生物活性

TAK-242 is a selective TLR4 signal transduction inhibitor initially used as a novelanti-sepsis agent capable of inhibiting inflammatory mediator production.

The IC50 values of LPS-induced NF-κB activationfor TAK-242 in HEK293 cells expressing human

TAK-242inhibits LPS (10ng/ml)- stimulated NO production in RAW264.7 with an IC50 valueof 5.5 nM.[1]

Inhibitory effect of TAK-242 against LPS -stimulatedTNF-α production

Effectof TAK-242 on NF-κB activation mediated by the intracellulardomain[1]

Cytokinesproduction inhibition of TAK-242 in mouse macrophages[3]

体外研究

体外研究表明，采用免疫共沉淀的方法，通过结合TLR4胞内结构域，抑制脂多糖诱导的炎症介质的产生。10种不同的人TLRs 中，选择性地结合TLR4。这些结果表明，可以选择性地结合TLR4，打乱TLR4与衔接分子的相互作用，从而抑制TLR4 信号转导及其下游信号。

体内研究

激酶实验

Binding assay with [³H]-TAK-242[2]

COS-7 and HEK293 cells were seeded in 10 cmdishes at 2 to4 x10⁵ and 0.6 to 1 x 10⁶ cells per dish,respectively, and incubated for 3 to 4 days. COS-7 cells were transiently transfectedwith expression vectors encoding FLAG-TLR2, FLAG-TLR4, FLAG-T2N-T4C,FLAG-T4N-T2C or TLR5-HA using Lipofectamine reagent and Plus reagent. HEK293cells were transiently transfected with expression vectors encoding FLAG-TLR4,TLR3-HA, TLR9-HA, FLAG-TRAM, FLAG-TRIF, HA-MyD88 or HA-TIRAP/Mal usingLipofectamine reagent and Plus reagent. After 2 days of transfection, cellswere incubated with 100 nM [³H]-TAK-242 at 37°C for 6 h. Forcompetition assays, the cells were incubated for 2 h with various concentrationsof nonradioactive TAK-242 and its enantiomer, then for 4 h with [³H]-TAK-242.After being washed with PBS, cells were lysed with lysis buffer [150mM NaCl, 0.1%Nonidet P40, 0.05% CHAPS, 30mM NaF, 1mM Na₃VO₄, 50mMTris-HCl (pH 7.5) plus a protease inhibitor cocktail]. For lysis of cellstransfected with TLR3-HA or TLR9-HA, the lysis buffer contained 100mM NaCl, 1mMEDTA, 1mM NaF, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X100, 1% glycerol,2mM Na₃VO₄, 10mM Tris-HCl (pH 7.5) plus a protease inhibitorcocktail. Cell lysates were incubated with anti- FLAG M2 antibody (Ab) oranti-HA 12CA5 Ab for 3 h at 4°C. Protein A Sepharose 6MB was then added to themixtures and further incubated overnight at 4°C. Immunoprecipitates were separatedby SDS-PAGE and transferred to polyvinylidene difluoride membranes. Themembranes were incubated with anti-FLAG M2 or anti-HA 12CA5 Abs as the firstAb, then ECL anti-mouse IgG, horseradish peroxidase-linked whole Ab (fromsheep) as the secondary Ab. Immunoprecipitates were detected using ECL westernblotting detection reagents. Radioactive imaging of theimmunoprecipitates was analysed with a BAStation.

细胞实验

Primary culture of pancreatic acinar cells[4]

Pancreatic acinar cells were isolated fromadult male (to avoid the effects of estrogen levels) C57BL/6J mice (weighing25-30 g). Briefly, the animals were sacrificed, and the pancreas was removedquickly and incubated in buffer solution (containing 130-mM NaCl, 4.7-mM KCl,1.3-mM CaCl₂, 1-mM MgCl₂, 1.2-mM KH2PO4, and 0.2% bovineserum albumin (BSA) at 37^oC for 10 min. Then, the cell suspension wascentrifugated at 30 g for 5 min at 4^oC, and the acinar cell clumpswere resuspended in Na-Hepes buffer without collagenase. To mimic AP in vitro,cultured pancreatic acinar cells were treated with taurocholate at theconcentration of 6mM under the conditions detailed in the following sentences.To investigate the potential protective effect of TAK-242, pancreatic acinarcells were treated with TAK-242(1μM) immediately after exposure totaurocholate, and the concentrations of taurocholate and TAK-242 were selected accordingto previous published data.

Lactatedehydrogenase release assay

The cytotoxicity in pancreatic acinar cellswas determined by measuring lactate dehydrogenase (LDH) release as a function ofmembrane integrity using a diagnostic kit according to the manufacturer’sinstructions. Briefly, 50 mL of supernatants were collected and incubated withnicotinamide adenine dinucleotide diaphorase (NADH) and 0.1% sodium pyruvate for15 min at 37^oC. Absorbance of the sample was monitored at 440 nm,and the activity of LDH was represented as the fold of control values.

TUNELstaining

Apoptosis in pancreatic acinar cellssubjected to taurocholate and/or TAK-242 treatment was detected by TUNELstaining, the method to observe DNA strand breaks in nucleus. Briefly, cellsseeded on glass slides were fixed by 4% methanol-free formaldehyde solution inPBS for 20 min and labeled with the fluorescein TUNEL reagent mixture for 1 hat 37^oC in the dark. Then, 2-(4-Amidinophenyl)-6- indolecarbamidinedihydrochloride (DAPI) was added to stain the nucleus before washing withphosphate buffered saline (PBS) three times. Fluorescence evaluation wasperformed under a confocal microscope, and the number of TUNEL-positive (green)cells was counted.

动物实验

Animal Modeling and Grouping[5]

Forty-five adult male Sprague-Dawley rats(250-300g) were randomized (random number) into three groups: sham, CME and CMEplus TAK-242 (n = 15 per group). The present study established a CME model byinjecting plastic microspheres into the left ventricle (LV). Briefly, a leftlateral thoracotomy was undertaken in rats at the third and fifth intercostalspace. The pericardium was opened and the ascending aorta was exposed fully. Asuspension of microspheres in saline solution containing about 3000microspheres (42μm in diameter) was injected into the LV during 10s occlusionof the ascending aorta. Rats in the CME plus TAK-242 group received anintravenous injection of TAK-242 (2 mg/kg) via the tail vein 30min beforesurgery, and rats in the sham group received an injection of the same volume ofnormal saline only.

CardiacFunction Monitoring

Because of cardiac function reached thepoorest at 6h after CME, the time point was selected to detect the cardiacfunction parameters. All echocardiographic examinations including leftventricular ejection fraction (LVEF), left ventricular fractional shortening(FS), cardiac output (CO) and left ventricular enddiastolic diameter (LVEDd) ofthe rats in each group were performed at a probe frequency of 10 MHz by anexperienced professional physician. All measures were expressed as the averageof three heart beat cycles.

Tissuesampling and sample treatment

After cardiac function detection, thehearts were arrested by injecting 2mL 10% potassium chloride into the tailvein. The hearts were isolated and cleaned with cold normal saline immediately.The atrial appendage and atrium cordis were removed, and parts of heartventricle were rapidly frozen in liquid nitrogen and stored at -80 °C forwestern blot analysis and qPCR. The others were fixed in 4% paraformaldehydefor12 h, embedded in paraffin and serially sectioned into slices of 4 μmthickness for TUNEL staining and hematoxylin-basic fuchsin-picric acid (HBFP)staining.

Apoptosisassay

Apoptotic cardiomyocytes were detectedusing TUNEL assay kit. TUNEL staining was performed according to themanufacturer’s instructions. The apoptotic nuclei were stained yellow-brown whilethe normal light blue. In each slice, 10 random high-power fields(magnification, x 400) were observed to count TUNEL-positive nuclei, and themyocardial apoptotic index was calculated as the number of TUNEL-positive cellnuclei / total nuclei ×100%.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Kawamoto T, Ii M, Kitazaki T, Iizawa Y, Kimura H. TAK-242 selectively suppresses Toll-like receptor 4-signaling mediated by the intracellular domain. Eur J Pharmacol. 2008;584(1):40-48.
[2] Takashima K, Matsunaga N, Yoshimatsu M, et al. Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model. Br J Pharmacol. 2009;157(7):1250-1262.
[3] Ii M, Matsunaga N, Hazeki K, et al. 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):1288-1295.
[more]

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

相关化合物库

使用AMQUAR产品发表文献后请联系我们