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Aprepitant-d4 is a stable isotope-labeled internal standard. Its unlabeled parent compound, Aprepitant, is a highly selective, high-affinity antagonist of the neurokinin-1 (NK1) receptor (human NK1 receptor: Ki = 0.09 nM, IC₅0 = 0.09 nM). The NK1 receptor is a G protein-coupled receptor (GPCR) for which the endogenous ligand is substance P (SP), a tachykinin peptide involved in emesis (vomiting) and pain perception. By binding to and blocking NK1 receptors in the central nervous system (specifically the area postrema and nucleus tractus solitarius) and in the periphery, Aprepitant prevents substance P from triggering the emetic reflex. This mechanism is complementary to that of serotonin (5-HT3) receptor antagonists and corticosteroids. The deuterated version mimics this high-affinity binding but is used for analytical quantification in research settings, not for therapeutic purposes.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
The in vitro biological activity of Aprepitant-d4 is presumed to be identical to its unlabeled parent, Aprepitant. In radioligand binding assays using human NK1 receptors expressed in CHO (Chinese hamster ovary) cells, Aprepitant binds with high affinity (Ki = 0.09 nM). It is highly selective for the human NK1 receptor over other tachykinin receptors (NK2 and NK3), with selectivity ratios of >1,000-fold. In functional assays measuring calcium mobilization (a downstream effect of NK1 receptor activation), Aprepitant acts as an antagonist, potently inhibiting the increase in intracellular calcium induced by substance P with an IC₅0 of 0.1-1 nM. In isolated guinea pig ileum preparations, Aprepitant (0.1-10 nM) concentration-dependently inhibits substance P-induced smooth muscle contraction. The labeled Aprepitant-d4 is used as an internal standard in LC-MS analysis to accurately quantify Aprepitant concentrations in these in vitro assay buffers and cell culture media, enabling precise characterization of the dose-response relationship. |
| ln Vivo |
The in vivo activity of Aprepitant-d4 is not directly evaluated; it is used as an internal standard. Its unlabeled parent, Aprepitant, is a clinically effective antiemetic. In ferrets (a standard animal model for emesis), oral administration of Aprepitant (0.1-10 mg/kg) dose-dependently prevents both the acute (0-2 hours) and delayed (2-24 hours) phases of emesis induced by the chemotherapeutic agent cisplatin (10 mg/kg i.v.), with an ED₅0 of 0.5-1 mg/kg for the acute phase and 0.2-0.5 mg/kg for the delayed phase. In the house musk shrew (Suncus murinus), another emesis model, Aprepitant (1-10 mg/kg p.o.) also blocks emesis induced by substance P itself (100 ug/kg i.v.). In humans, Aprepitant (125 mg on Day 1, then 80 mg on Days 2-3) is effective in reducing both acute and delayed CINV when given in combination with a 5-HT3 antagonist and dexamethasone. The labeled Aprepitant-d4 is used as an internal standard for LC-MS to accurately quantify the drug in plasma and tissues, enabling precise PK/PD modeling of the antiemetic effect.
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| Enzyme Assay |
A generic non-cell-based assay for Aprepitant-d4 involves its use as an internal standard in an LC-MS/MS method for quantifying Aprepitant in human plasma. Prepare a standard stock solution of unlabeled Aprepitant in DMSO (1 mg/mL). Prepare a separate stock solution of Aprepitant-d4 at the same concentration. Prepare calibration standards by spiking the unlabeled analyte into a blank matrix (e.g., charcoal-stripped human plasma) to achieve concentrations ranging from 0.5 to 1,000 ng/mL. Add a fixed concentration of the internal standard (e.g., 50 ng/mL) to each calibration standard. For sample preparation, perform liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE). Add 1 mL of MTBE to 100 uL of plasma, vortex for 5 minutes, centrifuge at 3,000 rpm for 10 minutes. Transfer the organic layer (top) to a clean tube and evaporate to dryness under nitrogen at 40degC. Reconstitute the residue in 150 uL of acetonitrile/water (50:50) with 0.1% formic acid. Analyze by LC-MS/MS in positive ESI mode. Monitor the mass transitions: m/z 535 → 277 (for Aprepitant, loss of the triazolone-morpholine moiety), and m/z 539 → 281 for Aprepitant-d4 (due to +4 Da shift from deuterium). Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. the nominal concentration. Method sensitivity should achieve a lower limit of quantification (LLOQ) of 0.5 ng/mL or better.
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| Cell Assay |
A standard in vitro cell-based protocol for the unlabeled Aprepitant involves measuring inhibition of substance P-induced calcium flux in NK1 receptor-expressing cells. Culture CHO cells stably expressing the human NK1 receptor in Ham‘s F12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 ug/mL streptomycin at 37degC in a 5% CO2 incubator. Seed cells in black-walled, clear-bottom 96-well plates at 3×10⁴ cells/well and allow to grow for 24 hours. On the day of the assay, wash the cells twice with HBSS (containing 20 mM HEPES, pH 7.4). Load the cells with the calcium-sensitive fluorescent dye Fluo-4 AM (2 uM in HBSS with 0.02% Pluronic F-127) for 30-45 minutes at 37degC. Wash the cells twice with HBSS to remove excess dye. Add increasing concentrations of unlabeled Aprepitant (0.01, 0.1, 1, 10, 100, 1,000 nM) or vehicle (DMSO, final concentration <0.1%) to the cells and measure baseline fluorescence (Ex/Em = 494/516 nm) for 1 minute. Then, add substance P at a submaximal concentration (e.g., 0.5-1 nM) to stimulate calcium influx. Record the change in fluorescence intensity (peak fluorescence after stimulation minus baseline) over 60 seconds. Calculate the percentage inhibition relative to the substance P-only control. Determine the half-maximal inhibitory concentration (IC₅0) from the dose-response curve (typically 0.1-1 nM). Use Aprepitant-d4 as an internal standard in an LC-MS analysis of the HBSS to confirm the exact drug concentrations used in the assay.
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| Animal Protocol |
A typical in vivo animal protocol for Aprepitant-d4 is a pharmacokinetic (PK) and tissue distribution study in rats or dogs. Use male Sprague-Dawley rats (250-300 g, n = 5-6 per time point). Administer a single oral dose of unlabeled Aprepitant (30 mg/kg) suspended in 0.5% methylcellulose/0.1% Tween 80. Collect blood samples via tail vein at various time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72 h) into heparinized tubes. Centrifuge at 3,000 rpm for 10 minutes to obtain plasma. At selected time points (e.g., 1, 4, 12, 24 h post-dose), euthanize separate groups (n = 3 per time point) and collect brain, liver, kidney, and fat tissues. For bioanalysis, spike plasma samples (50 uL) and tissue homogenates (50 uL, 1:3 w/v in PBS) with a fixed amount of Aprepitant-d4 internal standard. Extract samples by liquid-liquid extraction with MTBE. Evaporate to dryness and reconstitute in mobile phase. Analyze by LC-MS/MS. Calculate PK parameters including Cmax, Tmax, AUC(0-t), t½, oral clearance (CL/F), and volume of distribution (Vz/F). For tissue distribution, calculate tissue-to-plasma ratios. This protocol is used to characterize the PK and CNS penetration of aprepitant.
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| ADME/Pharmacokinetics |
Aprepitant-d4 is an analytical internal standard, so its PK is identical to its unlabeled parent, Aprepitant. Aprepitant is an oral NK1 receptor antagonist approved for chemotherapy-induced nausea and vomiting (CINV). In humans, Aprepitant is slowly absorbed after oral administration, with Tmax of approximately 3-6 hours. Oral bioavailability is moderate (approximately 60-70%) and is increased when taken with a high-fat meal. Aprepitant is highly plasma protein-bound (>99.5%), primarily to albumin and alpha1-acid glycoprotein. The volume of distribution is approximately 70 L in humans, indicating extensive tissue distribution, including penetration into the central nervous system. Aprepitant is extensively metabolized in the liver, primarily by CYP3A4, with minor contributions from CYP2C9 and CYP2C19. It is not a substrate for the efflux transporter P-glycoprotein (P-gp) to a significant degree, unlike some other NK1 antagonists. The elimination half-life is long, approximately 12-18 hours in healthy adults, allowing once-daily dosing. The major route of elimination is via the feces (>80%), with less than 10% excreted unchanged in the urine. The labeled Aprepitant-d4 is critical for accurate quantification of the drug in PK and bioequivalence studies.
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| Toxicity/Toxicokinetics |
Aprepitant-d4 is a research-grade internal standard, not a therapeutic agent. Its unlabeled parent, Aprepitant, is a safe and well-tolerated antiemetic drug. The most common adverse effects in clinical use are mild to moderate and include fatigue (15%), dizziness (10%), hiccups (5%), and constipation (5%). Aprepitant is a moderate inhibitor of CYP3A4 and can significantly increase plasma concentrations of other medications metabolized by this enzyme (such as midazolam, certain statins, and hormonal contraceptives). The oral LD₅0 of Aprepitant in rodents is >2,000 mg/kg, indicating low acute toxicity. In chronic toxicology studies in rats and dogs (6 months), no target organ toxicity was observed at exposures up to 40 times the human therapeutic exposure. Aprepitant is not genotoxic (Ames test negative) and is not carcinogenic in long-term rodent studies. For laboratory handling, standard safety precautions for pharmaceuticals (gloves, lab coat) are sufficient. Aprepitant-d4 should be stored as a powder at -20degC in a tightly sealed container, protected from light and moisture. For research use only, not for human consumption.
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| References | |
| Additional Infomation |
Aprepitant-d4 is the stable isotope-labeled version of Aprepitant, a highly selective, high-affinity antagonist of the neurokinin-1 (NK1) receptor. Aprepitant was the first drug in the NK1 antagonist class to be approved by the FDA (in 2003) and is used clinically for the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV) and for postoperative nausea and vomiting (PONV). It is typically administered as part of a three-drug regimen including a 5-HT3 antagonist (e.g., ondansetron) and dexamethasone. Aprepitant was originally developed by Merck & Co. (known as MK-0869 or L-754030). Its mechanism of action involves blocking substance P (SP) binding to NK1 receptors in the central nervous system, inhibiting the emetic reflex. The parent drug has a long half-life (12-18 hours) and can cross the blood-brain barrier. Aprepitant-d4 serves as a critical internal standard for LC-MS/MS bioanalysis in research applications, including pharmacokinetic studies, bioequivalence studies, and drug-drug interaction research. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C23H17D4F7N4O3
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| Molecular Weight |
538.45
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| Exact Mass |
538.175
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| CAS # |
1133387-60-6
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| PubChem CID |
135872779
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| Appearance |
White to off-white solid powder
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| Melting Point |
242-244°C
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| LogP |
4.89
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
37
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| Complexity |
810
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| Defined Atom Stereocenter Count |
3
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| SMILES |
[2H]C1=C(C(=C(C(=C1[C@H]2[C@H](OCCN2CC3=NNC(=O)N3)O[C@H](C)C4=CC(=CC(=C4)C(F)(F)F)C(F)(F)F)[2H])[2H])F)[2H]
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| InChi Key |
ATALOFNDEOCMKK-NSVJKUEGSA-N
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| InChi Code |
InChI=1S/C23H21F7N4O3/c1-12(14-8-15(22(25,26)27)10-16(9-14)23(28,29)30)37-20-19(13-2-4-17(24)5-3-13)34(6-7-36-20)11-18-31-21(35)33-32-18/h2-5,8-10,12,19-20H,6-7,11H2,1H3,(H2,31,32,33,35)/t12-,19+,20-/m1/s1/i2D,3D,4D,5D
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| Chemical Name |
3-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3-(2,3,5,6-tetradeuterio-4-fluorophenyl)morpholin-4-yl]methyl]-1,4-dihydro-1,2,4-triazol-5-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8572 mL | 9.2859 mL | 18.5718 mL | |
| 5 mM | 0.3714 mL | 1.8572 mL | 3.7144 mL | |
| 10 mM | 0.1857 mL | 0.9286 mL | 1.8572 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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