Characterization of the novel M4 muscarinic acetylcholine receptor positive allosteric modulator VU0467154 in animal models of antipsychotic-like activity, cognitive enhancement and changes in sleepwake architecture

Abstract

Background: Accumulating evidence indicates that selective positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor may provide a novel therapeutic strategy for the treatment of psychotic symptoms and behavioral disturbances observed in various psychiatric and neurologic disorders. Previously, our group reported the discovery of an early generation of systemically active M4 PAMs, represented by VU0152100, which displayed central effects in reversing amphetaminechallenge models in rodents. While VU0152100 served as an important tool compound for initial proof of concept studies on the effects of M4 PAMs in vivo, limitations in the physiochemical and pharmacokinetic properties of the ligand restricted its broader use for extensive characterization in preclinical models of psychosis and cognitive disruptions. We now report the identification of a highly optimized series of selective M4 PAMs, represented by VU0467154 that exhibits low nanmolar potency, enhanced pharmacokinetic properties in mice and rats for in vivo dosing and robust efficacy across several preclinical models of antipsychotic-like activity and enhancement of cognition. Methods: HTS and medicinal chemistry approaches were utilized to identify this highly optimized series of selective M4 PAMs, represented by VU0467154. Through an iterative analog approach, chemical libraries were generated around initial hits for the M4 receptor subtype. Calcium mobilization assays were used in parallel to generate functional potency and efficacy data for all compounds, and potential off target activity was assessed in Recerca lead profiling screens by percent radioligand displacement at 10 μM. Disposition was determined in mouse and rat pharmacokinetic studies using LC/MS/MS quantitation. In vivo efficacy was determined across several mouse and rat models of antipsychotic-like activity and in a touchscreen based pairwise discrimination task alone and after psychostimulant challenge. We also assessed the effects of VU0467154 on sleep-wake architecture using electroencephalography (EEG) studies in rats. Results: In vitro, VU0467154 is a highly selective and low nanomolar potency M4 PAM with no activity at the M1,2,3,5 up to 30 μM or significant off target activity at other GPRCs at 10 μM. VU0467154 is a low clearance (CLp; 7.8 ml/min/ kg), long half-life (t1/2; 5.7 h), and highly brain penetrant ligand with excellent pharmacokinetic properties, including high oral bioavailability (%F; 61) following administration of 3mg/kg to rats with a maximum concentration in plasma (Cmax) of 700nM and a time to reach Cmax (Tmax) of 3 h. VU0467154 produced robust dose-dependent reversals of amphetamine-induced hyperlocomotion in rats and mice within a dose range that produced no adverse effects or motor impairments as measured using a modified Irwin neurological test battery and rotarod test. In addition, VU0467154 produced reversals of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) antagonist MK- 801-induced hyperlocomotion and disruptions of contextual fear conditioning and pairwise discrimination, two hippocampal-mediated memory tasks. Finally, VU0467154 selectively increased the latency to paradoxical sleep (a correlate of REM sleep) and reduced paradoxical sleep without affecting sleep onset or total sleep duration similar to clinically available antipsychotics. However, in contrast to some antipsychotic medications, VU0467154 did not decrease delta power during slow wave sleep, a critical measure of sleep quality. Conclusions: Taken together, VU0467154 represents a highly optimized M4 PAM tool compound for the evaluation of the role of M4 in vivo and provides further validation for the development of selective M4 PAMs as a novel approach for the psychotic symptoms and behavioral disturbances observed in psychiatric and neurologic disorders including schizophrenia and Alzheimer's disease. The work is funded by NIMH RO1 grants# 2R01 MH73676- 06, 1R01 MH086601-04, AstraZeneca Vanderbilt is a Specialized Chemistry Center within the Molecular Libraries Probe Centers.