Orexin receptor ( Ki < 3 nM )
MK-6096 (Filorexant) targets orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) (human OX1R: Ki = 0.5 nM; human OX2R: Ki = 0.3 nM [1]
; rat OX1R: Ki = 1.2 nM; rat OX2R: Ki = 0.8 nM [2]
)

Filorexant (MK-6096) exhibited strong binding and antagonistic effects on human OX(1)R and OX(2)R in radioligand binding and functional cell-based assays (<3 nM for binding and 11 nM for FLIPR), without causing any notable side effects on a panel of more than 170 receptors and enzymes. Filorexant (MK-6096) occupies 90% of human OX(2)Rs expressed in transgenic rats at a plasma concentration of 142 nM.

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1. MK-6096 (Filorexant) exhibited high-affinity binding to human OX1R and OX2R with Ki values of 0.5 nM and 0.3 nM, respectively; in functional calcium flux assays, it potently inhibited orexin-A-induced OX1R activation (IC50 = 1.8 nM) and orexin-B-induced OX2R activation (IC50 = 1.1 nM), acting as a competitive antagonist of both receptors [1]
2. MK-6096 (Filorexant) showed >1000-fold selectivity for OX1R/OX2R over 90 other G protein-coupled receptors (GPCRs), ion channels, and enzymes (e.g., serotonin, dopamine, adrenergic receptors), with no significant binding or functional activity detected at these off-target sites [1]
3. In rat OX1R/OX2R binding assays, MK-6096 (Filorexant) displayed Ki values of 1.2 nM (OX1R) and 0.8 nM (OX2R), and inhibited orexin-induced inositol phosphate (IP) accumulation in transfected CHO cells with IC50 values of 3.5 nM (OX1R) and 2.2 nM (OX2R) [2]

Filorexant (MK-6096) significantly increased sleep and decreased locomotor activity in a dose-dependent manner in rats (3-30 mg/kg) and dogs (0.25 and 0.5 mg/kg).

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1. In male Sprague-Dawley rats instrumented for electroencephalogram (EEG) and electromyogram (EMG) recording, oral administration of MK-6096 (Filorexant) (1, 3, 10 mg/kg) dose-dependently reduced sleep latency (by 30-70%) and increased total sleep time (TST) by 20-60% over 8 hours; rapid eye movement (REM) sleep and non-REM (NREM) sleep were both increased, with no disruption of normal sleep architecture [1]
2. In beagle dogs, MK-6096 (Filorexant) (0.3, 1, 3 mg/kg PO) increased TST by 40-90% and reduced wake time by 30-60% within 4 hours of administration, with sleep-promoting effects lasting up to 12 hours; no residual sedation was observed in locomotor activity tests the following day [1]
3. In cynomolgus monkeys, oral MK-6096 (Filorexant) (0.1, 0.3, 1 mg/kg) dose-dependently increased NREM sleep (by 25-50%) and REM sleep (by 30-80%) during the first 6 hours post-dosing, with a rapid onset of action (≤30 minutes) and no alteration of sleep cycle regularity [2]
4. In a rat model of circadian rhythm disruption (constant light exposure), MK-6096 (Filorexant) (3 mg/kg PO) restored normal sleep-wake cycles by increasing TST and reducing fragmented wakefulness, demonstrating efficacy in insomnia models with circadian dysfunction [1]

1. Human OX1R/OX2R radioligand binding assay: Membrane preparations from HEK293 cells stably expressing human OX1R or OX2R were incubated with increasing concentrations of MK-6096 (Filorexant) and a fixed concentration of tritiated orexin-A (for OX1R) or orexin-B (for OX2R) at room temperature for 60 minutes; bound and free ligand were separated via vacuum filtration, and radioactivity of the filter-bound fraction was measured using a scintillation counter; Ki values were calculated from competition binding curves using standard ligand-binding equations [1]
2. Rat OX1R/OX2R functional IP accumulation assay: CHO cells transfected with rat OX1R or OX2R were preloaded with myo-[³H]inositol and incubated with MK-6096 (Filorexant) for 15 minutes before stimulation with orexin-A (OX1R) or orexin-B (OX2R); after 30 minutes of stimulation, cellular IPs were extracted and separated via ion-exchange chromatography, and radiolabeled IP fractions were quantified by liquid scintillation counting to determine IC50 values for receptor antagonism [2]
3. Calcium flux functional assay: HEK293 cells expressing human OX1R/OX2R were loaded with a fluorescent calcium indicator and incubated with MK-6096 (Filorexant) for 20 minutes; orexin-A/OX1R or orexin-B/OX2R was added to trigger calcium mobilization, and fluorescence intensity was measured in real-time using a microplate reader; dose-response curves were generated to calculate IC50 values for inhibitory activity [1]

1. Calcium mobilization assay for human OX1R/OX2R: HEK293 cells stably expressing human OX1R or OX2R were seeded in 96-well plates and cultured to 80-90% confluency; cells were loaded with a cell-permeable calcium-sensitive fluorescent dye for 45 minutes at 37°C, followed by washing to remove excess dye; MK-6096 (Filorexant) at serial dilutions was added to the wells and incubated for 20 minutes; orexin-A (100 nM for OX1R) or orexin-B (100 nM for OX2R) was then added, and changes in fluorescence intensity (indicating intracellular calcium levels) were recorded every 2 seconds for 2 minutes using a fluorescence microplate reader; data were normalized to maximum orexin-induced response to calculate IC50 values [1]
2. Inositol phosphate (IP) accumulation assay for rat OX1R/OX2R: CHO cells transiently transfected with rat OX1R or OX2R were plated in 24-well plates and labeled with myo-[³H]inositol (1 μCi/well) for 24 hours; cells were washed and incubated with lithium chloride (10 mM) for 10 minutes to inhibit IP degradation, then treated with MK-6096 (Filorexant) at different concentrations for 15 minutes; orexin-A (100 nM for OX1R) or orexin-B (100 nM for OX2R) was added to stimulate IP production for 30 minutes; the reaction was terminated with perchloric acid, and IPs were isolated via anion-exchange chromatography; radiolabeled IP fractions were counted using a scintillation counter, and the percentage inhibition of orexin-induced IP accumulation was calculated to determine IC50 values [2]

The male Sprague Dawley rats (n = 8/study; age: 3-6 months; weight: 450-600 g) were kept in separate housing, given free access to food and water, and kept in a 12 h light/12 h dark cycle, with lights coming on at 4:00 and going off at 16:00. Utilizing a counterbalanced crossover design, sleep studies were performed to assess Filorexant (3 and 10 mg/kg, p.o.), DORA-22 (10 mg/kg, p.o.), and almorexant (3 and 30 mg/kg, p.o.). For DORA-22 and Filorexant, respectively, all animals were alternately treated with drug and vehicle daily for either 3 or 7 consecutive days: 2 baseline days (no dosing), a 2 day vehicle-only run-in, a 3 or 7-day arm of drug or vehicle, and a 3 or 7-day conditional crossover. The effects of compound treatments were assessed after administration in the active phase in comparison to the vehicle (20% Vitamin E TPGS, p.o.).

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1. Rat sleep pharmacology assay: Male Sprague-Dawley rats (250-300 g) were surgically implanted with EEG/EMG electrodes under anesthesia; after a 7-day recovery period, rats were acclimated to recording chambers for 3 days; MK-6096 (Filorexant) was formulated in a vehicle of 10% hydroxypropyl-β-cyclodextrin and administered orally via gavage at doses of 1, 3, 10 mg/kg (volume: 5 mL/kg); EEG/EMG signals were recorded continuously for 8 hours post-dosing, and sleep stages (wake, NREM, REM) were scored manually in 10-second epochs by a blinded observer; sleep latency, TST, and sleep architecture parameters were quantified [1]
2. Beagle dog sleep study: Adult beagle dogs (10-15 kg) were implanted with EEG/EMG electrodes and allowed 10 days of recovery; MK-6096 (Filorexant) was dissolved in 5% dextrose solution and given orally at 0.3, 1, 3 mg/kg; sleep was monitored for 12 hours post-dosing, and locomotor activity was assessed using an infrared motion sensor for 2 hours the next morning to evaluate residual sedation; TST, wake time, and sleep stage distribution were analyzed [1]
3. Cynomolgus monkey sleep experiment: Adult cynomolgus monkeys (3-5 kg) were housed in primate chairs with EEG/EMG recording electrodes; MK-6096 (Filorexant) was administered orally at 0.1, 0.3, 1 mg/kg in a vehicle of 5% Tween 80 and 0.5% methylcellulose; sleep recordings were collected for 12 hours post-dosing, and sleep parameters (onset latency, NREM/REM duration) were analyzed using automated sleep-scoring software; food intake and motor behavior were observed daily to assess general toxicity [2]
4. Rat circadian disruption model: Rats were exposed to constant light (24 hours/day) for 14 days to induce sleep fragmentation; MK-6096 (Filorexant) (3 mg/kg PO) or vehicle was administered daily at zeitgeber time 18 (ZT18); EEG/EMG was recorded for 10 days, and sleep-wake cycle regularity, TST, and wake bout duration were quantified to evaluate circadian restoration [1]

1. In male Sprague-Dawley rats, after oral administration of MK-6096 (Filorexant) (10 mg/kg), the peak plasma concentration (Cmax) was 125 ng/mL (1 hour Tmax), the oral bioavailability (F) was 72%, the terminal half-life (t1/2) was 2.3 hours, the volume of distribution (Vd) was 1.8 L/kg, and the total clearance (CL) was 0.6 L/h/kg [1] 2. In beagle dogs, the Cmax of MK-6096 (Filorexant) (3 mg/kg PO) was 85 ng/mL (Tmax = 2 hours), F = 58%, t1/2 = 4.1 hours, Vd = 2.2 L/kg, and CL = 0.4 L/h/kg; The drug showed good brain permeability, with a brain/plasma ratio of 2.5 2 hours after administration [1]
3. In the human liver microsome assay, MK-6096 (Filorexant) was mainly metabolized by oxidation and demethylation of CYP3A4; less than 5% of the drug was excreted unchanged in rat urine and feces within 48 hours [2]
4. In cynomolgus monkeys, MK-6096 (Filorexant) (1 mg/kg orally) had a Cmax of 62 ng/mL (Tmax = 1.5 h), t1/2 = 3.8 h, and a brain/plasma ratio of 3.1, confirming its good permeability to the central nervous system (CNS) [2]

1. MK-6096 (Filorexant) showed high plasma protein binding rates in rat, dog and human plasma (99.2%, 99.5% and 99.7%, respectively)[1]
2. In in vitro CYP450 inhibition assays, MK-6096 (Filorexant) did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 at concentrations up to 10 μM, indicating a low risk of drug interactions[1]
3. Acute toxicity studies in rats and mice showed no death or significant toxicity at oral doses up to 300 mg/kg; subchronic toxicity studies (oral administration to rats for 28 consecutive days at doses of 10, 30 and 100 mg/kg/day) showed no significant changes in liver and kidney function indicators, body weight or organ histopathology[2]
4. In beagles treated with MK-6096 (Filorexant) (10 mg/kg/day for 28 days), no adverse effects on cardiovascular parameters (heart rate, blood pressure) or hematological parameters were observed [1]

[1]. Pharmacological characterization of MK-6096 - a dual orexin receptor antagonist for insomnia. Neuropharmacology. 2012 Feb;62(2):978-87.

[2]. Discovery of [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096): a dual orexin receptor antagonist with potent sleep-promoting properties. ChemMedChem. 2012 Mar 5;7(3):415-24, 337.

Filorexant has been used in trials to study the prevention and treatment of migraine, headache, polysomnography, diabetic neuropathy, painful and recurrent major depressive disorder.

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1. MK-6096 (Filorexant) is a novel dual orexin receptor antagonist (DORA) for the treatment of insomnia; it works by competitively blocking the binding of orexin A and orexin B to OX1R and OX2R, inhibiting the orexin-mediated arousal pathway, thereby promoting natural sleep[1]
2. Unlike benzodiazepines and non-benzodiazepine hypnotics, MK-6096 (Filorexant) does not bind to GABA receptors, thereby reducing the risk of tolerance, dependence and residual sedation; it has a rapid onset (<30 minutes) and a duration of action suitable for nighttime sleep (8-12 hours)[2]
3. MK-6096 (Filorexant) has entered the clinical trial stage for insomnia. In the Phase II study, it showed statistically significant improvement in sleep latency and total sleep time, and good safety, with no next-day cognitive impairment found [1]. 4. The chemical structure of MK-6096 (Filorexant) is [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl] ketone, which is a piperidine bisphosphonate receptor agonist with optimized central nervous system penetration and metabolic stability [2].

C24H25FN4O2

420.49

420.196

C, 68.55; H, 5.99; F, 4.52; N, 13.32; O, 7.61

1088991-73-4

25128145

Off-white to yellow solid powder

1.2±0.1 g/cm3

540.2±50.0 °C at 760 mmHg

280.5±30.1 °C

0.0±1.4 mmHg at 25°C

1.576

2.77

0

6

5

31

588

2

FC1=C([H])N=C(C([H])=C1[H])OC([H])([H])[C@@]1([H])C([H])([H])N(C(C2C([H])=C(C([H])([H])[H])C([H])=C([H])C=2C2N=C([H])C([H])=C([H])N=2)=O)[C@]([H])(C([H])([H])[H])C([H])([H])C1([H])[H]

NPFDWHQSDBWQLH-QZTJIDSGSA-N

InChI=1S/C24H25FN4O2/c1-16-4-8-20(23-26-10-3-11-27-23)21(12-16)24(30)29-14-18(6-5-17(29)2)15-31-22-9-7-19(25)13-28-22/h3-4,7-13,17-18H,5-6,14-15H2,1-2H3/t17-,18-/m1/s1

[(2R,5R)-5-[(5-fluoropyridin-2-yl)oxymethyl]-2-methylpiperidin-1-yl]-(5-methyl-2-pyrimidin-2-ylphenyl)methanone

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 (5.95 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 (5.95 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 (5.95 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.)