Brivanib Alaninate (BMS-582664) is the alaninate salt of a vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor with potential antineoplastic activity. Brivanib strongly binds to and inhibits VEGFR2, a tyrosine kinase receptor expressed almost exclusively on vascular endothelial cells; inhibition of VEGFR2 may result in inhibition of tumor angiogenesis, inhibition of tumor cell growth, and tumor regression.

VEGFR2:25 nM, FLK1:89 nM, VEGFR1:380 nM, FGFR1:148 nM

25 mg/kg BMS-582664在大鼠中AUC为13.4 μM×hr,Cmax为6.4 μM.50 mg/kg BMS-582664在小鼠中AUC为136 μM×hr,Cmax为41 μM.50 mg/kg和100 mg/kg BMS-582664作用于携带病患衍生的移植瘤06-0606 的小鼠,抑制肿瘤生长速度分别为55% 和13%.口服60 mg/kg BMS-582664显著降低携带病患衍生的移植瘤06-0606 的小鼠中肿瘤重量,促进细胞凋亡,降低微血管密度,抑制细胞增殖,并下调细胞周期调控.小鼠口服60 mg/kg BMS-582664,迅速吸收,Tmax为1小时,半衰期 (t1/2)为2.7小时,平均滞留时间为3.6小时.60 mg/kg和90 mg/kg BMS-582664剂量依赖性抑制携带H3396移植瘤的无胸腺小鼠中肿瘤生长,肿瘤生长抑制率分别为85%和97%.80 mg/kg和107 mg/kg.BMS-582664 处理携带L2987非小细胞肺癌移植瘤的无胸腺小鼠,剂量依赖性抑制肿瘤生长,肿瘤生长抑制率分别为85% 和 97%.100 mg/kg BMS-582664抑制两种携带移植瘤的小鼠模型（L2987和HCT116）中血管内皮细胞的增长.

BMS-582664固态稳定性较高,在50°C下放置干燥剂的12周期间仅有0.3％降解,在pH为6.5时也具有较好的液态稳定性。2 μM BMS-582664明显抑制VEGF和 bFGF刺激的SK-HEP1细胞和HepG-2细胞中VEGFR-2,FGFR-1,ERK1/2和Akt的磷酸化,而BMS-582664单独作用于未经刺激的细胞,则不影响ERK1/2,Akt,VEGFR-2和FGFR-1的磷酸化水平。BMS-582664抑制VEGF和FGF刺激的HUVECs 增殖,IC50分别为40 nM和276 nM。BMS-582664抑制CYP2C19,CYP3A4(BFC)和CYP3A4 (BzRes)细胞,IC50分别为2.4 μM,0.51 μM和1.6 μM。

Kinase inhibition assays: For the VEGFR2, Flk1 and FGFR1 kinase assays, BMS-582664 is dissolved in DMSO and diluted with water/10% DMSO to a final DMSO concentration of 2%. The kinase reactions consists of 8 ng of enzymes with GST tag, 75 μg/mL substrate, 1 μM ATP, and 0.04 μCi [γ-33P]ATP in 50 μL total reaction volume (kinase buffer:? 20 mM Tris, pH 7.0, 25 μg/mL BSA, 1.5 mM MnCl2, 0.5 mM dithiothreitol). In all cases, the reactions are incubated for 60 min at 27℃ and terminated with the addition of cold trichloroacetic acid (TCA) to a final concentration of 15%. The percent inhibition from the kinase assays is determined by nonlinear regression analyses, and data are reported as the inhibitory concentration required to achieve 50% inhibition relative to control reactions (IC50).

Cells are grown in 100 μL of minimal growth medium and 1.0% heat-inactivated fetal bovine serum in 96-well collagen IV coated plates at a density of 2 × 103 per well in a 37 ℃/5% CO2 environment. Twenty-four hours later, serum is adjusted to 10%, and BMS-582664 at various dilutions are added to each well in a final volume of minimal growth media that contains 10% serum. Forty-eight hours later, 0.5 μCi of [3H]thymidine is added in a volume of 20 μL of minimal media for 24 hours. Plates are washed once in PBS. Upon removal of PBS, Trypsin is added to cells which are subsequently harvested onto glass-fiber filters using an automated harvestor. Incorporated tritium is quantified using a β-counter. Dose?response curves are generated to determine the IC50 value, which is defined as the concentration of drug required to inhibit 50% of tritium incorporation when compared to untreated serum-stimulated cells.(Only for Reference)