International Union of Basic and Clinical Pharmacology. LXXXVI. Orexin Receptor Function, Nomenclature and Pharmacology

A. Orexin-A and -B Are Products of the Hypocretin Gene

Even before the genetic association with narcolepsy was discovered, orexin neuropeptides were simultaneously described by two different groups using distinct molecular and biochemical approaches. From mRNA enriched from rat hypothalamus, de Lecea et al. (1998) identified, cloned, and sequenced a 569-nucleotide transcript encoding a 130-amino acid prepro-peptide based on sequence similarity to secretin, a gut hormone involved in osmoregulation. Because of its CNS expression restricted to large cell bodies of the dorsal lateral hypothalamic area and its sequence similarity to the incretin family of peptide hormones, the gene was termed hypocretin (Hcrt). Two peptide products predicted from proteolytic cleavage sites were confirmed by immunohistochemistry to be present in cell bodies and efferent fibers, and one of the synthetically generated peptides was able to elicit depolarizing currents in primary cultures of hypothalamic neurons (de Lecea et al., 1998). In an effort to “deorphanize” a panel of G-protein-coupled receptors, a second group (Sakurai et al., 1998) identified one such receptor capable of mediating Ca²⁺ transients in response to crude rat brain extracts. Purification and sequencing of the biological activity capable of activating this receptor revealed a sequence encoding a precursor peptide processed into two related peptides. Because the mRNA was found to be expressed in the lateral hypothalamus (LH), an area implicated in feeding regulation (Bernardis et al., 1993; Bernardis and Bellinger, 1996), and because intraventricular administration of both peptides dose-dependently induced food intake, the peptides were designated orexin-A and -B (OX-A¹ and OX-B) after the Greek word orexis, for appetite. These deorphanized receptors were identified as orexin 1 and orexin 2 (OX₁ and OX₂) receptors (Sakurai et al., 1998). These findings ignited a large body of work aimed at deciphering the role of these signaling components in appetite control (Edwards et al., 1999; Sweet et al., 1999), energy homeostasis (Beck and Richy, 1999), and metabolism (Lubkin and Stricker-Krongrad, 1998; Takahashi et al., 1999) such that the term “orexin” seemed appropriate, yet the nomenclature debate had already begun (Nisoli et al., 1998). The subsequent discoveries of the genetic link to narcolepsy and the predominant role of orexin in arousal (Mieda et al., 2004), however, raised the possibility that orexin-induced feeding observed in preclinical models may be secondary to heightened wakefulness (Ida et al., 1999), further fueling the debate.

B. Nomenclature Recommendations

A parsimonious resolution to the nomenclature debate is to designate hypocretin the gene and mRNA name (human abbreviation: HCRT; rodent: Hcrt) and the precursor peptide and processed peptides after orexin (orexin A, Ox-A, orexin B, Ox-B) (Table 1). Likewise, the same is true for the two known G-protein-coupled receptors for these peptides; the HCRTR1 and HCRTR2 genes (Hcrtr1 and Hcrtr2 in rodents) encode the protein products OX₁ and OX₂ receptors, respectively. Although this nomenclature may be confusing to those new to the field, it does recognize the identification of the hypocretin mRNA and gene by molecular biology approaches (de Lecea et al., 1998) and the biochemical discovery of orexin peptides and their now deorphanized G-protein-coupled receptors (Sakurai et al., 1998). These distinct genetic and protein product designations are also now a matter of practical necessity. HCRT, HCRTR1, and HCRTR2 are now the accepted gene symbols in all genetic databases, including GenBank and HUGO. As for orexin-related protein products, the formal nomenclature from the International Union of Basic and Clinical Pharmacology Nomenclature Committee designates “OX-A” and “OX-B” as pharmacological ligands for “OX₁” and “OX₂” receptors, respectively (http://www.iuphar-db.org/DATABASE/FamilyMenuForward?familyId=51). The biological literature primarily uses the “orexin” designation, with references as well to “hypocretin” and/or the accepted gene name. Where the term “orexin” has been used much more exclusively, however, is in both the chemistry and patent literature, which has expanded substantially in the past 15 years as the therapeutic potential of modulating the orexin system has become increasingly evident (Fig. 1). Although early patents were filed for orexin peptide ligand derivatives and increasingly thereafter for molecular probes (e.g., microarray and quantitative polymerase chain reaction probes and primers), compound patents became more prominent with a growing interest in the development of small-molecule therapeutics. As seen in Fig. 1, the number of “orexin”-only patent filings has far exceeded that of “hypocretin”-only patents since 1999, the year the genetic link with narcolepsy was made. At the peak of patent filings in 2009, only 6 of a total of 965 patents filed mentioned “hypocretin” only, whereas 866 were exclusively for “orexin” and 93 for both. These figures demonstrate that the orexin and orexin receptor protein designations are the most widely accepted pharmacological designations.

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Fig. 1.

“Orexin” is used preferentially in the patent literature over “hypocretin.” The number of patent filings containing, in the full text, the words “orexin” (gray), “hypocretin” (black), or both (open) per year are shown. Results do not include patents in which the word “orexinergic” appears without either of the above.

C. Orexin-A and -B Structures Are Highly Conserved

Both OX-A and OX-B neuropeptides are derived from the same prepro-orexin precursor encoded by the HCRT gene. The structure and organization of the hypocretin gene has been largely conserved through evolution. In all vertebrates examined, the gene is composed of two exons with the intron splice falling within the early portion of the open reading frame encoding the secretory signal sequence (Fig. 2). Hcrt genes from multiple organisms, including teleost fish, avian, and mammalian species, are located within chromosomal loci having considerable synteny and, along with neighboring genes, may have experienced considerable pressure for functional conservation through evolution (Wong et al., 2011).

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Fig. 2.

OX-A and OX-B are encoded by the HCRT gene. Structures of the human gene [from UCSB genome browser (http://genome.ucsc.edu); intronic sequence is shown at 1/10th scale of exon sequence], mRNA, and protein gene products shown. IC₅₀ values for radioligand binding by OX-A and OX-B are depicted with the exception of the affinity of OX-B for OX₁ receptors (420 nM, not shown), which is ∼10-fold less than for OX₂ receptor (36 nM) (Sakurai et al., 1998).

The organization of prepro-orexin precursor peptide and the sequence of mature OX-A and OX-B ligands that are derived from it are highly conserved. The translated 131-amino acid human prepro-orexin peptide consists of nearly contiguous sequences encoding the secretory signal sequence, 33-amino acid OX-A, and 28-amino acid OX-B (Sakurai et al., 1999), and this organization, along with cleavage site sequences, are exactly conserved among all vertebrate organisms examined, including frog, chicken, and fish. This includes consensus “Gly-basic-basic” cleavage and C-terminal Gly-Lys-Arg amidation motifs, separating the OX-A and OX-B sequences, and Gly-Arg-Arg sequences, marking the termination of OX-B (Wong et al., 2011). Among mammals, the sequence of the mature OX-A ligand is entirely conserved among all species examined and contains two disulfide bridges, one formed by cysteines 6 and 12 and another between cysteines 7 and 14. These four residues are also 100% conserved from humans to amphibians (Wong et al., 2011). Mature OX-A is further post-translationally modified with an N-terminal pyroglutamic acid (Sakurai et al., 1998). Mammalian OX-B sequences, on the other hand, are very well conserved but have two points of differentiation: a serine reside at the second amino acid position in rodents, canines, and bovines is replaced by a proline in the human sequence, and a serine in the 18th position is replaced by an asparagine in rodents (Wong et al., 2011). Sequence diversity outside the secretory signal, OX-A, and OX-B sequences substantiates the functional importance of these defined regions.

OX-A and OX-B also share sequence similarity with one another, which is likely to underlie their ability to serve as ligands for both OX₁ and OX₂ receptors, albeit with differing affinities. In this regard, it is worth noting that mammalian OX-A and OX-B sequences are identical in the C-terminal portion of the mature peptides, including the nine-amino acid sequence Gly-Asn-His-Ala-Ala-Gly-Ile-Leu-Thr. They also share Arg-Leu and Leu-Leu sequences spaced two amino acids from one another and three amino acids N-terminal of the nine conserved positions mentioned above, suggesting that these residues may exist at one surface of an α-helical secondary structure (Sakurai et al., 1998; Wong et al., 2011). Because both peptides have measurable affinities for each of the OX₁ and OX₂ receptors, these observations indicate that these residues are essential for orexin receptor interaction. Human OX-A has nearly equal activity on both orexin receptors, with ligand binding affinities (IC₅₀) of 20 and 38 nM for OX₁ and OX₂ receptors, respectively, and EC₅₀ values of 30 and 34 nM in [Ca²⁺]_i mobilization assays of cells transfected to express human OX₁ and OX₂, respectively (Fig. 2) (Sakurai et al., 1998). OX-B, however, has markedly less activity toward OX₁ receptors with an IC₅₀ for radioligand binding of 420 nM and an EC₅₀ for [Ca²⁺]_i of 2500 nM. It is more selective for OX₂ receptors, exhibiting an IC₅₀ of 36 nM and EC₅₀ of 60 nM (Sakurai et al., 1998). This selectivity of OX-B for OX₂ has been used to interpret the relative roles of OX₂ and OX₁ receptors in biological functions, because differential responses to microinjection of these peptides into brain regions indicates OX₁ receptor function, whereas similar responses to both peptides may suggest OX₂ receptor function. Definitive receptor selective function, however, is demonstrated only with genetic and/or highly selective orexin receptor antagonist reagents.

D. Orexin Receptor Structure Is Evolutionarily Conserved

1. Orexin 1 Receptor.

Orexin 1 and 2 receptors are found throughout mammalian species, and the core regions of these proteins are highly conserved. The greatest diversity occurs between rat and human OX₁ receptor sequences, but even these proteins are 91% identical and 93% homologous (Table 2). As seen in the predicted structure of the OX₁ receptor based on a β₂-adrenergic receptor homology model (Fig. 3A), most of the divergent residues occur in the large cytoplasmic loop between transmembrane spanning helices five and six. Fewer residues within membrane spanning loops and the ligand-binding pocket diverge in rat, dog, and human sequences, and these changes are largely homologous. The exception is an Arg-to-His change at residue 205 within the second extracellular loop between transmembrane helices four and five of the predicted canine sequence, which may have the potential to affect ligand binding and antagonist activity. Differences in canine OX₁ receptor sequences relative to human and rodent sequences may be of interest given the possible difference in phenotypes displayed by disruptions in orexin signaling in these species. Truncation of the dog OX₂ receptor results in a narcoleptic phenotype accompanied by cataplexy (Lin et al., 1999), whereas deletion of the Hcrt gene or both orexin receptors in mice (Chemelli et al., 1999; Willie et al., 2003; Scammell et al., 2009) and orexin neuron loss in human narcoleptics is associated with this phenotype (Thannickal et al., 2003; Nishino et al., 2010). The Arg-to-His change in the dog OX₁ receptor, however, does not seem to affect ligand activation or the activity of two different dual orexin receptor antagonists, [2(R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096) and DORA-22, toward the dog OX₁ receptor (Winrow et al., 2012), indicating that any differences in the biological function of this receptor in canines may not be due to differences in receptor activity but might be explained by differential or regional expression changes.

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TABLE 2

Mammalian OX₁ receptor protein homology

The indicated annotated protein sequences were compared with the human OX₁ receptor sequence using a BLASTP algorithm with default comparative parameters (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Identity and similarity values are the percentage of identical or homologous amino acids shared with the core region of the human OX₁ receptor.

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Fig. 3.

Amino acid divergence in the structures of OX₁ and OX₂ receptors among human, rat, and canine sequences. Homology models were created using MOE software (Chemical Computing Group, Montreal, QC, Canada) based on the crystal structure of carazolol binding to the β₂-adrenergic receptor (Protein Date Bank code 2rh1) (Cherezov et al., 2007; Rosenbaum et al., 2007) used as the template. Sequence alignment of the transmembrane helices and the extracellular loop 2 (ECL2) of OX₁ and OX₂ receptors with the 2rh1 structure was performed according to Malherbe et al. (2010) and the best model selected from 10 intermediates. Backbone coordinates remained identical to the crystal structure such that no minimization was performed (structural data comparison and figure generated by M. Katharine Holloway, Ph.D., Chemical Modeling and Informatics, Merck Research Laboratories). The predicted structure of OX₁ (A) and OX₂ (B) receptors along with their ligand binding sites (lower panels) showing the peptide backbone (green) for sequence conserved among human, rat, and dog. Space-filling residues exhibiting sequence divergence are shown in magenta and the amino acid position and sequence substitutions are indicated in the lower panel (human/dog/rat amino acids at these positions). TM, transmembrane helices. The binding site is occupied by carazolol (yellow) used in homology modeling to the β₂-adrenergic receptor.

3. Evolutionary Origins.

Evolutionarily, OX₂ receptors seem to be a more ancient addition to class B G-protein-coupled receptors relative to the OX₁ receptor, which seems to have arisen from a more recent gene duplication (Wong et al., 2011).Hcrtr1 genes encoding OX₁ receptors have not been identified outside of the mammalian class (Fig. 4), suggesting that the function of these proteins represents a refinement of sleep, the control of vigilance or potentially other behavioral functions unique to mammals. Using the human orexin receptor sequences as queries in BLASTP searches, proteins exhibiting similarity to these receptors were identified, including neuropeptide FF receptors 1 and 2 (NPFF receptors 1 and 2), substance K receptors, GPR83, neuropeptide Y receptors, and QRF peptide receptors (QRFP receptor; also known as GPR103). Although NPFF receptors 1 and 2 share a greater number of identical amino acid positions with OX₂ receptors (31 and 33%) relative to QRFP receptor (27%), QRFP receptors retain a greater number of conserved or similar amino acid positions. When known QRFP receptors and NPFF receptors are included in phylogenetic analysis, QRFP receptors cluster nearer to orexin receptors (Fig. 4). This sequence similarity and lack of identity suggests that QRFP receptors diverged from orexin receptors earlier than NPFF receptors but experienced selective pressure to retain homologous sequence. Reported responses to central administration of QRF peptides, the ligands for QRFP receptor, include increases in arousal and locomotor activity, increased feeding, energy balance regulation, and the modulation of pain responses (Thuau et al., 2005; Moriya et al., 2006; Takayasu et al., 2006; Yamamoto et al., 2008; Lectez et al., 2009; Yamamoto et al., 2009), functions remarkably similar to that of orexin.

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Fig. 4.

Orexin receptor phylogeny and evolutionary conservation. Human OX₁ and OX₂ receptor sequences were used in BLASTP algorithm searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify orthologous orexin receptor sequences from other species as well as nonorexin receptor sequences sharing sequence identity and homology. Human NPFF receptor 1, NPFF receptor 2, and GPR103 protein sequences were then used to identify orthologous versions of these sequences in other species. Sequences were then aligned, and phylogenetic trees generated by using the Cobalt Phylo Tree tools at NCBI (http://www.ncbi.nlm.nih.gov/tools/cobalt), using human rhodopsin as the out-group. Note: human NPFF receptor 1 and NPFF receptor 2 sequences share greater identity with orexin receptors, whereas GPR103 (QRFP receptor) shares greater sequence similarity (residues of similar property) such that phylogenetic comparison, including GPR103 sequences from multiple species places them nearer orexin receptor sequences.

E. Orexin Receptor G Protein Signaling Modulating Neuronal Excitability

Orexin activation of OX₁ and OX₂ receptors results in target cell activation, generally reflected in increased intracellular Ca²⁺ levels and postsynaptic excitation that can last several minutes (Sakurai et al., 1998; van den Pol et al., 1998; Hagan et al., 1999; Bourgin et al., 2000; Van Den Pol et al., 2001; Liu et al., 2002; Arrigoni et al., 2010). Presynaptic modulation of neurotransmitter release and postsynaptic modulation of responses to other neurotransmitters have also been observed, particularly for OX₂ receptors, the cellular signaling mechanisms of which seem more diverse. The specific intracellular signaling pathways in which each of these receptors participate probably depend upon the cellular expression and subcellular localization (e.g., presynaptic versus postsynaptic) of other second-messenger signaling components.

Studies performed both in native primary neurons and in immortalized cell systems expressing recombinant orexin receptors indicate that these G-protein-coupled receptors increase intracellular Ca²⁺ through Gα_q/11 activation (Lund et al., 2000; Smart et al., 2000; Kukkonen and Akerman, 2001; Holmqvist et al., 2002; Zhu et al., 2003). Both orexin receptors seem to signal primarily through Gα_q/11, but some evidence suggests that both receptors are also capable of modulating cyclic nucleotide levels through Gα_s and Gα_i/o with varying ligand potencies, even though their predominant intracellular effect is to increase intracellular Ca²⁺ levels (Karteris et al., 2001; Holmqvist et al., 2002; Zhu et al., 2003). The most direct mechanism through which Gα_q/11 induces Ca²⁺ levels in orexin responsive cells is the activation of phospholipase C (PLC), triggering the liberation of inositol-1,4,5-trisphosphate (IP₃) and the release of Ca²⁺ from intracellular stores through IP₃ receptors (Smart et al., 1999). However, the full Ca²⁺ response mediated by OX₂ receptors requires extracellular Ca²⁺ conducted through either non–voltage-gated cation channels potentiating the PLC response (Lund et al., 2000; Kukkonen and Akerman, 2001; Holmqvist et al., 2002) or through L- and N-type Ca²⁺ channels, as indicated by a block in OX-A responsiveness of dopaminergic neurons of the ventral tegmental area (VTA) by Ω-conotoxin and nitrendipine (Uramura et al., 2001). Still other work has suggested a mechanism through which diacylglycerol, liberated by PLC, may directly activate transient receptor potential channels responsible for Ca²⁺ influx (Larsson et al., 2005; Näsman et al., 2006; Louhivuori et al., 2010).

The capacity of OX₂ receptors to both increase intracellular Ca²⁺ and regulate cAMP levels enables orexin signaling to modulate both the presynaptic release of neurotransmitters and the postsynaptic response to other transmitters. Orexin can regulate the presynaptic release of serotonin, GABA or glutamate (van den Pol et al., 1998; Liu et al., 2002). Postsynaptically in the VTA, the substantial Ca²⁺ responses induced by orexin can induce long-duration changes in N-methyl-d-aspartate receptor expression, thereby potentiating the response of these neurons for several hours (Borgland et al., 2006; Borgland et al., 2008). Orexin neurons also release dynorphin, which attenuates inhibitory postsynaptic potentials, an effect that ultimately potentiates orexin-induced postsynaptic activation of neurons in tuberomammillary nuclei (TMN) (Eriksson et al., 2004; Kantor et al., 2009; Williams and Behn, 2011). This synergistic effect of neuropeptides may provide an explanation for the more pronounced obesity and REM dysregulation phenotype of animal models in which orexin-secreting neurons are genetically ablated relative to Hcrt knockout animals harboring a mutation in the gene encoding the prepro-orexin peptide (Hara et al., 2005; Kantor et al., 2009).

A. Orexin Receptor Agonists/Potentiators

Advances in the study of orexin receptor agonism have been limited; fewer small-molecule tools are available compared with many antagonist compounds developed to date. Lack of an established positive control complicates any screening strategy to uncover a small-molecule agonist. Studies have thus been limited primarily to synthetic and modified versions of the endogenous neuropeptide agonists OX-A and OX-B.

In contrast to the narcoleptic phenotype in dogs and humans with dysfunctional orexin receptors or deficiencies in endogenous peptide ligands, (Lin et al., 1999; Nishino et al., 2000; Peyron et al., 2000; Wu et al., 2002), studies in rodents have demonstrated that intracerebroventricular administration of the orexin ligands serves to induce arousal and increase wakefulness (Hagan et al., 1999; Akanmu and Honda, 2005). Deadwyler et al. (2007) further reported that systemic and intranasal delivery of OX-A can decrease the effects of sleep deprivation on cognitive performance in nonhuman primates. The observation that systemic administration elicits an effect in these studies supports the finding that orexin-A crosses the blood-brain barrier by diffusion (Kastin and Akerstrom, 1999). No reports of behavioral responses to exogenously applied orexin peptides in humans, however, have appeared in the literature.

In addition to shedding light on the function of the endogenous orexin ligands, work with synthetic, modified peptides has allowed a greater understanding of ligand-receptor interactions. For example, the orexin-B analog [Ala¹¹,d-Leu¹⁵]orexin-B was generated by replacing l-leucine residues at positions 11 and 15. These particular substitutions resulted in an enhancement of selectivity toward the OX₂ receptor (compared with the OX₁ receptor) by approximately 400-fold (Asahi et al., 2003). In the same study, a systematic approach to residue replacement revealed that three leucine residues are important for OX-B's selectivity for the OX₂ receptor and determined the minimal peptide sequence required for orexin receptor activation.

In a recent patent disclosure, Yanagisawa (2010) reports a small-molecule agonist for OX₂ receptors. This represents the only published account of nonpeptidic orexin ligands to date. The chemical series reportedly induces a robust Ca²⁺ response in OX₂ receptor-expressing Chinese hamster ovary cells. There is one report of a small-molecule allosteric OX₂ receptor potentiator, a compound that binds to a site on the receptor other than the ligand-binding site and potentiates the response of the receptor to its cognate ligand (Lee et al., 2010). This compound, N-benzyl-N-(3,4-dimethoxybenzyl)glycyl-N²-(1-phenylethyl)glycinamide (OBPt-9), was identified via a microarray-based, two-color, cell-binding screen and was shown to potentiate the response to orexin-A in both OX₁ receptor- and OX₂ receptor-expressing cells (Lee et al., 2010). Further characterization of these molecules should prove informative.

B. Dual Orexin Receptor Antagonists

Intensive efforts to identify orexin receptor antagonists began soon after the discovery of these excitatory neuropeptides and their receptors. At least four structurally distinct dual orexin receptor antagonists (DORAs) have entered human trials including almorexant [Actelion Pharmaceuticals, now GlaxoSmithKline (GSK), Brentford, Middlesex, UK], N-[[(2S)-1-[[5-(4-fluorophenyl)-2-methyl-4-thiazolyl]carbonyl]-2-piperidinyl]methyl]-4-benzofurancarboxamide (SB-649868; GSK), suvorexant (Merck, Whitehouse Station, NJ), and MK-6096 (Merck). Others have reported the discovery and characterization of additional DORAs as well as selective OX₁ receptor and OX₂ receptor antagonists (SORAs) (Coleman and Renger, 2010). Both Actelion and Merck have reported clinical proof of concept for treating primary insomnia with their respective receptor antagonists.

In 2007, Actelion disclosed data for almorexant, a potent DORA, showing this compound to be effective in promoting sleep in preclinical species. When administered to rats, almorexant dose-dependently increased REM and NREM sleep, effects beginning within an hour of dosing and persisting for up to 12 h after treatment (Brisbare-Roch et al., 2007). With repeat dosing at 100 mg · kg⁻¹ · day⁻¹ in rats, no tolerance to the sleep effects were seen, and no rebound was observed upon discontinuation of drug treatment (Brisbare-Roch et al., 2008). It is noteworthy that when dosed during the inactive period, when endogenous levels of orexin are at their lowest, the compound had little effect beyond normal sleep (Brisbare-Roch et al., 2007). In dogs, 100 mg/kg almorexant significantly reduced mobility scores relative to vehicle-treated dogs; the subjects could be easily aroused by the presence of a familiar individual but quickly returned to sleep once the stimulus was withdrawn (Jenck et al., 2007). Cataplexy was not observed in either species at any dose (Brisbare-Roch et al., 2007).

In double-blind, placebo-controlled clinical studies, almorexant was well tolerated, with reports of somnolence, dizziness, disturbed attention, and fatigue at doses above 200 mg. The incidence of somnolence increased with dose over the range of 100 to 1000 mg. As in preclinical studies, no cataplexy-related side effects were reported (Hoever et al., 2010). In double-blind studies with zolpidem as an active control, almorexant increased sleep efficiency, reduced sleep latency, and increased total sleep time at doses greater than 200 mg in healthy volunteers. A phase II study in patients with primary insomnia demonstrated the efficacy of both the 200- and 400-mg doses in improving sleep efficiency. Significant effects on secondary endpoints including latency to persistent sleep (LPS) and wake after sleep onset (WASO) were observed at the 400-mg dose (Dingemanse et al., 2007). Actelion initiated a phase III study in adults with primary insomnia (RESTORA1) in 2007. In this study, almorexant met the primary endpoint of superiority compared with placebo on both objective and subjective measures of WASO. However, an undisclosed human tolerability issue resulted in termination of Phase III clinical development in January 2009 (Almorexant in adult subjects, NCT00608985, http://www.clinicaltrials.gov).

Researchers at GSK also discovered a series of piperidine-derived antagonists, including SB-649868 as potent DORAs. SB-649868 was reported to inhibit both OX₁ and OX₂ receptor activity and entered clinical trials in 2005. Preclinically, SB-649868 was shown to be sleep-promoting in rodent and primate studies (Di Fabio et al., 2011). In 2007, GSK announced that SB-649868 had advanced to phase II clinical trials. In initial single rising dose studies, SB-649868 was well tolerated and exhibited proportional increases in exposure across the dose range. After administration of SB-649868 to healthy volunteers, there were statistically significant improvements in total sleep time, reduced LPS, and WASO at both doses relative to placebo. Neither dose produced cognitive impairment the morning after evening drug administration (Bettica et al., 2009; Renzulli et al., 2011). Clinical studies, however, revealed that SB-649868 increased exposure of coadministered simvastatin in a drug-drug interaction study, consistent with the potent inhibition of CYP3A4 in vitro (Bettica et al., 2011). Phase II studies of SB-649868 were placed on clinical hold in late 2007 because of the emergence of a reported preclinical toxicity, and GSK entered into a collaborative agreement with Actelion in 2008 to codevelop almorexant and other potential back-up compounds.

Merck has developed a diverse portfolio of orexin receptor antagonists in several distinct structural classes (Coleman et al., 2011a). After completing a screening campaign to identify new leads, researchers at Merck disclosed proline bis-amides including DORA-1 as potent dual orexin receptor antagonists. Intracerebroventricular administration of orexin B to rats placed in a beam-break box produced significant increases in locomotor activity over several hours. When rats were pretreated with DORA-1 by intraperitoneal injection 30 min before neuropeptide administration, DORA-1 produced dose-dependent reductions in this locomotor activity relative to baseline (Bergman et al., 2008).

Merck has also reported N,N-disubstituted-1,4-diazepanes, including suvorexant (MK-4305) and DORA-12, to be potent dual orexin receptor antagonists. Both compounds are selective antagonists with excellent activity in cell-based assays (Cox et al., 2009, 2010). Suvorexant inhibits orexin induced Ca²⁺ levels in cells expressing human OX₁ or OX₂ receptors with IC₅₀ values of 50 and 56 nM, respectively, but has >6000-fold selectivity against a panel of 170 receptors and enzymes. Preclinically, suvorexant is orally bioavailable, has good brain penetrance, and demonstrates orexin receptor occupancy in rat brain (Cox et al., 2010; Winrow et al., 2011). An example of the sleep-promoting effects of DORA-12 in mice is seen in Fig. 5A. Active phase treatment is associated with dose-dependent reductions in active wake and augmentation of NREM and REM sleep, effects that diminish abruptly with the onset of the animals' normal inactive phase. These DORA-12-induced changes are mediated through OX₁ and OX₂ receptors, because these effects are absent in genetically modified mice lacking these receptors (Fig. 5B). In other rodent sleep studies, suvorexant dose dependently reduced active wake and increased REM and NREM sleep when administered orally at 10, 30, and 100 mg/kg. Suvorexant was also highly efficacious in promoting sleep in canines and rhesus monkeys (Winrow et al., 2011). Based on these and other efficacy studies as well as a favorable pharmacokinetic and safety profile, suvorexant was selected to advance into clinical development.

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Fig. 5.

The sleep-promoting effects of DORA-12 are absent in OX_1/2 receptor double-knockouts. Electrocorticogram and electromyogram were monitored in wild-type (A) and age-matched mice with targeted ablation of both OX₁ and OX₂ receptors (B) by radiotelemetry to determine mean time spent in the indicated sleep stages as described previously (Winrow et al., 2012). At times indicated by arrows, vehicle [20% vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate), by mouth, closed symbols] or DORA-12 at 60 or 100 mg/kg (open symbols) were administered in a balanced 5-day crossover paradigm. Treatment occurred during the late active phase, approximately 4 h before the onset of the inactive phase (10:00/10:30 AM; zeitgeber time, 08:00/08:30; black arrows). Closed and open bars below each plot represent dark (active) and light (inactive) phases, respectively. Plotted points are the mean time spent in each sleep state during 30-min intervals after treatment over 5 days of consecutive treatment as determined by automated scoring and analysis as described previously (Winrow et al., 2012). Error bars (where visible) depicting the S.E.M. for each point are included, and time points exhibiting significant differences between vehicle and DORA-12 responses are indicated by gray vertical lines and tick marks (short, p < 0.05; medium, p < 0.01; long, p < 0.001; linear mixed effects model for repeated measures applied t test).

Suvorexant was well tolerated in phase I studies, with peak plasma levels achieved at 1.5 to 4 h after dosing and a terminal plasma half-life of 8 to 14 h. In healthy volunteers, dose-dependent observations of somnolence were evident. Next-day residual sedation was not observed when suvorexant was administered at doses of 10 and 50 mg in the evening, whereas these same doses provided significant increases in overall sleep efficiency and reductions in WASO and LPS in a dose-dependent manner. Results from the phase IIb study demonstrated that suvorexant was superior to placebo in improving sleep efficiency on the first night of treatment as well as at the end of 4 weeks in patients with primary insomnia. These improvements in sleep efficiency were noted at all doses (10, 20, 40, and 80 mgs). Suvorexant also showed improvements in the secondary endpoints of reduced WASO at all doses and reduced LPS at 80 mg (Herring et al., 2010). In 2010, Merck announced that suvorexant had entered into phase III trials; it is currently the most advanced orexin antagonist in active clinical development.

Merck has disclosed an additional series of 2,5-disubstituted piperidine carboxamides including DORA-22 and MK-6096 as potent dual orexin receptor antagonists. The structure and preclinical pharmacology of MK-6096, a second dual orexin receptor antagonist in clinical development from Merck, has been disclosed (Coleman et al., 2012; Winrow et al., 2012). MK-6096 is structurally distinct from suvorexant and is highly efficacious in promoting sleep in rats (3–30 mg/kg) and dogs (0.25–0.5 mg/kg) (Winrow et al., 2012). MK-6096 was reported to have entered phase II clinical studies in 2009.

C. Orexin 2 Receptor-Selective Antagonists

Although dual orexin receptor antagonists have been shown to promote sleep in multiple species, SORAs have been used extensively to evaluate the relative role of each receptor subtype in the control of arousal and sleep, as well as other behaviors and physiology. An OX₂ SORA [1-(2,4-dibromophenyl)-3-[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl] urea (JNJ-10397049)] has been evaluated for sleep/wake effects, both alone and in conjunction with 1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea (SB-408124), an OX₁ SORA (McAtee et al., 2004; Dugovic et al., 2009). Both compounds are brain-penetrant, with JNJ-10397049 producing ∼80% cortical OX₂ receptor occupancy for more than 6 h, an OX₂ receptor occupancy matched by almorexant at this dose. Sleep-promoting effects of the OX₂ SORA JNJ-10397049 at 30 mg/kg were due to lengthening sleep bouts, whereas treatment with almorexant was associated with sleep-promoting effects marked by an increased number of REM and NREM sleep bouts. Both almorexant and JNJ-10397049 reduced histamine levels in the LH, whereas neither the histamine levels nor sleep parameters were affected by the relatively OX₁ receptor-selective SB-408124 (Dugovic et al., 2009).

Researchers at Roche have reported EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) as a selective OX₂ SORA structurally distinct from JNJ-10397049 that exhibits greater affinity for human OX₂ relative to OX₁ receptors (approximately 900-fold selective) (Malherbe et al., 2009). Autoradiography using [³H]EMPA in rat brain slices shows high specific binding in hypothalamus, tuberomammillary nuclei, hippocampus, and nucleus accumbens. In vivo, EMPA dose-dependently reversed OX-B–induced hyperlocomotion in mice, achieving full reversal at a dose of 300 mg/kg i.p. It is noteworthy that EMPA induced no psychomotor deficits when evaluated in a rat Rotorod assay at doses as high as 30 mg/kg i.p. (Malherbe et al., 2009). These results are consistent with earlier results reported for structurally distinct DORAs, including almorexant and SB-649868.

D. Orexin 1 Receptor-Selective Antagonists

The need for potent, selective preclinical research tools is highlighted by the wealth of studies using 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB-334867), described as a highly selective OX₁ SORA developed by SmithKline Beecham (now part of GlaxoSmithKline). More than 160 papers have reported the use of SB-334867 as an OX₁ SORA in behavioral models evaluating addiction, feeding, sleep, and other behaviors. This compound antagonizes OX-A-induced [Ca²⁺]_i signal mediated by OX₁ receptors expressed on Chinese hamster ovary cells (K_B, 40 nM), and shows ∼50-fold selectivity relative to OX₂ receptors evaluated in the same assay (K_B, 1995 nM) (Haynes et al., 2000; Smart et al., 2001) (Table 4). These results are consistent with binding affinities observed in our laboratories, which have found it to be ∼45-fold selective for OX₁ over OX₂ receptors (K_i = 18 and 835 nM, respectively) (A. Gotter, P. Coleman, J. Renger, and C. Winrow, unpublished observations). However, examination of SB-334867 potency against a panel of 170 other enzymes and receptors showed significant interactions with at least seven other targets at concentrations less than 10 μM. These included activities toward the adenosine A_2A receptor (K_i, 0.67 μM), 5-HT_2C receptor (K_i, 1.2 μM), monoamine transporter (K_i, 1.44 μM), norepinephrine transporter (K_i, 1.58 μM), adenosine transporter (K_i, 2.45 μM), adenosine A₃ receptor (K_i, 3 μM), and 5-HT_2B receptor (K_i, 3.47 μM) (A. Gotter, P. Coleman, J. Renger, and C. Winrow, unpublished observations). The favorable pharmacokinetic properties and commercial availability of SB-334867 have made this a popular tool compound for studying orexin signaling in vivo, and there is the potential for substantial central nervous system exposure with this compound. For example, in rats administered 20 mg/kg i.p. (25% 2-hydroxypropyl β-cyclodextrin), we observed a sustained concentration of 3.4 μM in plasma at both 30 minutes and 2 h after dosing. Given the selectivity profile of SB-334867, there exists a possibility for antagonism for not only OX₁ receptors but also OX₂ and several other targets at these doses. It is for these reasons that some caution should be exercised in interpretations of behavioral observations attributed to selective OX₁ receptor antagonism in studies using SB-334867 exclusively.

Two other OX₁ receptor-selective antagonists appearing in the literature are SB-408124 and 5-bromo-N-[(2S,5S)-1-(3-fluoro-2-methoxybenzoyl)-5-methylpiperidin-2-yl]methyl-pyridin-2-amine (GSK1059865). Disclosed in 2004 by SmithKline Beecham, SB-408124 has a published OX₁/OX₂ receptor selectivity of 63-fold (K_b, 22 versus 1405 nM in Ca²⁺ mobilization assays), slightly better than SB-334867 (Langmead et al., 2004). Like SB-334867, however, SB-408124 exhibits significant activity toward other receptors, notably 5-HT_2B (0.32 μM), dopamine D₁ (1.78 μM), 5-HT_2C (1.88 μM), adenosine A_2A (2.77 μM), and α_2b-adrenergic receptors (3.29 μM). SB-408124 has good pharmacokinetic properties, including bioavailability (∼80%) and brain penetration (1.7%) in rats (A. Gotter, P. Coleman, J. Renger, and C. Winrow, unpublished observations). However, these favorable properties allow SB-408124 to reach levels that may also affect other receptors. GSK1059865, a recently identified OX₁-selective antagonist (Gozzi et al., 2011), may offer an improvement over SB compounds, as it has a reported OX₁/OX₂ receptor selectivity of ∼79-fold (K_b, 1.6 versus 126 nM in IP₃ accumulation assays), with no significant activity at concentrations under 1 μM toward a panel of 113 different receptors with the exception of the κ-opioid receptor (K_i, 320 nM) (A. Gotter, P. Coleman, J. Renger, and C. Winrow, unpublished observations). More selective OX₁ SORAs will undoubtedly be developed, but currently available reagents used in combination with DORAs, 2-SORAs, and genetic models have nonetheless provided valuable insight into the function of this receptor in the control of vigilance state and other orexin-mediated physiology and behavior.

A. Neurological Pathways Regulating Sleep

The identification of orexin neuropeptides and their cognate receptors has provided an understanding of the network of neuronal pathways involved in the interplay between sleep and arousal-promoting centers and how these areas interact to control vigilance state. The predominant sleep-promoting influence in the brain is provided by the ventrolateral preoptic (VLPO) and adjacent median preoptic areas. Neurons of the VLPO are most active during NREM sleep, partially active during REM sleep, and silent during wakefulness. They send inhibitory projections to arousal promoting areas, including the tuberomammillary nuclei (TMN), laterodorsal and pedunculopontine tegmental (LDT, PPT) nuclei, the locus ceruleus (LC), dorsal raphe (DR) nuclei, the ventral tegmental area (VTA) (Saper et al., 2005b, 2010; España and Scammell, 2011) and notably, orexinergic neurons of the LH (Sakurai et al., 2005b; Yoshida et al., 2006). This inhibitory influence is primarily mediated by GABAergic influences. In fact, potentiation of GABA signaling from the VLPO is thought to underlie the mechanism of action of currently marketed sleep agents (e.g., zolpidem and eszopiclone) (España and Scammell, 2011).

Orexin-secreting neurons of the LH project to brainstem nuclei involved in promoting arousal (Fig. 6). The activity of these nuclei, which include the TMN, LDT/PPT, DR, and LC, is dependent upon the balance of influence imposed by both inhibitory signals from the VLPO and excitatory ones provided by orexin. Ultimately, the integration of these signals determines arousal and behavioral state. A primary orexinergic projection is sent to the TMN, which preferentially express OX₂ over OX₁ receptors (Trivedi et al., 1998; Marcus et al., 2001). TMN neurons project broadly to the prefrontal cortex (PFC), thalamus, and other subcortical structures and are normally active during wake and progressively less active during NREM and REM sleep. They represent the primary source of histaminergic (HA) neurons in the brain, and HA receptor agonists and antagonists promote and attenuate arousal, respectively (Monti et al., 1986; Lin et al., 1988; Mochizuki and Scammell, 2003). Up-regulation of this histaminergic activity underlies the mechanism of a new class of wake-promoting drugs that inhibit histamine H₃ receptors to counter the reduced HA levels observed in narcoleptics (Lin et al., 2008; España and Scammell, 2011).

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Fig. 6.

Orexin and OxR efferent pathways associated with arousal, vigilance state, and reward pathways. NAc, nucleus accumbens; HA, histaminergic; DA, dopaminergic; ACh, cholinergic; NE, noradrenergic; 5-HT, serotonergic. Green, orexinergic neuron projections; red, preferential OX₁ receptor expression; blue, preferential OX₂ receptor expression; violet, both OX₁ and OX₂ receptor expression.

Orexin neurons also send projections to cholinergic tegmental nuclei (the LDT and PPT) as well as noradrenergic LC and serotonergic DR neurons. OX₁ receptors are preferentially expressed in the LC, whereas both orexin receptors are detectable in LDT, PPT, and DR (Fig. 6) (Trivedi et al., 1998; Marcus et al., 2001). Along with arousal-promoting influences, these brainstem regions are also responsible for gating between vigilance states, particularly in and out of REM sleep. Orexin influences on neurons of the LDT and PPT also regulate muscular atonia that accompanies REM sleep, through both direct and indirect effects on ventromedial neurons of the medulla, which in turn inhibit spinal motor neurons through GABA projections. More extensive discussions of these mechanisms regulating vigilance state and sleep-dependent motor activity have been reviewed elsewhere (Saper et al., 2010; España and Scammell, 2011; Scammell and Winrow, 2011).

B. Orexin-A and -B Promote Arousal and Modulate Vigilance State

Orexins provide an arousal signal that is both necessary and sufficient for normal sleep/wake regulation. Over the course of the 24-h circadian cycle, changes in arousal match oscillating levels of orexin. In nocturnal animal models, orexin levels rise over the night-time hours, peaking late in the active phase, whereas in primates, OX-A levels in CSF accumulate over daytime hours, peaking just before the dark phase (Taheri et al., 2000; Zeitzer et al., 2003). OX-A applied exogenously (via intracerebroventricular injection) to rats results in increased locomotor activity, grooming and wakefulness, whereas the mean time spent in NREM and REM sleep is diminished. These effects are greatest when OX-A is administered during the inactive phase when endogenous orexin levels are at their lowest and barely detectable when applied during periods of wakefulness, as might be expected of an arousal signal (Hagan et al., 1999; Piper et al., 2000). Exogenously applied OX-A also promotes arousal in mice with selected genetic ablation of orexinergic neurons. In this case, the levels of wakefulness and NREM and REM suppression in mutant animals exceed those seen in wild-type animals treated identically (Mieda et al., 2004), indicating that downstream orexin signaling components, including orexin receptors, are intact and are up-regulated in these mutants and that the neuropeptide alone is sufficient as a wakefulness signal. Similar results are seen in narcoleptic dogs where OX-A rescues the cataplexy and hypersomnolence phenotype of an animal harboring a mutation in the Hcrt gene encoding prepro-orexin but has no effect in dogs with mutations in the gene for the OX₂ receptor (Fujiki et al., 2003). Artificial activation of orexin-secreting neurons is sufficient to drive arousal. In an elegant set of experiments, Adamantidis et al. (2007) and later Carter et al. (2009) used mice in which the expression of channelrhodopsin-2 was driven by the Hcrt promoter such that orexin-containing neurons could be photically stimulated by a fiber optic means. Activation of orexigenic neurons in the LH of these mice induced transitions from NREM or REM sleep into wakefulness (Adamantidis et al., 2007), but in the face of increased sleep pressure induced by sleep deprivation, these wake-promoting effects as well as orexin-dependent activation of TMN and LC neurons were diminished, indicating that homeostatic influences converge downstream of orexin neuron activation (Carter et al., 2009).

C. Modulation of Orexin Signaling

In general, the timing and extent of sleep and wakefulness is driven by two different influences; the circadian clock that aligns the timing of an organism's physiology and behavior with daily environmental timing cues, and the homeostatic drive for sleep aligning the restorative properties of rest with physiological and energy needs. The endogenous circadian pacemaker resides in the suprachiasmatic nuclei (SCN) of the hypothalamus, and controls daily cycles of arousal, locomotor activity, gut motility, and the timing of sleep even in the absence of external cues such as ambient lighting changes (Huang et al., 2011). When grown in culture, neurons of the SCN in themselves have periods of electrical activity of ∼24 h (Welsh et al., 1995). The activity of the SCN is primarily entrained to the environment by ambient lighting cues (Klein et al., 1991). The output from the SCN to the sleep network is conveyed through the ventral subparaventricular zone to the dorsomedial nucleus of the hypothalamus (DMH). From here the DMH projects to VLPO neurons via inhibitory GABA projections and sends excitatory connections to orexin containing neurons of the LH (Saper et al., 2005b). The DMH, which is largely active during wakefulness (Saper et al., 2005a), also receives appetite, feeding, and body temperature inputs, such that some level of homeostatic regulation may occur at this level (Saper et al., 2005b). Together, the DMH and the VLPO coordinate the activity of orexin neurons to regulate arousal aligned with circadian environmental cues.

The specific mechanism through which the homeostatic drive for sleep is mediated is less clear, however, but is thought to be associated with energy homeostasis and the need to conserve metabolic energy. The need for sleep accumulates with wakefulness such that the pressure for sleep accumulates with sleep deprivation. Evidence for orexin neurons being an integration site for homeostatic influences are the observations that orexin cell firing is increased by direct acetylcholine, glutamate, and ghrelin application and decreased by leptin, glucose, norepinephrine, 5-HT, and GABA (Burdakov, 2004). One attractive candidate for this regulation is adenosine, because during prolonged wakefulness, ATP is degraded to ADP, AMP, and eventually adenosine, which accumulates in parts of the brain (Porkka-Heiskanen et al., 2000). Orexin neurons express adenosine A₁ receptors, and application of the adenosine A1R antagonist 1,3,-dipropyl-8-phenylxanthine into the LH increases wake and suppresses both REM and NREM sleep (Thakkar et al., 2008). Microinfusion of the adenosine A_2A agonist 2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine(CGS21680) into the ventral striatum both promotes sleep measures and attenuates c-Fos production in orexin-producing cells, suggesting not only that adenosine's sleep promoting effect may be facilitated by reduced orexinergic cell firing but also that orexin signaling is not necessarily the exclusive mechanism for adenosine-mediated sleep promotion (Satoh et al., 2006).

Corticotropin-releasing factor (CRF) and dopamine also seem to modulate the influence of orexin on arousal. CRF signaling in response to stress represents a possible interaction with orexin signaling and sleep, because elevated levels of the hormone are associated with arousal, and intracerebroventricular administration of CRF induces wakefulness and locomotor activity (Sakurai and Mieda, 2011). Any interaction with CRF signaling, however, appears to be upstream of orexin, because orexin-induced arousal persists in CRF receptor knockouts as well as in the presence of CRF receptor antagonists (Fenzl et al., 2011). Evidence for the modulation of orexin signaling in arousal and vigilance state by dopamine has also recently emerged (Sakurai et al., 2010; Sakurai and Mieda, 2011). In orexin peptide knockout mice, pharmacological D₁ receptor activation decreases the prevalence of sleep attacks relative to wild-type animals. Conversely, the hypersomnolence of these animals is exacerbated with D₁ antagonism, whereas D₂ receptor modulation has little to no effect on arousal (Burgess et al., 2010). Cataplectic attacks, however, are affected by D₂ receptor activity; pharmacological D₂ activation and inhibition are associated with substantial increases and decreases, respectively, in behavioral arrest (Burgess et al., 2010). These studies further illustrate the existence of distinct pathways for arousal control and the regulation of vigilance state as well as the dopamine receptor subtype involvement in each.

A. Canine Narcolepsy

In dogs, narcolepsy is manifested as active-phase sleep attacks and, during the inactive phase, as short sleep latency and fragmented sleep with rapid EEG/polysomnographic transitions between sleep stages. In severe cases, cataplectic attacks are characterized by atonia with hind-limb buckling during which the animal retains consciousness, often with open eyes that are able to follow visual stimuli. Consciousness may give way to sleep, typically REM. These episodes are typically triggered by palatable food presentation or play, much as positive emotions do in human patients (Nishino, 2005).

Canine narcolepsy is classified into two forms: familial (genetic), associated with mutations in the OX₂ receptor gene, and sporadic, which is typically associated with loss of orexin-secreting neurons. The genetic form is transmitted in an autosomal-recessive fashion with complete penetrance. To date, OX₂ receptor mutations include truncations within transmembrane region five or just after transmembrane region six in Doberman pinschers and Labrador retrievers (Lin et al., 1999) and a point mutation in a dachshund family resulting in a glutamate to lysine change at amino acid position 54, rendering a receptor incapable of being bound and activated by OX-A or OX-B (Hungs et al., 2001). CSF and brain levels of OX-A and OX-B are normal in these mutants, and orexin-containing neurons are normal in appearance (Ripley et al., 2001). Although exogenously applied OX-A can reverse narcoleptic symptoms in sporadic narcoleptic dogs deficient in orexin neurons, high doses of intracerebroventricular and intravenous administration of OX-A have no effect on arousal or cataplectic symptoms of OX₂ receptor mutant narcoleptic Doberman pinschers (Fujiki et al., 2003). Sporadic canine narcolepsy (poodles, beagles, dachshunds, collies, fox terriers) does not seem to be associated with a single, highly penetrant gene mutation but is typically associated with symptoms more pervasive than the genetic form (Nishino, 2005). Unlike humans in which a leukocyte antigen gene is associated with sporadic narcolepsy, a canine allele specifically associated with the disorder remains elusive (Peyron et al., 2000; Nishino, 2005; De la Herrán-Arita et al., 2011).

B. Genetically Engineered Mouse and Rat Models

Genetic manipulation of rats and mice has been used to mimic the clinical etiology of narcolepsy associated with orexinergic neuron loss and to dissect the role of individual orexin signaling components. Efforts have included transgenic models in which the expression of a cellular toxic transgene is used to specifically ablate orexin-producing neurons to imitate the autoimmune loss of these neurons in patients with narcolepsy. Genetic models also include targeted gene knockouts of the Hcrt gene encoding the prepro-orexin peptide processed into OX-A and OX-B ligands (orexin peptide knockouts), the Hcrtr1 and Hcrtr2 genes encoding OX₁ and OX₂ receptors (OX₁ receptor and OX₂ receptor knockouts), and double receptor mutant mice lacking both of these receptors (OX_1/2 receptor double knockouts). Together, with pharmacological manipulation using selective receptor agonists and antagonists described in sections III and IV, these mutant animals have provided invaluable information regarding not only the role of orexin signaling in narcolepsy/cataplexy but also the general neuronal pathways regulating sleep and vigilance state (De la Herrán-Arita et al., 2011). Both approaches uncovered the importance of each of these signaling components in arousal, vigilance state, and the regulation of sleep. Overall, the importance of these signaling components to arousal may be summarized as follows: Ox neurons > orexin peptide KOs ≥ OX_1/2 receptor double KO > OX₂ receptor KOs ≫ OX₁ receptor KOs. It should be noted, however, that in many cases, these animal models have been evaluated by different laboratories using different methods (e.g., EEG versus visual observation) often using different criteria to make interpretations regarding the impact of a given mutation on arousal and/or cataplectic behavior. Efforts have been made, however, to standardize the criteria for the cataplectic behavior in mice. Based on observations from orexin peptide knockouts, the following criteria have been established on behalf of the International Working Group on Rodent Models of Narcolepsy (Chemelli et al., 1999; Willie et al., 2003; Scammell et al., 2009): 1) abrupt atonia lasting over 10 s, 2) animal immobility during the episode, 3) quantitative EEG (qEEG) dominated by θ activity during the episode, and 4) behavioral arrest preceded by a period of active wake lasting > 40 s. In addition, cataplectic behavior should be reversible, with administration of clomipramine or other anticataplectic (e.g., monoamine reuptake inhibitors such as desipramine) (Willie et al., 2003; Scammell et al., 2009). Nevertheless, this expression of the importance of these components on arousal can be concluded on the basis of the studies discussing these genetic models below, along with what has been determined from both dogs and humans.

C. Orexin Neurons Are Critical for Both Arousal and Vigilance State Gating

In theory, the best preclinical models for the majority of human cases exhibiting narcolepsy with cataplexy are transgenic mice and rats lacking orexin-producing neurons. These animals (Ox/Atx mice and rats) express an apoptotic Ataxin-3 transgene whose expression is driven by the human Hcrt gene promoter, resulting in programmed cell death of orexin-containing neurons while leaving neighboring neurons, including melanin-concentrating hormone-containing neurons of the LH, intact (Hara et al., 2001; Beuckmann et al., 2004). Despite incomplete loss of orexin neurons in hemizygous animals, OX-A levels within CSF, cortex, and brainstem are as much as 100-fold lower than that of wild-type animals (Zhang et al., 2007). The phenotype of Ox/Atx mice and rats resembles that seen in human patients with narcolepsy/cataplexy active-phase hypersomnolence, behavioral arrest/atonia episodes triggered by excited ambulation or grooming, and frequent wake-to-REM transitions never observed in wild-type animals that often enter REM through an intervening NREM transition (wake to NREM to REM). Inactive phase sleep is fragmented with reduced REM latency and frequent, but short bouts of wake and NREM with transitions occurring rapidly (Hara et al., 2001; Beuckmann et al., 2004). Remarkably, these animals remain sensitive to ectopically expressed or exogenously administered OX-A and OX-B, which reverse behavioral arrests and REM abnormalities, including wake-to-REM transitions of Ox/Atx mice, in some cases to greater effect than in wild-type mice (Mieda et al., 2004). One divergence from the accepted criteria for cataplexy, however, is the presence of quantitative EEG spectral pattern resembling wake during episodes of behavioral arrest in Ox/Atx rats (Beuckmann et al., 2004), as opposed to θ or REM activity characteristic of cataplexy. It remains to be seen whether this is a species-dependent divergence from mice or if more complete loss of orexin neurons in homozygous animals may affect these animals even more profoundly.

D. The Role of the Hcrt Gene and Hcrt Gene Product

Mice with a targeted deletion of the Hcrt gene encoding the prepro-orexin precursor peptide display a narcolepsy phenotype similar to transgenic Ox/Atx animals, with cataplectic episodes satisfying the criteria described above (Chemelli et al., 1999; Fujiki et al., 2009; Scammell et al., 2009). Furthermore, the abrupt behavioral arrests in orexin peptide knockouts are reversed by clomipramine, whereas caffeine only increased wakefulness and actually exacerbated cataplectic symptoms, demonstrating the specificity of this treatment (Willie et al., 2003). Although orexin peptide knockouts clearly exhibit cataplexy, their phenotype is not as pervasive as that observed in Ox/Atx animals. Direct comparisons revealed an even greater number of vigilance state transitions and time spent in REM for Ox/Atx animals (Kantor et al., 2009). These results indicate that orexin-containing neurons provide additional signals beyond orexin itself, perhaps glutamate or dynorphin, which can contribute to narcoleptic symptoms (Sakurai et al., 2005a; Kantor et al., 2009). Modeling of orexin neuron activity suggests that dynorphin is capable of modulating orexin responses, delaying its arousal effects at the sleep/wake transition by affecting the sensitization and firing rate of orexin-sensitive neurons (Williams and Behn, 2011). Nevertheless, orexin peptide knockout animals have provided a useful model in which to study the cataplectic episodes in detail, where both scheduled palatable food and running wheel presentation effectively increase the frequency of cataplectic episodes, presumably mimicking the effect of positive emotions in human patients (España et al., 2007; Clark et al., 2009). Consistent with this observation is the finding that both positive and negative olfactory stimuli (female and coyote urine) are also sufficient to induce narcoleptic episodes in male orexin peptide knockout mice (Morawska et al., 2011). These behavioral patterns indicate that orexin peptide knockouts are a particularly good model for early onset narcolepsy with cataplexy. Mutation of the human HCRT gene affecting peptide trafficking and processing is known to be associated with severe cataplexy (5–20 episodes/day) (Peyron et al., 2000).

E. Role of OX₂ Receptors in the Control of Arousal, Vigilance State

Arousal responses to orexin are primarily mediated by OX₂ receptors. Intracerebroventricular administration of OX-A, OX-B, or [Ala¹¹]OX-B, a modified signaling peptide having 120-fold selectivity for OX₂ receptors, similarly promote wakefulness and decrease the amount of time spent in REM and slow-wave sleep in a dose-dependent manner in rats (Hagan et al., 1999; Piper et al., 2000; Akanmu and Honda, 2005). Conversely, OX₂ receptor-selective antagonists have sleep-promoting effects similar to antagonists having equal potencies for both OX₁ and OX₂ receptors, including attenuated active wake, increased REM and NREM sleep, and decreased latencies to NREM and REM (Dugovic et al., 2008). As might be expected, OX₂ receptor knockouts display a narcoleptic phenotype similar to that of orexin peptide knockouts, including hypersomnolence, fragmented wakefulness and NREM sleep, increased active phase NREM sleep, and limited wake-to-REM transitions (Willie et al., 2003). The behavioral arrests exhibited by these mice, however, fall short of the cataplexy criteria established by Scammell et al. (2009). Although clomipramine reverses behavioral arrests exhibited by OX₂ receptor knockouts, these episodes are far less frequent than those observed in Hcrt knockouts, are typically more gradual in their onset, and are preceded by quiet wakefulness rather than “emotive” behaviors such as grooming or climbing that precede abrupt behavioral arrests. These gradual arrests are characterized by EEG power spectra similar to that of NREM sleep, as opposed to θ-rich REM sleep typical of abrupt cataplectic episodes (Willie et al., 2003). As such, the behavioral arrests displayed by OX₂ receptor knockouts do not seem cataplectic in nature but suggest that other signaling mechanisms may be required for these episodes.

F. Role of Orexin 1 Receptor in the Control of Vigilance State

As the only other known orexin receptor, the incomplete phenotype of OX₂ receptor knockouts relative to orexin peptide knockout animals indicates that the OX₁ receptor does participate in the mechanism of cataplexy. However, constitutive OX₁ receptor mutants reportedly display only a mild sleep phenotype with some increase in fragmentation (Kisanuki et al., 2000). Likewise, OX₁ receptor-selective antagonism with compounds of imperfect selectivity elicits little to no effect on sleep architecture (Dugovic et al., 2008) but has been reported to both increase extracellular dopamine in the PFC and to attenuate the sleep promoting effects of OX₂ receptor antagonism (Dugovic et al., 2009). Given its expression on locus ceruleus neurons involved in the control of REM and its activating effect on those neurons, a role for OX₁ receptor in gating transitions into REM remains possible (Bourgin et al., 2000; Ohno and Sakurai, 2008). In support of this assertion is the observation that small interfering RNA-mediated knockdown of OX₁ receptor expression in locus ceruleus is associated with inappropriate increases in REM sleep during the active period of rats for up to 4 days after treatment, a time-course coincident with reduced OX₁ receptor mRNA levels. Remarkably, neither wakefulness, NREM sleep, nor the qEEG power spectra of treated animals was affected, suggesting that this effect was specific for vigilance state gating and not a general effect on arousal (Chen et al., 2010).

G. Mechanisms Underlying Narcolepsy/Cataplexy

H. Genetic versus Pharmacological Manipulation: Complementary Interpretations

Independently, experiments using mutant animals and pharmacological manipulation of orexin receptor activity are largely complementary but have distinct caveats associated with these approaches. These differences stem from the constitutive nature of gene knockouts relative to acute transient pharmacological manipulation. Constitutive gene knockouts and Ox/ATX transgenics more closely mimic narcolepsy and varying aspects of the disorder than what might be expected from pharmacological manipulation. For example, behavioral arrests have been observed in Hcrt and OX₂ receptor knockouts (Chemelli et al., 1999; Willie et al., 2003) and in mice in which both orexin receptors have been mutated (Kalogiannis et al., 2011). Although it is currently unclear whether the behavioral episodes observed in the later model constitute cataplexy (Scammell et al., 2009), cataplectic episodes have not been observed in response to even high doses of dual orexin receptor antagonists across multiple species (Brisbare-Roch et al., 2007).

The fundamental difference between constitutive genetic manipulation and pharmacological perturbation is the likely development of molecular, cellular, neuronal, and/or behavioral mechanisms that compensate for the loss of a targeted gene product or orexin neuron loss. Indeed, the hypersomnolence exhibited by animals with a targeted disruption of the Hcrt gene is still punctuated by periods of wakefulness (Chemelli et al., 1999), and pharmacological orexin receptor antagonism is capable of acutely promoting sleep in wild-type animals to levels exceeding that seen in untreated mutant mice lacking both receptors (Winrow et al., 2012). Because of the likelihood of compensatory mechanisms, interpretations regarding the function of a gene product from genetically manipulated animal models should be made with this caveat in mind. In this regard, the availability of specific reagents targeting orexin receptors provides useful tools to specifically explore the function of orexin signaling within intact animals. Still, the interpretation of pharmacological perturbations depends not only on reagent specificity but also on the time of day at which they are applied, given oscillating endogenous orexin levels. Because OX-A and OX-B levels are highest during waking periods and reach a nadir during the inactive phase (Taheri et al., 2000; Zeitzer et al., 2003), the effectiveness of orexin receptor antagonists is expected to be greatest during periods of behavioral activity, a prediction confirmed experimentally (Brisbare-Roch et al., 2007; Li and Nattie, 2010; Winrow et al., 2011).

Further work combining genetic and pharmacological approaches will be particularly valuable. The most obvious experiments will examine the effectiveness of potential narcolepsy therapeutics in these knockout and Ox/ATX transgenic models of the disorder. These are anticipated to include both orexin agonists and other wake-promoting compounds such as those modulating histamine. Other studies have found orexin receptor knockouts to lack the response to orexin receptor antagonists, demonstrating their selectivity (Winrow et al., 2011, 2012). The use of small-molecule reagents specific for other neuronal targets in these animal models also has the potential to more clearly define the role of orexin in additional neurotransmitter pathways involved in modulating neurophysiology and behavior.

A. Central Modulation of Behavior and Physiology by Orexin Signaling

B. Orexin Influences on Peripheral Physiology

A growing body of evidence indicates that orexin signaling has the capacity to influence peripheral physiology. Many of these effects seem to be mediated by hypothalamic orexinergic neuron activity, modulating either autonomic nervous system tone or perhaps secondary to the peptides' effects on arousal and vigilance state. Clear demonstrations of the physiological role of orexin receptor activity in peripheral tissues are scarce, but the emergence of increasingly selective orexin receptor antagonists, however, should provide useful reagents to resolve this issue, because their concentrations are typically 10- to 100-fold higher in the periphery relative to the brain after administration. The potential roles for orexin signaling in the periphery, and whether these effects might be autonomic or secondary to its behavioral effects, remain to be determined.