7 nAChR PAMs ameliorate both social and repetitive behaviors.GABAAR and nAChR PAMs may address the core symptoms and comorbidities of autism.

Positive Allosteric Modulators of Non-Benzodiazepine γ-Aminobutyric acida Receptor Subtypes for the Treatment of Chronic Pain

Abstract 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 GABAAR-mediated currents by agonist ligands for &dgr; subunit–containing GABAARs. However, typical ligands that target &dgr; subunit–containing GABAARs 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 &agr;4&bgr;3&dgr; GABAARs optimized to be nonsedative by selective activation of &bgr;2/3-subunit–containing GABAARs over receptor subtypes incorporating &bgr;1 subunits. 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 GABAAR-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 &agr;4&bgr;3&dgr; GABAAR 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 &bgr;2/3-subunit–containing extrasynaptic GABAARs.