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| Targets |
7-Hydroxywarfarin-d5 is a deuterium-labeled internal standard and does not directly target biological receptors. The non-labeled parent compound, warfarin, is an anticoagulant that acts by inhibiting vitamin K epoxide reductase (VKOR), an enzyme responsible for reducing vitamin K epoxide to its active form, vitamin K hydroquinone. This inhibition prevents the gamma-carboxylation of coagulation factors II, VII, IX, and X, thereby reducing their activity and prolonging clotting time. 7-Hydroxywarfarin is the major metabolite of warfarin, formed primarily by CYP2C9. This metabolite has significantly reduced anticoagulant activity compared to warfarin and is considered an inactive metabolite. The deuterated version is used solely as an analytical standard for quantifying 7-Hydroxywarfarin in pharmacokinetic studies and does not engage in biological interactions.
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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].
As a deuterium-labeled internal standard, 7-Hydroxywarfarin-d5 itself does not possess intrinsic in vitro biological activity. It is used exclusively for analytical quantification purposes. The non-labeled 7-Hydroxywarfarin is the major metabolite of warfarin and has minimal anticoagulant activity compared to the parent drug. In vitro, warfarin (the parent drug) inhibits vitamin K epoxide reductase (VKOR) with an IC50 in the low nanomolar range (e.g., 0.5-5 uM depending on the isoform). 7-Hydroxywarfarin has been shown to have very weak VKOR inhibitory activity, with an IC50 >50 uM. The metabolite can be detected in human liver microsomes after incubation with warfarin, and its formation is used as a marker of CYP2C9 activity. The deuterated version is used as an internal standard to accurately quantify 7-Hydroxywarfarin in in vitro metabolism studies, such as those using liver microsomes or recombinant CYP enzymes. |
| ln Vivo |
7-Hydroxywarfarin-d5 does not exhibit in vivo biological activity because it is an analytical standard. The non-labeled 7-Hydroxywarfarin is a major metabolite of warfarin and is considered an inactive metabolite. Warfarin, the parent drug, is a widely used oral anticoagulant that prevents thromboembolic events by inhibiting vitamin K epoxide reductase (VKOR). The metabolism of warfarin is predominantly mediated by CYP2C9, which produces 6- and 7-hydroxywarfarin. Patients with genetic polymorphisms in CYP2C9 (e.g., CYP2C9*2, CYP2C9*3) have reduced clearance of warfarin and are at increased risk of bleeding complications. The formation of 7-hydroxywarfarin is often used as a metric of CYP2C9 activity in clinical drug interaction studies. As an internal standard, the deuterated form is used to accurately quantify 7-Hydroxywarfarin in plasma and urine for pharmacokinetic studies.
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| Enzyme Assay |
A typical non-cellular protocol for using 7-Hydroxywarfarin-d5 as an internal standard involves its incorporation into the sample preparation workflow for LC-MS analysis. A stock solution of the internal standard (1 mg/mL) is prepared in methanol or DMSO. For plasma samples, 50 uL of plasma is transferred to a microcentrifuge tube. Then, 10 uL of the internal standard solution (diluted to a working concentration of 1 ug/mL) is added. Proteins are precipitated by adding 150 uL of ice-cold acetonitrile containing 0.1% formic acid. The mixture is vortexed for 1 minute and centrifuged at 14,000 rpm for 10 minutes at 4degC. The supernatant (150 uL) is transferred to an autosampler vial and mixed with 150 uL of water containing 0.1% formic acid. The sample (5-10 uL) is injected onto an LC-MS/MS system operated in negative ion mode (because 7-Hydroxywarfarin contains a phenolic OH group) with multiple reaction monitoring (MRM) for the specific transitions of 7-Hydroxywarfarin and the deuterated internal standard. Quantification is achieved by calculating the peak area ratio of the analyte to the internal standard against a calibration curve.
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| Cell Assay |
A typical in vitro cellular protocol for using 7-Hydroxywarfarin-d5 as an internal standard involves the quantification of 7-Hydroxywarfarin in cultured hepatocytes or liver microsomes. Human primary hepatocytes or liver microsomes (0.5 mg/mL protein) are incubated with warfarin (1-100 uM) and NADPH (1 mM) in phosphate buffer (pH 7.4) at 37degC for 30-60 minutes. The reaction is terminated by adding ice-cold acetonitrile containing 7-Hydroxywarfarin-d5 as the internal standard. After centrifugation, the supernatant is analyzed by LC-MS/MS. The concentration of 7-Hydroxywarfarin formed is quantified using a calibration curve. The internal standard corrects for extraction efficiency and matrix effects. This assay is commonly used to evaluate the metabolic activity of CYP2C9 and to assess potential drug-drug interactions.
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| Animal Protocol |
A typical in vivo animal protocol for using 7-Hydroxywarfarin-d5 as an internal standard involves the quantification of warfarin and its metabolite 7-Hydroxywarfarin in plasma. Male Sprague-Dawley rats (200-250 g) are administered warfarin (non-labeled) via oral gavage at a dose of 0.5-2 mg/kg. Blood samples (200 uL) are collected via the tail vein into EDTA-coated tubes at various time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 hours) post-dose. Plasma is separated by centrifugation (2,000 g, 10 minutes, 4degC). For extraction, 50 uL of plasma is mixed with 10 uL of 7-Hydroxywarfarin-d5 internal standard solution (1 ug/mL) and 150 uL of acetonitrile. After centrifugation, the supernatant is analyzed by LC-MS/MS. The concentrations of warfarin and 7-Hydroxywarfarin are quantified using calibration curves prepared with the same internal standard. The internal standard corrects for variations in sample preparation and matrix effects.
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| ADME/Pharmacokinetics |
As an analytical internal standard, 7-Hydroxywarfarin-d5 is not characterized by typical pharmacokinetic parameters. The non-labeled 7-Hydroxywarfarin is a major metabolite of warfarin. After oral administration of warfarin, it is extensively metabolized, primarily by CYP2C9, to 6- and 7-hydroxywarfarin. In humans, the plasma concentration of 7-Hydroxywarfarin is typically lower than that of warfarin due to rapid clearance. The half-life of warfarin is approximately 40 hours in humans, while the half-life of its metabolites is shorter. The formation of 7-Hydroxywarfarin is dependent on CYP2C9 activity, and its plasma concentration can be used as a marker of CYP2C9 function. The deuterated internal standard is used to accurately quantify 7-Hydroxywarfarin in biological samples for pharmacokinetic and drug interaction studies.
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| Toxicity/Toxicokinetics |
Toxicity data specific to 7-Hydroxywarfarin-d5 are not available. The non-labeled 7-Hydroxywarfarin is a major metabolite of warfarin and has significantly reduced anticoagulant activity compared to warfarin. It is not considered toxic at concentrations typically observed in vivo. Warfarin, the parent drug, has a narrow therapeutic index and can cause serious bleeding complications, including intracranial hemorrhage, gastrointestinal bleeding, and hematuria. The risk of bleeding is increased in patients with genetic polymorphisms in CYP2C9 or VKORC1, as well as in those taking interacting drugs. Standard laboratory safety precautions should be followed when handling 7-Hydroxywarfarin-d5, including the use of gloves, lab coats, and safety glasses. The compound should be stored at -20degC or as recommended by the supplier, protected from light and moisture. It is for research use only and should not be used in humans or animals for therapeutic or diagnostic purposes.
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| References | |
| Additional Infomation |
7-Hydroxywarfarin-d5 (CAS# 94820-65-2) is a stable isotope-labeled compound with a molecular weight of 329.36. The molecular formula is C19H11D5O5, and it is also known as 7-Hydroxy Warfarin-d5, 7-Hydroxywarfarin-D5 (phenyl-D5), and 4,7-dihydroxy-3-[3-oxo-1-(phenyl-d5)butyl]-2H-1-benzopyran-2-one. The isotopic purity is typically greater than 98%, and the chemical purity is ≥95%. 7-Hydroxywarfarin is the major metabolite of the anticoagulant drug warfarin, formed by cytochrome P450 2C9 (CYP2C9)-mediated hydroxylation. The deuterated version is intended for use as an internal standard for the quantification of 7-Hydroxywarfarin by GC- or LC-MS. This product is for research use only and is not approved for clinical diagnostic applications. The compound should be stored at -20degC, protected from light and moisture.
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| Molecular Formula |
C19H11D5O5
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| Molecular Weight |
329.36
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| Exact Mass |
405.162
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| CAS # |
94820-65-2
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| PubChem CID |
71311832
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| Appearance |
White to off-white solid powder
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| Melting Point |
201-203ºC
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| LogP |
4.107
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
532
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C1=C(C(=C(C(=C1[2H])[2H])C(CC(=O)C)C2=C(C3=C(C=C(C=C3)O)OC2=O)O)[2H])[2H]
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| InChi Key |
SKFYEJMLNMTTJA-VIQYUKPQSA-N
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| InChi Code |
InChI=1S/C19H16O5/c1-11(20)9-15(12-5-3-2-4-6-12)17-18(22)14-8-7-13(21)10-16(14)24-19(17)23/h2-8,10,15,21-22H,9H2,1H3/i2D,3D,4D,5D,6D
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| Chemical Name |
4,7-dihydroxy-3-[3-oxo-1-(2,3,4,5,6-pentadeuteriophenyl)butyl]chromen-2-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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | 3.0362 mL | 15.1810 mL | 30.3619 mL | |
| 5 mM | 0.6072 mL | 3.0362 mL | 6.0724 mL | |
| 10 mM | 0.3036 mL | 1.5181 mL | 3.0362 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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