Suvorexant (Belsorma®) is the first orexin receptor antagonist approved by the US FDA (August 2014) for insomnia treatment. Following comprehensive Phase II/III studies, with up to 12 months of treatment in adult and elderly patients, there is little doubt that suvorexant induces and maintains sleep. However, the FDA and sponsor disagreed about effective versus safe doses (November 2012). The FDA considered that 5–15 mg were efficient and probably safe, whereas the sponsors had proposed 15–40 mg. The final approved doses are 5, 10, 15 and 20 mg. The major issues are next-morning somnolence and safety as seen in driving tests, with possible signs of muscle weakness, weird dreams, sleep walking, other nighttime behaviors and suicidal ideation. Despite its limitations, suvorexant's market entry offers a truly novel treatment for insomnia, paving the way for follow-up compounds and opening therapeutic avenues in other disorders for orexin receptor modulating compounds.

Introduction: The concept of an “immune privileged” CNS has been redefined to suggest that the CNS is a site of selective and modified immune reactivity [1]. The findings presented provide mechanistic and clinical evidence to suggest that phenotypes of neuropsychiatric disorders such as cognitive and affective dysfunction and neurodegeneration are influenced by pathological functional and morphological changes of the CNS induced by impaired immune functions, and by neuroinflammation in particular. Under immune challenged conditions such as Encephalitis and Multiple Sclerosis, inflammation in the CNS results in trafficking of B and T cells to the site of injury leading to impaired pathophysiological mechanisms relevant to neuropsychiatric disorders. Aim: This presentation will demonstrate the current understanding on the role of inflammation in neuropsychiatric disorders. Results: The activation of humoraland cell-mediated immunity is met with an upregulation of various inflammatory proteins such as cytokines and chemokines. It has been demonstrated that these immune cells traffic to various regions in the CNS including the hippocampus, a region regarded as essential for memory and learning. A chronic upregulation of such inflammatory proteins may result in dysfunction of key cytokine-mediated molecular mechanisms and neuronal–glial interactions that subserve synaptic plasticity and learning and memory processes in the brain. During pathological conditions microglial interact with neurons, possibly via P2x7 receptors, to induce a neuroinflammatory response characterized by an up-regulation of cytokines, such as, IL-1, IL-6 and TNF, which can then alter the function of cytokines in synaptic plasticity [2]. In addition, chronic inflammatory conditions in the CNS may lead to pathological morphological changes such as grey matter hypointensities and associated iron deposition in basal ganglia as seen in diseases like Multiple Sclerosis [3] associated with cognitive impairment and neuropsychiatric symptoms. Discussion: The above described mechanisms provide insight into shared pathologogical mechanisms of distinctly different neurological and psychiatric disorders such as depression, psychosis and cognitive decline / dementia.

Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various "dual" orexin receptor antagonists and the orexin receptors, OX1R and OX2R, over time using saturation and competition radioligand binding with [H-3]-BBAC ((S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX1R and OX2R using FLIPR (R) assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX2R, whereas SB-649868, suvorexant, and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX2R antagonist IPSU ((2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) bind rapidly and reach equilibrium very quickly in binding and/or functional assays. Overall, the "dual" antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the "dual" antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.

We have studied the interactions between human OX1 and OX2 receptors and various “dual” orexin receptor antagonists using radioligand binding with [3H] BBAC (S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). [3H] BBAC binds rapidly and reversibly to both hOX1 and hOX2 receptors. Competition experiments were performed at different time points (15 – 240 min) with [3H]BBAC and SB-649868, almorexant, suvorexant (MK4305) and filorexant (MK6096) in comparison to BBAC and IPSU (2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) a selective OX2 receptor antagonist, presented in the companion paper. Further, we have extended these studies to determine the kinetic features of the “dual” orexin receptor antagonists on calcium signaling using FLIPR® in cells expressing human, rat or mouse OX1 and OX2 receptors. The calcium signaling studies confirm what was observed in radioligand binding: almorexant reaches equilibrium very slowly at OX2 receptors, whereas SB-649868, suvorexant and filorexant may take hours to reach steady state at OX1 and/or OX2 receptors. On the other hand, compounds such as BBAC or IPSU bind rapidly and do reach equilibrium very quickly in both binding and / or functional assays. In summary, the “dual” antagonists tested here reach equilibrium very slowly; whereas they all tend to be rather unselective under non-equilibrium conditions, after equilibrium is reached, each ligand demonstrates a distinct selectivity profile. The data also suggest that since equilibrium will be reached slowly in vitro and probably in vivo, receptor occupancy may be longer lasting than predicted by pharmacokinetic studies measuring plasma or brain levels of these antagonists.

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.

Recent in vivo studies suggest a role for relaxin‐3 in feeding and stress. To further elucidate the function of relaxin‐3 in the central nervous system, we have employed a complementary approach, based on RNA interference, to modulate relaxin‐3 expression. We have designed, constructed, and characterized three microRNAs (miRNAs) targeting different regions of the relaxin‐3 transcript. These were tested to determine the amount of miRNA required to achieve the greatest knockdown and for their ability to reduce the expression of relaxin‐3 in transfected HEK293Tcells. All miRNA constructs significantly reduced relaxin‐3‐induced cAMP responses; however, miR499 was most effective. This should be a useful tool for in vitro studies and central targeting of relaxin‐3 in vivo using viral delivery systems.

Recent in vivo studies suggest a role for relaxin-3 in feeding and stress. To further elucidate the function of relaxin-3 in the central nervous system, we have employed a complementary approach, based on RNA interference, to modulate relaxin-3 expression. We have designed, constructed, and characterized three microRNAs (miRNAs) targeting different regions of the relaxin-3 transcript. These were tested to determine the amount of miRNA required to achieve the greatest knockdown and for their ability to reduce the expression of relaxin-3 in transfected HEK293Tcells. All miRNA constructs significantly reduced relaxin-3-induced cAMP responses; however, miR499 was most effective. This should be a useful tool for in vitro studies and central targeting of relaxin-3 in vivo using viral delivery systems.

The receptors for relaxin and insulin‐like peptide 3 (INSL3) are now well‐characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G‐protein‐coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine‐rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low‐density lipoprotein class A (LDL‐A) module at their N‐terminus. Ligand‐mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B‐chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL‐A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL‐A module is currently unknown. The specific understanding of the mechanisms underlying ligand‐mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.

Relaxin induces sustained physiological responses, which brings into question the deactivation processes typical of most G protein-coupled receptors (GPCR) for its receptor, relaxin family peptide receptor 1 (RXFP1). Here, we examined relaxin-dependent phosphorylation of RXFP1 and the related insulin-like peptide 3 (INSL3) receptor, RXFP2, as well as the capacity of these receptors to recruit beta-arrestins and internalize in response to ligand stimulation. We confirmed in human embryonic kidney (HEK)-293T cells, expressing RXFP1 or RXFP2, that both receptors elicit prolonged cAMP responses up to 6 h after stimulation. Receptors immunoprecipitated from (32)P metabolically labeled cells were used to investigate the agonist-specific phosphorylation. Rapid and robust receptor phosphorylation was not observed for either RXFP1 or RXFP2, although some (32)P-incorporation was observed at 30 min; however, this was not statistically significant. In accord with this result, RXFP1 and RXFP2 demonstrated poor internalization in response to relaxin or INSL3, as compared with the angiotensin II type 1 receptor (AT(1)R), which undergoes rapid and robust phosphorylation and internalization in response to angiotensin II. Additionally, coexpression of GPCR kinases has no effect on the rate of internalization for either RXFP1 or RXFP2. Confocal microscopy was used to follow the trafficking of green fluorescent protein-labeled beta-arrestins after receptor activation. Neither RXFP1 nor RXFP2 activation results in recruitment of beta-arrestins to the cell surface, whereas AT(1)R rapidly recruits both beta-arrestins-1 and -2. The apparent lack of classical regulation for RXFP1 and RXFP2 provides the molecular basis for the prolonged signaling and physiological actions of relaxin and related peptides.