Tubulin polymerization; microtubule

Eribulin (1-100 nM; 72 h) suppresses cell growth with IC50 values of 22.8 and 21.5 nM for LM8 and Dunn cells, respectively [1]. Eribulin (10-50 nM; 12-72 hours) significantly promotes early apoptosis in LM8 cells given at a dosage of 50 nM for 24 hours [1]. In LM8 cells, eribulin (10-50 nM; 12-72 hours) produced G2/M arrest at 50 nM for 12 hours, while long-term treatment (72 hours) at 10 nM Does not induce G2/M phase arrest[1]. Eribulin (1-50 nM; 12 hours) does not promote senescence in LM8 cells [1]. Eribulin (1-10 nM; 16 hours) produces morphological alterations and inhibits cell migration at low concentrations in LM8 cells [1].

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Therefore, in this study, we investigated the suppressive effect of the microtubule inhibitor eribulin mesylate (eribulin) on lung metastasis of osteosarcoma. At concentrations >proliferation IC50, eribulin induced cell cycle arrest and apoptosis in a metastatic osteosarcoma cell line, LM8. However, at concentrations Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. Using [(3)H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K(d) of 46 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 +/- 1.3 molecules per microtubule (K(d) = 3.5 microM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Preincubation of microtubules with 2 or 4 microM vinblastine induced additional lower-affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability.[2]

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Eribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase of microtubule dynamic instability in interphase cells, arrests mitosis, and induces apoptosis, suggesting that suppression of spindle microtubule dynamics induces mitotic arrest. To further test this hypothesis, we measured the effects of eribulin on dynamics of centromeres and their attached kinetochore microtubules by time-lapse confocal microscopy in living mitotic U-2 OS human osteosarcoma cells. Green fluorescent protein-labeled centromere-binding protein B marked centromeres and kinetochore-microtubule plus-ends. In control cells, sister chromatid centromere pairs alternated under tension between increasing and decreasing separation (stretching and relaxing). Eribulin suppressed centromere dynamics at concentrations that arrest mitosis. At 60 nmol/L eribulin (2 x mitotic IC(50)), the relaxation rate was suppressed 21%, the time spent paused increased 67%, and dynamicity decreased 35% (but without reduction in mean centromere separation), indicating that eribulin decreased normal microtubule-dependent spindle tension at the kinetochores, preventing the signal for mitotic checkpoint passage. We also examined a more potent, but in tumors less efficacious antiproliferative halichondrin derivative, ER-076349. At 2 x IC(50) (4 nmol/L), mitotic arrest also occurred in concert with suppressed centromere dynamics. Although media IC(50) values differed 15-fold between the two compounds, the intracellular concentrations were similar, indicating more extensive relative uptake of ER-076349 into cells compared with eribulin. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic arrest indicates that the primary mechanism by which eribulin blocks mitosis is suppression of spindle microtubule dynamics. [3]

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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 eribulin's 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 eribulin's 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 [4].

Eribulin (1 mg/kg; intravenously injected once weekly for 2 weeks) inhibits initial tumor development and lung metastasis of osteosarcoma in rats [1]. At low doses, eribulin (1 mg/kg; intravenously administered once) suppresses the development of circulating tumor cells (CTCs) [1].

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Higher doses of Eribulin administered on a standard schedule inhibited lung metastasis and primary tumor growth in a murine osteosarcoma metastasis model. Frequent low-dose eribulin administration (0.3 mg/kg every 4 days × 4) effectively inhibited lung metastasis but had little effect on primary tumor growth. Overall, our results indicate that eribulin could reduce osteosarcoma lung metastasis.[1]

Inhibition of lung metastasis by Eribulin in a murine model [1]
We first investigated whether Eribulin inhibits osteosarcoma lung metastasis in a mouse model using a clinical administration schedule. According to the package insert, eribulin was clinically administered at 1.4 mg/m2 on day 1 and day 8. The pharmacokinetics data for humans and mice revealed that 1 mg/kg eribulin administered to mice had similar pharmacokinetics to 1.4 mg/m2 eribulin in humans. Thus, we administered eribulin at 1 mg/kg every 7 days × 2 in the osteosarcoma metastatic model (Figure 1A). The body weights of mice in the treatment group were significantly lower than those in the control group (Figure 1B). Eribulin treatment significantly suppressed primary tumor growth (Figure 1C) and induced apoptosis in tumor cells (Figure 1D). We assessed lung metastasis by counting the metastatic foci (Figure 1E) and measuring their area in tissue sections (Figure 1F top). Eribulin clearly reduced lung metatasis. Histological images showed that in the control group, large metastatic foci infiltrated the lung parenchyma. In contrast, small metastatic foci were solitary within the normal alveolar structure in the treatment group (Figure 1F bottom). To determine whether eribulin reduced CTCs, blood samples were collected and cultured to form colonies. The colony number significantly decreased in the treatment group (Figure 1G) relative to that in the control group. These results indicate that eribulin reduced primary tumor growth and lung metastasis of osteosarcoma.

Eribulin Binding to Soluble Tubulin [2]
Phosphocellulose-purified tubulin (1.8 μM) in PEM 50 buffer (50 mM PIPES, 1 mM EGTA, and 1 mM MgSO4, pH 6.9) and 100 μM GTP was incubated with [3H]eribulin (0.1 μM-70 μM) for 20 min at room temperature (22 °C), then 100 μL of each sample was added to a Zeba™ microspin desalting column. Specific activity of each sample was measured immediately after eribulin addition. Columns were centrifuged for 2 min at 1500 × g following manufacturer’s instructions. The protein content and radioactivity in the tubulin flow-through were determined. Background radiolabel was assessed by centrifuging samples without protein at the concentrations of eribulin used. Background radioactivity levels correlated with eribulin concentration, with an average of 4.2% of the starting eribulin passing through the column with no protein present. This value was subtracted from all experimental radioactivity measurements.

Equilibrium binding data were quantified by measuring the fraction of tubulin bound with eribulin at various eribulin concentrations and a single fixed concentration of tubulin (2 μM). The following model for simple hyperbolic binding was fit to the data:
YD=Ymax⁢D/(Kd+D) In this model, D is the total concentration of eribulin, YD and Ymax are the ratios of bound eribulin to tubulin at subsaturating and infinite concentrations of D, respectively, and Kd is the equilibrium binding constant. The best-fit values for Ymax and Kd were determined by non-linear regression using KaleidaGraph® 3.5. Ymax and Kd values and standard errors of eribulin binding to soluble tubulin were calculated using all data points as a single data set.

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Microtubule Depolymerization [2]
Microtubule depolymerization was measured by turbidity using a temperature-controlled Beckman Coulter spectrophotometer. Polymerization of MAP-rich tubulin (3 mg/ml) was initiated by incubation at 30°C for 30 min in PEM 100 buffer (100 mM PIPES, 1 mM EGTA, and 1 mM MgSO4, pH 6.9) and 1 mM GTP. Microtubules were then sheared eight times through a 25 gauge needle and allowed to regain steady state for 15 min. Aliquots of microtubule suspension (100 μL) were added to cuvettes and placed in a warmed spectrophotometer at 30 °C. Eribulin (1 μM-20 μM) was added at time zero and absorbance readings were taken at a wavelength of 350 nm every minute for 30 min.

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Dynamic Instability of Microtubule Minus Ends [2]
The dynamic instability of microtubules in the presence of eribulin was measured as described previously. Briefly, microtubules (18 μM tubulin) were assembled from phosphocellulose-purified tubulin in PMEM buffer (86 mM PIPES, 26 mM MES, 1 mM EGTA, and 1.4 mM MgSO4, pH 6.8) and 1 mM GTP in the presence or absence of eribulin. Microtubules were nucleated using sea urchin flagellar axonemal fragments and incubated at 35 °C for 30 min to achieve steady state. The behavior of individual microtubules was recorded by video-enhanced differential interference contrast microscopy with an IX71 Olympus inverted microscope and analyzed for growth and shortening events. Minus ends were distinguished from plus ends as described previously. Between 45 and 50 growth and shortening events were measured for each condition.

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Eribulin Binding to Microtubules and Microtubule Length Determination [2]
Microtubules were assembled from MAP-rich tubulin (3 mg/ml) and sheared to obtain a large number of short microtubules as described above, then incubated another 20 min to regain steady state. [3H]Eribulin was added (0.1 μM-10 μM) to 375 μL of sheared microtubules and samples were immediately layered onto a microtubule stabilizing cushion (30% glycerol and 10% DMSO in PEM buffer) to minimize depolymerization at high eribulin concentrations (incubation time with drug was approximately 1-2 min). Microtubules were collected by centrifugation in an SW 50.1 swinging bucket rotor using a Beckman Coulter Optima ultracentrifuge (108,000 × g, 60 min, 30 °C). The soluble fraction was discarded, and microtubule pellets were carefully washed with PEM buffer and dissolved in 0.1 N NaOH at 4 °C overnight. Protein content and radioactivity of pellets were measured the following day. Background radiolabel was measured by performing parallel assays using 20 μM podophyllotoxin to inhibit microtubule polymerization. All results are reported with background subtracted.

Microtubule lengths were determined by electron microscopy for use in determination of the stoichiometry of eribulin binding to microtubules. Lengths of a minimum of 300 microtubules were measured for each experiment. For controls, mean microtubule length ranged from 2.4 to 4.2 μm per experiment, with an overall mean of ~3 μm. Microtubules, either without eribulin addition for controls, or after eribulin incubation as described below, were fixed with 0.2% glutaraldehyde, placed on electron microscopy grids, stained with cytochrome c (1 mg/mL) and 1% uranyl acetate, and imaged using a JEOL 1230 transmission electron microscope (80 kV).

To account for eribulin-induced changes in microtubule length in stoichiometry determination, microtubules were incubated with the stated eribulin concentrations for 1-2 min and centrifuged through a DMSO/glycerol stabilizing cushion into a denser layer of 70% sucrose. Pelleted microtubules were fixed and stained as above and their lengths measured by electron microscopy. At concentrations of 5 and 10 μM, eribulin reduced the mean microtubule length by 17% and 29% respectively. These length changes were factored into the calculations of bound eribulin per microtubule for these two concentrations.

The number of eribulin molecules bound per microtubule was calculated using the mass and radioactivity of each pellet, the mean measured value for microtubule length, and the factor of 1,690 tubulin heterodimers per μm. Microtubules per liter was calculated for each sample using the microtubule pellet mass for that sample. Non-linear regression of binding data was computed using KaleidaGraph® 3.5 using the above equation. Ymax (ratio of bound eribulin molecules per microtubule) and Kd values for eribulin binding to microtubules were calculated for individual experiments, and the mean and standard error for all experiments was determined. Effects of Vinblastine on Eribulin Binding to Microtubules [2]
Unlabeled vinblastine as added to sheared MAP-rich microtubules at a concentration of 2 μM or 4 μM for 15 min before [3H]eribulin was added, then samples were treated as above. Electron microscopy samples for length measurements were taken after vinblastine incubation, but before eribulin addition. Consistent with previous findings that vinblastine stabilizes microtubule ends against shortening, in the presence of vinblastine (2 or 4 μM), eribulin induced no significant changes in microtubule length; therefore, the stoichiometry of eribulin bound per microtubule in the presence of vinblastine was calculated using the vinblastine-treated microtubule length measured in each experiment.

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Morphology of Microtubule Ends [2]
To determine drug effects on microtubule ends, unsheared microtubules were incubated with no drug, 50 μM eribulin or 50 μM vinblastine for 15 min, then 5 μL of sample was gently added to 15 μL glutaraldehyde (0.1% final concentration) on an electron microscope grid and stained as above. Both ends of 50 microtubules each for control and eribulin-treated microtubules and 30 microtubules for vinblastine-treated microtubules were examined and categorized as blunt, slightly splayed, or extensively splayed/spiraled.

Cell proliferation assay[1]
Cell Types: LM8 cells and Dunn cells
Tested Concentrations: 0, 1, 10, 100 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of cell proliferation in a dose-dependent manner.

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Apoptosis analysis [1]
Cell Types: LM8 cells
Tested Concentrations: 0, 10, 50 nM
Incubation Duration: 12, 24, 48, 72 hrs (hours)
Experimental Results: 50 nM concentration induced early apoptosis after 12 hrs (hours). No apoptosis was detected at 10 nM concentration.

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Cell cycle analysis[1]
Cell Types: LM8 Cell
Tested Concentrations: 0, 10, 50 nM
Incubation Duration: 12, 24, 48, 72 hrs (hours)
Experimental Results: Treatment with 50 nM for 12 hrs (hours) induces G2/M arrest. 10 nM treatment did not induce G2/M arrest.

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Cell proliferation assay [1]
Cell viability was assessed with a CCK-8 WST assay kit according to the manufacturer's instructions. In brief, LM8 cells were plated in 96-well plates at 1 × 104 cells per well and incubated in DMEM with 10% FBS for 24 h. Then, the cells were treated with various Eribulin concentrations for 72 h. CCK-8 reagent was then added to the medium, and the cells were further incubated for 2 h at 37°C. Absorbance was measured at 450 nm on a plate reader.

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Flow cytometry analysis [1]
The cells were plated in 24-well plates at a concentration of 3 × 105 cells per well and were grown for 24 h. The cells were then treated with the indicated Eribulin concentrations for the specified time periods. They were stained with Annexin-V and 7-aminoactinomycin D (7-AAD) to identify apoptosis and stained with propidium iodide for cell cycle analysis. The samples were analyzed in a Guava EasyCyte Plus Flow Cytometry System.

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Immunofluorescence [1]
LM8 cells (1 × 103) were grown on eight-well culture slides coated with fibronectin (10 μg/mL) and incubated for 24 h. The cells were then treated with Eribulin for 16 h. They were fixed with 4% paraformaldehyde for 20 min and permeabilized with either 0.1% Triton X-100 (APC and p-FAK staining) or 0.1% Tween 20 (α-tubulin staining) for 10 min followed by blocking with 0.1% bovine serum albumin (BSA) and 0.1% Tween 20 for 60 min at room temperature. For vinculin staining, the cells were permeabilized at 4°C for 1 min with ice-cold permeabilization buffer (pH 6.9; 10 mM HEPES, 50 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, 300 mM sucrose, and 1 mM EGTA) before fixation. The cells were stained with primary antibodies (α-tubulin 1:500; p-FAK 1:250; APC 1:250; vinculin 1:50) overnight at 4°C followed by secondary antibody staining and counterstaining with Hoechst 33342 for 30 min at room temperature. For F-actin staining, the cells were incubated with rhodamine-phalloidin according to the manufacturer's instructions.

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Protrusion measurement [1]
LM8 cells were plated in eight-well culture slides at a rate of 1 × 103 cells per well and grown for 24 h. The cells were treated with Eribulin and incubated for 16 h. Microspikes >20 μm long were defined as protrusions. More than 150 cells from three biological replicates were analyzed.

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Migration assay [1]
The modified Boyden chamber migration and wound healing assays were performed as described previously. In brief, the modified Boyden chamber migration assay was performed for 12 h in 24-well Bio-Coat cell migration chambers. The lower surface of the membrane was coated with 30 μg/mL fibronectin for haptotactic migration. LM8 cells were applied to the upper chamber at a rate of 1 × 104 cells with Eribulin treatment for 12 h. Non-migratory cells were removed from the upper surface with cotton swabs. Migrated cells were fixed in 70% v/v methanol, stained with crystal violet, and counted. For the wound healing assay, confluent LM8 cells were scratched and throughly washed with phosphate buffered saline (PBS) to remove detached cells and debris. They were then treated with Eribulin for 12 h. Images of wounds were measured with Fiji/ImageJ. Cell directionality was evaluated by fixing the cells during wound healing with 100% ice-cold methanol for 10 min at room temperature then blocking them for 30 min with PBS containing 1% BSA and 0.1% Tween 20. MTOCs were stained with anti-pericentrin antibody (1:1,000) at 4°C overnight. The cells were then incubated with anti-Rabbit IgG and Hoechst for 30 min at room temperature. More than 100 cells from three separate experiments were analyzed.

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3D collagen culture [1]
Collagen gels were fabricated by diluting 3.8 mg/mL acid-solubilized rat-tail collagen in DMEM to 1.5 mg/mL and neutralizing to pH 7.4 with 1 mol/L NaOH. Cell suspensions were added to the wells at a density of 200 cells/well then immediately transferred to a 37°C incubator for 30 min to initiate polymerization. The collagen gels were then covered with culture media containing Eribulin. Cells or colonies were observed under a microscope on days 1, 4, and 6. Invadopodia lengths and colony sizes were analyzed using the Fiji/ImageJ software. Colony formation rates were calculated as the colony numbers on day 4 per applied cell number.

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Cell Proliferation and Mitotic Index [3]
Cells were seeded on poly-L-lysine-treated (50 mg/ml, 2 h, 37°C, washed once with sterile water) sterile glass coverslips in six-well plates at 1 × 105 cells/2 ml/well. One day later medium was replaced with fresh medium containing a range of Eribulin or ER-076349 concentrations (0.003–10,000 nmol/L) and further incubated for one cell cycle (28 h). Cells were harvested by combining floating cells with attached cells, which had been released by trypsinization (0.5 mg/ml in PBS: 137 mmol/L NaCl, 2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, 8.1 mmol/L Na2HPO4, 0.5 mmol/L EDTA, pH 7.2) (5 min, 37°C) and live cells were counted using a hemacytometer. Trypan blue dye was used to distinguish living from dead cells. To evaluate mitotic indices, cells were grown for 20 h in the absence and presence of drug. Mitotic indices were determined by microscopic examination of chromosomes and GFP-CENP-B centromeres in cells that were fixed in formalin/methanol (described below), stained with 4,6-diamidino-2-phenylindole (DAPI), and imaged using a Nikon Eclipse E800 microscope with 60× and 100× (numerical aperture 1.4 for both) objectives. Results are the mean and standard error of 5 independent experiments, in which a minimum of 1000 cells were counted for each condition in each experiment. IC50s were determined by linear regression of double-reciprocal plots of proliferation or mitotic index vs. drug concentration.

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Cellular uptake and washout/retention studies [4]
Evaluation of [3H]Eribulin, [3H]ER-076349, [3H]vincristine, and [3H]vinblastine uptake and washout/retention in U-937 cells was done as follows. Tritiated compounds were added to sterile 1.5 mL screw cap microcentrifuge tubes and air dried to remove solvent. After compound resuspension in 100 μL cell culture medium, 22.2 × 106 cells were added in 0.9 mL complete culture medium (including fetal bovine serum, glutamine, and antibiotics). Incubations were at 37°C with frequent vortexing. Compound uptake as a function of drug concentration was determined after 60 minutes, the empirically determined minimum time needed to reach maximal radioactivity uptake. Cell-associated radioactivity was determined as follows. Triplicate 25-μL samples of cells were removed and layered on top of 300 μL ice-cold 20% sucrose in 400 μL Sarstedt tubes, followed by centrifugation at 8,500 × g for 1 minute to separate cells from labeled media and to wash cells during centrifugal transit through the sucrose. Tubes with pelleted cells were immediately frozen in a dry ice/ethanol bath. Bottom portions of the still-frozen tubes containing pellets were immediately cut off directly into scintillation vials, followed by addition of scintillation fluid and radioactivity counting (Beckman LS 6000 counter). Because of unexpectedly large differences in cellular uptake between the paired drugs (Fig. 6), different preloading concentrations were used for washout/retention studies to start with equal intracellular drug levels (dashed lines, Fig. 6A and B). Thus, preloading concentrations for washout/retention studies used 800 nmol/L [3H]eribulin versus 100 nmol/L [3H]ER-076349, and 600 nmol/L [3H]vincristine versus 190 nmol/L [3H]vinblastine.

Animal/Disease Models: C3H/HeN mice (4 weeks old) were injected with LM8 cells [1]
Doses: 1 mg/kg
Route of Administration: intravenous (iv) (iv)injection once a week for 2 weeks
Experimental Results: Inhibited primary tumor growth and induced tumor cell apoptosis Death. Reduce lung metastasis. Lost weight.

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Four-week-old C3H/HeN mice were used. LM8 cells metastasize to the lungs by both subcutaneous and intraosseous transplantation. Researchers chose subcutaneous transplantation because of its high reproductivity of the metastatic process, such as CTC appearance and lung metastasis formation, compared to intraosseous transplantation. For the Eribulin metastasis reduction experiments, LM8 cells (5 × 106 per mouse) were injected into the subcutaneous tissue of the backs of syngeneic mice. The animals were randomized into control and treatment groups. Eribulin was injected into the tail veins at 1 mg/kg every 7 days × 2 or 0.3 mg/kg every 4 days × 4. Histological evaluations and pulmonary metastasis foci counts were performed four weeks after tumor cell injection because tumor-bearing mice die from lung metastasis around 5 weeks after subcutaneous transplantation. Lungs were fixed with 10% formalin, embedded in paraffin, cut into 8-μm sections, and stained with hematoxylin and eosin (H&E). Apoptosis in the primary tumors was assessed by a TUNEL assay of the paraffin sections using an In Situ Cell Death Detection Kit according to the manufacturer's instructions. For CTC quantification, Eribulin was injected into the tail veins at a rate of 1 mg/kg two weeks after LM8 transplantation as mentioned above. Peripheral blood samples (40 μL) were collected from the tail veins and maintained in DMEM supplemented with 10% FBS and penicillin (100 U/mL)-streptomycin (100 μg/mL) to form colonies as previously described. Colonies were fixed with 10% formalin, stained with crystal violet, and counted. [1]

Absorption, Distribution and Excretion
Eribulin is eliminated primarily in feces unchanged.
43 L/m2 to 114 L/m2
1.16 L/hr/m2 to 2.42 L/hr/m2 (dose range of 0.25 mg/m2 to 4.0 mg/m2). [FDA]

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Metabolism / Metabolites
There are no major human metabolites of eribulin, CYP3A4 negligibly metabolizes eribulin in vitro.

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Biological Half-Life
about 40 hours

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Eribulin (eribulin mesylate) is a synthetic analog of halichondrin B, a natural product derived from the marine sponge Halichondria okadai. It is a microtubule dynamics inhibitor used in the treatment of metastatic breast cancer and liposarcoma. Below is a summary of its pharmacokinetic (PK) characteristics based on available data:

Absorption
Eribulin is administered intravenously (IV) over 2–5 minutes, ensuring complete bioavailability.

Distribution
It has a large volume of distribution (~43–114 L/m²), indicating extensive tissue distribution.
Plasma protein binding is moderate, ranging from 49% to 65%.

Metabolism
Eribulin undergoes limited hepatic metabolism, primarily via CYP3A4 to a minor extent, but most of the drug is eliminated unchanged.
It is not a significant substrate, inhibitor, or inducer of major CYP enzymes, reducing the risk of drug-drug interactions.

Elimination
The elimination half-life is approximately 40 hours, allowing for a dosing schedule of Day 1 and Day 8 of a 21-day cycle.
Excretion occurs mainly via the feces (82%), with a smaller portion (9%) excreted unchanged in the urine.

Special Populations
Hepatic impairment: Patients with mild (Child-Pugh A) or moderate (Child-Pugh B) liver dysfunction require dose reductions due to increased exposure. Severe impairment (Child-Pugh C) has not been studied.
Renal impairment: Moderate renal impairment (CrCl 30–50 mL/min) also necessitates dose adjustment, while severe renal impairment (CrCl <30 mL/min) has limited data.

Key Considerations
Eribulin’s PK is not significantly affected by age, body weight, or race.
Due to its long half-life, accumulation can occur with repeated dosing, necessitating monitoring for toxicity (e.g., neutropenia, peripheral neuropathy).
These PK properties support its clinical use in advanced cancers, with adjustments needed for hepatic or renal dysfunction to minimize toxicity. For more details, refer to prescribing information or clinical studies.

Hepatotoxicity
Eribulin is a cytotoxic chemotherapeutic agent and serum aminotransferase and alkaline phosphatase elevations are common during cyclic therapy of breast cancer and liposarcoma. The reported rates of ALT elevations have ranged from 8% to 83% with values above 5 times the upper limit or normal (ULN) in 2% to 5%. Isolated mention of “toxic hepatitis” have been made in reports of clinical trials of eribulin but no details of the onset, clinical features and course have been published and the role of eribulin in these outcomes is uncertain. Nevertheless, despite high rates of serum enzyme elevations during treatment, cases of clinically apparent liver injury have not been reported in detail and must be rare.
Likelihood score: E* (unproven but suspected cause of liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of eribulin during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during eribulin therapy and for 2 weeks after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
49 to 65%.

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Eribulin (eribulin mesylate) is a microtubule inhibitor used in the treatment of metastatic breast cancer and liposarcoma. While effective, it is associated with several toxicities, primarily affecting the hematologic, neurological, and gastrointestinal systems. Below is a summary of its key toxicities:

1. Hematologic Toxicity
Neutropenia: The most common and dose-limiting toxicity, occurring in ≥25% of patients. Severe (Grade 3/4) neutropenia has been reported in up to 57% of cases, increasing the risk of infections.
Anemia: Frequently observed (≥25%), with some cases requiring transfusion support.
Thrombocytopenia: Less common but can lead to bleeding complications in severe cases.

2. Neurological Toxicity
Peripheral Neuropathy: A frequent adverse event (≥25%), manifesting as numbness, tingling, or pain in the extremities. It can be dose-dependent and sometimes irreversible.
Fatigue: Reported in a significant proportion of patients, impacting quality of life.

3. Gastrointestinal Toxicity
Nausea & Vomiting: Common (≥25%) but generally manageable with antiemetics.
Constipation: Frequently reported, sometimes requiring medical intervention.

4. Other Toxicities
Alopecia: Hair loss is common but typically reversible after treatment cessation.
Cardiac Effects: Rare cases of QT prolongation and arrhythmias have been reported, requiring monitoring in high-risk patients.
Hepatotoxicity: Elevated liver enzymes may occur, particularly in patients with pre-existing liver impairment.

Management Recommendations
Dose Adjustments: Required for severe neutropenia, neuropathy, or hepatic/renal impairment.
Prophylactic Measures: Growth factor support (e.g., G-CSF) may be needed for neutropenia, and dose delays are advised for unresolved toxicities.
Eribulin’s toxicity profile necessitates careful patient monitoring, particularly in those with pre-existing conditions. Dose modifications and supportive care are essential to mitigate adverse effects while maintaining therapeutic efficacy. For detailed guidelines, refer to prescribing information.

[1]. Low-dose eribulin reduces lung metastasis of osteosarcoma in vitro and in vivo. Oncotarget. 2019 Jan 4; 10(2): 161-174.

[2]. Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability. Biochemistry, 2010. 49(6): p. 1331-7.

[3]. Inhibition of centromere dynamics by eribulin (E7389) during mitotic metaphase. Mol Cancer Ther, 2008. 7(7): p. 2003-11.

[4]. Eribulin induces irreversible mitotic blockade: implications of cell-based pharmacodynamics for in vivo efficacy under intermittent dosing conditions. Cancer Res, 2011. 71(2): p. 496-505.

Eribulin is a fully synthetic macrocyclic ketone analogue of marine sponge natural products. Inhibits growth phase of microtubules via tubulin-based antimitotic mechanism, which leads to G2/M cell-cycle block, disruption of mitotic spindles, and, ultimately, apoptotic cell death after prolonged mitotic blockage It has a role as an antineoplastic agent and a microtubule-destabilising agent. It is a macrocycle, a polyether, a polycyclic ether, a cyclic ketone, a primary amino compound and a cyclic ketal. It is a conjugate base of an eribulin(1+).
Eribulin is a microtubule inhibitor indicated for the treatment of patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease. Eribulin was isolated from the marine sponge Halichondria okadai. Eribulin is also being investigated for use in the treatment of advanced solid tumors.
Eribulin is a Microtubule Inhibitor. The physiologic effect of eribulin is by means of Microtubule Inhibition.
Eribulin mesylate is an inhibitor of microtubule function and is used as an antineoplastic agent for refractory, metastatic breast cancer and liposarcoma. Despite its cytotoxic activity against cancer cells, eribulin has rarely been implicated in causing clinically apparent acute liver injury.
Eribulin is an analogue of halichondrin B, a substance derived from a marine sponge (Lissodendoryx sp.) with antineoplastic activity. Eribulin binds to the vinca domain of tubulin and inhibits the polymerization of tubulin and the assembly of microtubules, resulting in inhibition of mitotic spindle assembly, induction of cell cycle arrest at G2/M phase, and, potentially, tumor regression.
See also: Eribulin Mesylate (has salt form).

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Drug Indication
For the treatment of patients with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic cancer.
FDA Label
Halaven monotherapy is indicated for the treatment of patients with locally advanced or metastatic breast cancer who have progressed after at least one chemotherapeutic regimens for advanced disease (see section 5. 1). Prior therapy should have included an anthracycline and a taxane unless patients were not suitable for these treatments. Halaven is indicated for the treatment of adult patients with unresectable liposarcoma who have received prior anthracycline containing therapy (unless unsuitable) for advanced or metastatic disease (see section 5. 1).
Treatment of soft tissue sarcoma

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Mechanism of Action
Eribulin inhibits the growth phase of microtubules without affecting the shortening phase and sequesters tubulin into nonproductive aggregates. Eribulin exerts its effects via a tubulin-based antimitotic mechanism leading to G2/M cell-cycle block, disruption of mitotic spindles, and, ultimately, apoptotic cell death after prolonged mitotic blockage. [FDA]

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Pharmacodynamics
Linear

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Eribulin mesylate is a methanesulfonate salt obtained by reaction of eribulin with one equivalent of methanesulfonic acid. A fully synthetic macrocyclic ketone analogue of marine sponge natural products. Inhibits growth phase of microtubules via tubulin-based antimitotic mechanism, which leads to G2/M cell-cycle block, disruption of mitotic spindles, and, ultimately, apoptotic cell death after prolonged mitotic blockage. It has a role as an antineoplastic agent and a microtubule-destabilising agent. It contains an eribulin(1+).
Eribulin Mesylate is the mesylate salt of a synthetic analogue of halichondrin B, a substance derived from a marine sponge (Lissodendoryx sp.) with antineoplastic activity. Eribulin binds to the vinca domain of tubulin and inhibits the polymerization of tubulin and the assembly of microtubules, resulting in inhibition of mitotic spindle assembly, induction of cell cycle arrest at G2/M phase, and, potentially, tumor regression.
See also: Eribulin (has active moiety).

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ERIBULIN MESYLATE is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2010 and has 4 approved and 18 investigational indications.

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Our study has several limitations. We used C3H mice for our syngeneic metastasis model, however, the pharmacokinetics data we referred to in this study were measured using different strains (BALB/c and CF-1). We assume that the pharmacokinetics of eribulin in C3H mice is similar to that in other strains as most eribulin is excreted into the bile and urine without being metabolized after intravenous injection. Sampson et al. reported that some osteosarcoma cell lines regrew in the presence of eribulin, but remained sensitive to eribulin [42]. We did not examine the delayed growth of LM8 cells at metastatic sites. It is possible that lung metastasis of LM8 proliferate later during long-term administration of eribulin, therefore, we need to examine a combination strategy with other chemotherapeutic agents. Collectively, eribulin is a potential therapeutic option for lung metastasis of osteosarcoma. Its anti-metastatic effects at concentrations lower than IC50 suggest that it has a wide application range. Frequent low-dose eribulin administration can reduce lung metastasis for a long time with relatively few side effects.[1]

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By electron microscopy, we showed that eribulin at high concentration (50 μM) did not induce extensive protofilament spiraling at microtubule ends as occurs with vinblastine (Fig. 5). The ends of the microtubules were blunt or slightly splayed (Table 2). This finding supports the model that eribulin binds β-tubulin with high affinity and α-tubulin with low or no affinity, and thus eribulin does not “link” tubulin heterodimers together in the manner of vinblastine, which binds both subunits nearly equally. However, the presence of the slightly splayed microtubule ends suggests a possible low affinity binding to α-tubulin. Since 22% of eribulin-treated microtubules showed slight splaying at both ends, therefore it appears that at sufficiently high concentrations, eribulin may bind minus ends. Vinblastine inhibited eribulin binding to microtubules at low eribulin concentrations (≤ 5 μM), (Fig. 4A and B). Unexpectedly, vinblastine induced an increase in the number of eribulin binding sites per microtubule at higher eribulin concentrations, increasing from 11 to 14 and to 17 eribulin molecules per microtubule in the presence of 2 μM and 4 μM vinblastine, respectively (Fig. 4A). This slight increase in binding suggests that the vinblastine-induced splaying of protofilaments allows increased access of eribulin to additional sites at the microtubule end. [2]

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Eribulin is chemically distinct from the microtubule-targeted drugs paclitaxel and Vinca alkaloids, it binds differently to microtubules than paclitaxel, and it suppresses microtubule dynamics in a distinct manner from paclitaxel and Vinca alkaloids. However, at concentrations that induce mitotic arrest, all of these drugs suppress centromere dynamics. These results provide strong support for the idea that suppression of spindle microtubule dynamics by these drugs and the resulting disruption of tension on the chromosome kinetochores/centromeres is the primary mechanism responsible for their abilities to induce mitotic arrest. [3]

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In summary, our results point to eribulin's ability to induce irreversible mitotic blockade as a cell-based pharmacodynamic explanation for its superior in vivo efficacy relative to the more potent ER-076349. Eribulin's irreversible mitotic blockade leads to persistent inactivation of Bcl-2, leading to apoptosis and complete long-term loss of cell viability. Eribulin's irreversible mitotic blockade is associated with lower cellular drug uptake yet more persistent postwashout retention compared with ER-076349, although the precise mechanisms behind this remain unclear. Preferential binding of eribulin to small numbers of high affinity sites at microtubule ends may explain its persistent postwashout retention, although further studies are needed to confirm this. Regardless of mechanism, correlations between eribulin's irreversibility and long-term postwashout cell-viability loss provide a satisfying explanation for its in vivo superiority over ER-076349 in preclinical tumor models using intermittent dosing. In ongoing clinical trials, eribulin is administered using intermittent dosing. Its ability to induce irreversible mitotic blockade in cancer cells may thus have important pharmacodynamic implications for its clinical activity as well, in which fluctuations in circulating drug levels associated with intermittent dosing routinely occur. [4]

C₄₀H₅₉NO₁₁

729.90

729.408

C, 65.82; H, 8.15; N, 1.92; O, 24.11

253128-41-5

Eribulin mesylate;441045-17-6

11354606

White to off-white solid powder

1.29±0.1 g/cm3

1.584

3.88

2

12

4

52

1380

19

C[C@@H]1C[C@@H]2CC[C@H]3C(=C)C[C@@H](O3)CC[C@]45C[C@@H]6[C@H](O4)[C@H]7[C@@H](O6)[C@@H](O5)[C@@H]8[C@@H](O7)CC[C@@H](O8)CC(=O)C[C@H]9[C@H](C[C@H](C1=C)O2)O[C@@H]([C@@H]9OC)C[C@@H](CN)O

UFNVPOGXISZXJD-JBQZKEIOSA-N

InChI=1S/C40H59NO11/c1-19-11-24-5-7-28-20(2)12-26(45-28)9-10-40-17-33-36(51-40)37-38(50-33)39(52-40)35-29(49-37)8-6-25(47-35)13-22(42)14-27-31(16-30(46-24)21(19)3)48-32(34(27)44-4)15-23(43)18-41/h19,23-39,43H,2-3,5-18,41H2,1,4H3/t19-,23+,24+,25-,26+,27+,28+,29+,30-,31+,32-,33-,34-,35+,36+,37+,38-,39+,40+/m1/s1

(1S,3S,6S,9S,12S,14R,16R,18S,20R,21R,22S,26R,29S,31R,32S,33R,35R,36S)-20-[(2S)-3-amino-2-hydroxypropyl]-21-methoxy-14-methyl-8,15-dimethylidene-2,19,30,34,37,39,40,41-octaoxanonacyclo[24.9.2.13,32.13,33.16,9.112,16.018,22.029,36.031,35]hentetracontan-24-one

B1939; E7389; ER086526; Eribulin; 253128-41-5; Eribulin [INN]; eribuline; Eribulina; UNII-LR24G6354G; CHEBI:63587; LR24G6354G; B 1939; E 7389; ER 086526; B-1939; E-7389; ER-086526

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~200 mg/mL (~274.01 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (6.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (6.85 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (6.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)