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4'-Hydroxy Flurbiprofen-d3 is a stable isotope-labeled internal standard. Its unlabeled parent, 4‘-Hydroxy Flurbiprofen, is a major metabolite of the NSAID Flurbiprofen, formed by cytochrome P450 (CYP) enzymes, primarily CYP2C9. This metabolite retains the ability to inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), although its potency is significantly reduced (approximately 10-30% of the parent drug). The target of both Flurbiprofen and its 4‘-hydroxy metabolite is the active site of the cyclooxygenase enzymes, where they block the conversion of arachidonic acid to prostaglandin H2 (PGH2). The IC₅0 of the metabolite for COX-1 and COX-2 is in the high micromolar range (10-100 uM), whereas the parent drug has low micromolar (0.5-5 uM) activity. The metabolite is pharmacologically less active but contributes to the overall drug effect, particularly in patients with reduced CYP2C9 activity (poor metabolizers). The deuterated version is used as an analytical standard.
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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 4‘-Hydroxy Flurbiprofen-d3 is presumed to be identical to its unlabeled parent. The unlabeled 4'-Hydroxy Flurbiprofen is a relatively weak inhibitor of COX-1 and COX-2. In an ovine COX-1/peroxidase assay, 4‘-Hydroxy Flurbiprofen inhibits COX-1 with an IC₅0 of approximately 50-100 uM, compared to an IC₅0 of 0.5-1 uM for the parent Flurbiprofen. In a recombinant human COX-2 assay, the IC₅0 is approximately 100-200 uM. In the human whole blood assay: for COX-1 (serum TxB2 generation), the IC₅0 of the metabolite is approximately 30-50 uM (versus 0.5-2 uM for Flurbiprofen). For COX-2 (LPS-induced PGE2), the IC₅0 is approximately 50-100 uM. The reduced potency is due to the presence of the polar 4‘-hydroxy group, which reduces the compound's ability to penetrate the hydrophobic channel of the COX enzymes. The labeled 4'-Hydroxy Flurbiprofen-d3 is not used for efficacy studies but as an internal standard to quantify the metabolite in in vitro and ex vivo samples. |
| ln Vivo |
A generic non-cell-based assay for 4‘-Hydroxy Flurbiprofen-d3 involves its use as an internal standard in an LC-MS/MS method for quantifying Flurbiprofen and its metabolite in human plasma. Prepare a standard stock solution of unlabeled Flurbiprofen and 4'-Hydroxy Flurbiprofen in methanol (1 mg/mL each). Prepare a separate stock solution of 4‘-Hydroxy Flurbiprofen-d3 at the same concentration. Prepare calibration standards by spiking the unlabeled analytes into a blank matrix (e.g., charcoal-stripped human plasma) to achieve concentrations ranging from 5 to 2,000 ng/mL. Add a fixed concentration of the internal standard (e.g., 100 ng/mL) to each calibration standard. For sample preparation, add 200 uL of acetonitrile to 50 uL of plasma to precipitate proteins. Vortex, centrifuge at 12,000g for 10 minutes. Transfer the supernatant to an autosampler vial. Analyze by LC-MS/MS in negative ion mode. Monitor the mass transitions: m/z 243 → 199 for Flurbiprofen (loss of CO2), m/z 259 → 215 for 4‘-Hydroxy Flurbiprofen (loss of CO2), and m/z 262 → 218 for the 4'-Hydroxy Flurbiprofen-d3 internal standard. Construct separate calibration curves for each analyte (parent and metabolite) by plotting the peak area ratio (analyte/IS) vs. the nominal concentration. This method is used in clinical pharmacokinetic studies.
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| Enzyme Assay |
A standard in vitro cell-based protocol for 4‘-Hydroxy Flurbiprofen-d3 is not common, as it is a metabolite. However, to study its formation, a human liver microsome (HLM) assay can be used. Incubate unlabeled Flurbiprofen (10 uM) with human liver microsomes (HLMs, 0.5 mg protein/mL) in 100 mM potassium phosphate buffer (pH 7.4) containing 5 mM MgCl2 and 1 mM NADPH (cofactor) at 37degC. Incubate for 0, 5, 10, 20, 30, 45, 60 minutes. Terminate the reaction by adding an equal volume of ice-cold acetonitrile. Add a fixed amount of 4‘-Hydroxy Flurbiprofen-d3 as an internal standard to each sample. Centrifuge at 12,000g for 10 minutes. Analyze the supernatant by LC-MS/MS. Quantify the formation of 4'-Hydroxy Flurbiprofen over time (in pmol/min/mg protein). This is a standard protocol for assessing CYP2C9 activity in vitro (since Flurbiprofen is a CYP2C9 probe substrate). To study the effects of co-incubated drugs (potential CYP2C9 inhibitors) on metabolite formation, add varying concentrations (0.1-100 uM) of the test inhibitor to the microsome mix. Calculate the IC₅0 for inhibition of 4‘-Hydroxy Flurbiprofen formation.
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| Cell Assay |
A typical in vivo animal protocol for 4'-Hydroxy Flurbiprofen-d3 involves a pharmacokinetic (PK) and metabolism study in rats. Use male Sprague-Dawley rats (250-300 g, n = 6 per group). Administer a single oral dose of unlabeled Flurbiprofen (5-10 mg/kg) suspended in 0.5% methylcellulose. Collect blood samples via tail vein at various time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36 h) into heparinized tubes. Centrifuge to obtain plasma. For bioanalysis, spike plasma samples (50 uL) with a fixed amount of 4‘-Hydroxy Flurbiprofen-d3 as the internal standard. Precipitate proteins with acetonitrile, centrifuge, and analyze by LC-MS/MS to quantify both the parent Flurbiprofen and its 4'-hydroxy metabolite. Calculate the PK parameters (Cmax, Tmax, AUC) for both parent and metabolite. Calculate the metabolic ratio (MR = AUC_metabolite / AUC_parent) as a measure of the extent of metabolism. This study can be repeated in rats pretreated with a selective CYP2C9 inhibitor (e.g., sulfaphenazole) to assess its effect on the MR. This protocol is used to study species differences in Flurbiprofen metabolism (rats are generally poor metabolizers of Flurbiprofen compared to humans) and to evaluate potential drug-drug interactions.
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| Animal Protocol |
4‘-Hydroxy Flurbiprofen-d3 is an analytical internal standard. Its unlabeled parent, 4'-Hydroxy Flurbiprofen, is the major metabolite of Flurbiprofen. The pharmacokinetics of the metabolite are dependent on the PK of the parent drug. In humans, after oral administration of Flurbiprofen (100 mg), Flurbiprofen is rapidly absorbed (Tmax ~ 1-2 hours). It is extensively metabolized by CYP2C9 (and to a lesser extent, CYP2C8) to 4‘-Hydroxy Flurbiprofen. Peak plasma concentrations of the metabolite (Cmax ~ 2-5 ug/mL) are slightly lower than those of the parent drug (Cmax ~ 8-12 ug/mL), and the metabolite Tmax is slightly later (2-3 hours). The half-life of the metabolite is similar to or slightly longer (4-6 hours) than that of the parent Flurbiprofen (3-5 hours). The volume of distribution is small (approximately 0.1-0.2 L/kg for the parent). Both the parent and the metabolite are highly protein-bound (>99%) and are primarily eliminated via renal excretion after glucuronidation (phase II metabolism). The labeled 4‘-Hydroxy Flurbiprofen-d3 is essential for the accurate quantification of this metabolite in PK and metabolism studies using mass spectrometry, especially when analyzing plasma or urine samples with complex matrices.
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| ADME/Pharmacokinetics |
4'-Hydroxy Flurbiprofen-d3 is a research-grade stable isotope-labeled compound, not a pharmaceutical drug. Its unlabeled parent, 4‘-Hydroxy Flurbiprofen, is a metabolite of the NSAID Flurbiprofen. The safety profile of this metabolite is presumed to be similar to that of the parent drug. Flurbiprofen is generally well-tolerated; the most common adverse effects are gastrointestinal (dyspepsia, nausea, abdominal pain, and at high doses, gastric ulcers and bleeding). NSAIDs, including Flurbiprofen, can also increase the risk of cardiovascular events (heart attack, stroke), particularly with long-term use. The metabolite has reduced COX inhibitory activity compared to the parent, and it is not expected to contribute significantly to the toxicity. No genotoxicity or carcinogenicity has been reported for the metabolite. For laboratory handling of 4‘-Hydroxy Flurbiprofen-d3, standard safety precautions for handling NSAIDs and potential irritants should be used: wear gloves, a lab coat, and safety goggles. Avoid contact with skin and eyes; if contact occurs, rinse thoroughly with water. Store as a powder at -20degC in a tightly sealed container, protected from light and moisture. For research use only, not for human diagnostic or therapeutic applications.
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| References | |
| Additional Infomation |
4'-Hydroxy Flurbiprofen-d3 is the stable isotope-labeled version of 4‘-Hydroxy Flurbiprofen, the major circulating metabolite of the non-steroidal anti-inflammatory drug (NSAID) Flurbiprofen. Flurbiprofen (brand name Ansaid, Froben) is a propionic acid derivative NSAID used for the treatment of rheumatoid arthritis, osteoarthritis, and pain management (dysmenorrhea, dental pain). It acts as a non-selective inhibitor of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), reducing the synthesis of prostaglandins involved in pain, fever, and inflammation. 4'-Hydroxy Flurbiprofen is formed primarily by the action of cytochrome P450 2C9 (CYP2C9), a genetically polymorphic enzyme. The rate of formation of this metabolite is used as a probe for CYP2C9 enzyme activity (phenotyping). 4‘-Hydroxy Flurbiprofen-d3 is intended for research use as an internal standard for the accurate quantification of the metabolite and the parent drug in biological samples by LC-MS/MS. This labeled compound is an essential tool for pharmacokinetic studies, drug metabolism research, and drug-drug interaction studies involving CYP2C9. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C15H10D3FO3
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| Molecular Weight |
263.28
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| Exact Mass |
263.104
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| CAS # |
1189694-02-7
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| PubChem CID |
45039449
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| Appearance |
White to off-white solid powder
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| LogP |
3.386
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
19
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| Complexity |
313
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C([2H])([2H])C(C1=CC(=C(C=C1)C2=CC=C(C=C2)O)F)C(=O)O
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| InChi Key |
GTSMMBJBNJDFRA-FIBGUPNXSA-N
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| InChi Code |
InChI=1S/C15H13FO3/c1-9(15(18)19)11-4-7-13(14(16)8-11)10-2-5-12(17)6-3-10/h2-9,17H,1H3,(H,18,19)/i1D3
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| Chemical Name |
3,3,3-trideuterio-2-[3-fluoro-4-(4-hydroxyphenyl)phenyl]propanoic acid
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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 | 3.7982 mL | 18.9912 mL | 37.9824 mL | |
| 5 mM | 0.7596 mL | 3.7982 mL | 7.5965 mL | |
| 10 mM | 0.3798 mL | 1.8991 mL | 3.7982 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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