Osthole, a Natural Plant Derivative Inhibits MRGPRX2 Induced Mast Cell Responses.

Mast cells are tissue-resident innate immune cells known for their prominent role in mediating allergic reactions. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a promiscuous G-protein coupled receptor (GPCR) expressed on mast cells that is activated by several ligands that share cationic and amphipathic properties. Interestingly, MRGPRX2 ligands include certain FDA-approved drugs, antimicrobial peptides, and neuropeptides. Consequently, this receptor has been implicated in causing mast cell-dependent pseudo-allergic reactions to these drugs and chronic inflammation associated with asthma, urticaria and rosacea in humans. In the current study we examined the role of osthole, a natural plant coumarin, in regulating mast cell responses when activated by the MRGPRX2 ligands, including compound 48/80, the neuropeptide substance P, and the cathelicidin LL-37. We demonstrate that osthole attenuates both the early (Ca2+ mobilization and degranulation) and delayed events (chemokine/cytokine production) of mast cell activation via MRGPRX2 in vitro. Osthole also inhibits MrgprB2- (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Molecular docking analysis suggests that osthole does not compete with the MRGPRX2 ligands for interaction with the receptor, but rather regulates MRGPRX2 activation via allosteric modifications. Furthermore, flow cytometry and confocal microscopy experiments reveal that osthole reduces both surface and intracellular expression levels of MRGPRX2 in mast cells. Collectively, our data demonstrate that osthole inhibits MRGPRX2/MrgprB2-induced mast cell responses and provides a rationale for the use of this natural compound as a safer alternative treatment for pseudo-allergic reactions in humans.

Cell membrane chromatography coupled online with LC-MS to screen anti-anaphylactoid components from Magnolia biondii Pamp. targeting on Mas-related G protein-coupled receptor X2.

Mas-related G protein-coupled receptor X2 was a mast cell-specific receptor mediating anaphylactoid reactions by activating mast cells degranulation, and it was also identified as a target for modulating mast cell-mediated anaphylactoid and inflammatory diseases. The anti-anaphylactoid drugs used clinically disturb the partial effect of partial mediators released by mast cells. The small molecule of Mas-related G protein-coupled receptor X2 specific antagonists may provide therapeutic action for the anaphylactoid and inflammatory diseases in the early stage. In this study, the Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography was coupled online with liquid chromatography and mass spectrometry and successfully used to screen anti-anaphylactoid components from Magnolia biondii Pamp. Fargesin and pinoresinol dimethyl ether were identified as potential anti-anaphylactoid components. Bioactivity of these two components were investigated by ß hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit ß hexosaminidase and histamine release in a concentration-dependent manner. This Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography coupled online with liquid chromatography and mass spectrometry system could be applied for screening potential anti-anaphylactoid components from natural medicinal herbs. This study also provided a powerful system for drug discovery in natural medicinal herbs.

Inhibitory function of Shikonin on MRGPRX2-mediated pseudo-allergic reactions induced by the secretagogue.

BACKGROUND: Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China. PURPOSE: To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects. METHODS/STUDY DESIGNS: The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2. RESULTS: Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein. CONCLUSION: Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca2+ mobilization.

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies.

Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label-free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS-evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq-dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies.

Neuropeptide S increases motor activity and thermogenesis in the rat through sympathetic activation.

The central role of neuropeptide S (NPS), identified as the endogenous ligand for GPR154, now named neuropeptide S receptor (NPSR), has not yet been fully clarified. We examined the central role of NPS for body temperature, energy expenditure, locomotor activity and adrenal hormone secretion in rats. Intracerebroventricular (icv) injection of NPS increased body temperature in a dose-dependent manner. Energy consumption and locomotor activity were also significantly increased by icv injection of NPS. In addition, icv injection of NPS increased the peripheral blood concentration of adrenalin and corticosterone. Pretreatment with the ß1- and ß2-adrenergic receptor blocker timolol inhibited the NPS-induced increase of body temperature. The expression of both NPS mRNA in the brainstem and NPSR mRNA in the hypothalamus showed a nocturnal rhythm with a peak occurring during the first half of the dark period. To examine whether the endogenous NPS is involved in regulation of body temperature, NPSR antagonist SHA68 was administered one hour after darkness. SHA68 attenuated the nocturnal rise of body temperature. These results suggest that NPS contributes to the regulation of the sympathetic nervous system.

The orexin-1 receptor antagonist SB-334867 decreases anxiety-like behavior and c-Fos expression in the hypothalamus of rats exposed to cat odor.

Increasing evidence suggests that the orexin system is involved in modulating anxiety, and we have recently shown that cat odor-induced anxiety in rats is attenuated by the orexin receptor antagonist SB-334867. In the current experiment, c-Fos expression was used to map changes in neuronal activation following SB-334867 administration in the cat odor anxiety model. Male Wistar rats were exposed to cat odor with or without SB-334867 pre-treatment (10 mg/kg, i.p.). A naïve control group not exposed to cat odor was also used. Following cat odor exposure, brains were processed for c-Fos expression. Vehicle-treated rats showed an increase in anxiety-like behaviors (increased hiding and decreased approach toward the cat odor), and increased c-Fos expression in the posteroventral medial amygdala (MePV), paraventricular hypothalamus (PVN) and dorsal premammillary nucleus (PMd). In rats pretreated with SB-334867, approach scores increased and c-Fos expression decreased in the PVN and PMd. These results provide both behavioral and neuroanatomical evidence for the attenuation of cat odor-induced anxiety in rats via the orexin system.

Perifornical hypothalamic orexin and serotonin modulate the counterregulatory response to hypoglycemic and glucoprivic stimuli.

Previous reports suggested an important role for serotonin (5-hydroxytryptamine [5-HT]) in enhancing the counterregulatory response (CRR) to hypoglycemia. To elucidate the sites of action mediating this effect, we initially found that insulin-induced hypoglycemia stimulates 5-HT release in widespread forebrain regions, including the perifornical hypothalamus (PFH; 30%), ventromedial hypothalamus (34%), paraventricular hypothalamus (34%), paraventricular thalamic nucleus (64%), and cerebral cortex (63%). Of these, we focused on the PFH because of its known modulation of diverse neurohumoral and behavioral responses. In awake, behaving rats, bilateral PFH glucoprivation with 5-thioglucose stimulated adrenal medullary epinephrine (Epi) release (3,153%) and feeding (400%), while clamping PFH glucose at postprandial brain levels blunted the Epi response to hypoglycemia by 30%. The PFH contained both glucose-excited (GE) and glucose-inhibited (GI) neurons; GE neurons were primarily excited, while GI neurons were equally excited or inhibited by 5-HT at hypoglycemic glucose levels in vitro. Also, 5-HT stimulated lactate production by cultured hypothalamic astrocytes. Depleting PFH 5-HT blunted the Epi (but not feeding) response to focal PFH (69%) and systemic glucoprivation (39%), while increasing PFH 5-HT levels amplified the Epi response to hypoglycemia by 32%. Finally, the orexin 1 receptor antagonist SB334867A attenuated both the Epi (65%) and feeding (47%) responses to focal PFH glucoprivation. Thus we have identified the PFH as a glucoregulatory region where both 5-HT and orexin modulate the CRR and feeding responses to glucoprivation.

Discovery, synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists.

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.

Neuropeptide Y Y1 receptor knockdown can modify glutathione peroxidase and c-AMP response element-binding protein in phenylpropanolamine-treated rats.

It has been reported that antioxidative enzymes, neuropeptide Y (NPY), and c-AMP response element-binding protein (CREB) are involved in regulating phenylpropanolamine (PPA)-mediated appetite suppression. Here, we investigated whether Y1 receptor (Y1R) might be involved in this regulation. Rats were daily treated with PPA for 4 days. Changes in the contents of NPY, Y1R, glutathione peroxidase (GP), and CREB were assessed and compared. Results showed that Y1R, GP, and CREB increased, with a maximal increase about 100, 200, and 150 %, respectively, on Day 2. By contrast, NPY decreased with a biggest reduction about 48 % on Day 2 and the pattern of expression during PPA treatment was opposite to those of Y1R, GP, and CREB. Central knockdown (using antisense) or inhibition (using antagonist) of Y1R expression modulated the anorectic response of PPA and the reciprocal regulation between NPY and GP (or CREB), revealing an essential role of Y1R in regulating NPY, GP, and CREB. These results suggest that Y1R participates in the reciprocal regulation of NPY, GP, and CREB in the hypothalamus during PPA treatment in conscious rats. The present results may aid the therapeutic research of PPA and related antiobesity drugs.

The identification of neuropeptide Y receptor subtype involved in phenylpropanolamine-induced increase in oxidative stress and appetite suppression.

Hypothalamic neuropeptide Y (NPY) and superoxide dismutase (SOD) have been reported to participate in the regulation of appetite-suppressing effect of phenylpropanolamine (PPA), a sympathomimetic agent. This study explored whether Y1 receptor (Y1R) and/or Y5 receptor (Y5R) was involved in this regulation. Wistar rats were treated with PPA for 24 h. Changes in food intake and hypothalamic NPY, Y1R, Y5R, and SOD contents were assessed and compared. Results showed that food intake and NPY contents were decreased following PPA treatment, while Y1R and SOD contents were increased and Y5R contents remained unchanged. Moreover, although Y1R or Y5R knockdown by themselves could modify the food intake, Y1R but not Y5R knockdown could modify PPA-induced anorexia as well as NPY and SOD contents. In addition, selective inhibition of Y1R but not Y5R could modulate PPA-induced anorexia. It is suggested that Y1R but not Y5R participates in the anorectic response of PPA via the modulation of NPY and SOD. Results provide molecular mechanism of NPY-mediated PPA anorexia and may aid the understanding of the toxicology of PPA.

Blockade of orexin receptors with Almorexant reduces cardiorespiratory responses evoked from the hypothalamus but not baro- or chemoreceptor reflex responses.

Orexin neurons form a restricted group in the dorsal hypothalamus. The group is centered on the perifornical area within the classic hypothalamic defense area, an area which when activated produces marked cardiovascular and respiratory effects. Central administration of orexin can produce cardiorespiratory effects, but the extent to which orexin contributes to such responses evoked from the perifornical hypothalamus is not clear. To determine this, we used the dual orexin receptor antagonist Almorexant to challenge the cardiorespiratory effects evoked by disinhibition of the perifornical hypothalamus. Bicuculline (10 and 20 pmol) was microinjected in the perifornical area before and after administration of Almorexant (15 mg/kg iv) or vehicle in urethane-anesthetized rats. Almorexant significantly reduced the pressor, tachycardic, renal sympathoexcitatory, and tachypneic responses to bicuculline (10 pmol, by 55%, 53%, 28%, 77%; 20 pmol, by 54%, 27%, 51%, 72%, respectively). Reductions of similar magnitude were observed with bicuculline microinjections centered on more caudal sites just peripheral to the orexin neuron group, which would likely have activated fewer orexin neurons. In contrast, Almorexant had no effect on the cardiorespiratory response of the chemoreflex (sodium cyanide injection) or the sympathetic component of the baroreflex. Thus orexin makes a major contribution to the cardiorespiratory response evoked from the perifornical area even though orexin neurons represent only a fraction of the output of this area. Orexin neurons may also mediate cardiorespiratory responses from non-orexin neurons in the caudal hypothalamus. However, under resting conditions, blockade of orexin receptors does not affect the chemo- and baroreflexes.

Neuropeptides controlling energy balance: orexins and neuromedins.

In this chapter, we review the feeding and energy expenditure effects of orexin (also known as hypocretin) and neuromedin. Orexins are multifunctional neuropeptides that affect energy balance by participating in regulation of appetite, arousal, and spontaneous physical activity. Central orexin signaling for all functions originates in the lateral hypothalamus-perifornical area and is likely functionally differentiated based on site of action and on interacting neural influences. The effect of orexin on feeding is likely related to arousal in some ways but is nonetheless a separate neural process that depends on interactions with other feeding-related neuropeptides. In a pattern distinct from other neuropeptides, orexin stimulates both feeding and energy expenditure. Orexin increases in energy expenditure are mainly by increasing spontaneous physical activity, and this energy expenditure effect is more potent than the effect on feeding. Global orexin manipulations, such as in transgenic models, produce energy balance changes consistent with a dominant energy expenditure effect of orexin. Neuromedins are gut-brain peptides that reduce appetite. There are gut sources of neuromedin, but likely the key appetite-related neuromedin-producing neurons are in the hypothalamus and parallel other key anorectic neuropeptide expression in the arcuate to paraventricular hypothalamic projection. As with other hypothalamic feeding-related peptides, hindbrain sites are likely also important sources and targets of neuromedin anorectic action. Neuromedin increases physical activity in addition to reducing appetite, thus producing a consistent negative energy balance effect. Together with the other various neuropeptides, neurotransmitters, neuromodulators, and neurohormones, neuromedin and orexin act in the appetite network to produce changes in food intake and energy expenditure, which ultimately influences the regulation of body weight.

Stimulatory effect of intracerebroventricular administration of orexin A on food intake in the zebrafish, Danio rerio.

Orexin is a potent orexigenic neuropeptide implicated in feeding regulation of mammals. However, except for the case of goldfish, the involvement of orexin in the feeding behavior of teleost fish has not well been studied. Therefore, we investigated the role of orexin on food intake using a zebrafish (Danio rerio) model. We examined the effect of feeding status on orexin-like immunoreactivity and the expression level of orexin transcript in the brain. The number of neuronal cells showing orexin-like immunoreactivity in the hypothalamic region, including the posterior tuberal nucleus, was significantly increased in fish fasted for 7days. Orexin precursor mRNA levels in the brain obtained from fish fasted for 7 days were higher than those in fish that had been fed normally. We then investigated the effect of intracerebroventricular (ICV) administration of orexin A on food intake. Cumulative food intake was significantly increased by ICV administration of orexin A (at 0.3 and 3 pmol/g body weight, BW) during a 60-min observation period after treatment. The orexin A-induced orexigenic action (at 0.3 pmol/g BW) was blocked by treatment with an orexin receptor antagonist, SB334867, at 10 pmol/g BW. These results indicate that orexin A acts as feeding regulator in the zebrafish.

Orexin-1 receptor signalling within the ventral tegmental area, but not the paraventricular thalamus, is critical to regulating cue-induced reinstatement of cocaine-seeking.

Orexinergic signalling is critical to drug relapse-like behaviour; however, the CNS sites(s) of action remain unknown. Two candidate brain regions are the paraventricular thalamus (PVT) and ventral tegmental area (VTA). We assessed the effect of intra-PVT or -VTA administration of the orexin-1 receptor (OrxR1) antagonist SB-334867 on discriminative cue-induced cocaine-seeking. Animals received either PVT- or VTA-directed SB-334867 (0, 3 or 6 µg; 0, 1 or 3 µg, respectively) prior to reinstatement testing elicited by presenting cocaine-paired stimuli (S+). The effect of VTA-directed injections of SB-334867 (0 or 3 µg) on locomotor activity was also assessed. Intra-VTA, but not -PVT, SB-334867 dose-dependently attenuated S+-induced reinstatement (3 µg dose, p<0.01). Intra-VTA SB-334867 had no effect on locomotor activity. We conclude that OrxR1 signalling within the VTA, but not the PVT, mediates cue-induced cocaine-seeking behaviour. We hypothesize that blockade of VTA OrxR1 signalling may reduce nucleus accumbens dopamine in response to drug cue presentation.

Orexin receptors: pharmacology and therapeutic opportunities.

Orexin-A and -B (also known as hypocretin-1 and -2) are neuropeptides produced in the lateral hypothalamus that promote many aspects of arousal through the OX1 and OX2 receptors. In fact, they are necessary for normal wakefulness, as loss of the orexin-producing neurons causes narcolepsy in humans and rodents. This has generated considerable interest in developing small-molecule orexin receptor antagonists as a novel therapy for the treatment of insomnia. Orexin antagonists, especially those that block OX2 or both OX1 and OX2 receptors, clearly promote sleep in animals, and clinical results are encouraging: Several compounds are in Phase III trials. As the orexin system mainly promotes arousal, these new compounds will likely improve insomnia without incurring many of the side effects encountered with current medications.

Nicotine self-administration in the rat: effects of hypocretin antagonists and changes in hypocretin mRNA.

RATIONALE: The hypocretin (hcrt) system has been implicated in addiction-relevant effects of several drugs, but its role in nicotine dependence has been little studied. OBJECTIVES: These experiments examined the role of the hcrt system in nicotine reinforcement. METHODS: Rats were trained for nicotine self-administration (NSA) on fixed-ratio schedules. The effects of acute, presession treatments with the hcrtR1 antagonist SB334867 and the hcrtR1/2 antagonist almorexant were examined on NSA maintained on a fixed-ratio (FR) 5 schedule. Gene expression for the hcrt system (mRNA for hcrt, hcrtR1, and hcrtR2) was measured in animals following NSA on a FR 1 schedule for a 19-day period. RESULTS: The hcrtR1 antagonist SB334867 and the hcrtR1/2 antagonist almorexant both reduced NSA dose-dependently (significantly at doses of 30 and 300 mg/kg, respectively); SB334867 did not affect food-maintained responding whereas almorexant (at the 300 mg/kg) did. Tissue from animals collected 5 h after self-administration showed an increase in hcrtR1 mRNA in the arcuate nucleus compared to control subjects. In tissue collected immediately after a similar 19-day self-administration period, mRNA for hcrtR1 was decreased in the rostral lateral hypothalamus compared to controls. CONCLUSIONS: These data confirm a previous report (Hollander et al., Proc Natl Acad Sci U S A 105:19480-19485, 2008) that the hypocretin receptor hcrtR1 is activated in nicotine reinforcement and in addition show that both the arcuate nucleus and lateral hypothalamus are sites at which hcrt receptor mechanisms may influence reinforcement. Different patterns of mRNA expression at different times after NSA suggest that changes in the hcrt system may be labile with time.

Orexin receptor antagonists: a review of promising compounds patented since 2006.

IMPORTANCE OF THE FIELD: The orexin neuropeptide system plays a central role in maintaining arousal and wakefulness. It has been demonstrated that small molecule antagonists to the orexin receptors promote sleep in preclinical species and in patients with insomnia. AREAS COVERED IN THIS REVIEW: This review provides a summary of published patent applications claiming novel orexin antagonists from 2006 to mid-2009, covering both selective and dual orexin receptor antagonists. WHAT THE READER WILL GAIN: Readers will gain an overview of orexin biology focusing on genetic and pharmacological validation of this target for treating sleep disorders. Additionally, this review discusses the importance of receptor subtype selectivity and the potential role of subtype selective and dual orexin antagonists in treating psychiatric illnesses beyond insomnia. This review identifies companies that are significant contributors to the patent literature claiming novel orexin receptor antagonists. TAKE HOME MESSAGE: The study of the orexin system has emerged as one of the key new fields of investigation in neuroscience. The demonstration of clinical proof-of-concept for the treatment of primary insomnia by Actelion in early 2007 has spurred significant interest in this field and competition has markedly increased since 2006.

Almorexant, a dual orexin receptor antagonist for the treatment of insomnia.

Almorexant (ACT-078573) is an orally active dual orexin receptor antagonist that is being developed by Actelion Ltd, in collaboration with GlaxoSmithKline plc, for the treatment of primary insomnia. Almorexant is a first-in-class compound that targets the orexin system, which plays a key role in wake promotion and stabilization, in addition to having other regulatory functions. Decreasing orexin activity was hypothesized to have a sleep-promoting effect. Preclinical studies and phase I clinical trials have demonstrated that almorexant decreases alertness and increases sleep in healthy rats, dogs and humans when administered during the active phase of the circadian cycle, at peak endogenous orexin tone. No significant toxicological or safety concerns have been identified in studies in animals and humans, including no evidence of cataplexy, a sudden postural muscle tone weakening that is triggered by emotional stimuli and is considered unique to narcolepsy. The reported efficacy and safety data for almorexant support the continued development of the compound. At the time of publication, phase III clinical trials were underway, but no results had been reported; Actelion and GlaxoSmithKline were also investigating almorexant for other orexin-related neurological disorders. The use of an orexin receptor antagonist for the treatment of sleep disorders appears to be an approach that may provide unique benefits.

The orexin-1 receptor antagonist SB-334867 reduces amphetamine-evoked dopamine outflow in the shell of the nucleus accumbens and decreases the expression of amphetamine sensitization.

Orexin-expressing neurons are present in hypothalamic nuclei and send projections toward mesolimbic regions such as the nucleus accumbens (NAc), a key brain region implicated in the processing of the motivational significance of reinforcers. Recent evidence found that activation of the orexin system can lead to a state of hyperarousal that may facilitate drug craving or contribute to vulnerability to drug relapse. This study aimed at assessing the effects of the orexin-1 receptor antagonist SB-334867 [1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride] on amphetamine-induced dopamine (DA) release in the shell subregion of the NAc by means of in vivo microdialysis in freely moving rats. Since behavioral sensitization is thought to play a role in the maintenance of compulsive drug use, we also tested the effect of SB-334867 on the expression of sensitization to the locomotor activating effects of amphetamine. Acute administration of SB-334867 (30 mg/kg SC) significantly reduced the acute effects of amphetamine (1 mg/kg IP) on extracellular DA levels in the NAc shell. The expression of amphetamine sensitization was also significantly reduced by acute SB-334867 treatment. Altogether our findings show that selective orexin-1 antagonism both reduces the acute effects of amphetamine on DA outflow in the NAc shell and decreases the expression of locomotor sensitization to the repeated, intermittent administration of amphetamine.