BI 6727 , APolo-like Kinase Inhibitor with Improved Pharmacokinetic Profile and Broad

Purpose:Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced andmetastatic cancers.To identify novelmitosis-specific agents withhigher selectivity than approved tubulin-binding agents (taxanes,Vinca alkaloids), we have generated inhibitors of Polo-like kinase1, a target that functions predominantly inmitosis. Experimental Design:The first compound in this series, suitable for i.v. administration, has entered clinical development.To fully explore the potential of Polo-like kinase1inhibition inoncology, we have profiled additional compounds and now describe a novel clinical candidate. Results:BI 6727 is ahighly potent (enzyme IC50 = 0.87 nmol/L, EC50 = 11-37 nmol/L onapanel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (Vss = 7.6 L/kg, t1/2 = 46 h) and rats (Vss = 22 L/kg, t1/2 = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer.With oral and i.v. routes of administration, the totalweekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. Conclusion: These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated. In eukaryotes from yeast to men, orthologues of Polo-like kinase 1 (Plk1) control multiple essential steps of mitosis (reviewed in refs. 1, 2). In vertebrate cells, Plk1 has been implicated in the activation of Cdk1-cyclin B at mitotic entry (3), centrosome maturation (4), release of cohesin from chromosome arms (5), formation and maintenance of the bipolar spindle (6–8), mitotic exit (9), and cytokinesis (7, 10– 13). In addition, Plk1 is a target of the DNA damage checkpoint (14, 15) and seems to be required for mitotic reentry after DNA damage checkpoint recovery (16). Plk1 mRNA and protein expression, its catalytic activity, and subcellular localization are tightly regulated during cell cycle progression (17). Plk1 is located at centrosomes and kinetochores during early mitosis, and a fraction translocates to the midzone/midbody late in mitosis (18). Based on its temporal and spatial expression patterns as well as genetic and biological evidence, Plk1 seems to function uniquely in the regulation of the cell division cycle during late G2 and M phases. In previous studies, we have used small interfering RNA (siRNA) and a chemical biology approach with BI 2536 (6, 8, 13, 19) to validate this role of Plk1 in human cells. We observed that the cellular effects of down-regulating Plk1 mRNA and protein with siRNA are identical to selective inhibition of its serine/threonine kinase activity. Cellular effects of Plk1 inhibition include a distinctive ‘‘polo arrest’’ phenotype in prometaphase, accumulation of phospho–histone H3, and formation of aberrant mitotic spindles followed by a surge in apoptosis (6). Using BI 2536, these cellular phenotypes in tissue culture were linked to identical effects in vivo (19). Of note, three additional Polo-like kinase family members (Plk2/Snk, Plk3/Fnk, and Plk4/SAK) have been identified, but these are thought to function during the G1 and S phases of the cell cycle (reviewed in ref. 17) and their inhibition apparently does not contribute to the BI 2536–induced cellular phenotype. Consistent with its biological role, Plk1 mRNA and protein are highly expressed in proliferating tissues and Plk1 seems to be overexpressed in a broad spectrum of cancers, with highest expression levels being correlated with poor prognosis in several cancer types (20). Taken together, these observations highlight Plk1 as an attractive molecular target for the antimitotic approach to cancer therapy (21). Clearly, in this category of antiproliferative agents, the approved tubulinbinding taxanes and Vinca alkaloids serve as reference points for clinical efficacy, side effects, and convenience of drug administration. Three lines of continued drug development can be discerned. First, efforts are made to improve the formulation of existing microtubule-stabilizing (taxanes) and destabilizing (Vinca alkaloids) drugs, to develop close analogues suitable for Cancer Therapy: Preclinical Authors’Affiliations: Boehringer Ingelheim RCVGmbH&Co KG,Vienna, Austria and Boehringer Ingelheim Pharma GmbH& Co KG, Biberach, Germany Received9/22/08; revised1/19/09; accepted1/20/09;publishedOnlineFirst4/21/09. Requests for reprints: Dorothea Rudolph, Department of Pharmacology, Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria. Phone: 43-1-801-052780; Fax: 43-1-801-052366; E-mail: dorothea.rudolph@boehringer-ingelheim.com. F2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2445 www.aacrjournals. org Clin Cancer Res 2009;15(9) May1, 2009 3094 Research. on July 23, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals. org Downloaded from oral administration, and to develop novel chemical series of antimicrotubule agents, like epothilones, to overcome taxane resistance (22). Second, mitotic kinesins have been identified as microtubule-associated motor proteins and ATPases like Eg5 or CENP-E represent novel drug targets that provide specificity for the cell division–related function of microtubules (23, 24). Finally, the cell cycle–regulatory kinases CDK1, Aurora B, and Plk1 are tubulin-independent targets for mitotic inhibitors that are readily accessible to medicinal chemistry approaches (25). Of these three potential mitotic kinase targets, Aurora B has thus far been targeted extensively by drug candidates. The results of the first clinical trials with these Aurora B inhibitors are pending (26). Plk1 inhibitors have emerged more recently (7, 19, 27–29) with BI 2536 as the first compound to enter clinical studies. ON01910 has been reported earlier to act via Plk1 (30), but this notion was not corroborated in subsequent studies (6, 29) and its mode of action remains to be defined. While the first clinical studies of Plk1 inhibitors are ongoing, we and others (7, 19, 27–29) are continuing to broaden the range of clinical candidates targeting Plk1. These efforts are aided by the lessons learned from our previous program on the chemical optimization of dihydropteridinones and emerging X-ray crystallographic data regarding the basis of high Plk1 kinase selectivity (31, 32). Here, we report on BI 6727, a dihydropteridinone derivative with high potency and selectivity. In preclinical studies, a high volume of distribution, indicating good tissue penetration, and a long terminal half-life have emerged as distinct features of BI 6727, which may have a favorable effect on antitumor efficacy in vivo . The results of ongoing clinical studies in cancer patients will allow a comparison with human pharmacokinetic and pharmacodynamic parameters in the future. Materials and Methods Chemistry. The dihydropteridinone BI 6727 was derived from a chemical lead optimization program designed to generate Plk inhibitors (Patent Application WO 2004/076454). Crystal structure. Protein expression of the T210V mutant kinase domain was done using a baculovirus/insect cell expression system. A homogeneity of >95% as judged by Coomassie-stained SDS-PAGE was achieved after a chromatographic purification procedure including affinity, ion exchange, and gel filtration chromatography. Protein crystallization was done using the sitting drop method with a reservoir containing 3% to 16% PEG 3350-6000 and 0.2 to 0.5 mol/L sodium malonate (31). Protein-ligand complexation was done before crystallization by incubation of the ligand for 2 h at 4jC. Crystals were frozen using the Proteros Free Mounting System. Data were collected at the SLS beam line X06SA (Swiss Light Source, Paul Scherrer Institute) using the PILATUS 6M detector. Images were processed with XDS; structure solution was achieved by Patterson search methods and model building; and refinement was done with COOT and REFMAC. Protein crystallography was done by Proteros biostructures GmbH. The structure has been deposited in the Protein Data Bank under the access