EZM 2302 (GSK3359088) is a selective, and orally available arginine methyltransferase CARM1 inhibitor (IC50: 6 nM).[1]

CARM1:6 nM

方法：在多发性骨髓瘤 （MM） 细胞系 RPMI-8226 中通过免疫印迹测试EZM 2302(GSK3359088)(0.3/1/5/20/80/300/1250/5000nM)处理对细胞甲基化的影响。
结果：EZM 2302 (GSK3359088) 在体外抑制细胞 PABP1 和 SMB 甲基化。[1]

方法： 雄性CD-1小鼠和雄性Sprague-Dawley大鼠（n = 3）通过静脉注射（i.v.）以2mg / kg的单剂量EZM 2302 (GSK3359088)和10mg / kg的口服管饲给药（p.o.;仅小鼠）处理，另一组在颈静脉和门静脉中插管的大鼠通过口服管饲法（10mg / kg，在0.5%甲基纤维素的水中）给药。通过眶后出血（小鼠）、尾静脉（大鼠静脉注射）或颈静脉和门静脉取样（大鼠口服）以预先指定的时间间隔从动物身上采集约 110 μL 血液。将 2 小时样品分开用于平行测定血液和血浆浓度（离体比）。
结果：EZM 2302 (GSK3359088)在人肝细胞中稳定 （CL <3 mL/min/kg），与人、小鼠和大鼠血浆蛋白结合适度，平均未结合分数分别为 0.66、0.46 和 0.74。在小鼠和大鼠中，血浆清除率（CL）分别为43和91 mL/min/kg。在大鼠中，观察到与红细胞的低水平结合，而EZM 2302 (GSK3359088)小鼠中没有显示血液分配，因此，两个物种的血液CL是等效的。尽管大鼠表现出中等的平均生物利用度（F），但通过进行JVC-PVC大鼠PK实验测量，从胃肠道吸收的剂量比例（Fa * Fg）要高得多，为81%，反映了EZM2302高渗透性。因此，EZM2302具有口服生物利用度，适合体内研究。[1]
方法：CB-17 SCID小鼠使用随机组设计分配到组中。EZM2302或载体（0.5%甲基纤维素水溶液）以37.5、75、150或300mg/kg的剂量口服BID给药21天。在研究期间，每周测量两次体重。每周使用卡尺在二维空间中测量肿瘤大小两次。动物在最后一次给药后 3 小时被安乐死，收集血液和组织进行分析。
结果：在 RPMI-8226 异种移植模型中，EZM 2302 (GSK3359088)在 21 天后显示出剂量依赖性暴露和肿瘤生长抑制 ；与载体相比，第 21 天测量的所有 EZM 2302 (GSK3359088) 个剂量组的肿瘤都显示出肿瘤生长显着减少，肿瘤生长抑制范围从37.5mg/kg剂量组的45%到300mg/kg剂量组的63%；第 21 天收集的 RPMI-8226 异种移植肿瘤显示，所有测试的 CARM1 底物的甲基化呈剂量依赖性降低，在所有剂量组中检测到未甲基化的 SmB （SmBme0） 在统计学上显着增加，从 37.5 mg/kg 的 8 倍增加到 150 mg/kg 的 14 倍。aDMA水平在所有剂量组下同样显着降低，在75mg / kg剂量组下观察到65%的最大抑制。在异种移植组织中很难检测到总PABP1和甲基化PABP1的水平。[1]

CARM1 activity was measured as previously reported for the histone methyltransferases PRMT1/6/865 and PRMT558. Briefly, CARM1 was preincubated with compounds for 30?minutes at room temperature before reactions were initiated. Final assay conditions were 0.25?nM CARM1, 30?nM 3H-S-adenosyl-methionine (SAM), and 250?nM biotinylated peptide in buffer containing 20?mM bicine, 1?mM tris(2-carboxyethyl)phosphine, 0.005% bovine skin gelatin and 0.002% Tween-20, pH 7.5. The assays were quenched by the addition of 300?μM unlabeled SAM. The quantity of 3H-labeled peptide produced was measured by Flashplate.

Cultured cells in linear/log phase growth were split to a seeding density of 2e5 cells/mL in 2–20mLs of media, depending on the yield required at the end of the growth period. The compound was diluted in DMSO and added to each culture vessel with a final DMSO concentration of 0.2%. Cells were allowed to grow for 96?hours. At the conclusion of each treatment period, cells were harvested by centrifugation (5?minutes, 1200?rpm), and cell pellets were rinsed once with PBS before being frozen on dry ice pending further processing.

For the in vivo efficacy studies, there were 8 mice per dose group and each mouse was inoculated subcutaneously at the right flank. All cells were suspended in a 0.2?mL mixture of base media and Matrigel at 1:1 for tumor development. RPMI-8226 cells were inoculated at 5?×?10^6 cells/mouse and treatment began when the mean tumor sizes reached 120?mm3 (28 days post-inoculation). CB-17 SCID Mice were assigned into groups using a randomized block design. EZM2302 or vehicle (0.5% methylcellulose in water) was administered orally BID at a dose volume of 37.5, 75, 150, or 300?mg/kg for 21 days. Body weights were measured twice a week for the duration of the study. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in cubic millimeters. Animals were euthanized 3?hours post-final dose, with blood and tissues collected for analysis.