Orexin (hypocretin) receptor

Orexin (hypocretin) neuropeptides have, among others, been implicated in arousal/sleep control, and antagonizing the orexin signaling pathway has been previously demonstrated to promote sleep in animals and humans. This mechanism opens up a new therapeutic approach to curb excessive wakefulness in insomnia disorder rather than to promote sleep-related signaling. Here we describe the preclinical pharmacological in vitro and in silico characterization of lemborexant ((1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide)), a dual orexin receptor antagonist (DORA), as a novel experimental therapeutic agent for the symptomatic treatment of insomnia disorder and compare its properties to two other DORAs, almorexant and suvorexant. Lemborexant binds to both orexin receptors and functionally inhibits them in a competitive manner with low nanomolar potency, without any species difference apparent among human, rat, and mouse receptors. Binding and dissociation kinetics on both orexin receptors are rapid. Lemborexant is selective for both orexin receptors over 88 other receptors, transporters, and ion channels of important physiologic function. In silico modeling of lemborexant into the orexin receptors showed that it assumes the same type of conformation within the receptor-binding pocket as suvorexant, the π-stacked horseshoe-like conformation[2].

Lemborexant prevented the orexin-promoted increase in ACTH in rats, therefore demonstrating inhibition of the orexin signaling pathway. Furthermore, lemborexant promoted sleep in wild-type mice and rats. Lemborexant promoted REM and non-REM sleep at an equal rate (there was no change in the REM sleep ratio). In contrast, zolpidem reduced REM sleep. The sleep-promoting effect of lemborexant was mediated via the orexin-peptide signaling pathway as demonstrated by a lack of sleep promotion in orexin neuron-deficient mice. Chronic dosing was not associated with a change in effect size or sleep architecture immediately postdosing. Lemborexant did not increase the sedative effects of ethanol or impair motor coordination, showing good safety margin in animals. Pharmacokinetic/pharmacodynamic data for mice and rats were well aligned[1].

Measurement of Affinity by Receptor Binding Assay[2]
The binding affinity was assayed by receptor binding assay (RBA) using a 96-well Flashplate. The membrane fraction was prepared from Chinese Hamster Ovary (CHO) cells expressing human OX1R (hOX1R) or human OX2R (hOX2R). Membrane suspension of hOX1R or hOX2R (5 µg protein/assay) was mixed with test antagonists [lemborexant (0.6–200 nmol/l), almorexant (0.2–200 nmol/l), or suvorexant (0.2–60 nmol/l)], as well as OXA (10 µmol/l) solution or vehicle and [125I]OXA solution (0.2 nmol/l). The mixtures (final volume, 100 µl) were incubated for 30 minutes at room temperature on a 96-well Flashplate. All reaction mixtures were discarded, followed by two washing steps with 200 µl of 25 mmol/l HEPES buffer containing 525 mmol/l NaCl. The remaining radioactivity (in dpm) of each well was measured by TopCount, and inhibitory activity of the test antagonist was calculated using the following formula:
where T is reported in dpm in the presence of test antagonist (test), N is reported in dpm in the presence of 10 µmol/l OXA (nonspecific binding), and C is reported in dpm in the absence of compound (control).
Values in experiments were determined in triplicate (lemborexant, almorexant) or quadruplicate (suvorexant). Experiments with lemborexant were conducted three times in an identical fashion, and IC50 values were calculated for each experiment before averaging for the final IC50 value and its S.E.M. The experiments for almorexant and suvorexant were conducted once, with each value expressed as the mean ± S.E.M. for statistical analysis.

Cell-Based Functional Reporter Enzyme Assay[1]
HEK293 cells were stably transfected with human or mouse OX1R or OX2R and with a reporter system (Chen et al., 1995; Durocher et al., 2000) where a reporter enzyme [placental alkaline phosphatase (PLAP)] (Goto et al.,1996) could be induced upon functional OXR activation through an intracellular Ca2+-dependent reporter unit.
Cells were seeded into 96-well plates at a density of 10,000/well and cultivated overnight in culture medium. Next day, 5 µl of lemborexant solutions were added to cultured cells in 96-well plates to a final culture medium volume of 115 µl (23-fold dilution), resulting in 1, 3, 10, 30, 100, 300, and 1000 nmol/l end concentrations for the incubation of cells.
After the addition of lemborexant and incubation for approximately 2–3 hours at room temperature, orexin peptide agonists human/mouse OXA, human OXB (hOXB), mouse OXB (mOXB), or modified [Ala11, d-Leu15]-OXB were diluted in Dulbecco’s modified Eagle’s medium (containing 0.1% bovine serum albumin and 3.45 µmol/l forskolin), and 10 µl was added to cell wells, resulting in a 115-µl final volume. Final concentrations of peptide agonists ranged from 0.01 to 1000 nmol/l. After mixing by agitation of the plates, cells were incubated at 37°C for about 20 hours, with each respective concentration combination of lemborexant and peptide agonist having been applied to four cell wells. There are two amino acids different between hOXB and mOXB. For this reason, hOX2R was activated with hOXB, and mouse OX2R (mOX2R) was activated with mOXB. [Ala11, d-Leu15]-OXB has been described to be of higher selectivity for OX2R than natural OXB (Asahi et al., 2003).

Rodent (wild-type rats and wild-type and orexin neuron-deficient [orexin/ataxin-3 Tg/+] mice) studies were performed to evaluate the effects of single-dose oral lemborexant (1–300 mg/kg) on orexin-induced increases in plasma adrenocorticotropic hormone (ACTH), locomotor activity, vigilance state measures (wakefulness, nonrapid eye movement [non-REM] sleep, rapid eye movement [REM] sleep), ethanol-induced anesthesia, and motor coordination, and the effects of multiple-dose oral lemborexant (30 mg/kg) on vigilance state measures. Active comparators were almorexant and zolpidem. Pharmacokinetics were assessed after single-dose lemborexant in mice and rats.[1]

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lemborexant, almorexant, and zolpidem were suspended as free bases in the vehicle solution specified for each study. Doses for each compound in the mouse experiments were set based on the minimum necessary dose for sleep promotion in mice, and are therefore different for each compound.[1]

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Effect of single-dose lemborexant on the orexin-induced increase in ACTH in rats.[1]
Male F344 rats (age: 5 weeks; body weight: 85.8–103.5 g) were implanted with infusion cannulae into the left lateral ventricle for intracerebroventricular (i.c.v.) injection. Four to five days after surgery, rats were habituated for oral administration (p.o.) and handling once before the study. Six to seven days after cannula implantation, rats received p.o. vehicle (5% [v/v] dimethyl sulfoxide, 9.5% [v/v] cremophor in saline; n = 10) or lemborexant (5 mL/kg suspended in vehicle) 1, 3, 10, or 30 mg/kg (n = 5, 6, 6, and 5, respectively). One hour later, vehicle control rats received 5 µL of phosphate buffered saline (PBS) or [Ala11, D-Leu15]-orexin B (1 nmol/head, 0.2 mmol/L in PBS) via i.c.v. injection (n = 5 each). All lemborexant pretreated rats received [Ala11, D-Leu15]-orexin B via i.c.v. injection. Fifteen minutes later, rats were decapitated and blood samples were collected with Na2EDTA (100 mg/mL, 100 µL). Blood samples were then centrifuged (1000 × g, 10 min at 4°C) and supernatant plasma was stored at −80°C for later measurement of ACTH and lemborexant concentrations. After decapitation, blue ink was injected i.c.v. to confirm the correct placement of cannulae. Coronal cross-sections were made near the cannulae; placement was judged (by an experienced observer) to be correct if blue ink was visible in the lateral ventricle(s). Data from 3 out of 32 rats with incorrect cannula placement were excluded from analysis.

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Effect of single-dose lemborexant on spontaneous locomotor activity in wild-type and orexin neuron-deficient mice[1]
Wild-type male mice (age: 9 weeks; body weight: 19.4–22.5 g) were dosed p.o. with vehicle (5% [v/v] dimethyl sulfoxide, 10% [v/v] cremophor in 10 mmol/L HCl; 10 mL/kg; n = 16) or lemborexant (30 [n = 8] or 100 mg/kg [n = 7]) at Zeitgeber time 3:40 or 5:30. One hour after dosing, mice were placed in an open field arena and locomotor activity was automatically recorded as infrared light beam breaks as previously described. For activity values, all horizontal and vertical infrared light beam break counts were summed over 1 h after the start of locomotor activity recording.[1]
In a separate study, orexin neuron-deficient mice (age: 18–26 weeks; body weight: 27.9–36.0 g) were dosed p.o. with vehicle (as above; n = 8) or 100 mg/kg lemborexant (n = 8), the maximum dose tested in wild-type mice, at Zeitgeber time 3:40 or 5:30. Thirty minutes later, locomotor activity was recorded and summed over 1 h as described above.

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Effect of single-dose lemborexant on vigilance state measures in wild-type and orexin neuron-deficient mice[1]
Mice were allowed to recover and to habituate to the recording room for 1 week while being housed in recording cages, and were then divided into body-weight matched groups and habituated to p.o. dosing with vehicle (10 mL/kg 0.5% [w/v] methylcellulose 400) at Zeitgeber time 3:30–4:10 for 2 consecutive days. The next day, mice were dosed p.o. with vehicle (n = 5), lemborexant (1 [n = 5] or 10 mg/kg [n = 4]), almorexant (10 [n = 5] or 100 mg/kg [n = 4]), or zolpidem (3 [n = 5] or 30 mg/kg [n = 4]) at Zeitgeber time 3:30–4:10.

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Effect of single-dose lemborexant on vigilance state measures in rats[1]
Under deep sodium pentobarbital anesthesia, male Sprague Dawley rats (age: 7–10 weeks; body weight: 311–449 g) were intraperitoneally implanted with battery-driven, wireless telemetry devices (TL11M2-F40-EET) for EEG/EMG measurements. Two silver screws were fixed 2.0 mm left and right of lambda through the skull bone so as to touch the dura mater. EEG leads were knotted to the screws, while EMG leads were placed intranuchally into pockets formed by blunt dissection of neck muscle left and right of midline. After 9–11 days of recovery in home cages, rats were habituated to the recording room and p.o. dosing procedure for 2 days, while still being housed in the same home cages throughout the experiment. A total of 12 rats were then selected for the study and assigned to body-weight matched groups for five dosing and recording sessions. The sessions were conducted with 2–3 days of intermittent wash-out periods, with p.o. dosing taking place at Zeitgeber time 2:00–3:00 and subsequent recording of EEG/EMG signals for 4 h using a telemetry system for later off-line analysis. Continuous recordings were divided into 10-s epochs and automatically analyzed via an algorithm that determined vigilance states. Automated analysis results were then verified and, if necessary, corrected by a trained observer blinded to treatment. No animal received the same test compound at the same dose twice. There were a total of 10 treatment groups in the study; vehicle (10 mL/kg 0.5% [w/v] methyl-cellulose 400 [n = 6]), lemborexant (3, 10, 30, 100, or 300 mg/kg [all n = 6]), and zolpidem (3, 10, 30, or 100 mg/kg [all n = 6]).

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Effect of chronic-dose lemborexant on vigilance state measures in rats[1]
Male Sprague Dawley rats, fully habituated to experimental conditions from the previously described single-dose study, were used after a 5-day washout period. All rats were dosed p.o., once-daily at Zeitgeber time 2:00–3:00, with vehicle (0.5% [w/v] methylcellulose 400) on day 1–3, then vehicle (n = 2), lemborexant 30 mg/kg (n = 5), or zolpidem 100 mg/kg (n = 5) from day 4 to 24, and finally vehicle on days 25 and 26. EEG/EMG signals were recorded (as already described for the single-dose study) on days 1 and 2 (pretreatment), days 4, 7, 11, 14, 18, 21, and 24 (treatment), and days 25 and 26 (posttreatment) for nearly 3 hours. Lemborexant and zolpidem doses were chosen based on the maximum effects in the previous single-dose experiment.

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Effect of single-dose lemborexant on ethanol-induced anesthesia in wild-type mice[1]
Wild-type male mice (age: 13 weeks; body weight: 21.8–28.1 g) were dosed p.o. with vehicle (10 mL/kg 0.5% [w/v] methylcellulose 400), lemborexant (1, 3, or 10 mg/kg), almorexant (30, 100, or 300 mg/kg), or zolpidem (3, 10, or 30 mg/kg) (all groups, n = 6) during the light phase. After 5 min, mice received intraperitoneal injections of 3.0 g/kg ethanol (20% [w/v] in saline). The almorexant and zolpidem doses were based on those used in a previous rat study, where almorexant up to 300 mg/kg did not show interaction with ethanol, but zolpidem from 10 mg/kg upwards showed interaction with ethanol.

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Effect of single-dose lemborexant on motor coordination in wild-type mice [1]
For this study, male wild-type mice (age: 14 weeks; body weight: 22.1–29.1 g), previously trained on the treadmill for 3 consecutive days with subsequent 14 days rest, were allocated to body-weight equivalent groups to receive single p.o. dosing of vehicle (10 mL/kg 0.5% [w/v] methylcellulose 400), lemborexant (30, 100, or 300 mg/kg), or zolpidem (100 mg/kg) (all groups, n = 11). The starting lemborexant dose (30 mg/kg) was selected as this is approximately threefold the sleep-promoting dose, while zolpidem 100 mg/kg has previously been reported to impair motor coordination in rats

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Plasma and CSF concentrations of lemborexant after single dosing in rats[1]
Male Sprague Dawley rats (age: 9 weeks; body weight: 337–355 g) were dosed p.o. with lemborexant 30 mg/kg in 0.5% (w/v) methylcellulose 400 (5 mL/kg) at Zeitgeber time 4:30–5:00. Two hours later, rats were anesthetized and plasma and CSF samples were obtained from the abdominal aorta and cisterna magna, respectively, for the measurement of lemborexant concentrations by LC-MS/MS.

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Plasma concentrations of lemborexant after single dosing in mice[1]
Male C57BL/6N mice (age: 14 weeks; body weight: 26.7–31.7 g) were dosed p.o. with lemborexant 10 or 300 mg/kg in 0.5% [w/v] methylcellulose 400 (10 mL/kg) at Zeitgeber time 3:00–9:15. At 0.25, 0.5, 1, 3, 5, 6, 18, and 24 h after dosing, mice were anesthetized and plasma samples were obtained from the abdominal aorta for measurement of lemborexant concentrations by LC-MS/MS.

Absorption, Distribution and Excretion
Animal models of lemborexant disposition have demonstrated rapid absorption following oral administration. The Tmax of lemborexant is approximately 1-3 hours, or 3-5 hours following administration of a high-fat, high-calorie meal. Cmax and AUC0-24h increase at a rate slightly less than proportionate to the given dose. Following administration of a high-fat, high-calorie meal, Cmax is decreased by 23% and AUC0-inf is increased by 18%. AUC, Cmax, and terminal half-life are increased in the presence of moderate hepatic impairment, and AUC (but not half-life) is increased in the presence of mild hepatic impairment.
Following oral administration, 57.4% of the dose is found in the feces and 29.1% in the urine. Less than 1% of the dose recovered in the urine exists as unchanged parent drug, suggesting extensive metabolism.
The volume of distribution of lemborexant is 1970 L, indicating extensive tissue distribution.

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Metabolism / Metabolites
Given that less than 1% of an administered dose is recovered unchanged in the urine, it is likely that lemborexant is extensively metabolized - this has been confirmed in rat and monkey models, but its metabolism in humans has not been fully characterized. Prescribing information states that it is predominantly metabolized by CYP3A4, with a smaller contribution by CYP3A5. The major circulating metabolite is lemborexant's M10 metabolite, which is pharmacologically active and binds to orexin receptors with a similar affinity to the parent drug. The M10 metabolite has the potential to induce CYP3A and CYP2B6 enzymes, weakly inhibit CYP3A enzymes, and is a substrate of P-gp transporters.

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Biological Half-Life
The half-life for lemborexant at doses of 5mg and 10mg is 17 and 19 hours, respectively.

Hepatotoxicity
In several clinical trials, lemborexant was found to be well tolerated, with serum ALT elevations above 3 times the upper limit of normal in less than 1% of treated subjects and with similar rates in placebo recipients. The elevations were transient and asymptomatic, none required dose modification or discontinuation, and none were associated with a simultaneous elevation in serum bilirubin. Thus, in the registration trials of lemborexant, there were no reports of clinically apparent liver injury. Lemborexant has been available for a limited period of time, but has yet to be implicated in causing clinically apparent liver injury with its more widespread clinical use.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Amounts of lemborexant in milk appear to be low. If lemborexant is required by the mother, it is not a reason to discontinue breastfeeding. However, until more data become available, monitor the infant for sedation, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Lemborexant is approximately 94% protein-bound _in vitro_, though the specific proteins to which it binds in plasma have not been elucidated.

[1]. Preclinical in vivo characterization of lemborexant (E2006), a novel dual orexin receptor antagonist for sleep/wake regulation. Sleep. 2019 Mar 29;42(6):zsz076.

[2]. In Vitro and In Silico Characterization of Lemborexant (E2006), a Novel Dual Orexin Receptor Antagonist. J Pharmacol Exp Ther . 2017 Aug;362(2):287-295.

[3]. OREXIN\nRECEPTOR ANTAGONIST PROVEN EFFECTIVE FOR BOTH SLEEP ONSET AND SLEEP\nMAINTENANCE IN CLINICAL DEVELOPMENT PROGRAM OF MORE THAN 2,000 PATIENTS.\n2019.

[4]. HIGHLIGHTS OF PRESCRIBING INFORMATION

Lemborexant is a DEA Schedule IV controlled substance. Substances in the DEA Schedule IV have a low potential for abuse relative to substances in Schedule III. It is a Depressants substance.
Lemborexant is a novel dual orexin receptor antagonist used in the treatment of insomnia characterized by difficulties with sleep onset and/or sleep maintenance. Recent research in the field of sleep disorders has revealed that insomnia is likely driven not by the inability of the brain to "switch on" sleep-related circuits, but rather an inability to "switch-off" wake-promoting circuits. Whereas historically popular pharmacologic treatments for insomnia (e.g. [zopiclone], [zolpidem], benzodiazepines) focus on enhancing sleep drive via modulation of GABA and melatonin receptors, lemborexant and other orexin antagonists (e.g. [suvorexant]) act to counteract inappropriate wakefulness. This novel mechanism of action offers potential advantages over classic hypnotic agents, including a more favorable adverse effect profile and potentially greater efficacy, and may signal the beginning of a new wave of treatment options for patients suffering from insomnia.
Lemborexant is an Orexin Receptor Antagonist. The mechanism of action of lemborexant is as an Orexin Receptor Antagonist, and Cytochrome P450 2B6 Inducer.
Lemborexant is an orexin receptor antagonist used for the treatment of insomnia and sleep disorders. Lemborexant therapy is associated with rare occurrence of transient serum enzyme elevations, but has not been implicated in cases of clinically apparent liver injury.

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Drug Indication
Lemborexant is indicated for the treatment of adult patients with insomnia characterized by difficulties with sleep onset and/or sleep maintenance.
FDA Label

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Mechanism of Action
The orexin neuropeptide signaling system is involved in many physiologic functions, including sleep/wake control. Orexin-A and orexin-B activate post-synaptic G-protein coupled orexin-1 receptors (OX1R) and orexin-2 receptors (OX2R), which are found on neurons in the hypothalamus that project to numerous wake-controlling nuclei. Each receptor carries slightly different activity - activation of OX1R appears to suppress the onset of rapid eye movement (REM) sleep, whereas activation of OX2R appears to suppress non-REM sleep. Lemborexant is an competitive antagonist of OX1R and OX2R. By blocking the binding of wake-promoting orexin-A and -B at these receptors, lemborexant suppresses the wake-drive, thereby promoting sleep.

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Pharmacodynamics
Lemborexant promotes sleep by antagonizing the actions of wake-promoting chemicals in the brain. Episodes of complex sleep behaviors (e.g. eating food, having sex, making phone calls) have been reported in patients using lemborexant - these events may occur in hypnotic-naive and hyponotic-experienced patients, and patients are unlikely to remember these events. Patients exhibiting complex sleep behaviors should discontinue lemborexant immediately. Lemborexant may carry some risk of abuse, and should be used with caution in patients with a history of alcohol or drug addiction. Its controlled substance schedule is currently under review by the Drug Enforcement Administration.

C22H20F2N4O2

410.4248

410.155

1369764-02-2

1369764-02-2

56944144

Typically exists as solid at room temperature

1.3±0.1 g/cm3

596.1±50.0 °C at 760 mmHg

314.3±30.1 °C

0.0±1.7 mmHg at 25°C

1.619

3.16

1

7

6

30

612

2

O=C([C@H]1[C@@](C2=CC=CC(F)=C2)(COC3=CN=C(C)N=C3C)C1)NC4=NC=C(F)C=C4

MUGXRYIUWFITCP-PGRDOPGGSA-N

InChI=1S/C22H20F2N4O2/c1-13-19(11-25-14(2)27-13)30-12-22(15-4-3-5-16(23)8-15)9-18(22)21(29)28-20-7-6-17(24)10-26-20/h3-8,10-11,18H,9,12H2,1-2H3,(H,26,28,29)/t18-,22+/m0/s1

(1R,2S)-2-[(2,4-dimethylpyrimidin-5-yl)oxymethyl]-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropane-1-carboxamide

E-2006; Dayvigo; E 2006; 1369764-02-2; Dayvigo; UNII-0K5743G68X; 0K5743G68X; (1R,2S)-2-[(2,4-dimethylpyrimidin-5-yl)oxymethyl]-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropane-1-carboxamide; E2006; Lemborexant

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.)