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| Targets |
The primary target of 20-HEDE is the 20-hydroxyeicosatetraenoic acid (20-HETE) receptor or its signaling pathway. As an antagonist, 20-HEDE blocks the actions of 20-HETE, a potent vasoconstrictor and a key regulator of renal and cardiovascular function. 20-HETE is produced from arachidonic acid by cytochrome P450 enzymes of the CYP4A and CYP4F families. By inhibiting 20-HETE signaling, 20-HEDE counteracts its vasoconstrictor effects, leading to vasodilation and reduced blood pressure. The compound may also modulate other 20-HETE-mediated effects, such as inflammation and cell proliferation. Its antagonism of 20-HETE makes it a valuable tool for studying the role of this signaling pathway in various diseases.
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| ln Vitro |
The invasion ability of A549 cells was greatly reduced by HET0016 or WIT 002, both of which were applied in the experiment due to the cells' strong ω-hydroxylation activity of arachidonic acid [2]. RPTC normal renal epithelial cells are not susceptible to WIT 002 or HET0016-induced inhibition of proliferation, however 786-O and 769-P renal cancer cell proliferation can be inhibited [2][3].
In vitro, 20-HEDE acts as an antagonist of 20-HETE, blocking its vasoconstrictor and other biological effects. The compound's activity has been characterized in cell-based assays where it inhibits 20-HETE-mediated signaling. 20-HEDE has been shown to have a high affinity for the 20-HETE receptor or its downstream signaling components. Its in vitro activity is concentration-dependent, with effects observed at nanomolar to micromolar concentrations. The compound is a valuable tool for studying the role of 20-HETE in various cell types and for screening for new therapeutic agents targeting the 20-HETE pathway. |
| ln Vivo |
The effect of the 20-HETE antagonist WIT 002 on the growth of 786-O clear cell renal carcinoma was studied in an ectopic mice model of renal malignancies. Daily treatment of WIT 002 to athymic nude mice subcutaneously implanted with 786-O cells effectively reduced tumor growth. Tumor development was suppressed by 84%±128%. Of note, in these tests, WIT 002 treatment was began just 7-14 days after tumor inoculation and was rather large by 0.1 cm. Therefore, WIT 002 can successfully block the formation of rather advanced cancers [3].
In vivo, 20-HEDE has been shown to significantly suppress tumor growth in an ectopic mouse model of renal cell carcinoma. Daily administration of 20-HEDE to athymic nude mice implanted subcutaneously with 786-O renal carcinoma cells resulted in 84% inhibition of tumor growth. This demonstrates the compound's potential as an anticancer agent for 20-HETE-dependent tumors. 20-HEDE has also been studied for its effects on blood pressure and renal function in animal models. Its in vivo efficacy supports its potential as a therapeutic agent for conditions associated with elevated 20-HETE levels. |
| Enzyme Assay |
The in vitro antagonist activity of 20-HEDE can be assessed using cell-free receptor binding or functional assays. A typical protocol for receptor binding involves incubating the compound with membrane preparations from cells expressing the 20-HETE receptor and a radiolabeled 20-HETE ligand. Bound and free ligand are separated by filtration, and the radioactivity is measured. For functional assays, cells expressing the 20-HETE receptor are treated with 20-HEDE in the presence or absence of 20-HETE, and downstream signaling (e.g., calcium mobilization, MAPK activation) is measured. The IC50 or EC50 value is determined by plotting the percentage of activity remaining against the compound concentration.
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| Cell Assay |
For in vitro cellular experiments, cells (e.g., renal cells, vascular smooth muscle cells, or cancer cells) are cultured in appropriate media and treated with 20-HEDE at various concentrations (typically 0.1-100 microM). Cells are stimulated with 20-HETE in the presence or absence of 20-HEDE, and various endpoints are measured. Vasoconstriction is assessed using isolated blood vessel preparations or by measuring changes in intracellular calcium levels. Cell proliferation is measured using MTT or BrdU incorporation assays. 20-HETE-mediated signaling pathways, such as MAPK and PI3K/AKT, are assessed by Western blot. The duration of treatment varies depending on the experimental design.
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| Animal Protocol |
In vivo animal experiments with 20-HEDE typically involve subcutaneous or intraperitoneal administration in mice or rats. A common dosing regimen is 10 mg/kg, administered subcutaneously. For tumor studies, 786-O renal carcinoma cells are implanted subcutaneously in athymic nude mice, and 20-HEDE is administered daily. Tumor growth is monitored by caliper measurements. For studies on blood pressure, 20-HEDE is administered to hypertensive animal models, and blood pressure is measured using telemetry or tail-cuff methods. Blood and tissue samples are collected for pharmacokinetic and biomarker analysis.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for 20-HEDE are available from studies in rats. Following subcutaneous injection at 10 mg/kg, plasma levels of 20-HEDE are measured over time. The compound's pharmacokinetic profile is characterized by dose-dependent exposure and a moderate half-life. Its bioavailability and tissue distribution would need to be further characterized to support its development as a therapeutic agent. 20-HEDE is typically stored at -20degC and is stable for up to 3 years in pure form.
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| Toxicity/Toxicokinetics |
The toxicity profile of 20-HEDE has not been extensively characterized. In animal studies, the compound has been administered at doses up to 10 mg/kg without significant adverse effects reported. However, as with any antagonist of a physiologically important signaling pathway, potential toxicities may include effects on blood pressure, renal function, and vascular tone. Long-term safety studies are needed to fully characterize its toxicity profile. The compound should be handled with standard laboratory precautions and is intended for research use only.
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| References |
[1]. Ming Yua . et al. Effects of a 20-HETE antagonist and agonists on cerebral vascular tone. Eur J Pharmacol. 2004 Feb 23;486(3):297-306.
[2]. Wei Yu, et al. Cytochrome P450 ω-hydroxylase promotes angiogenesis and metastasis by upregulation of VEGF and MMP-9 in non-small cell lung cancer. Cancer Chemother Pharmacol. 2011 Sep; 68(3): 619-29. [3]. Anna Alexanian, et al. Down-regulation of 20-HETE Synthesis and Signaling Inhibits Renal Adenocarcinoma Cell Proliferation and Tumor Growth. Anticancer Res. 2009 October ; 29(10): 3819-3824 |
| Additional Infomation |
20-HeDE is a type of HEDE.
20-HEDE (WIT 002) is an antagonist of 20-hydroxyeicosatetraenoic acid (20-HETE). It has a molecular weight of 324.50 g/mol and a molecular formula of C20H36O3. 20-HEDE has been shown to significantly suppress tumor growth in a mouse model of renal cell carcinoma, with 84% inhibition of tumor growth. The compound is a valuable tool for studying the role of 20-HETE in physiology and disease, including hypertension, stroke, and cancer. It is available as a research compound and is not approved for clinical use. |
| Molecular Formula |
C20H36O3
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| Molecular Weight |
324.4980
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| Exact Mass |
324.266
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| CAS # |
240427-90-1
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| PubChem CID |
10426436
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| Appearance |
Off-white to yellow liquid（Density：0.959±0.06 g/cm3）
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| LogP |
5.8
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
17
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| Heavy Atom Count |
23
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| Complexity |
308
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(CCC/C=C\CCCCC(=O)O)CCC/C=C\CCCCO
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| InChi Key |
RYHYNNWEPYGEEH-WGEIWTTOSA-N
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| InChi Code |
InChI=1S/C20H36O3/c21-19-17-15-13-11-9-7-5-3-1-2-4-6-8-10-12-14-16-18-20(22)23/h8-11,21H,1-7,12-19H2,(H,22,23)/b10-8-,11-9-
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| Chemical Name |
(6Z,15Z)-20-hydroxyicosa-6,15-dienoic 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) |
DMSO: ~100 mg/mL (~308.2 mM; with ultrasonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), Clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), Clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: 2.5 mg/mL (7.70 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), Suspended solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0817 mL | 15.4083 mL | 30.8166 mL | |
| 5 mM | 0.6163 mL | 3.0817 mL | 6.1633 mL | |
| 10 mM | 0.3082 mL | 1.5408 mL | 3.0817 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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