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
Primarily excreted unchanged in feces. 43 L/m² to 114 L/m² 1.16 L/hr/m² to 2.42 L/hr/m² (dose range 0.25 mg/m² to 4.0 mg/m²). [FDA] Metabolisms/Metabolites Eribulin has no major human metabolites, and its metabolism by CYP3A4 in vitro is negligible. Biological Half-Life Approximately 40 hours. Eribulin (eribulin mesylate) is a synthetic analogue of Halichondria B, a natural product derived from the marine sponge Halichondria okadai. It is a microtubule dynamics inhibitor used to treat metastatic breast cancer and liposarcoma. The following are the pharmacokinetic (PK) characteristics summarized based on available data:
Absorption
Absorption occurs within 2-5 minutes after intravenous (IV) administration, ensuring complete bioavailability of eribulin.
Distribution
Large volume of distribution (approximately 43-114 L/m²), indicating its wide tissue distribution.
Metabolic binding is moderate, ranging from 49% to 65%.
Metabolic metabolism
Primarily metabolized by the liver, with a small amount metabolized by CYP3A4; the majority of the drug is excreted unchanged as eribulin.
It is not a significant substrate, inhibitor, or inducer of major CYP enzymes, thus reducing the risk of drug interactions.

Elimination
The elimination half-life is approximately 40 hours, therefore it can be administered on days 1 and 8 of a 21-day cycle.

Primarily excreted in feces (82%), with a small amount (9%) excreted unchanged in urine.

Special Populations
Hepatic Impairment: Patients with mild (Child-Pugh A) or moderate (Child-Pugh B) hepatic impairment require dose reduction due to increased drug exposure. Severe renal impairment (Child-Pugh C) has not been studied.

Renal Impairment: Moderate renal impairment (creatinine clearance 30–50 mL/min) also requires dose adjustment, while data for severe renal impairment (creatinine clearance <30 mL/min) are limited.
Key Considerations
The pharmacokinetics of eribulin are not affected by age, weight, or race.
Due to its long half-life, repeated dosing may lead to drug accumulation, therefore monitoring for toxicities (e.g., neutropenia, peripheral neuropathy) is necessary.
These pharmacokinetic characteristics support its clinical use in advanced cancer, but dosage adjustments are necessary based on hepatic and renal dysfunction to minimize toxicity. For more details, please refer to the prescribing information or clinical studies.

Hepatotoxicity
Eribulin is a cytotoxic chemotherapy drug. Elevated serum transaminases and alkaline phosphatases are common during cyclical treatment of breast cancer and liposarcoma. The incidence of eribulin elevation ranges from 8% to 83%, with 2% to 5% of patients having ALT values exceeding five times the upper limit of normal (ULN). Elevated ALT has been reported in eribulin clinical trial reports, but detailed information on its occurrence, clinical characteristics, and course has not been published, therefore the role of eribulin in these outcomes remains unclear. Only sporadic mentions of "toxic hepatitis" have been made. Despite the high incidence of elevated serum enzymes during treatment, there are no detailed reports of clinically significant liver injury, therefore such cases must be extremely rare. Probability Score: E (Unproven but suspected cause of liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Currently, there is no information regarding the clinical use of eribulin during lactation. The manufacturer recommends discontinuing breastfeeding during eribulin treatment and for 2 weeks after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information 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 to treat metastatic breast cancer and liposarcoma. While highly effective, it also has a variety of toxicities, primarily affecting the hematologic, nervous, and gastrointestinal systems. The following is an overview of its main toxicities:
1. Hematologic toxicities
Neutropenia: The most common and dose-limiting toxicity, occurring in ≥25% of cases. Severe (grade 3/4) neutropenia has been reported in up to 57% of cases, increasing the risk of infection.
Anemia: Common (≥25%), some cases require blood transfusion support.
Thrombocytopenia: Less common, but severe cases can lead to bleeding complications.
2. Neurotoxicity
Peripheral neuropathy: Common adverse events (≥25%), manifested as numbness, tingling, or pain in the extremities. Its toxicity may be dose-related and sometimes even irreversible.
Fatigue: A significant proportion of patients report fatigue, affecting their quality of life.
3. Gastrointestinal toxicity
Nausea and vomiting: Common (≥25%), but usually controllable with antiemetics.
Constipation: Common, sometimes requiring medical intervention.
4. Other toxicities
Alopecia: Alopecia is common, but usually reversible after discontinuation of the drug.
Cardiac Effects: Rare cases of QT interval prolongation and arrhythmias have been reported; high-risk patients require monitoring.
Hepatotoxicity: Elevated liver enzymes may occur, especially in patients with pre-existing liver impairment.

Management Recommendations

Dose Adjustment: Dose adjustment is required in cases of severe neutropenia, neuropathy, or liver/kidney impairment.


Precautions: Neutropenia may require growth factor support (e.g., granulocyte colony-stimulating factor, G-CSF). For unresolved toxicities, delayed administration is recommended.


The toxicity profile of eribulin necessitates close monitoring of patients, especially those with underlying medical conditions. Dose adjustment and supportive care are crucial for mitigating adverse reactions and maintaining therapeutic efficacy. Please refer to the prescribing information for detailed guidelines.

[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, a derivative of the marine sponge eribulin. It inhibits microtubule growth through a tubulin-based antimitotic mechanism, leading to G2/M phase cell cycle arrest, spindle disruption during mitosis, and ultimately apoptosis after prolonged mitotic arrest. Eribulin possesses antitumor and microtubule destabilizing effects. It is a macrocyclic compound belonging to the polyether, polycyclic ether, cyclic ketone, primary amino compound, and cyclic ketal classes. It is the conjugate base of eribulin (1+). Eribulin is a microtubule inhibitor indicated for the treatment of patients with metastatic breast cancer who have previously received at least two chemotherapy regimens. Eribulin was isolated from the marine sponge Halichondria okadai. Eribulin is also currently under investigation for the treatment of advanced solid tumors. Eribulin is a microtubule inhibitor. Its physiological action is achieved by inhibiting microtubules.
Eribulin mesylate is a microtubule function inhibitor used as an antitumor drug for refractory metastatic breast cancer and liposarcoma. Although eribulin has cytotoxic activity against cancer cells, clinically significant acute liver injury is rarely reported.
Eribulin is an analogue of Halilcondolin B (a substance with antitumor activity derived from marine sponges (Lissodendoryx sp.)). It binds to the vinca acid domain of tubulin, inhibiting tubulin polymerization and microtubule assembly, thereby inhibiting mitotic spindle assembly, inducing cell cycle arrest at the G2/M phase, and potentially leading to tumor regression.
See also: Eribulin mesylate (salt form).
Indications
For the treatment of patients with metastatic breast cancer who have previously received at least two prior chemotherapy regimens.
FDA Label
Indications for the treatment of patients with locally advanced or metastatic breast cancer whose disease has progressed after at least one prior chemotherapy regimen (see Section 5.1). Halavirin monotherapy. This should be included unless the patient is not suitable for anthracycline and taxane therapy. Prior treatment. For the treatment of adult patients with unresectable liposarcoma who have previously received anthracycline therapy (unless applicable) (see Section 5.1). Halavirin.
Treatment of soft tissue sarcomas

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Mechanism of action
Inhibits the growth phase of microtubules without affecting their shortening phase, and sequesters tubulin into nonfunctional aggregates. Eribulin acts through a tubulin-based antimitotic mechanism, leading to G2/M phase cell cycle arrest, mitotic spindle disruption, and ultimately apoptosis following prolonged mitotic arrest. Eribulin. [FDA]

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Pharmacodynamics
Linear

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is the mesylate obtained by reacting eribulin with an equivalent amount of mesylate. Eribulin mesylate. A fully synthetic macrocyclic ketone analog of a natural marine sponge product. It inhibits microtubule growth through a tubulin-based antimitotic mechanism, leading to G2/M phase cell cycle arrest, mitotic spindle disruption, and ultimately apoptosis after prolonged mitotic arrest. It possesses antitumor and microtubule destabilizing effects. It contains eribulin (1+). It is the mesylate salt of a synthetic analogue of Halicandrin B, a marine sponge (Lissodendoryx sp.) with antitumor activity. Eribulin binds to the vinblastic acid domain of tubulin, inhibiting tubulin polymerization and microtubule assembly, thereby inhibiting mitotic spindle assembly, inducing cell cycle arrest at the G2/M phase, and potentially leading to tumor regression. See also: Eribulin (containing the active moiety). Eribulin is a small molecule drug, with its highest clinical trial stage being Phase IV (covering all indications). It was first approved in 2010 and currently has 4 approved indications and 18 investigational indications.

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This study has some limitations. We used C3H mice as a homologous metastasis model; however, the pharmacokinetic data cited in this study were obtained using different strains (BALB/c and CF-1) of mice. We assumed that the pharmacokinetics of eribulin in C3H mice were similar to those in other strains, since most eribulin is excreted in bile and urine without being metabolized after intravenous injection. Sampson et al. reported that some osteosarcoma cell lines can regenerate in the presence of eribulin but remain sensitive to it [42]. We did not examine the delayed growth of LM8 cells at metastatic sites. LM8 lung metastases may proliferate after long-term use of eribulin, therefore, we need to investigate strategies for combination therapy with other chemotherapeutic drugs. In conclusion, eribulin is a potential option for the treatment of osteosarcoma lung metastases. Its anti-metastatic effect at concentrations below IC50 suggests broad application prospects. Frequent low-dose administration of eribulin can reduce lung metastases in the long term with relatively few side effects. [1]

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Electron microscopy revealed that high concentrations (50 μM) of eribulin did not induce extensive protofilament coiling at the microtubule tips, unlike vincristine (Fig. 5). The microtubule tips were blunt or slightly abducted (Table 2). This finding supports the model that eribulin binds with high affinity to β-tubulin and with low affinity or no affinity to α-tubulin, thus eribulin does not "link" the tubulin heterodimers together like vincristine (which binds both subunits with almost the same affinity). However, the slight abduction of the microtubule tips suggests that eribulin may bind with low affinity to α-tubulin. Since the microtubules treated with 22% eribulin showed slight opening at both ends, eribulin may bind to the negative ends of microtubules at sufficiently high concentrations. Vincristine inhibited the binding of eribulin to microtubules at low concentrations (≤ 5 μM) (Figs. 4A and B). Surprisingly, in the presence of high concentrations of eribulin, vincristine increased the number of eribulin binding sites per microtubule. In the presence of 2 μM and 4 μM vincristine, the number of eribulin molecules per microtubule increased from 11 to 14 and 17, respectively (Fig. 4A). This slight increase in binding suggests that vincristine-induced protonema opening makes it easier for eribulin to access more binding sites at the microtubule ends. [2]

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Compared with microtubule-targeting drugs paclitaxel and vinca alkaloids, eribulin is chemically distinct, binds to microtubules differently than paclitaxel, and inhibits microtubule dynamics differently than paclitaxel and vinca alkaloids. However, at concentrations that induce mitotic arrest, all of these drugs inhibit centromere dynamics. These results strongly support the view that the inhibition of spindle microtubule dynamics and the resulting disruption of chromosome kinetochord/centromere tension are the main mechanisms by which these drugs induce mitotic arrest. [3]

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In summary, our results indicate that eribulin induces irreversible mitotic arrest, which can explain its superior in vivo efficacy compared to the more potent ER-076349 at the cellular level. The irreversible mitotic arrest of eribulin leads to persistent inactivation of Bcl-2, which in turn induces apoptosis and ultimately results in long-term loss of cell viability. Compared to ER-076349, while the irreversible mitotic arrest of eribulin results in lower cellular drug uptake, it also leads to a longer drug retention time after elution, although the exact mechanism behind this remains unclear. Eribulin's preferential binding to a small number of high-affinity sites at the microtubule tips may explain its longer drug retention time after elution, but further investigation is needed to confirm this. Regardless of the mechanism, the correlation between the irreversibility of eribulin and the long-term loss of cell viability after elution provides a satisfactory explanation for its superior in vivo efficacy compared to ER-076349 in a preclinical tumor model using intermittent dosing. In the ongoing clinical trial, eribulin is administered intermittently. Therefore, the drug can induce irreversible mitotic arrest in cancer cells, which may have important pharmacodynamic significance for its clinical activity, since intermittent administration leads to fluctuations in circulating drug concentration. [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.)