Orexin receptors; Nemorexant targets both orexin receptors: OX1 receptor (OX1R) and OX2 receptor (OX2R) . It functions as a dual orexin receptor antagonist (DORA), competitively blocking the binding of the wake-promoting neuropeptides orexin A and orexin B to these receptors . The compound exhibits IC50 values of 2 nM for OX1 and 3 nM for OX2, with corresponding Ki values of 0.47 nM for OX1R and 0.93 nM for OX2R, indicating high affinity for both receptor subtypes . Additionally, nemorexant has been reported to inhibit BCRP (breast cancer resistance protein) with an IC50 of 3.0 μM .

In cell-based functional assays using Chinese hamster ovary (CHO) cells expressing human orexin receptors, nemorexant demonstrates potent antagonistic activity against both OX1 and OX2 receptors. The compound inhibits orexin A-induced calcium mobilization with IC50 values of 2 nM for OX1 and 3 nM for OX2 . The corresponding Ki values for receptor binding affinity are 0.47 nM for OX1R and 0.93 nM for OX2R . In additional in vitro studies, nemorexant hydrochloride has been shown to inhibit BCRP (breast cancer resistance protein) with an IC50 of 3.0 μM . The compound is formulated for oral administration and demonstrates oral bioavailability suitable for clinical use .

Nemorexant has demonstrated clinical efficacy for the treatment of insomnia disorder in Phase 2 and Phase 3 clinical trials. The compound improves difficulty falling asleep, difficulty maintaining sleep, and patient-reported total sleep time . Patient-reported daytime sleepiness has also been shown to be reduced with nemorexant treatment . At four times the maximum recommended dose, nemorexant does not prolong the QTc interval to any clinically significant extent . In animal studies assessing physical dependence, no withdrawal symptoms or signs were observed upon discontinuation after long-term use, indicating that the drug does not cause physical dependence . In a human abuse potential study, nemorexant showed some abuse potential at doses above the recommended range (100-150 mg), as evidenced by similar "drug preference" scores among recreational sedative users compared to zolpidem (30 mg) and suvorexant; however, at clinically relevant concentrations, nemorexant does not bind to abuse-related central nervous system targets .

The binding affinity of nemorexant for orexin receptors is typically assessed using competitive radioligand binding assays on membrane preparations from CHO cells stably expressing human OX1 or OX2 receptors. Cells are harvested and homogenized in ice-cold buffer (e.g., 50 mM Tris-HCl, pH 7.4, containing protease inhibitors). Membrane pellets are obtained by centrifugation and resuspended in assay buffer. For competition binding experiments, membranes are incubated with a radiolabeled orexin receptor ligand (e.g., [³H]-EMPA or [¹²⁵I]-orexin-A) and varying concentrations of nemorexant (ranging from 0.001 nM to 10 µM) for a predetermined period (typically 60-120 minutes) at room temperature. Nonspecific binding is determined using a high concentration of unlabeled orexin-A or a reference antagonist. Bound radioactivity is separated by rapid filtration through glass fiber filters using a cell harvester, and filter-bound radioactivity is quantified by liquid scintillation counting. The IC50 values are calculated from competition curves by nonlinear regression analysis, and Ki values are derived using the Cheng-Prusoff equation. Using this methodology, nemorexant demonstrates Ki values of 0.47 nM for OX1R and 0.93 nM for OX2R .

The functional antagonistic activity of nemorexant on orexin receptors is assessed using calcium mobilization assays in CHO cells stably expressing human OX1 or OX2 receptors. Cells are cultured in Ham F-12 medium with L-glutamine containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal calf serum. Cells are seeded at 20,000 cells per well into 384-well black clear-bottom sterile plates and incubated overnight at 37°C in 5% CO₂. On the assay day, cells are loaded with 50 μL of staining buffer containing 3 μM of the fluorescent calcium indicator Fluo-4 AM and incubated for 50 minutes at 37°C in 5% CO₂, followed by equilibration at room temperature for 30 minutes. Nemorexant is prepared as a 10 mM stock solution in DMSO, serially diluted, and transferred to assay plates. Within a Fluorescent Imaging Plate Reader (FLIPR), varying concentrations of nemorexant are added to the cells (10 μL/well) and incubated for 120 minutes. Human orexin-A (prepared as 1 mM stock in MeOH:water 1:1, diluted to a final concentration of 3 nM, approximately EC70) is then added (10 μL/well) to stimulate calcium mobilization. Fluorescence is measured at 1-second intervals, and the peak fluorescence height for each well is compared to vehicle-treated control wells. IC50 values are determined from concentration-response curves using nonlinear regression analysis .

Specific detailed animal experimental protocols for nemorexant are not fully described in the available literature. However, the compound has been evaluated in animal studies to assess physical dependence potential. In these studies, animals were administered nemorexant chronically, followed by discontinuation to evaluate withdrawal symptoms or signs. No withdrawal symptoms or signs were observed upon discontinuation after long-term use, indicating that the drug does not cause physical dependence . Additionally, the oral bioavailability and pharmacokinetic properties of nemorexant have been characterized in preclinical species, supporting its development as an orally administered insomnia treatment. For detailed dose-finding and efficacy studies, the compound was evaluated in standard sleep model动物 (e.g., rat EEG/EMG recordings), though specific protocols are not provided in the current search results.

Absorption, Distribution and Excretion
Dalidolex reaches peak plasma concentration within 1 to 2 hours. The absolute bioavailability of dalidolex is 62%. In healthy subjects, a high-fat, high-calorie meal delayed the time to peak concentration (Tmax) by 1.3 hours and reduced peak concentration (Cmax) by 16%, but did not affect total exposure (AUC). The primary route of excretion is feces, accounting for approximately 57% of drug excretion. Approximately 28% of the drug is excreted primarily as metabolites in the urine. Trace amounts of the original drug were detected in both feces and urine. The volume of distribution of dalidolex is 31 liters. The plasma concentration-to-plasma ratio is 0.64. It effectively crosses the blood-brain barrier. Limited information is available regarding its clearance rate. Metabolism/Metabolites Dalidolex is extensively metabolized via CYP3A4 (89%), primarily through oxidative transformation. Other CYP enzymes alone contribute less than 3% to the metabolic clearance of daridoresen.

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Biological half-life
The terminal half-life is approximately 8 hours.

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Nemorexant is an orally bioavailable compound formulated for oral administration as capsules . The recommended clinical dosing regimen is once daily in the evening . At four times the maximum recommended dose, nemorexant does not prolong the QTc interval to any clinically significant extent . The compound has a molecular weight of 450.92 g/mol and a calculated LogP (CLOGP) of 5.19, indicating lipophilic properties . The topological polar surface area (TPSA) is 88.93 Ų, and the compound has 8 hydrogen bond acceptors and 1 hydrogen bond donor . Based on Lipinski's rule of five analysis, nemorexant has 0 violations, suggesting favorable drug-like properties . For research purposes, nemorexant is soluble in DMSO, and stock solutions are recommended to be stored at -80°C for up to 6 months or -20°C for up to 1 month .

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Dalidoresen is present in very low amounts in breast milk. If the mother needs to take dalidoresen, breastfeeding does not need to be discontinued. The infant should be monitored for sedation, feeding difficulties, and poor weight gain until more data are available on breastfed infants. ◉ Effects on Breastfed Infants
No published information found as of the revision date. ◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
Dalidoresen binds to plasma proteins at a rate of 99.7%.

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Nemorexant has been evaluated for safety in clinical trials. At four times the maximum recommended dose, nemorexant does not prolong the QTc interval to any clinically significant extent . In a human abuse potential study, nemorexant showed some abuse potential at doses above the recommended range (100-150 mg), as evidenced by similar "drug preference" scores among recreational sedative users compared to zolpidem (30 mg) and suvorexant; however, at clinically relevant concentrations, nemorexant does not bind to abuse-related central nervous system targets . In animal studies and clinical trials assessing physical dependence, no withdrawal symptoms or signs were observed upon discontinuation after long-term use, indicating that the drug does not cause physical dependence . According to Safety Data Sheets, nemorexant is classified as not a hazardous substance or mixture, and no carcinogenicity has been identified by NTP, IARC, OSHA, or ACGIH . However, during combustion, may emit irritant fumes, and standard laboratory safety precautions (gloves, protective clothing, eye protection) should be followed when handling the compound .

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Hepatotoxicity: In two controlled trials conducted in support of approval of daridorexant in the United States, there were no changes in serum ALT, AST, alkaline phosphatase or bilirubin levels during treatment and no instances of drug induced liver injury. Since its approval and more widespread clinical use, there have been no published reports of clinically apparent liver injury attributed to daridorexant. Indeed, the two previously approved orexin receptor antagonists available in the United States were also found to be free of hepatic adverse effects. Likelihood score: E (unlikely cause of clinically apparent liver injury).

[1]. USE OF BENZIMIDAZOLE-PROLINE DERIVATIVES. WO2015083094A1.

Pharmacodynamics
Daridorexan binds to and antagonizes the effects of orexin receptors OX1R and OX2R (Ki values of 0.47 nM and 0.93 nM, respectively), with comparable potency. Clinical trials have shown that daridorexan improves difficulty falling asleep, difficulty maintaining sleep, and patient-reported total sleep time. Patient-reported daytime sleepiness has also been reported to be reduced. At four times the maximum recommended dose, daridorexan does not prolong the QTc interval to any clinically significant extent. Daridorexan is currently under evaluation for controlled substances in the United States. In a human abuse potential study, daridorexan showed some abuse potential at doses above the recommended dose (100-150 mg), as evidenced by similar "drug preference" scores among recreational sedative users to zolpidem (30 mg) and suvorexan. However, at clinically relevant concentrations, daridorexan does not bind to abuse-related central nervous system targets. In animal studies and clinical trials assessing physical dependence, no withdrawal symptoms or signs were observed upon discontinuation after long-term use of dalidoresen, indicating that the drug does not cause physical dependence.

C23H23CLN6O2

450.92

450.157

C, 61.26; H, 5.14; Cl, 7.86; N, 18.64; O, 7.10

1505484-82-1

1792993-84-0 (HCl);1505484-82-1;Daridorexant mesylate;

91801202

Solid powder

1.4±0.1 g/cm3

747.6±70.0 °C at 760 mmHg

405.9±35.7 °C

0.0±2.5 mmHg at 25°C

1.704

3.38

1

5

4

32

693

1

ClC1C=CC2=C(C=1C)N=C([C@]1(C)CCCN1C(C1=CC(=CC=C1N1N=CC=N1)OC)=O)N2

NBGABHGMJVIVBW-QHCPKHFHSA-N

InChI=1S/C23H23ClN6O2/c1-14-17(24)6-7-18-20(14)28-22(27-18)23(2)9-4-12-29(23)21(31)16-13-15(32-3)5-8-19(16)30-25-10-11-26-30/h5-8,10-11,13H,4,9,12H2,1-3H3,(H,27,28)/t23-/m0/s1

[(2S)-2-(5-chloro-4-methyl-1H-benzimidazol-2-yl)-2-methylpyrrolidin-1-yl]-[5-methoxy-2-(triazol-2-yl)phenyl]methanone

Nemorexant; Daridorexant; ACT-541468; Quviviq; ACT54146

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)