Plixorafenib (PLX8394) is an orally active inhibitor of the serine/threonine protein kinase B-Raf (BRAF) protein.Plixorafenib can selectively bind to and inhibit the activity of wild-type and mutant forms of BRAF, inhibiting the proliferation of tumor cells expressing mutant forms of BRAF.

Raf:23 nM, B-Raf (V600E):3.8 nM, BRAF:14 nM

方法：Plixorafenib （PLX8394 ）（1 μM，24小时）处理亲本 1205LuTR 报告细胞和 PRT#3 和 #4 ，然后裂解细胞并通过蛋白质印迹法分析指示的细胞周期蛋白；（PLX8394 ）（1 μM，24小时）处理1205Lu、PRT #3 和 PRT #4 ，最后 16 小时加入 EdU，通过流式细胞术分析 EdU 掺入。
结果： Plixorafenib 有效降低了亲本细胞1205Lu中细胞周期蛋白 D3 和细胞周期蛋白 D1、视网膜母细胞瘤蛋白磷酸化和细胞周期蛋白 A2的表达；在 PRT #3 和 PRT #4 细胞中未出现降低的表现；Plixorafenib 抑制了PRT #3 和 PRT #4 细胞中 EdU 掺入S 期。[1]

方法：Plixorafenib （PLX8394 ）（75,150,300 mg/kg，口服）治疗HCC364细胞模型小鼠中，治疗后 0、1、2、4 和 8 小时测量血清浓度 (ng/mL)；将稳定表达萤火虫荧光素酶的HCC364细胞手术植入免疫缺陷小鼠中，使用Plixorafenib（150 mg/kg，口服）治疗小鼠并使用基于生物发光的成像（BLI）系统监测肿瘤生长和对RAF抑制剂治疗的反应。
结果： Plixorafenib（150 mg/kg）产生血浆浓度>150μM，且对动物无明显毒性；Plixorafenib显着抑制肿瘤生长，同时在治疗中观察到更快速和实质性的初始抗肿瘤反应，这种效果与增强和持久抑制 ERK 磷酸化和肿瘤细胞增殖有关。[2]

Dissolvent: DMSO. For MTT assays, 2×103?cells are seeded in triplicate in 96 wells in their regular culture medium (containing PLX4720 for PRT lines). Next day, cells are washed twice with PBS and then the medium is replenished containing the indicated RAF inhibitor. Medium is changed 48 hours later and after a further 48 hours, 10 μL of 5 mg/mL MTT reagent is added to wells and incubated for three hours. Formazan crystals are then solubilized overnight with a 1:10 dilution of 0.1 M glycine (pH 10.5) in DMSO. Wells are then analyzed at 450 nM in a Multiskan Spectrum spectrophotometer. Results depicted are normalized to DMSO conditions and are a composite of three independent experiments. Error bars shown are representative of the standard error of mean (SEM).

PLX8394 is dissolved in PEG 400 [20% (v/v)], TPGS [5% (v/v)], and water [75% (v/v)].H1755 tumor xenografts are generated by injection of 5×106 cells in a 50/50 mixture for matrigel and PBS into 6- to 8-wk-old female NOD/SCID mice. Mice are randomized to treatment groups once tumors reach an average size of 150 mm3. H1755 cells are s.c. implanted and allowed to grow to appr 200 mm3 (4 wk after implantation). Mice are then treated with vehicle, vemurafenib, or PLX8394 for 15 d. The vehicle for daily oral gavage is PEG 400 [20% (vol/vol)], tocopheryl polyethylene glycol succinate (TPGS) [5% (vol/vol)], water [75% (vol/vol)]. PLX8394 is dissolved in PEG 400 [20% (vol/vol)], TPGS [5% (vol/vol)], and water [75% (vol/vol)] and vortexed continuously throughout the dosing period. PLX8394 (p.o.) is given at a dose of 150 mg/kg/d.