生物活性

SR1 is an antagonist of the aryl hydrocarbon receptor.SR1 is the first small molecule that promotes robust expansion/self-renewal of human CD34+ peripheral blood and cord blood hematopoietic stem cells (HSCs). Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. SR1 can be potentially used for ex vivo expansion of normal HSCs or leukemic stem/progenitor cells.

SR1 is apotent AhR antagonist with an IC50 of 127 nM.[1]

SR1inhibits PAL binding with an IC50 of 40 nM.[1]

SR1 increasesthe number of CD34+ cells with an EC50 of ~120 nM.[1]

体外研究

体内研究

激酶实验

细胞实验

Cellculture[2]

Leukemia cell lines Jurkat, Kasumi-1, NB4and K562 were cultured in RPMI-1640 medium containing 10% fetal bovine serum(FBS) and 10U/ml penicillin and 10 μg/ml streptomycin at 37˚C in 5% CO2.Cells were subcultured every 2-3 days, and maintained at a cell density of0.5-1.0×106cells/ml.

Treatmentwith SR1 and cell counting

SR1 was diluted in dimethyl sulfoxide(DMSO) to a final concentration of 1.5 mM. NB4 cells were cultured in a 24-wellplate at 5.0×105 cells/ml in the presence of three different SR1 concentrations(0.375 μM, 0.75 μM and 1.5 μM, containing 0.1% DMSO), or 0.1% DMSO only(control). Under all conditions, cell culture was performed in triplicatewells. Morphology of the cells was observed under a phase-contrast OlympusCKX41 microscope and recorded using ZEN 2 (blue edition) software. After 2 and4 days of culture, cells were collected and washed with PBS (−). The cells weremixed with Trypan Blue Solution (Wako Pure Chemical Industries) and the numberof live and dead cells was counted by using Bürker Türk blood corpuscle countingchamber.

动物实验

Engraftmentof CD34+ cells in NSG mice[1]

A quantity of 250,000 CB-derived CD34+cells were cultured with control conditions (DMSO, 0.01%) or SR1 (0.75 μM) for3 weeks. At this point control cultures had expanded 1080-fold and SR1 treatedcells expanded 2024-fold relative to starting cell numbers. A quantity of 30 to30,000 uncultured CD34+CB-derived cells or a fraction of the finalculture equivalent to 30 to 10,000 starting cells was transplanted. The cellswere injected intravenously via the retro-orbital route into sub-lethally irradiated6- to 10-week-old NSG mice. Engraftment was performed within 24 h afterirradiation. Engraftment was monitored by flow cytometric analysis of bloodobtained via retro-orbital sinus or bone marrow using anti-human CD45 andanti-mouse CD45 antibodies. Fifteen weeks after transplantation, bone marrowwas harvested from the secondary mice and analyzed by flow cytometry.

FlowCytometry

Multicolor analysis for progenitor and stemcell phenotyping was performed on a LSR II flow cytometer. Cells were stainedin staining media (HBSS supplemented with FBS [2%] and EDTA [2 mM]) at 4°C for1 h with APC anti-CD90, PerCP anti-CD34, FITC anti-CD38, PECy7 anti-CD45RA, andPE anti-CD133, then washed with staining media and analyzed. Cells isolatedfrom the bone marrow, blood, spleen and thymus were stained for 1 h with APC orpacific blue antihuman CD45, FITC anti-mouse CD45, PeCy7 anti-human CD33, PEanti-human CD11b, FITC anti-human CD19, PeCy7 anti-human CD3, PE anti-humanGlycophorin A, FITC anti-human CD41, and PeCy7 anti-human CD56, washed withstaining media and analyzed.

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] Boitano AE WJ, Romeo R, Bouchez LC, Parker AE, Sutton SE, Walker JR, Flaveny CA, Perdew GH, Denison MS, Schultz PG, Cooke MP. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010 329(5997):1345-1348.
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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。

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