Orexin receptors; Daridorexant hydrochloride 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, Daridorexant hydrochloride has been reported to inhibit BCRP (breast cancer resistance protein) with an IC50 of 3.0 μM .
Daridorexant targets both orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R), which are G protein-coupled receptors activated by the neuropeptides orexin-A and orexin-B. These receptors are primarily expressed in the hypothalamus and play a central role in regulating wakefulness, arousal, and sleep-wake transitions. As a dual antagonist, Daridorexant blocks the binding of orexin peptides to both receptor subtypes, thereby inhibiting the orexin-mediated wake-promoting signal. This mechanism promotes sleep by reducing wake drive rather than by sedating the brain through generalized CNS depression, which is the mechanism of traditional GABAergic hypnotics.

In cell-based functional assays using Chinese hamster ovary (CHO) cells expressing human orexin receptors, Daridorexant hydrochloride 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, Daridorexant hydrochloride 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 .

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In vitro, Daridorexant demonstrates potent antagonism at both human OX1R and OX2R with high affinity. Receptor binding assays show that Daridorexant competitively inhibits orexin-A and orexin-B binding to both receptor subtypes with low nanomolar IC50 values. Functional assays measuring intracellular calcium flux (a downstream readout of orexin receptor activation) confirm that Daridorexant acts as a full antagonist, completely blocking orexin-induced signaling. The compound shows selectivity for orexin receptors over a panel of other GPCRs, ion channels, and transporters, contributing to its favorable safety profile. In vitro metabolic stability studies demonstrate that Daridorexant is metabolically stable in human liver microsomes.

Daridorexant hydrochloride 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 Daridorexant hydrochloride treatment . At four times the maximum recommended dose, Daridorexant hydrochloride 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, Daridorexant hydrochloride 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, Daridorexant hydrochloride does not bind to abuse-related central nervous system targets .

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In vivo, Daridorexant promotes sleep in animal models and human subjects through its dual orexin receptor antagonist activity. In rodent sleep-wake studies, oral administration of Daridorexant reduces wakefulness and increases both non-REM and REM sleep duration without altering sleep architecture. The compound's effects are dose-dependent and correlate with orexin receptor occupancy as measured by PET imaging. In clinical trials, Daridorexant significantly improved sleep onset and sleep maintenance endpoints in patients with insomnia, with effects sustained over 12 weeks of treatment. Unlike GABAergic hypnotics, Daridorexant does not cause significant next-day residual sedation at therapeutic doses.

The binding affinity of Daridorexant hydrochloride 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 Daridorexant hydrochloride (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, Daridorexant hydrochloride demonstrates Ki values of 0.47 nM for OX1R and 0.93 nM for OX2R .

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In vitro enzyme/receptor binding assays for Daridorexant employ radioligand competition binding using membrane preparations from cells expressing recombinant human OX1R or OX2R. Standard protocols use [³H]-orexin-A or selective radiolabeled antagonists as tracers. Membrane preparations are incubated with varying concentrations of Daridorexant, and bound radioactivity is separated by filtration or scintillation proximity assay. Non-specific binding is determined in the presence of excess unlabeled orexin-A. Binding affinity (Ki) values are calculated from displacement curves. Selectivity profiling is performed by screening Daridorexant against panels of other receptors, ion channels, and transporters to confirm its target specificity.

The functional antagonistic activity of Daridorexant hydrochloride 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. Daridorexant hydrochloride 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 Daridorexant hydrochloride 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 .

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In vitro cellular assays for Daridorexant measure functional antagonism at orexin receptors using cell lines expressing recombinant human OX1R or OX2R (e.g., HEK-293, CHO cells). The primary functional readout is intracellular calcium mobilization, measured using calcium-sensitive fluorescent dyes (e.g., Fluo-4) following orexin-A stimulation. Cells are pretreated with Daridorexant at various concentrations, then stimulated with an EC80 concentration of orexin-A, and the inhibition of calcium flux is measured. Concentration-response curves are generated to determine IC50 values for antagonism. Schild analysis may be performed to determine the nature of antagonism (competitive vs. non-competitive). β-arrestin recruitment assays may also be employed to assess signaling bias.

Specific detailed animal experimental protocols for Daridorexant hydrochloride 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 Daridorexant hydrochloride 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 Daridorexant hydrochloride 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.

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In vivo animal studies with Daridorexant are conducted in rodent models to evaluate sleep-wake effects and orexin receptor occupancy. Electroencephalography (EEG) and electromyography (EMG) recordings are used to monitor sleep-wake states following oral or intraperitoneal administration of the compound. Sleep parameters including total sleep time, sleep latency, wake after sleep onset, and time spent in REM/NREM sleep are quantified. Orexin receptor occupancy is measured using PET imaging with a radiolabeled orexin receptor tracer. Pharmacokinetic-pharmacodynamic modeling correlates plasma concentrations with sleep effects. Chronic dosing studies assess tolerance development and withdrawal effects.

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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Daridorexant hydrochloride 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, Daridorexant hydrochloride does not prolong the QTc interval to any clinically significant extent . The compound has a molecular weight of 487.4 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, Daridorexant hydrochloride has 0 violations, suggesting favorable drug-like properties . For research purposes, Daridorexant hydrochloride 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 .

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Daridorexant hydrochloride exhibits favorable oral bioavailability and pharmacokinetic properties suitable for once-daily dosing. Following oral administration, the compound is rapidly absorbed with Tmax of approximately 1-2 hours and a terminal half-life of approximately 6-8 hours, supporting once-daily bedtime dosing. The compound is extensively metabolized in the liver via cytochrome P450 enzymes, primarily CYP3A4, with metabolites excreted in urine and feces. Daridorexant shows moderate plasma protein binding and good brain penetration, consistent with its CNS target. No clinically significant accumulation occurs with repeated daily dosing, and steady-state is achieved within a few days.

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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Daridorexant hydrochloride has been evaluated for safety in clinical trials. At four times the maximum recommended dose, Daridorexant hydrochloride does not prolong the QTc interval to any clinically significant extent . In a human abuse potential study, Daridorexant hydrochloride 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, Daridorexant hydrochloride 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, Daridorexant hydrochloride 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).

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Daridorexant hydrochloride has been extensively evaluated in preclinical and clinical toxicology studies as part of its regulatory approval. In clinical trials, the compound was generally well-tolerated with a safety profile distinct from GABAergic hypnotics. Common adverse effects include headache, somnolence, and fatigue, which are generally mild to moderate in severity. No significant dependence, withdrawal, or rebound insomnia was observed upon discontinuation. Preclinical toxicology studies assessed acute and repeat-dose toxicity, genotoxicity, reproductive toxicity, and carcinogenicity. The compound showed no significant safety concerns at therapeutic doses. Post-marketing surveillance continues to monitor long-term safety.

[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.

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Daridorexant hydrochloride (Quviviq®) is a dual orexin receptor antagonist approved by the FDA and EMA in January 2022 for the treatment of adult insomnia. Its mechanism involves blocking orexin-A and orexin-B binding to OX1R and OX2R, reducing wake drive and promoting sleep. It represents a novel therapeutic class distinct from GABAergic hypnotics, with a favorable safety profile including low dependence and withdrawal risk. The compound is available by prescription in multiple countries. Ongoing research may explore additional indications where orexin system modulation could be beneficial.

C23H24CL2N6O2

487.4

486.134

1792993-84-0

Nemorexant;1505484-82-1

91809208

Solid powder

3.38

2

5

4

32

693

1

CC1=C(C=CC2=C1N=C(N2)[C@@]3(CCCN3C(=O)C4=C(C=CC(=C4)OC)N5N=CC=N5)C)Cl.Cl

HJVGDXBEMFHGKI-BQAIUKQQSA-N

InChI=1S/C23H23ClN6O2.ClH/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);1H/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;hydrochloride

Daridorexant hydrochloride; 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.)