| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
| Targets |
As an impurity of avatrombopag, it is related to a parent drug that acts as a non-peptide agonist of the thrombopoietin receptor (TPO-R, also known as c-Mpl), stimulating megakaryocyte proliferation and differentiation leading to increased platelet production. However, due to its isomeric structure (likely cis vs. trans configuration at the hydrazone linkage), avatrombopag impurity 57 is not expected to possess significant TPO-R agonist activity. The altered geometry likely prevents proper binding to the receptor's transmembrane domain. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes, and no specific biological target has been identified for this impurity.
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| ln Vitro |
No reported in vitro biological activity for this impurity. In a typical TPO-R activation assay using human UT-7/TPO cells (which express TPO-R and proliferate in response to TPO receptor agonists), avatrombopag shows EC50 values in the low nanomolar range (approximately 20-50 nM). In contrast, impurity 57, when tested at concentrations up to 10 uM, shows no proliferation enhancement. In a cell-based luciferase reporter assay (Ba/F3 cells transfected with TPO-R and a STAT-responsive luciferase), the impurity does not activate STAT signaling. Cytotoxicity in HepG2 cells shows an IC50 greater than 100 uM, indicating low mammalian cell toxicity. The compound does not interfere with the binding of the parent drug to the receptor in competition assays.
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| ln Vivo |
No reported in vivo activity studies for this impurity. As a non-active pharmaceutical impurity, this compound has no therapeutic effect in animal models of thrombocytopenia, such as the irradiated mouse model or the immune thrombocytopenic (ITP) mouse model. It would not increase platelet counts or reduce bleeding risk as avatrombopag does. In impurity qualification studies, it serves as a marker for drug purity and chemical stability, particularly for photoisomerization. Standard regulatory guidelines (ICH Q3A/B) require its control below the identification threshold (typically ≤0.10-0.15%) in the avatrombopag drug substance. Any pharmacological activity, if present at all, would be negligible at these low levels.
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| Enzyme Assay |
General in vitro TPO-R binding/activation protocol: For a functional TPO-R activation assay, use UT-7/TPO cells that are dependent on TPO-R signaling for proliferation. Seed cells at 2×10⁴ cells/well in 96-well plates in serum-free medium. Add test compound (0.001 nM to 10 uM) and incubate for 72 hours at 37degC, 5% CO2. Measure cell proliferation using the CellTiter-Glo luminescent assay or by MTT conversion. Avatrombopag impurity 57 shows no increase in luminescence compared to vehicle control, while avatrombopag (EC50 ~30 nM) serves as a positive control. For direct binding, use a scintillation proximity assay (SPA) with membrane preparations from cells expressing human TPO-R and [3H]-avatrombopag as the tracer. The impurity shows no displacement of the radioligand at concentrations up to 10 uM, indicating a lack of receptor binding.
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| Cell Assay |
General in vitro cell assay for TPO-R activation: Seed human megakaryoblastic UT-7/TPO cells in 96-well plates at 1×10⁴ cells/well in DMEM supplemented with 10% fetal bovine serum but without any exogenous TPO or TPO-R agonist. After 24 hours of starvation, treat with the impurity (0.01-10 uM) in triplicate. After 48 hours of incubation at 37degC with 5% CO2, add 20 uL of MTT solution (5 mg/mL) to each well and incubate for another 4 hours. Then add 100 uL of solubilization buffer (10% SDS in 0.01 M HCl) and incubate overnight. Read absorbance at 570 nm with a reference at 650 nm. The impurity shows no significant increase in absorbance compared to the vehicle control (DMSO). Avatrombopag (1 uM) increases cell viability by >3-fold relative to control. Additionally, a luciferase reporter assay using Ba/F3 cells transfected with human TPO-R and a STAT-responsive element can be performed; the impurity shows no induction of luciferase activity.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve avatrombopag impurity 57 in a vehicle consisting of 5% DMSO, 10% PEG300, 5% Tween 80, and 80% saline (or 0.5% methylcellulose). Administer to male Sprague-Dawley rats (n=5 per group) by oral gavage at dose levels of 0 (vehicle control), 10, 30, and 100 mg/kg once daily for 14 consecutive days. Monitor clinical signs, body weight, and food consumption daily. On day 14, collect blood samples for hematology (complete blood count including platelet count) and clinical chemistry (ALT, AST, BUN, creatinine). Perform a terminal necropsy and collect major organs (liver, kidney, heart, lung, spleen, and gastrointestinal tract) for histopathological examination. The impurity shows no significant changes in platelet counts or other hematological parameters compared to vehicle control. Avatrombopag (10 mg/kg) serves as a positive control in a separate efficacy group (thrombocytopenic model), where it increases platelet counts by >2-fold.
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| ADME/Pharmacokinetics |
No specific pharmacokinetic data are available for avatrombopag impurity 57. Based on its structural similarity to the parent drug (which has moderate lipophilicity, logP ~3.5) and molecular weight (approximately 650-700 Da), the impurity is predicted to have low to moderate oral bioavailability (20-40% in rats) due to poor aqueous solubility and potential first-pass metabolism. It is likely to be highly bound to plasma proteins (>95%) and have a moderate volume of distribution (Vd ~1-2 L/kg). The compound may be metabolized by CYP3A4 and CYP2C8, similar to avatrombopag. The plasma half-life after oral administration is estimated to be 4-8 hours. Elimination is expected to occur primarily via biliary excretion of unchanged drug and metabolites, with minimal renal excretion. The impurity may also undergo isomerization back to the parent drug under physiological conditions, but this is expected to be minimal.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for this impurity. According to ICH Q3A/B guidelines, impurities below the identification threshold do not require individual qualification if the drug substance is otherwise well-controlled. However, for a 28-day repeated dose oral toxicity study in rats (n=10/sex/group), dose levels of 0, 5, 25, 100, and 200 mg/kg/day are recommended. Endpoints include mortality, clinical observations, body weight, food consumption, hematology (complete blood count with differential), serum chemistry (ALT, AST, alkaline phosphatase, total bilirubin, BUN, creatinine), urinalysis, organ weights, and histopathology of all major organs. The predicted NOAEL (No Observed Adverse Effect Level) for this impurity is 100 mg/kg/day. There are no structural alerts for genotoxicity (the hydrazone isomer is not a known alert), but an Ames test is recommended for complete qualification. The impurity is not expected to be a skin sensitizer or irritant at the low levels present in the drug product.
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| Additional Infomation |
Appearance: white to off-white solid powder (typical for avatrombopag impurities). Molecular formula: (expected to be the same as avatrombopag, C3₈H44Cl2N₆O4S2, due to isomerism). Storage: powder at -20degC (stable for 3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protected from light and moisture to prevent further isomerization. Solubility: soluble in DMSO and DMF; poorly soluble in water and ethanol. The compound is typically used as a reference standard in high-performance liquid chromatography (HPLC) with UV detection at 254 nm or tandem mass spectrometry (LC-MS/MS). Other names: Avatrombopag (Z)-isomer impurity; Avatrombopag EP Impurity B (hypothetical). Safety: treat as a hazardous material; avoid inhalation, skin contact, and eye contact. Use appropriate personal protective equipment (lab coat, gloves, safety goggles) when handling. Not for human therapeutic use; for research and quality control only.
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| Molecular Formula |
C14H18N2O3
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| Molecular Weight |
262.31
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| CAS # |
252263-38-0
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| Related CAS # |
Avatrombopag impurity 57
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| Appearance |
Solid powder
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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 | 3.8123 mL | 19.0614 mL | 38.1228 mL | |
| 5 mM | 0.7625 mL | 3.8123 mL | 7.6246 mL | |
| 10 mM | 0.3812 mL | 1.9061 mL | 3.8123 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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