TAK-242

Assay for LPS binding to PBMCs

PBMCs were suspended in BSA solution (phosphate-buffered saline containing 0.1% BSA and 0.01% sodium azide). In a total volume of 50 μL, PBMCs (3 × 105 cells) were incubated with TAK-242, anti-human CD14 monoclonal antibody (MAb) MEM-18, or anti-human CC-chemokine receptor 5 (CCR5) MAb as a negative control for 30 mins at 4 °C. The cells were further incubated with 50 ng/mL LPS from E. coli serotype O55:B5 conjugated with Alexa Fluor 488 per milliliter in the presence of human serum at a final concentration of 1% for 45 mins at 37 °C. After washing twice with BSA solution, 1 × 104 cells were analyzed by flow cytometry using CytoACE300 cytofluorometer. The assays were performed in triplicate for each preparation of PBMCs obtained from four different donors. Specific LPS binding was estimated by subtracting the percentage of LPS-binding cells in the absence of LPS from that in the presence of LPS.

Cell lines

RAW264.7 cells

Preparation method

This compound is soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

1, 10, 100 and 1000 nM; 15 mins

Applications

In RAW264.7 cells, TAK-242 inhibited the phosphorylation of IRAK-1 induced by LPS.

Animal models

Wistar Hannover rats

Dosage form

0.5 mg/kg; i.v.

Applications

TAK-242 prevented the accumulation of potentially deleterious inflammatory and oxidative/nitrosative mediators in the brain frontal cortex of rats.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Ii M, Matsunaga N, Hazeki K, Nakamura K, Takashima K, Seya T, Hazeki O, Kitazaki T, Iizawa Y. A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex- 1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling. Mol Pharmacol. 2006;69(4):1288-95.

[2]. Naoko Matsunaga, Noboru Tsuchimori, Tatsumi Matsumoto, and Masayuki Ii. TAK-242 (Resatorvid), a Small-Molecule Inhibitor of Toll-Like Receptor (TLR) 4 Signaling, Binds Selectively to TLR4 and Interferes with Interactions between TLR4 and Its Adaptor Molecules. Mol Pharmacol 79:34–41, 2011.

[3] Iciar Gárate, Borja García-Bueno, José Luis Mu?oz Madrigal, Javier R Caso, Luis Alou, María Luisa Gómez-Lus and Juan Carlos Leza. Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. Journal of Neuroinflammation 2014, 11:8.