Abstract Recently, we disclosed our M1 PAM that had advanced into Phase I clinical studies, VU0467319, as well as a deuterated back-up compound, VU6045422, to address a major metabolite produced in man and improve tox profile. Ultimately, this series failed to overcome its liabilities and was terminated. However, the team was challenged with developing chemically distinct backup candidates without performing a new HTS campaign. De novo design strategies based upon the other two M1 ago-PAMs that had previously entered clinical development, MK-7622 and TAK-071, led to the discovery of a fundamentally new chemotype based on a 3,4-dihydrobenzo[f][1,4]oxazepin-5-(2H)-one core, providing VU6069863. VU6069863 proved to be an M1 PAM (EC50 = 248 nM, 87% ACh Max) with minimal M1 agonism, (EC50 > 30 μM, 19% ACh Max) despite being derived from potent M1 ago-PAM chemotypes, with good CNS penetration (rat Kp = 0.59, Kp,uu = 0.13, MDCK-MDR1 ER = 1.8, Papp = 32.8 × 10–6 cm/s) and pro-cognitive efficacy in both contextual fear conditioning (minimally effective dose [MED] = 0.1 mg/kg PO) and novel object recognition (MED = 1 mg/kg PO) in rats.

Herein, we report the discovery and development of an optimized mGlu2 Negative Allosteric Modulator (NAM) in vivo tool compound, VU6066098, based on a novel, structurally distinct chemotype. VU6066098 is a potent, selective, and CNS-penetrant mGlu2 NAM with excellent rat PK (CLp = 23.9 mL/min/kg, t1/2 = 2.2 h, %F = 100, Kp = 1.28, Kp,uu = 0.25), making it ideal to explore the therapeutic potential of selective mGlu2 inhibition in preclinical rat models. In a rat forced swim test, VU6066098 displayed an oral minimum effective dose (MED) of 1 mg/kg and was equi-efficacious to ketamine. In amphetamine-induced hyperlocomotion, VU6066098 displayed an oral minimum effective dose (MED) of 30 mg/kg. While in the preclinical cognitive tasks of rat novel object recognition and acquisition of contextual fear conditioning, VU6066098 produced robust dose-dependent effects at oral minimum effective doses (MED) of 3 mg/kg and 0.3 mg/kg, respectively. In a blast-related traumatic brain injury (TBI) model, administration of VU6066098 at a dose of 10 mg/kg IP was effective acutely, and the effect on NOR memory was sustained up to 30 days postdose. Thus, mGlu2 NAMs show therapeutic potential for the treatment of a broad range of affective and cognitive symptoms associated with Major Depressive Disorder, Alzheimer's disease, TBI, and acute psychosis; moreover, these data strongly support further optimization of mGlu2 NAMs for future clinical development.

Herein, we report the structure-activity relationship to develop novel tricyclic M4 positive allosteric modulator scaffolds with improved pharmacological properties. This endeavor involved modifying a 5-amino-3,4-dimethylthieno[2,3-c]pyridazine-6-carboxamide core via a "tie-back" strategy to discover a novel tricyclic 3,4-dimethylpyrimido[4',5':4,5]thieno[2,3-c]pyridazine core. From this exercise, VU6008055/AF98943 was identified as a preclinical candidate, which displays low nanomolar potency against both human and rat M4. Moreover, VU6008055 is highly brain penetrant, has an overall superior pharmacological and DMPK profile to previously reported M4 PAMs, and demonstrates efficacy in preclinical models of antipsychotic-like activity.

The development of cholinergic neurotransmitter based cognitive enhancers for Alzheimer’s disease and other neuropsychiatric disorders have focused recently on allosteric modulation of specific muscarinic acetylcholine receptor (mAChR) subtypes to reduce dose-limiting side-effects that have been the hallmark of earlier orthosteric mAChR agonists. VU0467319 (VU319) is an investigational positive allosteric modulator of the M1 mAChR. A Phase 1 first-in-human study was conducted assessing safety and brain activity utilizing cognitive tasks and event-related potentials (ERPs) in single-ascending dose and food effect studies. VU319 was given orally to 52 healthy volunteers aged 18–55 years. The single ascending dose study tested 40 participants in five dose escalating cohorts (60, 120, 240, 400, 600 mg; 6 VU319/2 placebo per dose). The food effect study involved 12 participants, 10 VU319 (120 mg)/2 placebo. Exploratory cognitive and electrophysiological tasks were examined pre-dosing and at 5 h post-dose. Tolerability was good with no observed dose limiting side effects throughout the full dose range tested. In the single ascending dose study, there were 47 TEAEs reported across the 5 cohorts, 14 in the placebo group and 33 across the 5 active dose cohorts. In the food effect study, there were 20 TEAEs reported, 6 in the placebo group and 14 in the fed and fasted conditions. Drug exposure increased with dose in a less than dose-proportional manner with a half-life ranging from 30 to 55 h. Peak concentration was observed between 5 and 9.5 h across the dosage groups. Absorption was increased with food. Exploratory cognitive/ERP testing showed evidence for drug-induced CNS activity on higher doses of VU319 compared to placebo. Single dose VU319 across five ascending cohorts appeared to have a favorable safety profile and a PK profile consistent with once daily dosing. Target engagement results suggest stimulation of the cholinergic system functioning in healthy adults following a single dose of VU319. These results provide a strong foundation for further studies of positive allosteric modulators of muscarinic M1 receptors for potential cognitive or behavioral benefits.

We recently disclosed VU0467319, a muscarinic acetylcholine receptor subtype 1 (M1) Positive Allosteric Modulator (PAM) clinical candidate that had successfully completed a Phase I Single Ascending Dose (SAD) clinical trial, but the identification of an inactive metabolite constituting a major portion of the total plasma AUC detracted from the molecules' pharmacokinetic profile and contributed to clinical development discontinuation. Attempts to block metabolism with the incorporation of deuterium atoms proved successful in vitro and in vivo at low exposures; however, in high-dose nonclinical toxicology studies, the degree of oxidative metabolism and metabolite accumulation was comparable to that of the proteo-congener. Here, we describe a second-generation back-up effort based on the VU0467319 scaffold to discover VU6052254. Strategic placement of a tertiary hydroxyl moiety afforded VU6052254, a potent M1 PAM (EC50 = 59 nM, 79% ACh max), with high CNS exposure (rat Kp = 1.07; Kp,uu = 1.27; P-gp ER = 1.97, Papp = 23 × 10-6 cm/s), reduced metabolism across species, excellent pharmacodynamic responses (MED in rat NOR = 1 mg/kg PO; MED in rat CFC = 0.3 mg/kg PO), excellent multispecies PK (Clps < 10 mL/min/kg, %F > 65), and favorable human PK and dose projections. Based on these beneficial attributes, VU6052254 was nominated for further nonclinical development. However, possible CYP450 induction liability as well as uncertain projected margins for human efficacy at those systemic concentrations where dose/exposure-related clinical and anatomic pathology kidney findings were observed in a 14-day exploratory toxicity study in male rats, precluded further development.

Herein we describe the chemical optimization of a selective and CNS penetrant series of TREK inhibitors (the K2P family of potassium ion channels), culminating in the discovery of ONO-9517601 (VU6022856) and ONO-7927846 (VU6024391). Optimization of ONO-TR-772 focused on replacements for the N-Boc aniline moiety and identified N-acyl piperidine pyrazoles as attractive surrogates, affording excellent potency, PK profiles, CNS penetration and ion channel selectivity. ONO-9517601 and ONO-7927846 displayed robust efficacy in an MK-801 challenge rat NOR paradigm, with MEDs of 1 mg/kg and 0.3 mg/kg, respectively. These ligands represent valuable preclinical research tools for exploring selective TREK inhibition in vitro and in vivo.

While the muscarinic acetylcholine receptor mAChR subtype 5 (M5) has been studied over decades, recent findings suggest that more in-depth research is required to elucidate a thorough understanding of its physiological function related to neurological and psychiatric disorders. Our efforts to identify potent, selective, and pharmaceutically favorable next-generation M5 antagonist tool compounds have led to the discovery of a novel triazolopyridine-based series. In particular, VU6036864 (45) showed exquisite potency (human M5 IC50 = 20 nM), good subtype selectivity (>500 fold selectivity against human M1–4), desirable brain exposure (K p = 0.68, K p,uu = 0.65), and high oral bioavailability (%F > 100%). VU6036864 (45) and its close analogues will support further studies of M5 as advanced antagonist tool compounds and play an important role in the emerging biology of M5.

Herein, we report structure-activity relationship (SAR) studies to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace a 5-amino-2,4-dimethylthieno[2,3-d]pyrimidine-6-carboxamide core, which led to the discovery of two novel tricyclic cores. While both tricyclic cores displayed low nanomolar potency against both human and rat M4 and were highly brain-penetrant, the 2,4-dimethylpyrido[4',3':4,5]thieno[2,3-d]pyrimidine tricycle core provided lead compound, VU6016235, with an overall superior pharmacological and drug metabolism and pharmacokinetics (DMPK) profile, as well as efficacy in a preclinical antipsychotic animal model.

The central cholinergic system plays an important role in normal arousal and sleep/wake architecture, and is known to decline in non-pathological aging and Alzheimer’s disease (AD). The M 4 muscarinic acetylcholine receptor (mAChR) is expressed in cortical, limbic, and thalamic brain regions, crucial for the control of arousal and sleep/wake architecture. While previous clinical studies have identified M 4 as an exciting target for the potential treatment of behavioral disturbances and other symptoms associated with AD, there remains limited data on its role in the control of sleep/wake architecture and arousal in non-pathological aging and AD. In the current studies, we evaluated the effects of the selective M 4 positive allosteric modulator (PAM) VU0467154 on sleep disruptions observed in non-pathologically aged mice across the circadian rhythm. HD-X02 telemetry devices (DSI) were implanted in young (4-5-year-old) and aged (20-21-month-old) C57/BL6 mice, followed by administration of vehicle or a dose of the M 4 PAM VU0467154 during either the inactive and active phase. Time in wake, non-rapid eye movement (NREM), and rapid eye movement sleep (REM) sleep were assessed across 24-hours post- dosing. NREM sleep fragmentation was also assessed for the 8-hours post-dosing. Arousal, as measured by gamma power during wake, and sleep quality, as measured by delta power during NREM sleep, were expressed relative to a 1-hour predose baseline. In addition, young mice were concurrently dosed with a maximally efficacious dose of VU0467154 (30 mg/kg) in combination with the M 4 antagonist VU6028418 to confirm that the observed effects were mediated through an M 4 -dependent mechanism. VU0467154 increased NREM sleep and decreased arousal following dosing during either phase; and reduced NREM sleep fragmentation and REM sleep following dosing during the inactive phase in young mice. These effects are attenuated by the M 4 antagonist VU6028418. In aged mice, similar effects of VU0467154 are observed, with NREM sleep quality also enhanced during the inactive phase. When evaluated across the circadian rhythm, the M 4 PAM VU0467154 normalized deficits in NREM sleep fragmentation and increased NREM sleep duration and quality in non-pathological aged mice in the inactive phase.

Historically, animal models have been routinely used in the characterization of novel chemical entities (NCEs) for various psychiatric disorders. Animal models have been essential in the in vivo validation of novel drug targets, establishment of lead compound pharmacokinetic to pharmacodynamic relationships, optimization of lead compounds through preclinical candidate selection, and development of translational measures of target occupancy and functional target engagement. Yet, with decades of multiple NCE failures in Phase II and III efficacy trials for different psychiatric disorders, the utility and value of animal models in the drug discovery process have come under intense scrutiny along with the widespread withdrawal of the pharmaceutical industry from psychiatric drug discovery. More recently, the development and utilization of animal models for the discovery of psychiatric NCEs has undergone a dynamic evolution with the application of the Research Domain Criteria (RDoC) framework for better design of preclinical to clinical translational studies combined with innovative genetic, neural circuitry-based, and automated testing technologies. In this chapter, the authors will discuss this evolving role of animal models for improving the different stages of the discovery and development in the identification of next generation treatments for psychiatric disorders.

Recent evidence suggests that inhibition of the M5 muscarinic acetylcholine receptor (mAChR) may provide a novel non-opioid mechanism for the treatment of opioid use disorder (OUD). Previous studies from our group and others have demonstrated that acute administration of the long-acting M5 negative allosteric modulator (NAM) ML375 attenuates established self-administration of cocaine, ethanol, oxycodone, and remifentanil in rats. In the present study, we characterized the effects of acute and repeated administration of the novel, short-acting M5 NAM VU6008667 on the reinforcing effects of oxycodone and reinstatement of oxycodone-seeking behaviors in male Sprague-Dawley rats, as well as on physiological withdrawal from oxycodone. Acute VU6008667 decreased oxycodone self-administration under both fixed ratio 3 (FR3) and progressive ratio (PR) schedules of reinforcement and attenuated cue-induced reinstatement of lever pressing following extinction from oxycodone self-administration, a commonly used relapse model. When administered daily to opioid-naïve rats, VU6008667 prevented acquisition of oxycodone self-administration behavior. VU6008667 had minimal effects on naloxone-precipitated withdrawal. After acute administration, VU6008667 did not inhibit sucrose self-administration and, when given chronically, delayed but did not prevent acquisition of sucrose maintained self-administration. VU6008667 also did not impact oxycodone induced anti-nociception or motor coordination, but mildly decreased novelty exploration. Finally, acute or daily VU6008667 administration did not impair cued fear conditioning. Overall, these results suggest that inhibition of the M5 mAChR may provide a novel, non-opioid based treatment for distinct aspects of OUD by inhibiting opioid intake in established OUD, reducing relapse during abstinence, and by reducing the risk of developing OUD.

Declining basal forebrain cholinergic integrity has been associated with disturbances in sleep-wake architecture in Alzheimer’s Disease (AD). Acetylcholinesterase inhibitors (AChEIs) provide symptomatic treatment for the cognitive deficits in AD through nonselective enhancement of cholinergic signaling. Additionally, AChEIs promote wakefulness, but reduce NREM sleep quality and produce dose limiting side effects through nonselective activation of peripheral muscarinic acetylcholine receptors (mAChR). Selectively targeting different mAChRs with positive allosteric modulators (PAMs) provides an alternate approach. In the current study, we assessed the effects of the M 1 PAM VU0453595 and the M 4 PAM VU0467154 on sleep-wake architecture and arousal following dosing across the circadian cycle in aged mice. Young (3-4 month-old, n = 13-14 per group) and aged (19-21 month-old, n = 14 per group) C57/BL6 mice were implanted with HD-X02 telemetry devices (DSI) for electroencephalography (EEG) recording. The M 1 PAM VU0453595 (3-30mg/kg IP) or the M 4 PAM VU0467154 (1-30mg/kg IP) were dosed 2-hours into the active or inactive phases of the circadian cycle. EEG was recorded for 24-hours, with sleep stages scored in 5-second epochs. Arousal was assessed through changes in gamma power during wake, and sleep quality was measured through changes in delta power during NREM sleep. Statistical comparisons involved repeated measures one- or two-way ANOVAs as appropriate, with Dunnett’s or Sidak’s multiple comparisons applied in all cases. The M 1 PAM VU0453595 increased wakefulness and arousal with inactive phase dosing in young and aged mice. During the active phase, VU0453595 increased wakefulness and arousal only in aged mice. The M 4 PAM VU0467154 increased NREM sleep in young and aged mice when dosed in both phases. In the inactive phase, suppression of REM sleep in young and aged mice is also observed. During NREM sleep, VU0467154 increased delta power in young and aged mice. These findings suggest that M 1 and M 4 PAMs may be valuable as treatments in AD at different phases of the circadian cycle. M 1 PAMs could be beneficial with morning dosing in clinical populations as they enhanced wakefulness and arousal. M 4 PAMs could be advantageous with evening dosing in clinical population as they enhanced NREM sleep quantity and quality.

The muscarinic acetylcholine receptor (mAChR)subtype 5 (M5) represents a novel potential target for thetreatment of multiple addictive disorders, including opioid usedisorder. Through chemical optimization of several functionalhigh-throughput screening hits,VU6019650(27b) was identifiedas a novel M5orthosteric antagonist with high potency (human M5IC50= 36 nM), M5subtype selectivity (>100-fold selectivity againsthuman M1???4) and favorable physicochemical properties forsystemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies,27bblocked the nonselectivemuscarinic agonist oxotremorine-M-induced increases in neuronalfiring rates of midbrain dopamine neurons in the ventraltegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover,27balso inhibited oxycodone self-administrationin male Sprague-Dawley rats within a dose range that did not impair general motor output.

Degeneration of basal forebrain cholinergic signaling in aging and Alzheimer's disease (AD) is linked with abnormalities in arousal, sleep/wake architecture, and cognition. While acetylcholinesterase inhibitors (AChEIs) are approved for the treatment of AD, they produce dose‐limiting adverse effects due to nonselective activation of peripheral muscarinic acetylcholine receptors (mAChRs). Selectively targeting the M1 mAChR using M1 positive allosteric modulators (PAMs) has shown promise for boosting cognitive performance and arousal across preclinical species. We evaluate the effects of the M1 PAM VU0453595 on arousal and sleep/wake architecture in aged and young mice across both the active and inactive phases of the circadian cycle. We hypothesize that dosing an M1 PAM during the active phase in aged mice will produce a greater enhancement of arousal and wakefulness, normalizing deficits observed in aged mice.

Degeneration of basal forebrain cholinergic circuitry represents an early event in the development of Alzheimer's disease (AD). These alterations in central cholinergic function are associated with disruptions in arousal, sleep/wake architecture, and cognition. Changes in sleep/wake architecture are also present in normal aging and may represent a significant risk factor for AD. M(1)muscarinic acetylcholine receptor (mAChR) positive allosteric modulators (PAMs) have been reported to enhance cognition across preclinical species and may also provide beneficial effects for age- and/or neurodegenerative disease-related changes in arousal and sleep. In the present study, electroencephalography was conducted in young animals (mice, rats and nonhuman primates [NHPs]) and in aged mice to examine the effects of the selective M(1)PAM VU0453595 in comparison with the acetylcholinesterase inhibitor donepezil, M-1/M(4)agonist xanomeline (in NHPs), and M(1)PAM BQCA (in rats) on sleep/wake architecture and arousal. In young wildtype mice, rats, and NHPs, but not in M(1)mAChR KO mice, VU0453595 produced dose-related increases in high frequency gamma power, a correlate of arousal and cognition enhancement, without altering duration of time across all sleep/wake stages. Effects of VU0453595 in NHPs were observed within a dose range that did not induce cholinergic-mediated adverse effects. In contrast, donepezil and xanomeline increased time awake in rodents and engendered dose-limiting adverse effects in NHPs. Finally, VU0453595 attenuated age-related decreases in REM sleep duration in aged wildtype mice. Development of M(1)PAMs represents a viable strategy for attenuating age-related and dementia-related pathological disturbances of sleep and arousal.

Opioid use disorder (OUD) is a debilitating neuropsychiatric condition characterized by compulsive opioid use, dependence, and repeated relapse after periods of abstinence. Given the high risk of developing OUD following prescription opioid use, the continued need for opioid-induced analgesia, and the limitations of current OUD treatments, it is necessary to develop novel, non-opioid-based treatments for OUD and decrease abuse potential of prescription opioids. Recent evidence suggests that negative allosteric modulation (NAM) of the M5 muscarinic acetylcholine receptor (M5 mAChR) may provide an alternative therapeutic approach for the treatment of OUD. Previous studies demonstrated localization of M5 mAChR expression within the mesocorticolimbic reward circuitry and that the selective M5 NAM ML375 attenuates both cocaine and alcohol self-administration in rats. In the present study, the effects of ML375 were evaluated in rats self-administering the μ-opioid agonists oxycodone or remifentanil on a progressive ratio (PR) schedule or on cue reactivity (a rodent model of relapse) in the absence of oxycodone following 72 h of abstinence. ML375 reduced the PR break point for oxycodone and remifentanil self-administration and attenuated cue-elicited responding. Importantly, ML375 did not affect sucrose pellet-maintained responding on a PR schedule or opioid-induced antinociception using the hot-plate and tail-flick assays. We also confirm expression of M5 mAChR mRNA in the ventral tegmental area and show that this is primarily on dopamine (tyrosine hydroxylase mRNA-positive) neurons. Taken together, these findings suggest that selective functional antagonism of the M5 mAChR may represent a novel, non-opioid-based treatment for OUD.

This letter describes progress towards an M-4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno [2,3-c] pyridine core to an equipotent, non-CNS penetrant thieno [2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M-4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.