Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts

BPR0L075, 6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity in vitro. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels in vitro and ex vivo. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.

BPR0L075，即6-甲氧基-3-(3′,4′,5′-三甲氧基苯甲酰基)-1H-吲哚（6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-indole），是一种微管蛋白结合剂（tubulin-binding agent），可通过结合秋水仙碱结合位点（colchicine-binding site）抑制微管蛋白聚合（tubulin polymerization）。BPR0L075在体外已展现出抗有丝分裂（antimitotic）与抗血管生成（antiangiogenic）活性。本研究采用动态生物发光成像（dynamic bioluminescence imaging），评估了BPR0L075在人乳腺癌乳腺脂肪垫异种移植瘤（human breast cancer mammary fat pad xenografts）中的血管破坏活性。单次腹腔内（intraperitoneally, i.p.）注射50 mg/kg的BPR0L075，可诱导肿瘤血管快速、暂时性闭塞（给药后2、4、6小时均可观测到此效应）；该现象可通过表达荧光素酶的原位MCF7与MDA-MB-231乳腺癌肿瘤，在给予荧光素底物（luciferin substrate）后发光信号的快速且可重复下降得到验证。通过灌注标志物Hoechst 33342与肿瘤血管标志物CD31的免疫组织化学染色（immunohistological staining），证实BPR0L075给药后肿瘤灌注呈时间依赖性降低。给药后24小时内，肿瘤血管即可实现显著恢复。单次腹腔注射50 mg/kg的BPR0L075，最初可在6小时内达到微摩尔级的血浆药物浓度，但后续的药物分布与清除过程可使其血浆水平在24小时内快速降至纳摩尔级。针对人脐静脉内皮（human umbilical vein endothelial, HUVEC）细胞与体外培养的肿瘤细胞的实验显示，BPR0L075对肿瘤细胞与增殖性内皮细胞均具有细胞毒性（cytotoxic），并可在体外与离体（ex vivo）条件下破坏已形成的血管结构。综上，BPR0L075可诱导肿瘤血管快速但暂时性的闭塞，并降低原位人乳腺癌异种移植瘤的肿瘤灌注，提示这种抗有丝分裂剂有望作为血管破坏性癌症治疗手段。