Boris M. Seletsky

Eribulin Induces Irreversible Mitotic Blockade: Implications of Cell-Based Pharmacodynamics for In vivo Efficacy under Intermittent Dosing Conditions

Eribulin (E7389), a mechanistically unique microtubule inhibitor in phase III clinical trials for cancer, exhibits superior efficacy in vivo relative to the more potent compound ER-076349, a fact not explained by different pharmacokinetic properties. A cell-based pharmacodynamic explanation was suggested by observations that mitotic blockade induced by eribulin, but not ER-076349, is irreversible as measured by a flow cytometric mitotic block reversibility assay employing full dose/response treatment. Cell viability 5 days after drug washout established relationships between mitotic block reversibility and long-term cell survival. Similar results occurred in U937, Jurkat, HL-60, and HeLa cells, ruling out cell type-specific effects. Studies with other tubulin agents suggest that mitotic block reversibility is a quantifiable, compound-specific characteristic of antimitotic agents in general. Bcl-2 phosphorylation patterns parallel eribulin and ER-076349 mitotic block reversibility patterns, suggesting persistent Bcl-2 phosphorylation contributes to long-term cell-viability loss after eribulins irreversible blockade. Drug uptake and washout/retention studies show that [3H]eribulin accumulates to lower intracellular levels than [3H]ER-076349, yet is retained longer and at higher levels. Similar findings occurred with irreversible vincristine and reversible vinblastine, pointing to persistent cellular retention as a component of irreversibility. Our results suggest that eribulins in vivo superiority derives from its ability to induce irreversible mitotic blockade, which appears related to persistent drug retention and sustained Bcl-2 phosphorylation. More broadly, our results suggest that compound-specific reversibility characteristics of antimitotic agents contribute to interactions between cell-based pharmacodynamics and in vivo pharmacokinetics that define antitumor efficacy under intermittent dosing conditions.

Case History: Discovery of Eribulin (HALAVEN™), a Halichondrin B Analogue That Prolongs Overall Survival in Patients with Metastatic Breast Cancer

Publisher Summary This chapter focuses on the discovery and development of eribulin. Eribulin mesylate (HALAVEN) is an antitubulin antimitotic agent with distinct microtubule end-binding properties that result in inhibition of microtubule dynamics in ways that differ from those of vinblastine and paclitaxel. This agent was recently approved by the U.S. Food and Drug Administration (FDA) for use in patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease. HALAVEN represents a new and exciting treatment option that for the first time has been shown to improve overall survival in heavily pretreated women with late stage breast cancer. The path leading to the discovery and development of eribulin included close three-way collaboration among the Kishi group at Harvard University, the National Cancer Institute (NCI), and Eisai. At many points along the drug discovery path, setbacks and roadblocks arose that threatened to derail the program. Nevertheless, each of the problems was solved in turn, thereby demonstrating that a structurally complex molecule could be optimized through total synthesis to successfully deliver a marketed drug that meets all pharmacological, toxicological, and physicochemical requirements.

Novel second generation analogs of eribulin. Part II: Orally available and active against resistant tumors in vivo.

Eribulin mesylate is a newly approved treatment for locally advanced and metastatic breast cancer. We targeted oral bioavailability and efficacy against multidrug resistant (MDR) tumors for further work. The design, synthesis and evaluation of novel amine-containing analogs of eribulin mesylate are described in this part. Attenuation of basicity of the amino group(s) in the C32 side-chain region led to compounds with low susceptibility to PgP-mediated drug efflux. These compounds were active against MDR tumor cell lines in vitro and in xenograft models in vivo, in addition to being orally bioavailable.

Novel second generation analogs of eribulin. Part I: Compounds containing a lipophilic C32 side chain overcome P-glycoprotein susceptibility

Eribulin mesylate (Halaven™), a totally synthetic analog of the marine polyether macrolide halichondrin B, has recently been approved in the United States as a treatment for breast cancer. It is also currently under regulatory review in Japan and the European Union. Our continuing medicinal chemistry efforts on this scaffold have focused on oral bioavailability, brain penetration and efficacy against multidrug resistant (MDR) tumors by lowering the susceptibility of these compounds to P-glycoprotein (P-gp)-mediated drug efflux. Replacement of the 1,2-amino alcohol C32 side chain of eribulin with fragments neutral at physiologic pH led to the identification of analogs with significantly lower P-gp susceptibility. The analogs maintained low- to sub-nM potency in vitro against both sensitive and MDR cell lines. Within this series, increasing lipophilicity generally led to decreased P-gp susceptibility. In addition to potency in cell culture, these compounds showed in vivo activity in mouse xenograft models.

Novel second generation analogs of eribulin. Part III: Blood-brain barrier permeability and in vivo activity in a brain tumor model.

Novel second generation analogs of eribulin mesylate, a tubulin agent recently approved for the treatment of breast cancer, are reported. Our recent efforts have focused on expanding the target indications for this class of compounds to other tumor types. Herein, we describe the design, synthesis and evaluation of eribulin analogs active against brain tumor cell lines in vitro and corresponding brain tumor models in mice. Attenuation of basicity of the amino group(s) in the C32 side-chain region led to compounds with lower susceptibility to P-gp mediated drug efflux, allowing these compounds to permeate through the blood-brain barrier. In preclinical in vivo studies, these compounds showed significantly higher levels in the brain and cerebrospinal fluid as compared to eribulin. In addition, analogs within this series showed antitumor activity in an orthotopic murine model of human glioblastoma.

Abstract 4493: Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Eribulin (E7389) is an antimitotic drug that inhibits microtubule assembly in vitro and in vivo by binding to tubulin. Several studies have been performed on eribulin mechanism of action; however, the mode of interaction with soluble tubulin (αβ-heterodimer) and binding site differences with vinca alkaloids (vinblastine and vincristine) and paclitaxel have not been well characterized. To understand binding differences, we performed an in vitro conformational change assays and established a surface plasmon resonance (SPR)-based direct binding assay using chemical probes and soluble tubulin. Unlike vinca alkaloids and paclitaxel, binding of eribulin with tubulin has little or no effect on conformational change of tubulin which is consistent with previous observations in the following three independent assays: 1) limited proteolysis pattern, 2) intrinsic tryptophan fluorescence and 3) exposure of hydrophobic area on the tubulin molecule. We therefore examined whether eribulin binds to the vinca or paclitaxel site of tubulin in competitive inhibition assay on SPR. Vinca alkaloids and paclitaxel failed to inhibit the binding of tubulin to immobilized eribulin chemical probe. Similarly, eribulin and paclitaxel did not inhibit the binding of tubulin to immobilized vinblastine chemical probe. Thus, the eribulin binding site on tubulin is different from that of vinca alkaloids and paclitaxel. In addition, we further investigated the binding affinities and characteristics of interaction of eribulin with GTP- or GDP-forms of tubulin, respectively. Eribulin chemical probe binds both forms of tubulin specifically with significantly higher affinity (∼40 nM) as compared with vinblastine chemical probe (∼1 μM). Interestingly, we found that eribulin chemical probe preferentially recognizes GTP- over GDP-tubulin and dissociation of GTP-form from eribulin chemical probe was slower than that of GDP-form of tubulin. In contrast, vinblastine chemical probe did not show significant difference in binding to the two nucleotide states of tubulin. This indicates that eribulin distinguishes different nucleotide state of tubulin and binds to GTP-tubulin more tightly. These characteristic features of eribulin differ from those of vinca alkaloids and paclitaxel. Overall, our results suggest that eribulin exerts its unique antimitotic activity by inhibiting microtubule assembly by binding with high affinity at a novel tubulin site at the plus-end of the growing microtubule. This may potentially relate to the mechanism of eribulin clinical efficacy and diminished neuronal toxicity. Citation Format: Kishan Lal Agarwala, Kenji Kubara, Koji Sagane, Boris M. Seletsky, Bruce A. Littlefield. Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2013-4493