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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.

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 [(3)H]-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® 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.

Relaxin-3 is a neuropeptide that is abundantly expressed by discrete brainstem neuron populations that broadly innervate forebrain areas rich in the relaxin-3 G-protein-coupled-receptor, RXFP3. Acute and subchronic central administration of synthetic relaxin-3 or an RXFP3-selective agonist peptide, R3/I5, increase feeding and body weight in rats. Intrahypothalamic injection of relaxin-3 also increases feeding. In this study, we developed a recombinant adeno-associated virus 1/2 (rAAV1/2) vector that drives expression and constitutive secretion of bioactive R3/I5 and assessed the effect of intrahypothalamic injections on daily food intake and body weight gain in adult male rats over 8 weeks. In vitro testing revealed that the vector rAAV1/2-fibronectin (FIB)-R3/I5 directs the constitutive secretion of bioactive R3/I5 peptide. Bilateral injection of rAAV1/2-FIB-R3/I5 vector into the paraventricular nucleus produced an increase in daily food intake and body weight gain (P<0.01, ~23%, respectively), relative to control treatment. In a separate cohort of rats, quantitative polymerase chain reaction analysis of hypothalamic mRNA revealed strong expression of R3/I5 transgene at 3 months post-rAAV1/2-FIB-R3/I5 infusion. Levels of mRNA transcripts for the relaxin-3 receptor RXFP3, the hypothalamic 'feeding' peptides neuropeptide Y, AgRP and POMC, and the reproductive hormone, GnRH, were all similar to control, whereas vasopressin and oxytocin (OT) mRNA levels were reduced by ~25% (P=0.051) and ~50% (P<0.005), respectively, in rAAV1/2-FIB-R3/I5-treated rats (at 12 weeks, n=9/8 rats per group). These data demonstrate for the first time that R3/I5 is effective in modulating feeding in the rat by chronic hypothalamic RXFP3 activation and suggest a potential underlying mechanism involving altered OT signalling. Importantly, there was no desensitization of the feeding response over the treatment period and no apparent deleterious health effects, indicating that targeting the relaxin-3-RXFP3 system may be an effective long-term therapy for eating disorders.

To study the specific actions of relaxin through RXFP1 in human cells, it would be advantageous to develop cell populations with permanent RXFP1 knockdown (KD). We have developed and assessed four microRNA against human RXFP1. One of the four designed microRNA displayed significant RXFP1 KD as assessed by reduced relaxin binding when co-transfected with human RXFP1 into HEK-293T cells. The selected microRNA sequence was subsequently retrovirally delivered into the human dermal fibroblast cell line BJ3 which natively expresses RXFP1. The RXFP1 KD BJ3 cells displayed diminished RXFP1 mRNA expression and complete loss of ability of relaxin treatment to reduce collagen deposition after TGF-beta1 stimulation. The retroviral expression of miRNA to successfully silence RXFP1 expression is an invaluable tool to investigate receptor specificity, signalling and possible off-target effects of newly developed relaxin analogs.

There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1-4 (RXFP1-4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.

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.

Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.

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.

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.

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.