Steven V. Fox

Promotion of Sleep by Suvorexant—A Novel Dual Orexin Receptor Antagonist

Abstract: Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX1R and OX2R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX2R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 μM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.

Design, Synthesis, and Evaluation of a Novel 4-Aminomethyl-4-fluoropiperidine as a T-Type Ca2+ Channel Antagonist

The novel T-type antagonist ( S)- 5 has been prepared and evaluated in in vitro and in vivo assays for T-type calcium ion channel activity. Structural modification of the piperidine leads 1 and 2 afforded the fluorinated piperidine ( S)- 5, a potent and selective antagonist that displayed in vivo CNS efficacy without adverse cardiovascular effects.

Discovery of [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096): a dual orexin receptor antagonist with potent sleep-promoting properties.

Insomnia is a common disorder that can be comorbid with other physical and psychological illnesses. Traditional management of insomnia relies on general central nervous system (CNS) suppression using GABA modulators. Many of these agents fail to meet patient needs with respect to sleep onset, maintenance, and next‐day residual effects and have issues related to tolerance, memory disturbances, and balance. Orexin neuropeptides are central regulators of wakefulness, and orexin antagonism has been identified as a novel mechanism for treating insomnia with clinical proof of concept. Herein we describe the discovery of a series of α‐methylpiperidine carboxamide dual orexin 1 and orexin 2 receptor (OX1R/OX2R) antagonists (DORAs). The design of these molecules was inspired by earlier work from this laboratory in understanding preferred conformational properties for potent orexin receptor binding. Minimization of 1,3‐allylic strain interactions was used as a design principle to synthesize 2,5‐disubstituted piperidine carboxamides with axially oriented substituents including DORA 28. DORA 28 (MK‐6096) has exceptional in vivo activity in preclinical sleep models, and has advanced into phase II clinical trials for the treatment of insomnia.

In Vitro Characterization of T-Type Calcium Channel Antagonist TTA-A2 and In Vivo Effects on Arousal in Mice

T-type calcium channels have been implicated in many behaviorally important neurophysiological processes, and altered channel activity has been linked to the pathophysiology of neurological disorders such as insomnia, epilepsy, Parkinsons disease, depression, schizophrenia, and pain. We have previously identified a number of potent and selective T-type channel antagonists (Barrow et al., 2007; Shipe et al., 2008; Yang et al., 2008). Here we describe the properties of the antagonist TTA-A2 [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide], assessed in patch-clamp experiments. TTA-A2 blocks T-type channels (Cav3.1, 3.2, 3.3) voltage dependently and with high potency (IC50 ∼100 nM). Stimulation at 3 Hz revealed additional use dependence of inhibition. A hyperpolarized shift of the channel availability curve and delayed channel recovery from inactivation suggest that the compound preferentially interacts with and stabilizes inactivated channels. The compound showed a ∼300-fold selectivity for Cav3 channels over high-voltage activated calcium channels. Inhibitory effects on native T-type currents were confirmed in brain slice recordings from the dorsal lateral geniculate nucleus and the subthalamic nucleus. Furthermore, we demonstrate that in vivo T-type channel inhibition by TTA-A2 suppresses active wake and promotes slow-wave sleep in wild-type mice but not in mice lacking both Cav3.1 and Cav3.3, suggesting the selective effect of TTA-A2 on recurrent thalamocortical network activity. The discovery of the potent and selective T-type channel antagonist TTA-A2 has enabled us to study the in vivo effects of pharmacological T-channel inhibition on arousal in mice, and it will help to explore the validity of these channels as potential drug targets for sleep-related and other neurological diseases.

Orexin receptor antagonism prevents transcriptional and behavioral plasticity resulting from stimulant exposure

Orexin is a key neurotransmitter of central arousal and reward circuits in the CNS. Two receptors respond to orexin signaling, Orexin 1 Receptor (OX1R) and Orexin 2 Receptor (OX2R) with partially overlapping brain distributions. Genetic and pharmacological studies suggest orexin receptor antagonists could provide therapeutic benefit for insomnia and other disorders in which sleep/wake cycles are disrupted. Preclinical data has also emerged showing that the orexin system is involved in the behavioral and neurological effects of drugs of abuse (Aston-Jones et al., 2009; Harris et al., 2005). Here we report sleep promoting effects of a recently described small molecule dual orexin receptor OX1R and OX2R antagonist. This dual orexin receptor antagonist (DORA) also inhibits the ability of subchronic amphetamine to produce behavioral sensitization measured 10 days following pre-treatment. Transcriptional profiling of isolated reward and arousal circuits from brains of behaviorally sensitized animals showed that the DORA blocked the significant alteration of gene expression levels in response to amphetamine exposure, particularly those associated with synaptic plasticity in the VTA. Further, DORA attenuates the ability of nicotine to induce reinstatement of extinguished responding for a reinforcer, demonstrating selectivity of the effect to reward pathways and not to food intake. In summary, these data demonstrate efficacy of a dual orexin receptor antagonist for promotion of sleep and suggest that pharmacological inhibition of the orexin system may play a role in both prevention of drug-induced plasticity and drug-relapse.

Orexin Receptor Antagonists Differ from Standard Sleep Drugs by Promoting Sleep at Doses That Do Not Disrupt Cognition

Selective orexin receptor antagonism promotes sleep at doses lower than those that impair attention and memory in contrast to the GABA receptor–positive allosteric modulators currently in use. Sleep Without the After Effects Currently available treatments for insomnia can produce a number of central nervous system–based cognitive side effects, including the potential to impair memory and attention. Recently, selective dual orexin receptor antagonists, such as suvorexant and almorexant, have been shown to promote sleep onset and maintenance in clinical trials for patients with insomnia. In new work, Uslaner and colleagues compared sleep-promoting doses to the cognitive-impairing doses for an orexin receptor antagonist, DORA-22, versus sleep drugs currently in use: zolpidem, diazepam, or eszopiclone. At doses that produced equivalent amounts of sleep in rat and rhesus monkey, zolpidem, diazepam, and eszopiclone significantly disrupted attention and memory, whereas DORA-22 promoted sleep at doses that did not exert measurable effects on cognition. Furthermore, when compared to the other insomnia treatments that modulate γ-aminobutyric acid (GABA) receptor function, the authors saw greater separation for orexin receptor antagonism between doses that promoted sleep and doses that reduced expression of a hippocampal gene involved in synaptic plasticity called Arc. These findings suggest that dual orexin receptor antagonists might provide an effective treatment for insomnia with a greater therapeutic margin for sleep versus cognitive disturbances compared to the GABAA-positive allosteric modulators currently available. Current treatments for insomnia, such as zolpidem (Ambien) and eszopiclone (Lunesta), are γ-aminobutyric acid type A (GABAA)–positive allosteric modulators that carry a number of side effects including the potential to disrupt cognition. In an effort to develop better tolerated medicines, we have identified dual orexin 1 and 2 receptor antagonists (DORAs), which promote sleep in preclinical animal models and humans. We compare the effects of orally administered eszopiclone, zolpidem, and diazepam to the dual orexin receptor antagonist DORA-22 on sleep and the novel object recognition test in rat, and on sleep and two cognition tests (delayed match to sample and serial choice reaction time) in the rhesus monkey. Each compound’s minimal dose that promoted sleep versus the minimal dose that exerted deficits in these cognitive tests was determined, and a therapeutic margin was established. We found that DORA-22 has a wider therapeutic margin for sleep versus cognitive impairment in rat and rhesus monkey compared to the other compounds tested. These data were further supported with the demonstration of a wider therapeutic margin for DORA-22 compared to the other compounds on sleep versus the expression of hippocampal activity–regulated cytoskeletal-associated protein (Arc), an immediate-early gene product involved in synaptic plasticity. These findings suggest that DORAs might provide an effective treatment for insomnia with a greater therapeutic margin for sleep versus cognitive disturbances compared to the GABAA-positive allosteric modulators currently in use.

Positive allosteric interaction of structurally diverse T-type calcium channel antagonists.

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.

Discovery of 5′′-Chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2′:5′,3′′-terpyridine-3′-carboxamide (MK-1064): A Selective Orexin 2 Receptor Antagonist (2-SORA) for the Treatment of Insomnia

The field of small‐molecule orexin antagonist research has evolved rapidly in the last 15 years from the discovery of the orexin peptides to clinical proof‐of‐concept for the treatment of insomnia. Clinical programs have focused on the development of antagonists that reversibly block the action of endogenous peptides at both the orexin 1 and orexin 2 receptors (OX1R and OX2R), termed dual orexin receptor antagonists (DORAs), affording late‐stage development candidates including Merck’s suvorexant (new drug application filed 2012). Full characterization of the pharmacology associated with antagonism of either OX1R or OX2R alone has been hampered by the dearth of suitable subtype‐selective, orally bioavailable ligands. Herein, we report the development of a selective orexin 2 antagonist (2‐SORA) series to afford a potent, orally bioavailable 2‐SORA ligand. Several challenging medicinal chemistry issues were identified and overcome during the development of these 2,5‐disubstituted nicotinamides, including reversible CYP inhibition, physiochemical properties, P‐glycoprotein efflux and bioactivation. This article highlights structural modifications the team utilized to drive compound design, as well as in vivo characterization of our 2‐SORA clinical candidate, 5′′‐chloro‐N‐[(5,6‐dimethoxypyridin‐2‐yl)methyl]‐2,2′:5′,3′′‐terpyridine‐3′‐carboxamide (MK‐1064), in mouse, rat, dog, and rhesus sleep models.

Discovery of a Pharmacologically Active Antagonist of the Two-Pore-Domain Potassium Channel K2P9.1 (TASK-3)

TWIK‐related acid‐sensitive K+ (K2P9.1, TASK‐3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK‐3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK‐3 channel modulation exists, there are only a few documented examples of potent and selective small‐molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK‐3 channel antagonists based on a 5,6,7,8‐tetrahydropyrido[4,3‐d]pyrimidine high‐throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.

Quantitative electroencephalography within sleep/wake states differentiates GABAA modulators eszopiclone and zolpidem from dual orexin receptor antagonists in rats.

Dual orexin receptor antagonists (DORAs) induce sleep by blocking orexin 1 and orexin 2 receptor-mediated activities responsible for regulating wakefulness. DORAs represent a potential alternative mechanism to the current standard of care that includes the γ-aminobutyric acid (GABA)A receptor-positive allosteric modulators, eszopiclone and zolpidem. This work uses an innovative method to analyze electroencephalogram (EEG) spectral frequencies within sleep/wake states to differentiate the effects of GABAA modulators from DORA-22, an analog of the DORA MK-6096, in Sprague–Dawley rats. The effects of low, intermediate, and high doses of eszopiclone, zolpidem, and DORA-22 were examined after first defining each compound’s ability to promote sleep during active-phase dosing. The EEG spectral frequency power within specific sleep stages was calculated in 1-Hz intervals from 1 to 100 Hz within each sleep/wake state for the first 4 h after the dose. Eszopiclone and zolpidem produced marked, dose-responsive disruptions in sleep stage-specific EEG spectral profiles compared with vehicle treatment. In marked contrast, DORA-22 exhibited marginal changes in the spectral profile, observed only during rapid eye movement sleep, and only at the highest dose tested. Moreover, while eszopiclone- and zolpidem-induced changes were evident in the inactive period, the EEG spectral responses to DORA-22 were absent during this phase. These results suggest that DORA-22 differs from eszopiclone and zolpidem whereby DORA-22 promotes somnolence without altering the neuronal network EEG activity observed during normal sleep.