Zibotentan (ZD4054) (ZD4054) is a specific Endothelin (ET)A antagonist with IC50 of 21 nM, exhibiting no activity at ETB. Phase 3.

ETA:21 nM

Zibotentan专门抑制ETA介导的抗凋亡效应，而不影响ETB介导的促凋亡效应，在人类和大鼠的平滑肌细胞中，Zibotentan高亲和力地结合于内皮素A受体(ETA)，Ki值为13 nM，对内皮素B受体(ETB)无亲和力，IC50值>10 μM。[1] 1 μM的Zibotentan处理抑制在分泌ET-1且表达ETA和ETB mRNA的卵巢癌细胞系HEY和OVCA 433中ET-1诱导的有丝分裂活性。[2] ZD4054（1 μM）抑制ET-1在HEY和OVCA 433细胞中诱导的EGFR转激活。Zibotentan（1 μM）通过增强E-钙黏蛋白表达和启动子活性，以及抑制血管内皮生长因子(VEGF)分泌和侵袭性，逆转ET-1介导的上皮-间质转换(EMT)。[3] Zibotentan还强效地抑制在SKOV-3和A-2780细胞中基础和ET-1诱导的细胞增殖，这与AKT和p42/44MAPK磷酸化的抑制、bcl-2的抑制和caspase-3及多聚(ADP-核糖)聚合酶蛋白的激活增加的凋亡有关。[4]

给予小鼠Zibotentan 10 mg/kg/日，连续21天的处理显著抑制HEY卵巢癌异种移植瘤的增长，抑制率达69%，且未观察到相关毒性。这与通过Ki-67表达的37%抑制评估的细胞增殖阻断，以及62%的肿瘤诱导血管生成抑制有关。一致地，Zibotentan处理显著抑制基质金属蛋白酶-2（MMP-2）和VEGF的表达，以及p42/44 MAPK和EGFR的激活，并强力增强E-cadherin的表达。[3]

Receptor-binding assays: The inhibition by Zibotentan (varying concentrations) of 125iodine-ET-1 binding to cloned human ETA is assessed using standard radioligand-binding techniques. Human recombinant ETA is expressed in mouse erythroleukaemic cells, and cell membranes prepared for competitive binding studies using 125iodine-ET-1 as the radioligand. Incubations are carried out in triplicate in the presence of Zibotentan, 100 pM to 100 μM in half-log increments, and inhibition of ET-1 binding is expressed as the geometric mean pIC50 value (concentration to inhibit 50% of binding) with a 95% confidence interval (CI). The affinity of Zibotentan for cloned human ETA is also assessed using the equation of Cheng and Prusoff to determine the equilibrium dissociation constant (Ki) in a further receptor-binding screen utilizing a greater number of concentration-response curves determined in three separate studies.

Cells are serum starved by incubation for 24 hours in serum-free DMEM before exposed to Zibotentan for 48 hours. After the treatment, cells are lysed and the supernatant is recovered and assayed for histone-associated DNA fragments, at 405 nm by the use of a microplate reader. For detection of early apoptotic events, floating and adherent cells are collected. Cells are double stained with FITC-conjugated Annexin V and propidium iodide using the Vybrant Apoptosis Kit and are immediately analyzed by cytofluorometric analysis.(Only for Reference)