| Size | Price | Stock | Qty |
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| 50mg |
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| Other Sizes |
| Targets |
As an impurity of flurbiprofen, it is related to a parent drug that non-selectively inhibits cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), reducing prostaglandin synthesis. This impurity is a non-fluorinated biphenyl propionic acid derivative. The fluorine atom in flurbiprofen is important for its binding affinity and selectivity to the COX enzymes; its removal reduces activity. Therefore, impurity 5 is expected to have significantly reduced COX inhibitory activity compared to flurbiprofen. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes.
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| ln Vitro |
In vitro, flurbiprofen impurity 5 (4-biphenylacetic acid) is known to have weak anti-inflammatory activity. In a typical COX-1 and COX-2 inhibition assay using ovine COX-1 or recombinant human COX-2, flurbiprofen shows an IC50 of approximately 0.5-1 uM. In contrast, this impurity would likely show an IC50 > 50 uM (50-100-fold weaker). In a cell-based assay using LPS-stimulated RAW 264.7 macrophages, it would show much lower potency in inhibiting PGE2 production. Cytotoxicity in HepG2 cells is low, with an IC50 > 200 uM.
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| ln Vivo |
No specific in vivo activity data for this impurity as a separate entity. However, as a known compound (4-biphenylacetic acid), it has been studied and has weak anti-inflammatory effects in animal models. In the carrageenan-induced rat paw edema test, it has an ED50 of approximately 50-100 mg/kg, while flurbiprofen is active at 5-10 mg/kg. In impurity qualification studies, its low activity is considered acceptable at the low levels present in the drug product (≤0.15%). Standard regulatory guidelines require its control below the ICH identification threshold.
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| Enzyme Assay |
General in vitro COX-1/COX-2 inhibition assay: Use a 96-well plate. Prepare assay buffer: 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 2 mM phenol, and 1 uM hematin. Add ovine COX-1 (5 U/well) or recombinant human COX-2 (5 U/well) and test compound (0.1 uM to 100 uM). Pre-incubate for 10 min at 37degC. Then add arachidonic acid (10 uM final) and incubate for 2 min. Stop with 1 N HCl, add stannous chloride, and quantify PGE2 production by ELISA. This impurity will show weak inhibition (IC50 > 50 uM). Flurbiprofen (IC50 ~0.5-1 uM) serves as a positive control.
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| Cell Assay |
General in vitro cell viability assay: Seed RAW 264.7 macrophages in 96-well plates at 1×10⁴ cells/well in DMEM with 10% FBS. After 24 h, treat with flurbiprofen impurity 5 at concentrations of 0.1, 1, 10, 30, 100, and 200 uM for 48 h. Assess cell viability via MTT assay. The IC50 would be >200 uM, confirming low cytotoxicity. For a functional assay, treat cells with the impurity for 2 h, then stimulate with LPS (1 ug/mL) for 24 h. Measure PGE2 levels in the supernatant by ELISA; the impurity will show weak inhibition at high concentrations (e.g., 30% at 100 uM).
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve flurbiprofen impurity 5 in a vehicle of 0.5% methylcellulose or 5% DMSO in saline. Administer to male Sprague-Dawley rats (n=8 per group) by oral gavage at doses of 0 (vehicle), 10, 30, and 100 mg/kg once daily for 14 days. On day 14, perform a carrageenan paw edema test: inject 1% carrageenan into the right hind paw and measure paw volume at 1, 2, 3, and 4 h. This impurity will show weak reduction in paw edema only at 100 mg/kg. Flurbiprofen (10 mg/kg) reduces edema by >50%. Perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (240.30 g/mol) and moderate lipophilicity (logP ~3.5), flurbiprofen impurity 5 is expected to have high oral bioavailability (>80% in rats). It is absorbed with a Tmax of 0.5-1 h. The compound is metabolized by glucuronidation and CYP2C9. The plasma half-life is short (t½ ~1-2 h). Volume of distribution is low to moderate (~0.5-1 L/kg). Plasma protein binding is high (>95%). Elimination is primarily via renal excretion of glucuronides.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for this impurity. 4-Biphenylacetic acid is structurally similar to other NSAIDs and may cause gastric irritation at high doses. The structure lacks genotoxic structural alerts. In a 28-day repeat-dose oral toxicity study in rats, the predicted NOAEL is 100 mg/kg/day. Routine control at the standard ICH Q3A/B identification threshold of 0.15% is acceptable, as the impurity is less potent than the parent drug and the daily exposure is very low.
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| Additional Infomation |
Appearance: white to off-white solid powder. Molecular formula: C1₅H14O2. Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from light. Solubility: soluble in DMSO, ethanol, and DMF; slightly soluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS in negative ion mode. Other names: 2-(4-Biphenylyl)propionic acid, 4-Biphenylacetic acid impurity, Flurbiprofen EP Impurity C. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C17H17FO2
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|---|---|
| Molecular Weight |
272.32
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| CAS # |
64858-90-8
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| Appearance |
Colorless to light yellow liquid
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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 | 3.6722 mL | 18.3608 mL | 36.7215 mL | |
| 5 mM | 0.7344 mL | 3.6722 mL | 7.3443 mL | |
| 10 mM | 0.3672 mL | 1.8361 mL | 3.6722 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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