Tubulin polymerization; microtubule

ER-076349 inhibits the proliferation of various cancer cells with IC50 of 0.59 nM (MDA-MB-435), 2.4 nM (COLO 205), 7.3 nM (DLD-1), 3.6 nM (DU 145), 1.8 nM (LNCaP), 3.2 nM (LOX ), 2.6 nM (HL-60), 4.0 nM (U937)[1].

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Halichondrin B is a highly potent anticancer agent originally found in marine sponges. Although scarcity of the natural product has hampered efforts to develop halichondrin B as a new anticancer drug, the existence of a complete synthetic route has allowed synthesis of structurally simpler analogues that retain the remarkable potency of the parent compound. In this study, we show that two macrocyclic ketone analogues of halichondrir B, ER-076349 and ER-086526, have sub-nM growth inhibitory activities in vitro against numerous human cancer cell lines. ER-076349 and ER-086526 induce G2-M cell cycle arrest and disruption of mitotic spindles, consistent with the tubulin-based antimitotic mechanism of halichondrin B. This is supported further by direct binding of the biotinylated analogue ER-040798 to tubulin and inhibition of tubulin polymerization in vitro by ER-076349 and ER-086526. Retention of the extraordinary in vitro and in vivo activity off halichondrin B in structurally simplified, fully synthetic analogues establishes the feasibility of developing halichondrin B-based agents as highly effective, novel anticancer drugs [1].

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Effects of Eribulin and ER-076349 on Mitosis in U-2 OS Cells [2]
The goal of these experiments was to measure the movements of centromeres due to the growing and shortening dynamics of their attached dynamic microtubules in living U-2 OS human osteosarcoma cells at drug concentrations that inhibited proliferation and arrested mitosis. Thus, we first determined the effects of eribulin and ER-076349 on cell proliferation (28 h, one cell cycle) and mitotic progression. Cell proliferation was inhibited with IC50s of 30 nmol/L eribulin and 3 nmol/L ER-076349 (Fig. 1B).

The effects of the drugs on mitotic progression were measured by incubating the cells with drug (3 pmol/L – 10 µmol/L, 20 h), and determining the mitotic index by microscopy (Materials and Methods). At concentrations >3 nmol/L for eribulin and >0.1 nmol/L for ER-076349, cells accumulated in mitosis reaching a maximum of 60 % at 1 µmol/L eribulin and at 100 nmol/L ER-076349 (Fig. 1C). Half-maximal arrest occurred at 30 nmol/L eribulin and at 2 nmol/L ER-076349, nearly the same as the IC50s observed for proliferation. ER-076349 was thus 15 times more potent than eribulin in U-2 OS cells.

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Spindle and Microtubule Organization and Localization of GFP-CENP-B [2]
The localization of GFP-CENP-B, microtubules and chromosomes in the presence and absence of drugs (4 h incubation) is shown in fixed and immunostained U-2 OS cells in Fig. 2, A–D. In control cells at metaphase (Fig. 2A), chromosomes were completely congressed to the metaphase plate, and centromere pairs (arrows) were oriented parallel to the spindle axis. During anaphase, sister chromatids and their associated centromeres separated and the centromeres appeared as single, spherical dots (Fig. 2B). After 4 h incubation with 30 nmol/L eribulin or 2 nmol/L ER-076349, spindles were somewhat smaller but were often bipolar with all chromosomes congressed to the metaphase plate. However, many spindles were bipolar but with “lagging” chromosomes or chromosomes stuck at one or both poles (arrow, Fig. 2C). Some spindles were tripolar or multipolar (Fig. 2D).

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The Effects of Eribulin and ER-076349 on Centromere Dynamics [2]
We examined centromere dynamics at the mitotic IC50 and twice the mitotic IC50 for both compounds (30 and 60 nmol/L eribulin and 2 and 4 nmol/L ER-076349), after incubation with drug for 4 h. As shown in Table I, both drugs suppressed centromere dynamics at concentrations that arrested mitosis. The most prominent and concentration-dependent changes were reductions in the percentages of time spent stretching and relaxing, increases in the percentage of time spent in a paused state, and suppression of the overall dynamicity (by 6% and 35% for 30 nM and 60 nM eribulin, respectively, and by 5% and 32% by 2 nM and 4 nM ER-076349, respectively)(Table I). Although effects on the stretching rate were minimal, both compounds significantly suppressed the relaxing rate, by 21% at 2× IC50. The transition frequency was not affected at the IC50 but was reduced at 2× IC50 (by 24% and 35% for eribulin and ER-076349, respectively). In contrast with the effects of vinblastine and paclitaxel on centromere dynamics, eribulin and ER-076349 had no significant effect on the mean separation distance.

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Intracellular Levels of Eribulin and ER-076349 [2]
The time courses for uptake of eribulin and ER-076349 into U-2 OS cells at their IC50s and 2× IC50s are shown in Fig. 4. Eribulin (30 nmol/L and 60 nmol/L) was taken up rapidly into the cells, attaining intracellular concentrations of 1 µM and 3.5 µM, respectively, 4 h after addition, and remaining at approximately those levels at least to 20 h. Thus eribulin accumulated in the cells 30–60 fold over its concentration in the medium. ER-076349 (2 nmol/L and 4 nmol/L) was taken up more rapidly, attaining intracellular concentrations of 300 nmol/L and 3.2 µM, respectively, 4 h after addition, and high levels were retained at least to 20 h. Thus the intracellular drug concentrations that induced mitotic arrest were equivalent for the two drugs (3.5 µmol/L for eribulin and 3.2 µmol/L for ER-076349) and these intracellular concentrations affected microtubule dynamics similarly. The extent of accumulation was greater for ER-076349 than for eribulin, (i.e.,150-fold and 800-fold for 2 and 4 nmol/L ER076349, respectively). With both drugs (eribulin and ER-076349), washing of the cells at 20 h and incubation in drug-free medium induced a rapid loss of drug from the cells, although less than half the intracellular eribulin was lost whereas nearly all the intracellular ER-076349 was lost during the first hour after washing. Twenty-four h after washing, intracellular concentrations of both drugs were equivalent to background (data not shown).

In this study, we show that two macrocyclic ketone analogues of halichondrir B, ER-076349 and ER-086526, have marked in vivo activities at 0.1-1 mg/kg against four human xenografts: MDA-MB-435 breast cancer, COLO 205 colon cancer, LOX melanoma, and NIH: OVCAR-3 ovarian cancer. Retention of the extraordinary in vitro and in vivo activity off halichondrin B in structurally simplified, fully synthetic analogues establishes the feasibility of developing halichondrin B-based agents as highly effective, novel anticancer drugs [1].

Cell Proliferation and Mitotic Index [2]
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 (Nikon, Melville, NY) 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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Intracellular Drug Concentration [2]
U-2 OS cells were seeded into poly-lysine-treated scintillation vials (1 × 105 cells, 2ml). After 24 h, media was replaced with fresh media containing 30 –60 nmol/L [3H] eribulin (final specific activity after dilution with unlabeled eribulin: 2 mCi/mol) or 2–4 nmol/L [3H] ER-076349 (final specific activity: 18–24 mCi/mol) or unlabeled drug (for determination of cell number). Media were removed from vials from 30 min to 20 h after drug addition, cells were rapidly rinsed twice with 2 ml PBS, and intracellular drug concentration was determined by scintillation counting. Background radioactivity was determined after washing vials that contained only radiolabeled drug in medium. Cell number was determined by manual cell counting using a hemacytometer at the time of drug addition and 20 h later in vials treated in parallel using unlabeled drug. The intracellular drug concentration was determined by dividing the moles of intracellular eribulin by the average cell volume times the number of cells/vial. The mean cell volume was calculated from the mean diameter of cells rounded up after trypsinization (n=38; mean cell volume, 3.2 × 10−12 L). Additionally, after 20 h, cells were washed with 2 ml PBS and fresh media was added for 1 h and 24 h to determine how readily the drugs are washed out of cells. All time points were measured in duplicate, and results are the means and standard errors of 5 experiments.

Eribulin (E7389) is a microtubule inhibitor with a unique mechanism of action and is currently in Phase III clinical trials for cancer treatment. Compared to the more potent compound ER-076349, eribulin exhibits superior efficacy in vivo, and this difference in efficacy is not due to differences in their pharmacokinetic properties. Flow cytometry analysis of the reversibility of mitotic arrest (using full dose/response treatment) revealed that eribulin (not ER-076349)-induced mitotic arrest is irreversible, suggesting a cell-based pharmacodynamic explanation. Cell viability analysis 5 days after drug clearance confirmed the relationship between the reversibility of mitotic arrest and long-term cell survival. Similar results were observed in U937, Jurkat, HL-60, and HeLa cells, ruling out cell type-specific effects. Studies of other microtubule inhibitors have shown that the reversibility of mitotic arrest is a quantifiable, compound-specific characteristic commonly found in antimitotic drugs. The Bcl-2 phosphorylation pattern parallels the reversible mitotic arrest patterns of eribulin and ER-076349, suggesting that persistent Bcl-2 phosphorylation is one of the reasons for the decreased long-term cell viability following irreversible arrest by eribulin. Drug uptake and clearance/retention studies showed that [3H]eribulin accumulated at lower levels in cells than [3H]ER-076349, but retained for longer periods and at higher levels. Similar results were observed with irreversible and reversible vincristine, indicating that persistent cell retention is a component of its irreversibility. Our results suggest that the in vivo advantage of eribulin stems from its ability to induce irreversible mitotic arrest, an ability that appears to be related to persistent drug retention and persistent Bcl-2 phosphorylation. More broadly, our results indicate that the compound-specific reversible characteristics of antimitotic drugs contribute to the interaction between cellular pharmacodynamics and in vivo pharmacokinetics, thereby determining antitumor efficacy under intermittent dosing conditions. Cancer Res; 71(2); 496–505.

[1]. In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer Res. 2001 Feb 1;61(3):1013-21.

[2]. Inhibition of Centromere Dynamics by Eribulin (E7389) during Mitotic Metaphase. Mol Cancer Ther. 2008 Jul;7(7):2003–2011.

Eribulin (E7389) is a synthetic Halilcondolin B analogue currently in a Phase III clinical trial for breast cancer. It binds to tubulin and microtubules. At low concentrations, eribulin inhibits the microtubule dynamic instability phase in interphase cells, arrests mitosis, and induces apoptosis, suggesting that inhibition of spindle microtubule dynamics can lead to mitotic arrest. To further verify this hypothesis, we used time-lapse confocal microscopy to measure the effects of eribulin on centromere and its associated kinetochore microtubule dynamics in U-2 OS human osteosarcoma cells during in vivo mitosis. Green fluorescent protein-labeled centromere-binding protein B labeled the apical ends of centromeres and kinetochore microtubules. In control cells, sister chromatid centromere pairs alternately exhibited states of increased and decreased separation (stretching and relaxation) under tension. Eribulin inhibited centromere dynamics at concentrations sufficient to arrest mitosis. At a concentration of 60 nmol/L eribulin (2 times the IC50 for mitosis), spindle relaxation decreased by 21%, arrest time increased by 67%, and dynamics decreased by 35% (but mean centromere spacing did not decrease), indicating that eribulin reduced normal microtubule-dependent spindle tension at the kinetochore, thereby preventing the transmission of mitotic checkpoint signals. We also tested ER-076349, an antiproliferative halliculin derivative with stronger efficacy but poorer tumor response. At a concentration of 2 times the IC50 (4 nmol/L), mitotic arrest also occurred simultaneously with the inhibition of centromere dynamics. Although the culture medium IC50 values of the two compounds differed by 15 times, their intracellular concentrations were similar, indicating that ER-076349 was more readily taken up by cells than eribulin. There was a strong correlation between the inhibition of kinetochore microtubule dynamics and mitotic arrest, suggesting that the main mechanism by which eribulin blocks mitosis is the inhibition of spindle microtubule dynamics. [2]

C40H58O12

730.88

730.393

253128-15-3

11297138

White to off-white solid powder

2

12

4

52

1370

19

O1[C@H](C[C@H](O)CO)[C@@H](OC)CC(=O)C[C@]2([H])CC[C@@]([H])(C2)C[C@]2([H])O[C@@]3(C[C@@]2([H])CC3)CC2O[C@@]3([H])O[C@]([H])(C(=C)C2C3)CCO2O[C@@]3([H])O[C@@]4([H])C1C2CO(C)C4C3=C

SOGFSQSGBFDQFA-JBQZKEIOSA-N

InChI=1S/C40H58O12/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/h19,23-39,41,43H,2-3,5-18H2,1,4H3/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)-2,3-dihydroxypropyl]-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

253128-15-3; (1S,3S,6S,9S,12S,14R,16R,18S,20R,21R,22S,26R,29S,31R,32S,33R,35R,36S)-20-[(2S)-2,3-Dihydroxypropyl]-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; (2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-2-[(2S)-2,3-Dihydroxypropyl]hexacosahydro-3-methoxy-26-methyl-20,27-bis(methylene)-11,15:18,21:24,28-triepoxy-7,9-ethano-12,15-methano-9H,15H-furo[3,2-i]furo[2 inverted exclamation marka,3 inverted exclamation marka:5,6]pyrano[4,3-b][1,4]dioxacyclopentacosin-5(4H)-one; ER-076349; 67XUM42NP8; CHEMBL1683522; Eribulin mesylate intermediate 3; SCHEMBL16765869;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). 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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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