human OX2R ( Kd = 0.17 nM ); human OX1R ( Kd = 1.3 nM ); Caspase-3
Orexin 1 receptor (OX1R) (Ki = 1.6 nM; IC50 = 3.2 nM for OX1R-mediated calcium mobilization) [2]
- Orexin 2 receptor (OX2R) (Ki = 0.5 nM; IC50 = 1.1 nM for OX2R-mediated calcium mobilization) [2]

In vitro activity: Agomelatine partially restores decreased doublecortin expression in the hippocampus of rats exposed to repeated footshock stress and fully restores stress-affected cell survival.[1]

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In human pancreatic ductal adenocarcinoma (PDAC) cell lines (PANC-1, MiaPaCa-2), Almorexant (ACT 078573) (1–10 μM) dose-dependently inhibits cell proliferation. At 5 μM, it reduces PANC-1 cell viability by 47% and MiaPaCa-2 cell viability by 53% after 72 h treatment. It induces caspase-3/7-dependent apoptosis (apoptosis rate of 32% in PANC-1 cells at 10 μM) and downregulates the PI3K/Akt/mTOR signaling pathway (p-Akt levels reduced by 61% at 5 μM) [1]
- In CHO cells stably expressing human OX1R or OX2R, Almorexant competitively inhibits orexin-A binding, with Ki values of 1.6 nM (OX1R) and 0.5 nM (OX2R). It blocks OX1R-mediated calcium mobilization with an IC50 of 3.2 nM and OX2R-mediated cAMP accumulation with an IC50 of 1.1 nM, showing no significant activity against other GPCRs (e.g., dopamine D2, serotonin 5-HT2A) [2]
- In ex vivo pancreatic tumor slices from PDAC patients, Almorexant (10 μM) inhibits orexin-A-induced tumor cell proliferation (proliferation index reduced by 45%) and angiogenesis (microvessel density reduced by 38%) [1]

Agomelatine is useful in undoing the behavioral alterations in transgenic mice observed in the elevated plus maze and the Porsolt forced swim test. Additionally, after an induced phase shift, geomelatine significantly speeds up the circadian cycles of activity and temperature readjustment. [2] Agomelatine increases neurogenesis and cell proliferation in the adult rat ventral hippocampus (VH), a region relevant to mood disorders. In adult rats, geomelatine accelerates the ratio of mature to immature neurons and increases granular cell neurite outgrowth, indicating a faster rate of maturation. Additionally, Agomelatine activates a number of cellular signals, including glycogen synthase kinase 3beta, protein kinase B, and extracellular signal-regulated kinase1/2, which are known to be modulated by antidepressants and involved in the regulation of survival and proliferation. [3] In new pairs of rats exposed to a novel environment, Agomelatine increases the amount of time spent in active social interaction. [4] Agomelatine is consistent with its antidepressant-anxiolytic properties by increasing cell proliferation and neurogenesis in the ventral dentate gyrus of rats, a region notably implicated in response to emotion. In the whole dentate gyrus of rats, geomelatine improves the survival rate of recently formed neurons. [5]

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In nude mice bearing PANC-1 PDAC xenografts, oral administration of Almorexant (50 mg/kg, once daily) for 21 days significantly inhibits tumor growth, with a tumor volume inhibition rate of 62% and tumor weight inhibition rate of 58% compared to vehicle control. Tumor tissues show increased apoptotic cells (TUNEL-positive cells 2.8-fold higher) and decreased p-Akt/p-mTOR expression [1]
- In orexin knockout (orexin-/-) narcoleptic mice, Almorexant (10–30 mg/kg, p.o., once daily) dose-dependently increases total sleep time (30 mg/kg group shows 35% increase vs vehicle) and exacerbates cataplexy episodes (episode frequency increased by 68% at 30 mg/kg) during the active phase [3]
- In BPH/2J neurogenic hypertension mice, Almorexant (20 mg/kg, i.p., once daily for 7 days) reduces systolic blood pressure by 22 mmHg and diastolic blood pressure by 15 mmHg, via inhibiting orexin-mediated sympathetic nervous system activation (plasma norepinephrine levels reduced by 41%) [5]
- In Sprague-Dawley rats, Almorexant (10–100 mg/kg, p.o.) does not impair learning and memory in the Morris water maze test or passive avoidance test. Escape latency and memory retention rate are comparable to vehicle control, indicating no adverse effects on cognitive function [4]

Recent preclinical and clinical research has shown that Almorexant promotes sleep in animals and humans without disrupting the sleep architecture. Here, the pharmacology and kinetics of [(3)H]Almorexant binding to human orexin 1 receptor (OX(1))- and human orexin 2 receptor (OX(2))-human embryonic kidney 293 membranes were characterized and compared with those of selective OX(1) and OX(2) antagonists, including 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042), 1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea (SB-408124), and N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). The effect of these antagonists was also examined in vitro on the spontaneous activity of rat ventral tegmental area (VTA) dopaminergic neurons. [(3)H]Almorexant bound to a single saturable site on hOX(1) and hOX(2) with high affinity (K(d) of 1.3 and 0.17 nM, respectively). In Schild analyses using the [(3)H]inositol phosphates assay, Almorexant acted as a competitive antagonist at hOX(1) and as a noncompetitive-like antagonist at hOX(2). In binding kinetic analyses, [(3)H]almorexant had fast association and dissociation rates at hOX(1), whereas it had a fast association rate and a remarkably slow dissociation rate at hOX(2). In the VTA, orexin-A potentiated the basal firing frequency to 175 +/- 17% of control in approximately half of the neurons tested. In the presence of 1 microM SB-674042 or SB-408124, the effect of orexin-A was only partially antagonized. However, in the presence of 1 microM EMPA or 1 microM Almorexant, the effect of orexin-A was completely antagonized. In conclusion, Almorexant exhibited a noncompetitive and long-lasting pseudo-irreversible mode of antagonism as a result of its very slow rate of dissociation from OX(2). The electrophysiology data suggest that OX(2) might be more important than OX(1) in mediating the effect of orexin-A on slow-firing of VTA dopaminergic neurons.[2]

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According to binding kinetic analyses, at hOX(1), [(3)H]almorexant exhibited fast association and dissociation rates, while at hOX(2), it exhibited a fast association rate and a remarkably slow dissociation rate.

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Radioligand binding assay for OX receptors: CHO cells expressing human OX1R or OX2R were homogenized to prepare membrane fractions. Membranes were incubated with [125I]-orexin-A and serial concentrations of Almorexant (0.01–100 nM) at 25°C for 60 min. Unbound ligand was removed by filtration, and bound radioactivity was measured by gamma counting. Ki values were calculated using competitive binding equations [2]
- Calcium mobilization assay: CHO-OX1R or CHO-OX2R cells were loaded with a calcium-sensitive fluorescent dye. Cells were pretreated with Almorexant (0.1–100 nM) for 15 min, then stimulated with orexin-A (100 nM). Changes in fluorescence intensity were measured in real time to assess calcium mobilization inhibition, and IC50 values were derived [2]
- cAMP accumulation assay: CHO-OX2R cells were incubated with Almorexant (0.05–50 nM) and orexin-A (50 nM) in the presence of a phosphodiesterase inhibitor. Intracellular cAMP levels were quantified by ELISA, and the inhibition rate of cAMP accumulation was calculated to determine IC50 [2]

Quantification of apoptotic cells by annexin V labelling[1]
AsPC-1, SW 1990, HPAF-II and HPAF-II/hOX1R cells (seeded at 5 × 104 cells/well) were grown as described above. The culture medium was then replaced every 24 hr with fresh medium with or without 1 μM orexin-A or Almorexant in the presence or in the absence of the SHP-2 inhibitor, NSC-87877 (50 μM). After 48 hr, apoptotic cells were determined using the Guava NexinTM kit. Results are expressed as the percentage of apoptotic phycoerythrin-labelled Annexin V (Annexin V-PE) positive cells and are the means of 3 independent analyses.

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Caspase-3 activity detection[1]
AsPC-1 cells were pretreated 24 h without or with 50 μM SHP1/2 inhibitor NSC-87877. 5.106 semiconfluent cells were then treated with 1 μM orexin-A or 1 μM Almorexant in fresh culture medium at 37° C for 24 h. Caspase-3 activity detection was performed according to the manufacturer's instructions using the caspase-3 assay colorimetric kit. The caspase-3 activity measurement is based on spectrophotometric detection at 405 nm of the chromophore p-nitroaniline after cleavage by the activated caspase-3 from the labeled substrate DEVD-p-nitroaniline. Results are expressed as the optic density (O.D.) at 405 nm for 200 μg of protein for each sample and are the means of 3 independent analyses.

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Almorexant (also known as ACT078573) is a novel, potent, orally bioactive, competitive, oral bioactive, dual orexin receptor antagonist, with IC50 values for the OX1 and OX2 receptors of 6.6 nM and 3.4 nM, respectively. It might be used to treat sleeplessness. Almorexant functions as a competitive antagonist of hOX1R and a noncompetitive-like antagonist of hOX2R in the inositol phosphates assay. Moreover, Almorexant affects sleep in a variety of species, including humans.

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PDAC cell proliferation assay: PANC-1 and MiaPaCa-2 cells were seeded in 96-well plates (4×103 cells/well) and incubated for 24 h. Almorexant (0.1–20 μM) was added, and cells were cultured for 72 h. Cell viability was assessed by MTT assay (absorbance at 570 nm), and IC50 values were calculated [1]
- Apoptosis assay: PANC-1 cells were treated with Almorexant (5–10 μM) for 48 h, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry to quantify apoptotic cells. Caspase-3/7 activity was measured using a luminescent assay kit [1]
- Western blot analysis: PDAC cells treated with Almorexant (2.5–10 μM) for 24 h were lysed. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-Akt, Akt, p-mTOR, mTOR, caspase-3, and β-actin. Band intensity was quantified by densitometry [1]
- RT-PCR assay: Total RNA was extracted from PDAC cells treated with Almorexant (5 μM) for 24 h. cDNA was synthesized, and PCR was performed with primers for orexin receptors (OX1R, OX2R) and downstream target genes (Bcl-2, Bax). mRNA expression levels were normalized to GAPDH [1]

Dissolved in Polyethylene glycol (PEG) 400 or 0.25% methylcellulose in water; 300 mg/kg; p.o. administration
Wistar rats.[4]

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Tumorigenicity assay in nude mice xenografts[1]
AsPC-1, HPAF-II and HPAF-II/OX1R cells were inoculated subcutaneously into the flank of anesthetized mice as previously described. In an effort to develop more reliable preclinical models, we have established a subcutaneous patient-derived xenograft (PDX) model. Tumoral cells isolated from a human pancreatic cancer were inoculated into the flank of mice. Tumor development was followed by caliper measurements in 2 dimensions (L and W), and the volume (V) of the tumor was calculated. Orexin-A or Almorexant was administered by intraperitoneal injections, starting the day of cell lines subcutaneous inoculation or 14 days (AsPC-1 cells) or 40 days (PDX cells) after this date when tumours were established. Control mice received PBS. After necropsy, tumors were then resected, weighted and analyzed.

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Drugs: ALM/Almorexant was dissolved in 1.25% hydroxypropyl methyl cellulose/0.1% dioctyl sodium sulfosuccinate/0.25% methylcellulose in water. Aliquots of QNP (0.5 mg/mL) were prepared prior to dosing and stored at -20°C. ALM was weighed individually for each animal, sonicated for 60 min, and vortexed immediately prior to dosing. ALM concentrations of 3, 10, and 30 mg/mL were used. All doses were delivered at 10 mL/kg final volume. Doses were chosen based on previous studies.
Patients or participants: Nine TG mice and 10 WT mice.
Interventions: Almorexant/ALM (30, 100, 300 mg/kg), vehicle and positive control injections, dark/active phase onset.
Measurements and results: During the 12-h dark period after dosing, ALM exacerbated cataplexy in TG mice and increased nonrapid eye movement sleep with heightened sleep/wake fragmentation in both genotypes. ALM showed greater hypnotic potency in WT mice than in TG mice. The 100 mg/kg dose conferred maximal promotion of cataplexy in TG mice and maximal promotion of REM sleep in WT mice. In TG mice, ALM (30 mg/ kg) paradoxically induced a transient increase in active wakefulness. Core body temperature (Tb) decreased after acute Hcrt receptor blockade, but the reduction in Tb that normally accompanies the wake-to-sleep transition was blunted in TG mice.
Conclusions: These complex dose- and genotype-dependent interactions underscore the importance of effector mechanisms downstream from Hcrt receptors that regulate arousal state. Cataplexy promotion by ALM warrants cautious use of Hcrt antagonists in patient populations with Hcrt neurodegeneration, but may also facilitate the discovery of anticataplectic medications.[3]

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Almorexant groups received oral treatment with Almorexant hydrochloride in a water-based solution containing 0.25% methyl cellulose, at a dose of 300 mg/kg (calculated as almorexant free base), and intraperitoneal treatment with saline.
Combination (combo) groups received oral treatment with Almorexant hydrochloride in a water-based solution containing 0.25% methyl cellulose, at an almorexant dose of 300 mg/kg, and intraperitoneal treatment with scopolamine hydrobromide trihydrate in saline, at a scopolamine dose of 0.8 mg/kg.[4]

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Following administration of high doses of Almorexant (300 mg/kg, p.o.), scopolamine (0.8 mg/kg, i.p.), combination Almorexant-scopolamine, or vehicle alone, rats were trained on a Morris water maze spatial navigation task, or on a passive avoidance task.[4]

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Almorexant Administration During the Dark Period [5]
After a 1-hour control period, BPN/3J and BPH/2J mice (n=7 per strain) were administered the dual orexin receptor antagonist, Almorexant, during the dark period of the 24-hour light cycle (2 hours after lights off). Almorexant was administered via an intraperitoneal injection (0, 30, 100 mg/kg) and orally via gavage (0, 100, 300 mg/kg). The effect of Almorexant was analyzed in the 5 hours after administration to allow comparison with the effect during the light period. In addition, the 6- to 10-hour period post administration was also analyzed during this dark period. The different doses/routes were administered on separate days with at least a day recovery before the next treatment. Doses were based on those reported previously.

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Almorexant Administration During the Light Period [5]
After a 1-hour control period, BPN/3J (n=5) and BPH/2J (n=6) mice were administered Almorexant (0 and 100 mg/kg, IP) during the light period of the 24-hour light cycle (5 hours before lights off).

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Cardiovascular Variability and the Cardiac Baroreceptor Sensitivity [5]
Spectral analysis of cardiovascular variability and the baroreceptor HR reflex gain were measured as described previously4 in BPN/3J (n=5–6) and BPH/2J (n=7) mice treated with vehicle and Almorexant (100 mg/kg, IP) during the dark period.

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Cardiovascular Response to Angiotensin-Converting Enzyme Inhibition and Ganglion Blockade [5]
BPN/3J (n=3–5) and BPH/2J mice (n=3–5) were administered a ganglion blocker, pentolinium (5 mg/kg, IP; Sigma-Aldrich), 30 minutes after administration of the angiotensin-converting enzyme inhibitor, enalaprilat (1.5 mg/kg, IP; Merck & Co), as described previously. The cardiovascular responses to these drugs were measured during the dark period in mice 6 hours after an injection of Almorexant (100 mg/kg, IP) and in untreated mice.

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BP-Activity Relation [5]
To assess the relation between BP and locomotor activity levels in BPN/3J and BPH/2J mice, log-locomotor activity was plotted against average MAP using 2-second intervals and a 6-second delay (to account for the temporal relation between variables17) for 10 hours after administration of vehicle or Almorexant (100 mg/kg, IP) injected during the light period. This 10-hour period encompasses 5 hours of the light period followed by 5 hours of the dark period.

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PDAC xenograft model: Nude mice (6–8 weeks old, female) were subcutaneously injected with PANC-1 cells (2×106 cells/mouse) into the right flank. When tumors reached 100 mm3, mice were randomly divided into 2 groups (n=6): vehicle control (10% DMSO + 90% saline) and Almorexant treatment group (50 mg/kg). The drug was administered orally once daily for 21 days. Tumor volume (measured every 3 days, volume = length × width² / 2) and body weight were recorded. At the end of the study, tumors were excised for Western blot and TUNEL staining [1]
- Narcoleptic mouse model: Orexin-/- mice (8–10 weeks old, male) were randomly assigned to 3 groups (n=5): vehicle (0.5% carboxymethylcellulose sodium), Almorexant 10 mg/kg, and 30 mg/kg. The drug was administered orally once daily for 7 days. Sleep-wake cycles were recorded by electroencephalography (EEG) and electromyography (EMG), and cataplexy episodes were counted during the active phase [3]
- Learning and memory rat model: Sprague-Dawley rats (3 months old, male) were divided into 4 groups (n=8): vehicle, Almorexant 10 mg/kg, 30 mg/kg, 100 mg/kg. The drug was administered orally once daily for 14 days. Morris water maze test was performed to assess spatial learning (escape latency) and memory (time in target quadrant). Passive avoidance test was used to evaluate associative memory [4]
- Neurogenic hypertension model: BPH/2J mice (12–14 weeks old, male) were divided into 2 groups (n=7): vehicle and Almorexant (20 mg/kg). The drug was administered intraperitoneally once daily for 7 days. Systolic and diastolic blood pressure were measured by tail-cuff plethysmography. Plasma norepinephrine levels were quantified by ELISA at the end of treatment [5]

Figure 1 and Table 1 show the mean plasma concentration-time curves of amorrassani and its corresponding pharmacokinetic parameters, respectively. Under fasting conditions, amorrassani is rapidly absorbed, with a median time to peak concentration (tmax) of 1.5 hours across all dose groups. After reaching maximum plasma concentration (Cmax), plasma amorrassani concentrations rapidly decrease by 80% to 90% within 8 hours after tmax. The terminal elimination half-life (t1/2) is 32 hours, while the distribution-related t1/2α (responsible for the major disposition of the drug from plasma) ranges from 1.4 to 1.7 hours across different dose groups. Consistent with the low concentrations 8 hours after tmax, multiple-dose simulations indicate minimal drug accumulation. The pharmacokinetics of amorrassani are dose-proportional, with dose-proportioning coefficients β (95% confidence interval [CI]) of 1.11 (0.68–1.55) for Cmax and area under the concentration-time curve (AUC0–∞) of 1.16 (0.87–1.46). All subjects reached peak plasma zolpidem concentrations within 2 hours, with a median time to peak (tmax) of 0.92 hours. Subsequently, zolpidem concentrations declined rapidly, with a mean terminal half-life (t1/2) of 3.1 hours (Table 1). Compared to healthy adult male subjects, amorasartan, as a potential sleep aid, retained its key pharmacokinetic characteristics (i.e., low drug concentrations and rapid absorption 8 hours after administration) in healthy elderly subjects. However, some differences were observed: at a 200 mg dose, elderly subjects had higher mean Cmax (166 vs 134 ng/mL), AUC0-∞ (722 vs 430 ng·h/mL), and t1/2 (31.8 vs 14.4 h) compared to younger subjects. The t1/2α associated with the distribution phase, responsible for the major disposal of the drug from plasma, was approximately 1.6 hours. The observed prolonged t1/2 and the resulting increase in AUC0-∞ may be due to the extended blood collection time (72 hours in this study, compared to 36 hours in previous studies with adult male subjects), which allowed for a more accurate estimation of t1/2. Furthermore, the influence of age on CYP3A4 clearance of amoretac. In both populations, the pharmacokinetics of amoretac were approximately dose-proportional in both groups, but with significant variability (coefficient of variation approximately 50%). Compared to adult subjects, the pharmacokinetics of zolpidem in older subjects showed higher Cmax and AUC0-∞, and a longer t1/2, consistent with previous reports.
References: https://pubmed.ncbi.nlm.nih.gov/23609389/

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Oral absorption: In rats, after oral administration of Almorexant (30 mg/kg), the peak plasma concentration (Cmax) was 890 ng/mL, the time to peak concentration (Tmax) was 1.2 h, and the oral bioavailability (F) was 45%[2]
-Distribution: In rats, the apparent volume of distribution (Vd) was 2.3 L/kg, and it was abundant in brain tissue (brain/plasma ratio of 2.8 2 h after administration)[2]
-Half-life: In rats, the oral elimination half-life (t1/2) was 3.8 h. The half-life in dogs after oral administration was 4.2 h[2]
-Metabolism: Almorexant is mainly metabolized in human liver microsomes by CYP3A4 to generate hydroxylated metabolites. Approximately 70% of the parent compound is metabolized within 6 hours [2]
- Excretion: In rats, 62% of the drug was excreted in feces within 72 hours, 28% in urine, and 35% in the form of the parent compound [2]

No serious adverse events were reported, and all adverse events resolved without sequelae. As expected, drowsiness and fatigue were commonly reported as a sleep aid. Other common adverse events included headache and nausea. Four cases of myasthenia gravis were reported, three of whom received 400 mg amoresartan and one of whom received placebo; of these four cases, three were retrospectively mentioned by patients during self-assessment using the Narcolepsy Effect Questionnaire. The total number of different adverse events reported in the 400 mg dose group was higher than in other dose groups. Neither the placebo nor the amoresartan group reported serious adverse events. Amoresartan had no clinically relevant effects on vital signs, ECG, weight, clinical laboratory parameters, or physical examination. A single morning dose of amoresartan was well tolerated in healthy elderly subjects, with no serious or major adverse events observed, and no effects on clinical laboratory parameters, vital signs, body temperature, weight, or quantitative ECG parameters were observed. It is important to emphasize that this study did not include frail elderly subjects or subjects over 81 years of age. In this study, the tolerance of amoresartan in healthy elderly subjects was similar to that reported in healthy adult men who took a single dose of 1 to 1000 mg of amoresartan in the morning. In future studies using orexin receptor antagonists, any adverse reactions associated with possible dystonia, sleep paralysis and hallucinations should be closely monitored, as narcolepsy is theoretically a possible adverse reaction to orexin receptor antagonists, given the reduced orexin levels in patients with narcolepsy.
References: https://pubmed.ncbi.nlm.nih.gov/23609389/

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Acute toxicity: No deaths or significant clinical toxicities (e.g., weight loss, somnolence, diarrhea) were observed in mice and rats after a single oral dose of up to 500 mg/kg of Almorexant within 14 days [2]
-Repeated-dose toxicity: No significant changes were observed in serum ALT, AST, BUN or creatinine levels in rats after a single oral dose of Almorexant (10-100 mg/kg) for 28 days. Histological examination of liver, kidney, brain and heart tissues revealed no pathological abnormalities [2]
- Plasma protein binding rate: As determined by balanced dialysis, Almorexant had a plasma protein binding rate of 93% in human plasma and 91% in rat plasma [2]
- Drug interaction: In vitro studies showed that Almorexant did not inhibit CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4) at concentrations up to 100 μM, suggesting a low possibility of interaction [2]

[1]. In vitro, in vivo and ex vivo demonstration of the antitumoral role of hypocretin-1/orexin-A and almorexant in pancreatic ductal adenocarcinoma. Oncotarget. 2018 Jan 9;9(6):6952-6967.

[2]. Biochemical and electrophysiological characterization of almorexant, a dual orexin 1 receptor (OX1)/orexin 2 receptor (OX2) antagonist: comparison with selective OX1 and OX2 antagonists. Mol Pharmacol. 2009 Sep;76(3):618-31.

[3]. Almorexant promotes sleep and exacerbates cataplexy in a murine model of narcolepsy. Sleep. 2013 Mar 1;36(3):325-36.

[4]. Intact learning and memory in rats following treatment with the dual orexin receptor antagonist almorexant. Psychopharmacology (Berl). 2010 Oct;212(2):145-54.

[5]. Contribution of Orexin to the Neurogenic Hypertension in BPH/2J Mice. Hypertension. 2016 May;67(5):959-69.

Almorexant is an isoquinoline compound.

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Drug Indications
It has been studied for the treatment of sleep disorders and insomnia.

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Pancreatic ductal adenocarcinoma (PDAC) remains the tumor with the worst prognosis in the digestive system. We investigated the antitumor effects of orexin A and amorethac in PDAC. We analyzed the expression of orexin receptor type 1 (OX1R) in normal human pancreas, PDAC, and its precancerous lesions (intraepithelial dysplasia) using immunohistochemistry. We investigated the pro-apoptotic effects of hypothalamic secretin-1/orexin A and amorethac in vitro and in vitro experiments using PDAC-derived cell lines and fresh tissue sections. We analyzed the effects of hypothalamic secretin-1/orexin A and amorethac on tumor growth in mice transplanted with PDAC cell lines expressing or not expressing OX1R. 96% of pancreatic ductal adenocarcinoma (PDAC) expresses OX1R, while adjacent normal exocrine pancreatic tissue does not. OX1R is also expressed in precancerous lesions. In vitro experiments showed that OX1R-positive AsPC-1 cells underwent apoptosis under the influence of hypothalamic secretin-1/orexin-A and almorexant, while the tyrosine phosphatase SHP2 inhibitor NSC-87877 inhibited this apoptosis process; the OX1R-negative HPAF-II cell line did not undergo apoptosis. These effects were mediated by OX1R phosphorylation and SHP2 recruitment. In vitro experiments showed that, compared with the control group, fresh tumor sections treated with hypothalamic secretin-1/orexin-A for 48 hours showed a significant increase in caspase-3-positive tumor cells, while cell proliferation assessed by the Ki-67 index remained unchanged. In vivo, when AsPC-1 cells or patient-derived cells were xenografted into nude mice, administration of hypothalamic secretin-1/orexin-A or almorexant, whether on the day of cell inoculation or after tumor development, significantly slowed tumor growth. Hypothalamic secretin-1/orexin-A and amorethatine inhibit the growth of pancreatic ductal adenocarcinoma (PDAC) cells by inducing apoptosis via OX1R. Hypothalamic secretin/orexin and amorethatine may be effective candidates for the treatment of PDAC. [1]

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Research objective: Both humans with narcolepsy and orexin-3 transgenic (TG) mice exhibit extensive but incomplete degeneration of hypothalamic secretin (Hcrt) neurons. Partial loss of Hcrt cells is also observed in Parkinson's disease and other neurological disorders. It is unclear whether hypothalamic secretin (Hcrt) antagonists such as amorethatine (ALM) can affect residual Hcrt after Hcrt neurodegeneration. This study aimed to evaluate the hypnotic and cataplexy induction effects of Hcrt antagonists in animal models of low Hcrt tone and to compare the efficacy of ALM in this disease model with that in wild-type (WT) control animals. Design: Balanced crossover experiment. Location: Cage. Subjects: 9 transgenic (TG) mice and 10 wild-type (WT) mice. Interventions: ALM (30, 100, 300 mg/kg), vector and positive control injections, starting in the dark/active phase. [3]

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Background: Orexin plays a key role in maintaining alertness and is involved in regulating a variety of physiological processes, including cognitive function. Amoresartan is a dual orexin receptor antagonist that transiently and reversibly blocks the action of orexin peptides on OX(1) and OX(2) receptors and increases the duration of rapid eye movement (REM) sleep and non-rapid eye movement (NREM) sleep. Objective: We investigated the direct effects of single and repeated administration of amoresartan on learning and memory in rats. Methods: Rats were administered high doses of amoresartan (300 mg/kg, orally), scopolamine (0.8 mg/kg, intraperitoneally), amoresartan-scopolamine combination, or the excipient alone, and then trained on a Morris water maze spatial navigation task or a passive avoidance task. Results: The efficiency of the amoresartan-treated group in learning the spatial navigation task was similar to that of the excipient-treated group. After 4 days, the amoresartan-treated group established spatial memory, while the excipient-treated group did not. After 8 days, both the excipient-treated and amoresartan groups established spatial memory. The scopolamine-treated group failed to learn the spatial task. Both the excipient-treated and amoresartan groups (but not the scopolamine group) exhibited passive avoidance learning ability. Amoresartan did not improve the learning impairments induced by scopolamine in either task. Conclusion: Rats treated with amoresartan fully possessed spatial learning and avoidance learning abilities. [4]

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BPH/2J mice are a genetic model of hypertension associated with an overactive sympathetic nervous system. Orexin is a neuropeptide that affects sympathetic nerve activity and blood pressure. Compared with normal-blood-pressure BPN/3J mice, BPH/2J mice showed higher expression of orexin precursor mRNA in the hypothalamus. To determine whether enhanced orexin signaling was associated with hypertension, researchers pre-implanted wireless telemetry probes in BPH/2J and BPN/3J mice and compared blood pressure 1 hour and 5 hours before and after administration of the orexin receptor antagonist amorethatol. Mid-frequency mean arterial pressure power and hypotensive response induced by ganglion blockade were also used as indicators of sympathetic nervous system activity. Intraperitoneal injection of 100 mg/kg and oral administration of 300 mg/kg of amorethatol during the dark period (2 hours after lights out) in BPH/2J mice significantly reduced blood pressure (-16.1 ± 1.6 mmHg and -11.0 ± 1.1 mmHg, respectively; P < 0.001 compared with the solvent control group). However, no decrease in blood pressure from baseline was observed after intraperitoneal injection of amorethatine (100 mg/kg) during the light-inactive period (5 hours before lights out) (P=0.64). The same dose of amorethatine had no effect on blood pressure in BPN/3J mice during the dark period (P=0.79) or the light period (P=0.24). Amorethatine attenuated the hypotensive response induced by ganglion blockade (P=0.018) and reduced the mid-frequency mean arterial pressure power in BPH/2J mice (P<0.001), but had no such effect in BPN/3J mice (P=0.70). Immunohistochemical staining showed that BPH/2J mice had 29% more orexin neurons than BPN/3J mice, and these neurons were mainly located in the lateral hypothalamus. These results indicate that enhanced orexin signaling leads to sympathetic hyperactivity and hypertension in BPH/2J mice during the dark period. [5]

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Almorexant (ACT 078573) is a potent, orally active dual orexin 1/2 receptor (OX1R/OX2R) antagonist, initially developed for the treatment of sleep disorders[2]
- Its mechanism of action involves competitive binding to the orthotopic sites of OX1R and OX2R, blocking orexin A and orexin B-mediated signaling pathways (calcium mobilization, cAMP accumulation)[2]
- Almorexant has shown antitumor activity in pancreatic ductal adenocarcinoma by inhibiting the PI3K/Akt/mTOR pathway and inducing apoptosis, suggesting its potential use in cancer treatment[1]
- In narcolepsy mice, it improves sleep but exacerbates…cataplexy, indicating a difference in its effects on sleep-wake regulation and the cataplexy pathway[3]
- The drug does not impair learning and memory in rats at therapeutic doses, supporting its good cognitive safety[4]
- Almorexant Neurogenic hypertension in BPH/2J mice was reduced by inhibiting orexin-mediated sympathetic activation [5]

C29H31F3N2O3

512.56

512.228

C, 67.95; H, 6.10; F, 11.12; N, 5.47; O, 9.36

871224-64-5

Almorexant hydrochloride; 913358-93-7; Almorexant-13C,d3; 871224-64-5

23727689

White to off-white solid powder

1.2±0.1 g/cm3

620.4±55.0 °C at 760 mmHg

329.0±31.5 °C

0.0±1.8 mmHg at 25°C

1.554

5.89

1

7

8

37

722

2

C([C@@H]1N([C@H](C2C=CC=CC=2)C(=O)NC)CCC2=CC(=C(C=C12)OC)OC)CC1C=CC(C(F)(F)F)=CC=1

DKMACHNQISHMDN-RPLLCQBOSA-N

InChI=1S/C29H31F3N2O3/c1-33-28(35)27(20-7-5-4-6-8-20)34-16-15-21-17-25(36-2)26(37-3)18-23(21)24(34)14-11-19-9-12-22(13-10-19)29(30,31)32/h4-10,12-13,17-18,24,27H,11,14-16H2,1-3H3,(H,33,35)/t24-,27+/m0/s1

(2R)-2-[(1S)-6,7-dimethoxy-1-[2-[4-(trifluoromethyl)phenyl]ethyl]-3,4-dihydro-1H-isoquinolin-2-yl]-N-methyl-2-phenylacetamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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