Treating Cancer by Spindle Assembly Checkpoint Abrogation: Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase.

Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.

Discovery of Vilaprisan (BAY 1002670): A Highly Potent and Selective Progesterone Receptor Modulator Optimized for Gynecologic Therapies.

Progesterone plays an important role in the female reproductive system. However, there is also evidence that gynecologic disorders/diseases such as uterine fibroids and endometriosis are progesterone-dependent. Steroidal and non-steroidal selective progesterone receptor modulators (SPRMs) have shown potential for the treatment of such diseases. Steroidal SPRMs, including mifepristone and ulipristal acetate, have proven effective in clinical trials. However, several steroidal SPRMs containing a dimethylamino substituent have been associated with elevated liver enzymes in patients. An earlier drug discovery program identified lonaprisan as a highly selective SPRM that did not show drug-related change in liver enzyme activity. Building on data obtained from that work, here we describe the research program that culminated in the discovery of a novel steroidal SPRM, vilaprisan, which combines an extremely high potency with very favorable drug metabolism and pharmacokinetic properties. Vilaprisan has entered clinical development and is currently undergoing phase 3 clinical trials.

Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR.

Biocatalysis at Work: Applications in the Development of Sagopilone.

For the antitumour agent sagopilone, an epothilone analogue, a large-scale synthesis was developed to synthesise the active pharmaceutical ingredient for clinical trials, exploring enzymatic and microbial methods to produce chiral building blocks on a multi-kilogram scale. The three building blocks were identified as key intermediates in the synthesis and needed to be produced with high optical purity in yields higher than those previously published. The improved syntheses of two of these building blocks are detailed herein. For building block A, the chemical research synthesis was abandoned, and a novel chemical route was developed leading to building block A via an enzymatic hydrolysis process. For building blocks C, replacement of a chemical catalytic procedure by a microbial process meant that the development of a new starting material could be avoided, thereby accelerating the development process. For the clinical development process, a human metabolite of sagopilone was required as a reference. To accelerate the synthesis of the metabolite, no chemical synthesis was investigated; rather, we relied solely on oxidoreductases. The human metabolite of sagopilone was synthesised on a multi-gram scale in a single-step process using genetically engineered E. coli expressing human cytochrome P450 enzyme 2C19. The integration of enzymatic and microbial processes provided tools that enable the synthesis of highly functionalised intermediates and metabolites.

Molecular mode of action and role of TP53 in the sensitivity to the novel epothilone sagopilone (ZK-EPO) in A549 non-small cell lung cancer cells.

Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but--compared to high concentrations (40 nM)--only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone.

Evaluation of activity and combination strategies with the microtubule-targeting drug sagopilone in breast cancer cell lines.

Sagopilone, a fully synthetic epothilone, is a microtubule-stabilizing agent optimized for high in vitro and in vivo activity against a broad range of tumor models, including those resistant to paclitaxel and other systemic treatments. Sagopilone development is accompanied by translational research studies to evaluate the molecular mode of action, to recognize mechanisms leading to resistance, to identify predictive response biomarkers, and to establish a rationale for combination with different therapies. Here, we profiled sagopilone activity in breast cancer cell lines. To analyze the mechanisms of mitotic arrest and apoptosis and to identify additional targets and biomarkers, an siRNA-based RNAi drug modifier screen interrogating 300 genes was performed in four cancer cell lines. Defects of the spindle assembly checkpoint (SAC) were identified to cause resistance against sagopilone-induced mitotic arrest and apoptosis. Potential biomarkers for resistance could therefore be functional defects like polymorphisms or mutations in the SAC, particularly in the central SAC kinase BUB1B. Moreover, chromosomal heterogeneity and polyploidy are also potential biomarkers of sagopilone resistance since they imply an increased tolerance for aberrant mitosis. RNAi screening further demonstrated that the sagopilone-induced mitotic arrest can be enhanced by concomitant inhibition of mitotic kinesins, thus suggesting a potential combination therapy of sagopilone with a KIF2C (MCAK) kinesin inhibitor. However, the combination of sagopilone and inhibition of the prophase kinesin KIF11 (EG5) is antagonistic, indicating that the kinesin inhibitor has to be highly specific to bring about the required therapeutic benefit.

Comparative profiling of the novel epothilone, sagopilone, in xenografts derived from primary non-small cell lung cancer.

PURPOSE: Characterization of new anticancer drugs in a few xenograft models derived from established human cancer cell lines frequently results in the discrepancy between preclinical and clinical results. To take the heterogeneity of tumors into consideration more thoroughly, we describe here a preclinical approach that may allow a more rational clinical development of new anticancer drugs. EXPERIMENTAL DESIGN: We tested Sagopilone, an optimized fully synthetic epothilone, in 22 well-characterized patient-derived non-small cell lung cancer models and correlated results with mutational and genome-wide gene expression analysis. RESULTS: Response analysis according to clinical trial criteria revealed that Sagopilone induced overall responses in 64% of the xenograft models (14 of 22), with 3 models showing stable disease and 11 models showing partial response. A comparison with response rates for established drugs showed the strong efficacy of Sagopilone in non-small cell lung cancer. In gene expression analyses, Sagopilone induced tubulin isoforms in all tumor samples, but genes related to mitotic arrest only in responder models. Moreover, tumors with high expression of genes involved in cell adhesion/angiogenesis as well as of wild-type TP53 were more likely to be resistant to Sagopilone therapy. As suggested by these findings, Sagopilone was combined with Bevacizumab and Sorafenib, drugs targeting vascular endothelial growth factor signaling, in Sagopilone-resistant models and, indeed, antitumor activity could be restored. CONCLUSION: Analyses provided here show how preclinical studies can provide hypotheses for the identification of patients who more likely will benefit from new drugs as well as a rationale for combination therapies to be tested in clinical trials.

Sagopilone inhibits breast cancer bone metastasis and bone destruction due to simultaneous inhibition of both tumor growth and bone resorption.

PURPOSE: Bone metastases have a considerable impact on quality of life in patients with breast and other cancers. Tumors produce osteoclast-activating factors, whereas bone resorption promotes the growth of tumor cells, thus leading to a "vicious cycle" of bone metastasis. Sagopilone, a novel, fully synthetic epothilone, inhibits the growth of breast cancer cells in vitro and in vivo, and here we report its activity in the MDA-MB-231(SA) breast cancer bone metastasis mouse model. EXPERIMENTAL DESIGN: The potency of sagopilone was determined in treatment models simulating the adjuvant (preventive) and metastatic (therapeutic) settings in the clinic. RESULTS: We showed that sagopilone inhibited tumor burden and bone destruction, in addition to reducing tumor-induced cachexia and paraplegia. The reduction in osteolytic lesions, tumor growth in bone, and weight loss was statistically significant in the preventive model compared with the vehicle group. In the therapeutic model, sagopilone treatment significantly lowered the number of activated osteoclasts and significantly reduced the osteolytic lesion area, bone volume loss, and bone resorption compared with vehicle treatment while simultaneously inhibiting tumor burden. An in vitro assay confirmed that sagopilone inhibited osteoclast activation without cytotoxic effects, whereas paclitaxel resulted in lower inhibition and high levels of cytotoxicity. CONCLUSIONS: Sagopilone seems to inhibit the vicious cycle at both the tumor growth and bone resorption stages, suggesting the possibility for substantial benefit in the treatment of patients with breast cancer at risk from bone metastases or with bone lesions already present. Phase II clinical trials with sagopilone in patients with breast cancer are ongoing.

Organofluorosilanes as model compounds for 18F-labeled silicon-based PET tracers and their hydrolytic stability: experimental data and theoretical calculations (PET = positron emission tomography).

Silicon chemistry has only recently been discovered by radiochemists as a straightforward tool for the introduction of (18)F into biomolecules for positron emission tomography (PET) imaging. (18)F-labeled PET tracers must be stable towards defluorination under physiological conditions, but it is known that the hydrolytic stability of the silicon-fluorine bond is determined by the nature of the substituents on silicon. In the presented study we performed an extensive investigation on the hydrolytic stability of various synthesized organofluorosilane model compounds. By means of density functional theory (DFT) methods a theoretical model of organofluorosilane hydrolysis, which correlates with the experimentally determined hydrolytic half-lives, is developed. The calculation of the difference of Si-F bond lengths between the optimized structures of the starting material A and the intermediate structure C allows the estimation of the hydrolytic stability of newly designed compounds. This model permits the facilitated development of improved building blocks for the synthesis of novel (18)F-silyl-modified biomolecules for PET imaging.

Sagopilone crosses the blood-brain barrier in vivo to inhibit brain tumor growth and metastases.

The aim of this study was to determine the efficacy of sagopilone (ZK-EPO), a novel epothilone, compared with other anticancer agents in orthotopic models of human primary and secondary brain tumors. Autoradiography and pharmacokinetic analyses were performed on rats and mice to determine passage across the blood-brain barrier and organ distribution of sagopilone. Mice bearing intracerebral human tumors (U373 or U87 glioblastoma, MDA-MB-435 melanoma, or patient-derived non-small-cell lung cancer [NSCLC]) were treated with sagopilone 5-10 mg/kg, paclitaxel 8-12.5 mg/kg (or temozolomide, 100 mg/kg) or control (vehicle only). Tumor volume was measured to assess antitumor activity. Sagopilone crossed the blood-brain barrier in both rat and mouse models, leading to therapeutically relevant concentrations in the brain with a long half-life. Sagopilone exhibited significant antitumor activity in both the U373 and U87 models of human glioblastoma, while paclitaxel showed a limited effect in the U373 model. Sagopilone significantly inhibited the growth of tumors from CNS metastasis models (MDA-MB-435 melanoma and patient-derived Lu7187 and Lu7466 NSCLC) implanted in the brains of nude mice, in contrast to paclitaxel or temozolomide. Sagopilone has free access to the brain. Sagopilone demonstrated significant antitumor activity in orthotopic models of both glioblastoma and CNS metastases compared with paclitaxel or temozolomide, underlining the value of further research evaluating sagopilone in the treatment of brain tumors. Sagopilone is currently being investigated in a broad phase II clinical trial program, including patients with glioblastoma, NSCLC, breast cancer, and melanoma.

Sagopilone (ZK-EPO): from a natural product to a fully synthetic clinical development candidate.

BACKGROUND: Tubulin is among the most established and clinically validated targets in oncology. The taxanes, paclitaxel and docetaxel, stabilize microtubules and have shown significant clinical activity, but factors such as the development of resistance can limit their clinical use. The epothilones are a novel class of natural microtubule-stabilizing products with potential activity in an expanded spectrum of tumour indications. OBJECTIVE: In an extensive lead optimization programme, we selected sagopilone from 350 compounds produced by total synthesis because of its combination of potent activity and good tolerability in tumour models. It is the first fully synthetic epothilone in clinical development. METHODS: Here we review the directed optimization of the natural product epothilone B to produce sagopilone, along with its mechanism of action, preclinical data and emerging clinical results. RESULTS/CONCLUSIONS: We show how this optimization process translated into superior preclinical activity, coupled with a favourable tolerability profile. Activity has been determined in a number of animal models, including those from tumours resistant to other systemic treatments. The approach used to develop sagopilone may become more common as structure-driven research is increasingly employed to exploit the enormous potential of natural products, in parallel with other targeted approaches, heralding a new era of anticancer therapy.

Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks.

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.

Improved cellular pharmacokinetics and pharmacodynamics underlie the wide anticancer activity of sagopilone.

Sagopilone (ZK-EPO) is the first fully synthetic epothilone undergoing clinical trials for the treatment of human tumors. Here, we investigate the cellular pathways by which sagopilone blocks tumor cell proliferation and compare the intracellular pharmacokinetics and the in vivo pharmacodynamics of sagopilone with other microtubule-stabilizing (or tubulin-polymerizing) agents. Cellular uptake and fractionation/localization studies revealed that sagopilone enters cells more efficiently, associates more tightly with the cytoskeleton, and polymerizes tubulin more potently than paclitaxel. Moreover, in contrast to paclitaxel and other epothilones [such as the natural product epothilone B (patupilone) or its partially synthetic analogue ixabepilone], sagopilone is not a substrate of the P-glycoprotein efflux pumps. Microtubule stabilization by sagopilone caused mitotic arrest, followed by transient multinucleation and activation of the mitochondrial apoptotic pathway. Profiling of the proapoptotic signal transduction pathway induced by sagopilone with a panel of small interfering RNAs revealed that sagopilone acts similarly to paclitaxel. In HCT 116 colon carcinoma cells, sagopilone-induced apoptosis was partly antagonized by the knockdown of proapoptotic members of the Bcl-2 family, including Bax, Bak, and Puma, whereas knockdown of Bcl-2, Bcl-X(L), or Chk1 sensitized cells to sagopilone-induced cell death. Related to its improved subcellular pharmacokinetics, however, sagopilone is more cytotoxic than other epothilones in a large panel of human cancer cell lines in vitro and in vivo. In particular, sagopilone is highly effective in reducing the growth of paclitaxel-resistant cancer cells. These results underline the processes behind the therapeutic efficacy of sagopilone, which is now evaluated in a broad phase II program.