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| Targets |
AFP464 targets HIF-1α, a transcription factor that plays a central role in cellular response to hypoxia by regulating the expression of genes involved in angiogenesis, metabolism, and survival. Inhibition of HIF-1α by AFP464 leads to reduced expression of its downstream target genes, including vascular endothelial growth factor (VEGF). Additionally, AFP464 activates the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved in xenobiotic metabolism and immune regulation.
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| ln Vitro |
In vitro, AFP464 dihydrochloride demonstrates potent inhibition of HIF-1α with an IC₅₀ of 0.25 μM. The compound effectively reduces HIF-1α protein levels and its transcriptional activity in various cancer cell lines. It also activates the aryl hydrocarbon receptor (AhR), leading to the induction of AhR target genes such as CYP1A1. These activities contribute to its anti-cancer and anti-angiogenic effects in preclinical studies.
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| ln Vivo |
In vivo, AFP464 has shown anti-tumor activity in animal models of cancer, including xenograft models. By inhibiting HIF-1α, the compound reduces tumor angiogenesis and growth. Its activation of the aryl hydrocarbon receptor (AhR) may also contribute to its anti-tumor effects through modulation of the immune response and induction of metabolic enzymes. Detailed in vivo efficacy data are limited to preclinical studies.
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| Enzyme Assay |
In vitro enzyme/receptor binding assays for AFP464 typically involve measuring its ability to inhibit HIF-1α activity. Cell-free assays may include HIF-1α DNA-binding activity using electrophoretic mobility shift assays (EMSA) or ELISA-based DNA-binding assays. The compound's ability to activate AhR can be assessed using reporter gene assays in cell-free systems. Standard radioligand binding assays are not typically performed for this compound.
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| Cell Assay |
In vitro cellular assays for AFP464 are conducted in cancer cell lines such as HeLa, HepG2, or other HIF-1α-expressing cells. Cells are treated with various concentrations of AFP464 (typically 0.1–10 µM) for 24–72 hours. HIF-1α protein levels are measured by Western blot. HIF-1α transcriptional activity is assessed using a luciferase reporter construct containing hypoxia response elements (HRE). AhR activation is measured by CYP1A1 mRNA expression using qPCR. Cell viability is assessed using MTT or CellTiter-Glo assays.
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| Animal Protocol |
In vivo efficacy studies for AFP464 are conducted in mouse xenograft models of human cancers. Tumor-bearing mice are administered AFP464 via intraperitoneal or intravenous injection at various doses. Tumor growth is monitored by caliper measurement. HIF-1α target gene expression and angiogenesis markers are assessed in tumor tissues by immunohistochemistry and qPCR. The compound's ability to inhibit tumor growth and reduce vascularization is evaluated.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for AFP464 are limited. As a small molecule with a molecular weight of 521.36, the compound is expected to have reasonable bioavailability. It is soluble in DMSO (approximately 50 mg/mL) and water (approximately 16.67 mg/mL). Standard pharmacokinetic studies in rodents would involve administration via intraperitoneal or intravenous routes, with plasma concentration measured by LC-MS/MS. Further studies are needed to fully characterize its PK profile.
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| Toxicity/Toxicokinetics |
Toxicological data for AFP464 are limited. As an investigational compound, its safety profile has not been fully established. The compound is intended for research use only and is not for human therapeutic use. Standard safety precautions for handling small-molecule inhibitors apply, including the use of appropriate personal protective equipment and working in a well-ventilated area.
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| References | |
| Additional Infomation |
AFP464 dihydrochloride (NSC710464 dihydrochloride) is a research compound that inhibits HIF-1α and activates the aryl hydrocarbon receptor (AhR). It is used to study hypoxia signaling pathways and their role in cancer and other diseases. The compound is supplied as a yellow to orange solid powder and should be stored at -20°C for long-term stability. This product is for research use only and is not intended for diagnostic or therapeutic use.
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| Molecular Formula |
C22H25CL2F3N4O3
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| Molecular Weight |
521.36
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| Appearance |
Yellow to orange 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 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) |
DMSO :~50 mg/mL (~95.90 mM; with sonication)
H2O :~16.67 mg/mL (~31.97 mM; with sonication (<60°C)) |
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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 | 1.9181 mL | 9.5903 mL | 19.1806 mL | |
| 5 mM | 0.3836 mL | 1.9181 mL | 3.8361 mL | |
| 10 mM | 0.1918 mL | 0.9590 mL | 1.9181 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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