OX₁ Receptor

Orexin A (human, rat, mouse) exhibits a high affinity for OX1R, with 38 nM IC50 and 34 nM EC50 values in the the [Ca^2﹢]i transient assay[1].

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Characterization of Orexin Receptors [1]
Figure 2c shows the deduced amino acid sequences of the original HFGAN72 receptor, which we now call OX1 receptor (OX1R). Among various classes of GPCR, OX1R is structurally most similar to certain neuropeptide receptors, most notably to the Y2 neuropeptide Y (NPY) receptor (26% identity), followed by the TRH receptor, cholesystokinin type-A receptor, and NK2 neurokinin receptor (25%, 23%, and 20% identity, respectively). This is consistent with our hypothesis that OX1R is the receptor for orexins, another class of small regulatory peptides. In order to characterize further their pharmacological interactions, we performed in vitro functional assays using transfected cell lines expressing the receptor. Mock transfected CHO cells did not exhibit detectable levels of specific binding of radio-iodinated [125I-Tyr17]Orexin A. Stable transfection of CHO cells with an expression vector containing the human OX1R cDNA (CHO/OX1R) conferred the ability to bind [125I]orexin-A (Figure 3A). The radioligand binding was inhibited by nanomolar concentrations of unlabeled synthetic orexin-A in a dose-dependent manner, but not by any of several unrelated peptides tested, including human NPY and endothelin-1 at up to 10 μM (data not shown). The concentration of unlabeled Orexin A required to displace 50% of specific radioligand binding (IC50) was 20 nM as calculated by the LIGAND program (Munson and Rodbard 1980). Orexin-A also induced a transient increase in [Ca2+]i in CHO/OX1R cells in a dose-dependent manner (Figure 3C), but failed to induce detectable [Ca2+]i transients in mock transfected CHO cells. We feel that the calcium mobilization is likely caused by the activation of the Gq class of heterotrimeric G proteins (Hepler et al. 1994). The calculated concentration of Orexin A required to induce half-maximum response (EC50) was 30 nM. We obtained similar results in radioligand binding and [Ca2+]i transient assays performed with stably transfected HEK293 cells (data not shown). These findings confirm that Orexin A is indeed a specific, high-affinity agonist for OX1R.
To characterize functionally the OX2R further, we repeated competitive radioligand binding assays and [Ca2+]i transient dose-response studies using stably transfected CHO cells expressing the human OX2R cDNA. The results demonstrated that OX2R is indeed a high-affinity receptor for human orexin-B, with an IC50 of 36 nM in the binding assay and an EC50 of 60 nM in the [Ca2+]i transient assay (Figure 3B and Figure 3D). Orexin-A also had high affinity for this receptor, with 38 nM IC50 and 34 nM EC50 values, which are similar to the values for orexin-B. Thus, we conclude that OX2R is a nonselective receptor for both orexin-A and -B, while OX1R is selective for Orexin A.
In radiation hybrid mapping, the MIT markers showing tightest linkage to the human OX1R and OX2R genes are the STS markers D1S195 and D1S443, and WI-5448 and CHLC.GATA74F07, respectively. The inferred cytogenetic locations between these markers are 1p33 for OX1R, and 6cen (p11-q11) for OX2R (accurate cytogenetic locations are often difficult to interpret from radiation hybrid maps in which the gene lies near the centromere).

Orexin A (human, rat, and mouse) (3–30 mg/kg; i.v.; 5-min pre-test) dramatically lengthens the time to response at 10 and 30 mg/kg i.v. when administered to vehicle-treated mice, increasing it from 24.8±2.0 s to 35.0±3.7 s and 45.7±4.5 s, respectively[2]. Orexin A (human, rat, mouse) (3, 10 and 30 mg/kg; i.v.) was administered right before phenylp-quinone (PPQ), and in mice treated with vehicle, it increased the latency to the first PPQ-induced constriction from 357.4±35.2 s to 500.3±31.2 s at 10 mg/kg and 594.5±5.5 s at 30 mg/kg[2].

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Orexin-A and orexin-B (also known as hypocretin-1 and hypocretin-2) are hypothalamic peptides and regulate feeding behaviour, energy metabolism and the sleep-wake cycle. Orexin-A binds equally to both orexin-1 and orexin-2 receptors, while orexin-B has a preferential affinity for orexin-2 receptors.

Orexins are also known to be concentrated in superficial laminae of the spinal dorsal horn, and Orexin-A and orexin-1 receptors are found in the dorsal root ganglion cells.

In the present study, the authors examined the effect of intrathecal injection of either orexin-A or orexin-B in the rat formalin test (a model of inflammatory pain) and in the rat hot plate test. The paw formalin injection induces biphasic flinching (phase 1: 0–6 min; phase 2: 10–60 min) of the injected paw.

Intrathecal injection of Orexin-A, but not orexin-B, decreased the sum of flinches in phases 1 and 2 in the formalin test and increased the hot plate latency. These effects of orexin-A were completely antagonized by pre-treatment with SB-334867, a selective orexin-1 receptor antagonist. Intrathecal injection of SB-334867 alone had no effect in the formalin test or in the hot plate test.

Intrathecal injection of Orexin-A suppressed the expression of Fos-like immunoreactivity (Fos-LI), induced by paw formalin injection, in laminae I-II of L4–5 of the spinal cord.

These data suggest that the spinal orexin-1 receptor is involved in the nociceptive transmission and that the activation of the spinal orexin-1 receptor produces analgesic effects in the rat formalin test and in the rat hot plate test [3].

Radioligand Binding Assay [1]
Synthetic human Orexin A was 125I-labeled at Tyr17 by Chloramine-T oxidation in the presence of Na125I (2,000 Ci/mmol), and monoiodinated peptide was purified by C18 reverse-phase HPLC as described (Takigawa et al. 1995). Stable transfectant CHO cell lines expressing human OX1R or OX2R were each seeded onto 12-well plates at a density of 3 × 105 cells per well. After an overnight culture, medium was discarded and cells were incubated at 20°C for 90 min with binding buffer (HEPES-buffered saline/0.5% bovine serum albumin) containing 10−10 M [125I]Orexin A plus designated concentrations of unlabeled competitors. Cells were then washed three times with ice-cold phosphate-buffered saline, lysed in 0.1 N NaOH, and cell-bound radioactivity was determined by a γ-counter.

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In Situ Hybridization [1]
A 0.29 kb segment of rat cDNA encoding Gln33-Ser128 of prepro-orexin was generated as described above and subcloned into pBluescript II SK(+) vector. Sense and anti-sense riboprobes were generated with T7 and T3 RNA polymerases, respectively, using the Maxiscript kit in the presence of 35S-CTP. In situ hybridization to adult rat brain sections was performed as described (Benjamin et al. 1997).

Intracellular Calcium Transient Assay [1]
Stable transfectant HEK293 cells expressing orphan GPCRs were loaded with Fura-2 AM in suspension, and [Ca2+]i transients evoked by agonists were monitored by a Model CAF-110 Intracellular Ion Analyser in 500 μl cuvette as previously described. For in vitro pharmacological characterization (Figure 3), the same procedures were performed using stably transfected CHO cells expressing human OX1R or OX2R.

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Immunohistochemistry [1]
Anti-orexin-A antiserum was raised in rabbits by immunization with synthetic Orexin A[14–33], CRLYELLHGAGNHAAGILTL-amide, conjugated to keyhole limpet hemocyanin using 3-maleimidobensoic acid N-hydroxysuccimide ester. Antiserum was affinity purified on an Orexin A-conjugated Sepharose CL-4B column and used for immunohistochemistry. Male Sprague-Dawley rats (∼300 g) were anesthetized and perfused via the left cardiac ventricle with phosphate-buffered saline (PBS). Brain was taken out, directly embedded in OCT compound, and frozen in liquid nitrogen. Cryostat sections (15 μm) were cut and mounted on silane-coated glass sides. The sections were post-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 1 hr and washed three times in PBS. The sections were incubated with 1% bovine serum albumin in PBS for 1 hr and then incubated with affinity-purified antiserum in the same solution for 1 hr at room temperature. After washing three times in PBS, the sections were incubated with FITC-conjugated goat anti-rabbit IgG antibody for 1 hr at room temperature. Slides were then washed three times in PBS and examined under fluorescence microscope.

Female mice (mouse carrageenan-induced thermal hyperalgesia test) [2]
3, 10 and 30 mg/kg
i.v.; 5 min pre-test

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Mouse abdominal constriction test [2]
Male mice were randomly assigned into groups of ten and placed in Perspex cages for approximately 10 min for habituation. They were dosed with vehicle or Orexin A (1, 3, 10 and 30 mg/kg i.v.) immediately before administration of phenyl-p-quinone (PPQ, 0.25 mg/kg i.p.). The latency to first constriction was measured, after which they were immediately killed by cervical dislocation. A cut-off time of 600 s was used and any mice not responding in this time were killed once this time had elapsed.

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Mouse hotplate test [2]
Male mice were randomly assigned to five groups of ten and given vehicle or orexin (1, 3, 10 or 30 mg/kg i.v.). Five minutes later they were placed on the hotplate maintained at 50°C and the latency to response was measured (licking or fanning of fore- or hindpaws). At this point mice were immediately removed from the thermal stimulus and killed by cervical dislocation. All dosing and testing were done blind. A cut-off time of 60 s was used to prevent tissue damage and any animal not responding within this time was removed from the thermal stimulus and killed as above. In a separate experiment, morphine (1.25, 2.5 or 5 mg/kg s.c.) was given 30 min pre-test and the experiment was repeated as above. In another experiment, Orexin A (30 mg/kg, i.v.) was dosed 15, 30 and 60 min pre-test and the latency to response was measured as above. In a separate experiment, a similar protocol was followed using a hotplate temperature of 55°C and a cut-off time of 40 s. The Orexin A receptor antagonist SB-284422, the 5-HT2B/2C receptor antagonist SB-228357 (1, 3, 10 and 30 mg/kg i.p.) and morphine (2.5, 5 and 10 mg/kg s.c.) were also given 30 min pre-test and latencies were measured as above.

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Mouse carrageenan-induced thermal hyperalgesia [2]
Female mice were placed in the test apparatus for 10 min once a day for 3 days for habituation. Withdrawal latencies of the left and right hindfeet to a focused thermal stimulus (Hargreaves et al., 1988) were measured on a further 2 days and the last measurement was taken as a baseline. The intensity of the thermal stimulus was set to give a baseline withdrawal latency of approximately 9 s. The range for the experimental groups reported here was 10.3–10.5 s for test 1 and 8.9–9.3 s for test 2 for the orexin experiments, and 8.4–9.2 s for the morphine experiments. Carrageenan (2% w/v in saline, 0.25 ml) was injected into the plantar surface of the left foot and withdrawal latencies were measured 240 min post-carrageenan. Orexin A was given at 10 and 30 mg/kg i.v., and in a separate experiment at 3, 10 and 30 mg/kg, 5 min pre-plantar test. In another experiment, morphine (2.5, 5 and 10 mg/kg s.c.) was tested 30 min pre-plantar test.
The orexin-A receptor antagonist SB-284422 and the 5-HT2B/2C receptor antagonist SB-228357 (1, 3, 10 and 30 mg/kg i.p.) were also given 30 min pre-test and latencies were measured as above.

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Rat ICV studies: hotplate test [2]
Sprague–Dawley rats (Charles River, 250–300 g at the time of surgery) were implanted under anaesthesia (Domitor® (medetomidine HCl, 0.4 mg/kg s.c), Sublimaze® (fentanyl, 0.45 mg/kg i.p.) with an indwelling cannula directed towards either the left or right lateral ventricle (co-ordinates: ±1.6 mm from midline, 0.8 mm caudal from bregma, −4.1 mm from skull surface, incisor bar at −3.2 mm below zero) (Paxinos and Watson, 1986). Anaesthesia was reversed by Antisedan® (atipamezole HCl, 2.5 mg/kg s.c.) and post-operative analgesia was provided by Nubain® (nalbuphine HCl, 2 mg/kg s.c., Du Pont Pharmaceuticals, Letchworth Garden City, UK). All rats were singly housed after surgery and for the duration of the study to avoid damage to the guide and dummy cannulae. Rats were handled frequently during the studies to prevent the development of hyperactivity and aggression. Following 7 days recovery from surgery, during which rats were fed soaked food pellets, weighed and health-checked daily by a veterinary technician, correct cannula placement was verified by an intense drinking response to angiotensin II (100 ng ICV) (Simpson et al., 1978). At least 7 days later Orexin A (3, 10 and 30 μg/rat) was given ICV in a volume of 5 μl over 60 s. The injection needle (extending 1 mm from the end of the guide cannula) was left in place for a further 90 s to allow complete diffusion of the drug. Five minutes later rats were placed on the hotplate maintained at 50°C and the latency to response was measured (licking or fanning of fore- or hindpaws). At this point rats were immediately removed from the thermal stimulus and killed by cervical dislocation. All dosing and testing were done blind. A cut-off time of 60 s was used to prevent tissue damage and any animal not responding within this time was removed from the thermal stimulus and killed as above.

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Orexin A pharmacokinetics and brain penetration studies in the rat and mouse [2]
The pharmacokinetics, oral bioavailability and steady-state brain penetration of Orexin A were investigated in the conscious rat. The steady-state brain penetration of orexin-A was investigated in the conscious mouse. Chronic cannulation of the jugular vein for blood sampling (both species) and the vena cava for drug administration (via cannulation of the femoral vein in the rat and via laparotomy in the mouse) was performed using methods described by Griffiths et al. (1996).
The pharmacokinetic and oral bioavailability study in the rat (n=3) was conducted as a crossover design on two study days 3 days apart. On study day 1, Orexin A (6 mg/ml dissolved in normal saline) was infused at a target dose of 30 mg/kg administered over 30 min. On study day 2, orexin-A (3 mg/ml dissolved in purified water) was administered orally at a target dose of 30 mg/kg. Serial blood samples (50 μl) were collected over 10 h on both study days, diluted with an equal volume of water, mixed and then extracted immediately (see below).
The brain penetration of Orexin A was evaluated under steady-state conditions after intravenous infusion of orexin-A to rats and mice (n=3, both species). Orexin A (2 mg/ml dissolved in normal saline) was infused at a target dose level of 10 mg/kg per h for 2 h. Serial blood samples (50 μl rats, 25 μl mice, prepared as described above) were obtained during the last hour of the infusion to establish steady-state blood concentrations and at 2 h the animals were killed, exsanguinated and had their brains removed. Whole brains were diluted with two volumes of water, homogenized and then extracted immediately (see below).

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Intracerebroventricular Administration of Orexins [1]
Male Wistar rats (180–200 g on arrival) were housed under controlled lighting (12 hr light-dark cycle) and temperature (22°C) conditions. Food (standard chow pellets) and water were available ad libitum. Rats (200–220 g) were anesthetized with pentobarbital (50 mg/kg i.p.), positioned in a Koph Model 900 stereotaxic frame, and implanted with a guide cannula into the left lateral ventricle under sterile conditions using a MEDIBIO Optical Brain Tracer (Ikeda and Matsushita 1980). The coordinates used to map the correct positioning of the implants were: 6.1 mm anterior to the lambda, 1.5 mm lateral from the midline, and ∼3.4 mm (guided by MEDIBIO) ventral to the skull surface, with the incisor bar set 3.3 mm below the interauricular line. Rats were then housed singly under the same conditions as above for a recovery period of at least 7 days, and body weights were monitored daily for the duration of the study. After recovery from surgery, rats were transferred to grid-floor cages and fed with powdered chow so that food intake measurements could be made. The rats were acclimated to the new environment at least for 1 day. The position of the cannula was verified by central administration of human NPY (3 nmol in sterile water); for a positive test, at least 8 g of food was eaten over a 4 hr period postinjection. Only positively testing animals (n = 8–10) were used. The studies were conducted according to a multidose, crossover design, with the order of dosing determined using the Latin square principle, leaving at least one rest day between administrations. All doses were delivered in a volume of 5 μl in sterile water over 30 s, and the injector remained in position for a further 30 s to ensure complete dispersal of the peptide. All intracerebroventricular administrations began at 2 h into light cycle, and food intake was measured at 1, 2, and 4 h intervals. All peptides were dissolved in sterile water, initially at 6 mM, and diluted in water as needed. Water alone was used for the vehicle control.

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The intrathecally administered drugs were delivered in a total volume of 10 μl followed by 10 μl of saline to flush the catheter.The intraperitoneally administered drugs were delivered in a total volume of 1 ml. The agents used in this study were orexin-A (molecular weight=3561, Peptide Institute), orexin-B (molecular weight=2936), SB-334867 (1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride, molecular weight=356), a selective orexin-1 receptor antagonist (Smart et al., 2001; Porter et al., 2001) and naloxone hydrochloride (molecular weight=364).[3]

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Experimental protocol [3]
Formalin test [3]
For the dose-response study, agents were administered intrathecally 10 min before the formalin injection. To obtain control data, the vehicle (saline) was injected intrathecally. In a separate group of rats, to verify whether the effects of intrathecally administered orexin-A on the formalin test were mediated by the action of the drugs in the spinal cord, a most-effective dose (0.3 nmol) of orexin-A was administered intraperitoneally 10 min before the foramlin injection and the effect on the formalin test was examined (intraperitoneal injection study). To obtain control data, the vehicle (saline) was injected intraperitoneally. To verify that the effect of intrathecally administered orexin-A on the formalin test was produced by an interaction between orexin-A and spinal orexin-1 receptor, 30 nmol of SB-334867 were administered intrathecally 5 min before the intrathecal injection of 0.3 nmol of orexin-A and formalin was injected subcutaneously into the right hindpaw 10 min after the orexin-A administration. The effect of intrathecal administration of 30 nmol of SB-334867 on the formalin test was also examined. To verify that the effect of intrathecally administered orexin-A is due to the modulation of release of endogenous opioids at the spinal level, 28 nmol of naloxone were administered intrathecally 5 min before the intrathecal injection of 0.3 nmol of orexin-A and formalin was injected subcutaneously 10 min after the orexin-A injection.

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Hot plate test [3]
Two baseline measurements were made before the drug injection. For the dose-response study, agents were administered intrathecally and the hot plate latency was measured at 5, 15, 30 and 60 min after the drug injection. To obtain control data, the vehicle (saline) was injected intrathecally. In a separate group of rats, to verify whether the effects of intrathecally administered orexin-A on the hot plate test were mediated by the action of the drugs in the spinal cord, a most-effective dose (0.1 nmol) of orexin-A was administered intraperitoneally and the effect on the hot plate test was examined (intraperitoneal injection study). To obtain control data, vehicle (saline) was injected intraperitoneally. To verify that the effect of intrathecally administered orexin-A on the hot plate test was produced by an interaction between orexin-A and spinal orexin-1 receptor, 10 nmol of SB-334867 was administered intrathecally 5 min before the intrathecal injection of 0.1 nmol of orexin-A and the effect of 0.1 nmol of orexin-A on the hot plate test was examined. The effect of intrathecal injection of 10 nmol of SB-334867 on the hot plate test was also examined.

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Immunohistochemical study [3]
A most-effective dose (0.3 nmol) of orexin-A or saline was administered intrathecally 10 min before the formalin injection, and the expression of Fos-LI was examined 2 h after the formalin injection.

Short half-life and lack of brain penetration of orexin-A [2]
Non-compartmental pharmacokinetic parameters for orexin-A following intravenous and oral administration to the rat are shown in Table 2. Following intravenous administration of orexin-A to rats to a target dose of 30 mg/kg, the compound was slowly cleared (CLb, 17 ml/min per kg) and had a small volume of distribution (<0.2 l/kg, suggesting minimal tissue distribution) resulting in a short terminal half-life of less than 0.2 h. Following oral administration of orexin-A, all blood concentrations were non-detectable and therefore oral bioavailability in the rat was effectively zero.
The relatively short duration of action (<30 min) is mirrored by the pharmacokinetic profile, with a terminal half-life of 12 min for orexin-A in rat. Moreover, orexin-A has similar efficacy to morphine (%MPE=23.9–82.1%), with a %MPE of between 29.4 and 136.2%, depending on the type of test, dose used and the species involved. Importantly, its analgesic efficacy does not appear to involve activation of endogenous opiate systems as shown by the lack of effect of naloxone on the orexin-A-mediated responses.

[1]. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell. 1998 Feb 20;92(4):573-85.

[2]. Orexin-A, an hypothalamic peptide with analgesic properties. Pain. 2001 May;92(1-2):81-90.

[3]. Analgesic effect of intrathecally administered orexin-A in the rat formalin test and in the rat hot plate test. Br J Pharmacol . 2002 Sep;137(2):170-6.

Neuropeptide hormones that play a role in regulating a variety of behavioral and physiological processes in response to motivational stimuli.

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The hypothalamus plays a central role in the integrated control of feeding and energy homeostasis. We have identified two novel neuropeptides, both derived from the same precursor by proteolytic processing, that bind and activate two closely related (previously) orphan G protein-coupled receptors. These peptides, termed orexin-A and -B, have no significant structural similarities to known families of regulatory peptides. prepro-orexin mRNA and immunoreactive orexin-A are localized in neurons within and around the lateral and posterior hypothalamus in the adult rat brain. When administered centrally to rats, these peptides stimulate food consumption. prepro-orexin mRNA level is up-regulated upon fasting, suggesting a physiological role for the peptides as mediators in the central feedback mechanism that regulates feeding behavior.[1]

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The hypothalamic peptide orexin-A and the orexin-1 receptor are localized in areas of the brain and spinal cord associated with nociceptive processing. In the present study, localization was confirmed in the spinal cord and demonstrated in the dorsal root ganglion for both orexin-A and the orexin-1 receptor. The link with nociception was extended when orexin-A was shown to be analgesic when given i.v. but not s.c. in mouse and rat models of nociception and hyperalgesia. The efficacy of orexin-A was similar to that of morphine in the 50 degrees C hotplate test and the carrageenan-induced thermal hyperalgesia test. However, involvement of the opiate system in these effects was ruled out as they were blocked by the orexin-1 receptor antagonist SB-334867 but not naloxone. Orexin-1 receptor antagonists had no effect in acute nociceptive tests but under particular inflammatory conditions were pro-hyperalgesic, suggesting a tonic inhibitory orexin drive in these circumstances. These data demonstrate that the orexinergic system has a potential role in the modulation of nociceptive transmission.[2]

C152H247N47O44S4

3565.13610768318

3559.713843

205640-90-0

Orexin A (human, rat, mouse) (TFA); Orexin A (human, rat, mouse) (acetate)

56842143

White to off-white solid powder

-10.1

49

53

94

247

8430

33

N1(CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1C=CC(=CC=1)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(N)=O)C(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCC(=O)N1

OFNHNCAUVYOTPM-IIIOAANCSA-N

InChI=1S/C152H243N47O44S4/c1-20-76(14)118(146(239)188-96(51-74(10)11)138(231)197-119(80(18)201)147(240)180-92(121(156)214)47-70(2)3)195-115(209)62-166-123(216)78(16)171-124(217)79(17)172-131(224)99(55-84-59-162-69-169-84)186-136(229)100(56-111(155)205)174-114(208)61-165-122(215)77(15)170-113(207)60-167-125(218)98(54-83-58-161-68-168-83)185-134(227)95(50-73(8)9)183-132(225)93(48-71(4)5)182-129(222)90(38-41-116(210)211)179-135(228)97(53-82-32-34-85(203)35-33-82)184-133(226)94(49-72(6)7)181-127(220)88(29-24-44-164-152(159)160)177-140(233)104-64-244-245-65-105-141(234)176-87(28-23-43-163-151(157)158)126(219)178-89(36-39-110(154)204)128(221)175-86(27-21-22-42-153)130(223)196-120(81(19)202)148(241)194-107(142(235)190-103(63-200)139(232)192-104)67-247-246-66-106(143(236)193-105)191-137(230)101(57-117(212)213)187-144(237)108-30-26-46-199(108)150(243)102(52-75(12)13)189-145(238)109-31-25-45-198(109)149(242)91-37-40-112(206)173-91/h32-35,58-59,68-81,86-109,118-120,200-203H,20-31,36-57,60-67,153H2,1-19H3,(H2,154,204)(H2,155,205)(H2,156,214)(H,161,168)(H,162,169)(H,165,215)(H,166,216)(H,167,218)(H,170,207)(H,171,217)(H,172,224)(H,173,206)(H,174,208)(H,175,221)(H,176,234)(H,177,233)(H,178,219)(H,179,228)(H,180,240)(H,181,220)(H,182,222)(H,183,225)(H,184,226)(H,185,227)(H,186,229)(H,187,237)(H,188,239)(H,189,238)(H,190,235)(H,191,230)(H,192,232)(H,193,236)(H,194,241)(H,195,209)(H,196,223)(H,197,231)(H,210,211)(H,212,213)(H4,157,158,163)(H4,159,160,164)/t76-,77-,78-,79-,80+,81+,86-,87-,88-,89-,90-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-,105-,106-,107-,108-,109-,118-,119-,120-/m0/s1

(4S)-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-[[2-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S,3S)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-2-oxoethyl]amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(1R,4S,7S,10S,13S,16R,21R,24S,31R)-7-(4-aminobutyl)-10-(3-amino-3-oxopropyl)-13-(3-carbamimidamidopropyl)-31-[[(2S)-3-carboxy-2-[[(2S)-1-[(2S)-4-methyl-2-[[(2S)-1-[(2S)-5-oxopyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]propanoyl]amino]-4-[(1R)-1-hydroxyethyl]-24-(hydroxymethyl)-3,6,9,12,15,23,26,32-octaoxo-18,19,28,29-tetrathia-2,5,8,11,14,22,25,33-octazabicyclo[14.10.7]tritriacontane-21-carbonyl]amino]-5-carbamimidamidopentanoyl]amino]-4-methylpentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-5-oxopentanoic acid

Hypocretin-1 (human, rat, mouse); Orexin A; orexin-A; Orexine A; UNII-8RDY08V4VC; ...; Orexin A (human, rat, mouse); Orexin A (human, rat, mouse)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ≥ 50 mg/mL (~14.0 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)