Volasertib (BI 6727) (BI-6727) is a potent inhibitor of PLK1 (IC50: 0.87 nM), inducing mitotic arrest and apoptosis. It also inhibits PLK2/PLK3 (IC50s: 5/56 nM).

PLK2:5 nM (cell free), PLK1:0.87 nM (cell free), PLK3:56 nM (cell free)

Volasertib（BI 6727）有效抑制了Plk1及其两个密切相关的激酶Plk2和Plk3（IC50值分别为0.87、5、56 nmol/L）。BI 6727能够抑制来自多种癌症组织的多种细胞系的增殖，包括结肠癌（HCT 116，EC50=23 nmol/L）和肺癌（NCI-H460，EC50=21 nmol/L），黑色素瘤（BRO，EC50=11 nmol/L）以及血液癌症（GRANTA-519，EC50=15 nmol/L；HL-60，EC50=32 nmol/L），其EC50值范围为11至37 nmol/L [1]。BI 6727对NB TICs显示出nM级别的活性，EC50为21 nmol/L，并具有出色的选择性，SKPs的EC50为2.8 μmol/L [2]。在测试的所有40种细胞系中，Volasertib抑制了增殖，平均半最大抑制浓度为313 nmol/l（范围：4-5000 nmol/l）[3]。

BI 6727具有良好的理化性质和药代动力学性质，能够进行静脉注射(i.v.)以及口服制剂的体内测试，为给药计划提供了灵活性。此外，BI 6727在多种癌症模型中显示出显著的抗肿瘤活性，包括对紫杉醇耐药性结肠癌模型[1]。Volasertib对RMS-1泡状肌肉瘤移植瘤表现出高度活性，实现了100%的肿瘤消退。其活性与G2/M期阻滞的完全和持久相关，并随后引起凋亡细胞死亡。Volasertib与长春新碱表现出协同作用，但与依托泊苷表现出拮抗效应[3]。

Recombinant human Plk1 (residues 1-603) was expressed as NH2-terminal, GST-tagged fusion protein using a baculoviral expression system and purified by affinity chromatography using glutathione-agarose. Enzyme activity assays for Plk1, Plk2, and Plk3 were done in the presence of serially diluted inhibitor using 20 ng of recombinant kinase and 10 μg casein from bovine milk as substrate. Kinase reactions were done in a final volume of 60 μL for 45 min at 30°C [15 mmol/L MgCl2, 25 mmol/L MOPS (pH 7.0), 1 mmol/L DTT, 1% DMSO, 7.5 μmol/L ATP, 0.3 μCi γ-32P-ATP]. Reactions were terminated by the addition of 125 μL of ice-cold 5% TCA. After transferring the precipitates to MultiScreen mixed ester cellulose filter plates, plates were washed with 1% TCA and quantified radiometrically. Dose-response curves were used for calculating IC50 values. To establish a kinase selectivity profile, additional kinase assays were done by contract research organizations or reagents were purchased from commercial sources and assays were done according to the supplier's instructions. Appropriate positive and negative controls were included in the assay design [1].

Cell proliferation assays were done by incubating cells in the presence of various concentrations of BI 6727 for 72 h and cell growth was assessed by measuring Alamar blue dye conversion in a fluorescence spectrophotometer. Effective concentrations at which cellular growth was inhibited by 50% (EC50) were extrapolated from the dose-response curve fit [1].

Female BomTac:NMRI-Foxn1nu mice were grafted s.c. with 2 × 10^6 HCT 116 human colon carcinoma cells (ATCC CCL-247), 1 × 10^6 NCI-H460 non–small cell lung cancer cells (ATCC HTB-177), or CXB1 human colon carcinoma tumor pieces derived from patient material by serial transplantation in nude mice. When tumors had reached a volume of ～50 to 100 mm^3, animals were randomized into treatment and control groups of 10 mice each. BI 6727 was formulated in hydrochloric acid (0.1 N), diluted with 0.9% NaCl, and injected i.v. into the tail vein at the indicated dose and schedule. For oral treatment, BI 6727 was resuspended in 0.5% Natrosol 250 hydroxyethyl-cellulose and given intragastrally via gavage needle. An administration volume of 10 mL per kilogram of body weight was used for both administration routes. Tumor volumes were determined thrice a week using a caliper. The results were converted to tumor volume (mm^3) by the formula length × width2 × π/6. The weight of the mice was determined as an indicator of tolerability on the same days. Median tumor volumes on the last day of the experiment were used to calculate treated versus control values (= tumor volume treated mice × 100/tumor volume control mice) [1].