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Experimental Therapeutics |
Departments of Anticancer Research [M. J. T., K. A. S., J. B., B. F. W., G. K., K. K. A., S. W., B. A. L.] and Medicinal Chemistry [W. Z., B. M. S., M. H. P., G. J. H., L. A. S., L. V. D., Y. W., J. J. C., D. A. Q., M. J. Y.] and Advisory Board [Y. K.], Eisai Research Institute, Andover, Massachusetts 01810; Cytoskeleton, Incorporated, Denver, Colorado 80206 [A. D.]; and Discovery Research Laboratories II, Tsukuba Research Laboratories, Eisai Company, Limited, Tsukuba-shi, Ibaraki 300-26, Japan [K. Y.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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found in a variety of marine sponges, including Halichondria
okadai (1
, 2)
, Axinella sp.
(3)
, Phakellia carteri (4)
, and
Lissondendryx sp. (5)
. In 1986, halichondrin B
was shown to have remarkable in vitro and in vivo
anticancer activities against murine melanomas and leukemias
(2)
. Subsequent studies by several groups
(6, 7, 8, 9)
confirmed the sub-nM in
vitro potency of halichondrin B and identified a tubulin-based
antimitotic mechanism. Although halichondrin B is classified as a
tubulin depolymerizer (similar to Vinca alkaloids,
dolastatins, cryptophycin, and so on), the patterns of its interaction
with tubulin are highly specific and place it in its own unique class
within this group (6, 7, 8, 9)
. Although speculative, unique
interactions with tubulin may help explain the marked in
vivo anticancer potency of halichondrin B, now well documented in
several human tumor models including melanoma, osteogenic sarcoma, lung
cancers, and colon cancers (10)
. More importantly, unique
tubulin interactions relative to existing tubulin-based drugs suggest
the possibility of unique tumor specificities or other desirable
clinical characteristics.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and Cell Culture.
The following human tumor cell lines were obtained from the American
Type Culture Collection (Rockville, Maryland) and grown under American
Type Culture Collection-recommended conditions: COLO 205 and DLD-1
colon cancer, HL-60 promyelocytic leukemia, U937 histiocytic lymphoma,
and LNCaP and DU 145 prostate cancer. LOX human melanoma cells were
obtained from the Division of Cancer
Treatment-NCI4
Tumor Repository (Frederick, Maryland) and were grown in RPMI 1640
medium supplemented with 10% heat-inactivated fetal bovine serum and 2
mM L-glutamine. MDA-MB-435 human breast cancer
cells were generously provided by Dr. Mary J. C. Hendrix
(University of Iowa College of Medicine, Iowa City, Iowa) and were
grown in DMEM (high glucose) supplemented with 10% heat-inactivated
fetal bovine serum, 20 mM
N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid and 1
mM sodium pyruvate.
All of the cell lines were grown at 37°C in a humidified atmosphere containing 5% CO2. Seeding densities for each cell line were empirically optimized for passaging twice weekly. Quantification of cell number was by hemocytometer counting, and viability determinations were made by standard trypan blue exclusion techniques. Routine harvesting of monolayer cultures was by standard trypsinization procedures.
Cell Growth Inhibition Assays.
Cells were plated in 96-well plates at 7,500 cells/well (except LNCaP
cells, which were at 10,000 cells/well) and grown in the continuous
presence of test compounds for 4 days. For monolayer cultures (DU 145,
LNCaP, LOX, and MDA-MB-435), growth was assessed using modifications
(16)
of a methylene blue-based microculture assay
(17)
. For suspension cultures (HL-60, U937) or loosely
adherent monolayers (COLO 205), growth was assessed using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-based
assay (18)
modified as follows. After 4 days of incubation
with test compounds, sterile-filtered
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma
Chemical Co., St. Louis, MO) was added to each well (final
concentration, 0.5 mg/ml), and plates were incubated at 37°C for
4 h. Acid-isopropanol (0.1 N HCl in isopropanol, 150
µl) was then added to each well, and the resultant formazan crystals
were dissolved by gentle mixing. Absorbances at 540 nm were measured on
a Titertek Multiscan MCC/340 plate reader.
Cell Cycle Analysis.
DNA content analysis of U937 cells was performed by single channel flow
cytometry as follows. U937 cells were exposed for 0–14 h to 30
nM ER-076349 or 100 nM ER-086526;
concentrations were roughly 10-fold higher than those that minimally
induce complete G2-M blocks based on
dose/response studies. Typically, 2–4 x 106
cells were collected by centrifugation at
300 x g for 10 min and resuspended in 3 ml
of saline, followed by the immediate addition of 7 ml of ice-cold 100%
ethanol while vortexing. After fixing overnight at 4°C in the
saline/ethanol solution, cells were recovered by centrifugation, washed
in PBS, and resuspended in 0.5 ml PBS containing 200 µg/ml RNase A
(Sigma; preboiled 5 min as 10 mg/ml stock in PBS; stored at -80°C).
RNase digestion was at 37°C for 30 min, followed by the addition of
0.5 ml of 10 µg/ml propidium iodide (Sigma) in PBS at least 15 min
before flow analysis. Single channel flow cytometry was performed on a
Becton Dickinson FACScan with a Macintosh computer; the collection and
analysis of data were performed using Becton Dickinson CELLQuest
software. Doublet events were eliminated from analyses by proper gating
on FL2-W/FL2-A primary plots before histogram analysis of DNA content
(measured as FL2-A).
Fluorescence Microscopy.
For simultaneous anti-tubulin and DAPI staining of DU 145 cells, cells
were plated in 4-well plastic Lab-Tek chamber slides (Nunc, Inc.,
Naperville, IL) at 7.5 x 104
cells/chamber in 0.9 ml of complete medium. After culturing overnight,
100 µl of 10 x concentrates of test compounds in
complete media were added to a final concentration equal to three times
the empirically determined IC50 of each compound;
control wells received 100 µl of complete media only. After 20 h, cell monolayers were fixed with ice-cold anhydrous methanol for 6
min at -20°C, followed by two washes with 0.1% (v/v) Tween 20 in
PBS and two washes with PBS alone. Fixed cells were then incubated with
1:50 diluted (v/v in PBS) anti-ß-tubulin TUB 2.1 monoclonal
antibody (Sigma) for 1 h at room temperature with rocking,
followed by three 5-min washes with PBS and incubation with 1:50
diluted (v/v in PBS) FITC-conjugated goat F(ab')2
antimouse immunoglobulin (BioSource International, Camarillo, CA) for
1 h at room temperature with rocking. Cells were washed 3x with
PBS, incubated with 2 µg/ml DAPI in PBS for 10 min at room
temperature, followed by one 5-min wash with PBS and two 5-min washes
with 2 mM Tris, 0.1 mM EDTA (pH 7.5). Chamber
walls and gaskets were then removed, and the slides were air-dried.
Coverslips were mounted with Gel/Mount (Biomeda, Foster City, CA), and
fluorescence microscopy was performed using a Nikon Labophot-2
microscope equipped with 100 x oil immersion objective
and B-2A and UV-2A filter cubes for FITC and DAPI staining,
respectively. Images were acquired with a Photometrics NU 200 CCD
camera and digitized using IPLab Spectrum software (Scanalytics,
Fairfax, VA).
Affinity Binding of Tubulin to Biotinylated Macrocyclic Analogue
ER-040798.
UltraLinkTM Immobilized NeutrAvidin Plus (Pierce Chemical Co.,
Rockford, IL) was charged with ER-040792, ER-040798 (135
µM final concentrations), or buffer alone at room
temperature for 60 min in 20 mM Tris, 0.15 M
NaCl, 1 mM Na2EDTA (pH 7.5;
wash-elution buffer). After five washes in wash-eluted buffer,
matrices were incubated in the same buffer with 375 µg/ml bovine
brain tubulin (Molecular Probes, Eugene, OR) at room temperature for 30
min, followed by three washes in wash-elution buffer and elution with
20% acetonitrile (v/v in water). Eluted proteins were dried in a
Speed-Vac, resuspended in SDS sample buffer, and subjected to modified
discontinuous SDS PAGE on 8% gels as described by Matthes
(19)
for separation of 
- and ß-tubulin subunits.
Gels were silver-stained (20)
or transferred to
polyvinylidene difluoride membranes (Sigma) for Western immunoblotting
(21)
with subtype-specific anti--tubulin or
anti-ß-tubulin monoclonal antibodies (clones B-5-1-2 and TUB 2.1,
respectively; Sigma) and horseradish peroxidase-conjugated sheep
antimouse immunoglobulin secondary antibodies (Amersham Corp.,
Arlington Heights, IL) using a chemiluminescence protocol (ECL Western
Blotting Detection System; Amersham).
In Vitro Tubulin Polymerization Studies.
ER-076349, ER-086526, and vinblastine in 10 mM anhydrous
DMSO stocks were diluted into 10% DMSO, 90% PEM. Substocks were made
to final concentrations of 1, 10, 100, and 1000 µM; these
were diluted into 100-µl volumes of 3.0 mg/ml bovine brain tubulin
(Cytoskeleton, Inc., Denver, CO) in PEM buffer plus 1 mM
ATP, 3% (v/v) glycerol to achieve desired test compound
concentrations. The amount of 10% DMSO, 90% PEM was made up to 11
µl in all of the samples to achieve 1% final DMSO concentrations.
Microtubule polymerization was initiated by raising the temperature
from 4°C to 37°C over a 3-min period. Absorbance
(A340 nm) was measured once/min for
60 min.
In Vivo Xenograft Studies.
Anticancer effects of ER-076349 and ER-086526 were evaluated in the
MDA-MB-435 human breast cancer, COLO 205 human colon cancer, and LOX
human melanoma xenograft models using 5–6-week-old female Swiss nude
mice and in the NIH:OVCAR-3 human ovarian cancer model using 7-week-old
female BALB/c nude mice. All of the studies using laboratory animals
were approved either by the Eisai Research Institute (Andover, MA) or
Tsukuba Research Laboratories (Tsukuba, Japan) Institutional Animal
Care and Use Committees and adhered to all of the applicable
institutional and governmental guidelines for the humane care and use
of laboratory animals.
On day 0 of each experiment, mice received injections s.c. with
1 x 106
cells (MDA-MB-435, COLO
205, and LOX) or 6 x 105
cells
(NIH:OVCAR-3; previously adapted for enhanced penetrance via tumor
passage in vivo). For COLO 205 and MDA-MB-435 models, mice
received injections with 200 µl of test compound in saline on
Monday/Wednesday/Friday i.p. (COLO 205) or i.v. (MDA-MB-435)
schedules, respectively, beginning on day 13 for four weekly cycles.
Treatment of mice bearing LOX tumors was by daily i.p. injection (200
µl) in saline on days 3–7 and 10–14 inclusive. In the NIH:OVCAR-3
model, test compounds were injected i.v. (200µl/20g body weight) in
4% DMSO/saline beginning on day 40 and continuing
Monday/Wednesday/Friday for three weekly cycles. Control groups in all
of the studies received appropriate vehicle injections. Paclitaxel at
its empirically determined MTD for each treatment regimen was included
in all of the experiments and was administered using Diluent 12 (1:1
Cremophor EL/ethanol diluted into 5% glucose in water) as described by
others (22
, 23)
. Group sizes were 10 mice for all
of the tumor models except NIH:OVCAR-3, which used 6 mice/group. Tumor
volumes were determined by caliper measurements and were
calculated according to V = 0.67
[(L + W)/4]3
(i.e., volume
[V] of a semisphere with diameter equal to the mean of
tumor length [L] and width [W]). Body weights
and water consumption were measured in all of the experiments to assess
toxicity.
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RESULTS |
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. The two analogues themselves differ
only at C35, with hydroxyl and primary amine substituents for ER-076349
and ER-086526, respectively.
On the basis of the known anti-tubulin mechanism of halichondrin B
(6, 7, 8, 9)
, in vitro growth inhibitory effects of
ER-076349 and ER-086526 were compared with those of the microtubule
destabilizer vinblastine and the microtubule stabilizer paclitaxel in
MDA-MB-435 human breast cancer cells (Fig. 2)
. In this experiment, ER-076349 and ER-086526 inhibited cell growth at
sub-nM levels (IC50 values
of 0.15 nM and 0.07 nM,
respectively) with greater potency than vinblastine (0.54
nM) or paclitaxel (2.6 nM).
Similar low- or sub-nM potencies were observed in
several other human cancer cell lines, including COLO 205 and DLD-1
colon cancers, DU 145 and LNCaP prostate cancers, LOX melanoma, HL-60
monocytic leukemia, and U937 histiocytic lymphoma (Table 1)
. Interestingly, DLD-1 cells were about 10-fold less sensitive to
ER-086526 compared with the other seven lines (overall mean without
DLD-1 data, 0.7 ± 0.2 nM); this
was not seen for ER-076349. The reasons for the relative insensitivity
of DLD-1 cells to ER-086526 are unknown, although it is noted that
these cells were also less sensitive to vinblastine and paclitaxel.
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Despite sub-nM growth inhibition of several human cancer cell types, neither ER-076349 nor ER-086526 showed cytotoxic effects against quiescent IMR-90 human fibroblasts at concentrations up to 1 µM, as assessed using an ATP-dependent luciferase-based viability assay (data not shown). Thus, growth inhibition by low- or sub-nM levels of ER-076349 and ER-086526 is specific for proliferating cells and not secondary to nonspecific cytotoxicity.
Cell Cycle Effects: G2-M Phase Block.
Cell cycle analysis of U937 human histiocytic lymphoma cells was
performed after 0–14 h-incubation with 30 nM ER-076349 or
100 nM ER-086526. As shown in Fig. 3
, treatment with either ER-076349 or ER-086526 for as little as 2 h
led to increased numbers of G2-M phase cells. In
both groups, G2-M cells accumulated at the
expense of G1, which was almost completely
depleted by 8 h. Progressive depletion of S phase then followed
beginning at 10 h because of cessation of new cells entering S
from G1. Depletion of S phase was essentially
complete by 14 h, at which time almost all of the living cells
were blocked in G2-M. For both compounds,
increased numbers of hypodiploid events were seen after 8–10 h,
indicating apoptosis of cells after prolonged
G2-M blockage. The fact that both
G1 and S phases became progressively depleted
under continuous ER-076349 or ER-086526 exposure confirms normal
movement of cells through these phases even in the presence of a drug.
Thus, ER-076349 and ER-086526 are rapid and effective
G2-M phase blockers that do not affect cell cycle
progression through the G1 or S phases or the
G1-S transition point. Similar
G2-M blockage of U937 cells was observed
previously using synthetic halichondrin B (24)
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. In this study, cells in log phase growth were treated for 20 h
with vehicle, 2.1 nM ER-076349, 2.7 nM
ER-086526, 11 nM vinblastine, or 28 nM
paclitaxel, representing empirically determined 3 x IC50 concentrations in this cell line
(preliminary studies comparing 0.3, 1, 3, and 10 x IC50 levels established 3 x IC50 as optimal; data not shown). After drug
exposure, cells were fixed and dual stained with anti-ß-tubulin
monoclonal antibodies and DAPI to label microtubules and DNA,
respectively. As shown in Fig. 4
, treatment with either ER-076349
(B) or ER-086526 (C) led to marked disruption of
normal mitotic spindle architecture (A). As expected,
mitotic spindle disruption was also seen with the anti-tubulin agents
vinblastine (D) and paclitaxel (E). With all of
the four drugs, cells with disrupted spindles showed the condensed but
disorganized chromosomal material characteristic of prometaphase
blockage. Occasional ER-076349- and ER-086526-treated cells were seen
that had not yet reached the mitotic block point during the 20-h drug
treatment period. These interphase (G1, S, or
G2) cells, identified by intact DAPI-stained
nuclei, appeared essentially normal with respect to filamentous
cytoplasmic microtubules and intact, well-defined nuclear
perimeters, consistent with the lack of effect of ER-076349 and
ER-086526 on progression through G1 and S seen by
flow cytometry (Fig. 3)
. Similar disruption of DU 145 mitotic spindles
was observed previously using synthetic halichondrin B
(24)
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are related to mechanistic differences.
Binding of Biotinylated Analogue ER-040798 to Tubulin.
To directly address whether macrocyclic halichondrin B analogues were
capable of tubulin binding, the ability of the biotinylated analogue
ER-040798 (Fig. 1)
to bind tubulin under affinity chromatography
conditions was tested. As shown in Fig. 5
, immobilized ER-040798, but not the biotinylated negative control
ER-040792, retained two Mr
50,000–55,000 protein bands from bovine brain tubulin
(Lanes A–E). These bands were
recognized by subtype-specific anti--tubulin (Lanes F–J)
and anti-ß-tubulin (Lanes K–O) monoclonal antibodies,
confirming their identities as - and ß-tubulin, respectively.
Thus, ER-040798 directly binds tubulin, suggesting that the biological
activity of halichondrin B macrocyclic analogues derives from
mechanisms similar or identical to parental halichondrin B. It should
be noted that this study did not identify the tubulin subtype to which
ER-040798 binds, because binding to either subtype in /ß-tubulin
heterodimers would lead to retention of both. Finally, specific
retention of - and ß-tubulin by immobilized ER-040798 was also
seen using U937 whole cell lysates (data not shown), which demonstrated
that the retention of - and ß-tubulin seen in Fig. 5
was not
simply because of the presence of only these two proteins in the
purified bovine brain tubulin preparations used.
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, ER-076349, ER-086526, and vinblastine inhibited both rates and extents
of tubulin polymerization in dose-dependent fashions. The inhibition of
polymerization rates and extents was similar for each drug, with
calculated IC50s for ER-076349, ER-086526, and
vinblastine of 5.7 µM, 6.9
µM, and 1.8 µM for rate
inhibition, and 5.5 µM, 6.0
µM, and 2.1 µM for
extent inhibition, respectively. By either criterion, ER-076349 and
ER-086526 were about 3–4-fold less effective as inhibitors of in
vitro tubulin polymerization compared with vinblastine.
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Inhibition of Human Tumor Xenograft Growth in Vivo.
The abilities of ER-076349 and ER-086526 to inhibit tumor growth
in vivo were tested in several human tumor xenograft models,
including MDA-MB-435 breast cancer, COLO 205 colon cancer, LOX
melanoma, and NIH:OVCAR-3 ovarian cancer (Fig. 7)
. In all of the cases, treatment with ER-076349 and ER-086526 in the
0.05–1 mg/kg range led to significant antitumor effects, with
ER-086526 showing greater efficacy in all of the models. Thus, in the
MDA-MB-435 model (Fig. 7, A and B)
, treatment
with 0.25–1.0 mg/kg ER-076349 led to 60–70% inhibition at day 42,
whereas ER-086526 at the same levels led to >95% inhibition,
including actual regression of measurable tumors present on day 14. For
both compounds, tumor regrowth rates after cessation of dosing allowed
efficacy comparisons with paclitaxel at its empirically determined MTD
of 25 mg/kg. Again, ER-086526 showed superiority over ER-076349;
regrowth in all of the ER-076349 groups preceded the paclitaxel group
by about 5 weeks, whereas all of the doses of ER-086526 were either
equally efficacious (0.25, 0.5 mg/kg) or superior (1 mg/kg) to 25 mg/kg
paclitaxel. Interestingly, the therapeutic window of ER-086526 seemed
unusually large for a cytotoxic drug; >95% tumor suppression occurred
over the 4-fold dosing range of 0.25–1.0 mg/kg with no evidence of
toxicity based on body weight losses or decreased water consumption
(data not shown). In contrast, complete tumor suppression by paclitaxel
with this dosing regimen is only seen between 15–25 mg/kg, with 10
mg/kg inducing only partial inhibition (data not shown); the
therapeutic window for paclitaxel in this model is thus just 1.7-fold.
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. As in the MDA-MB-435 study, ER-086526
showed superior efficacy, with frank regression and long-term
suppression of tumor regrowth at 0.5 mg/kg and small but measurable
decreases in growth rates at 0.125 mg/kg. Suppression with 0.5 mg/kg
ER-086526 was superior to that of paclitaxel at its empirically
determined MTD of 20 mg/kg, both in rapidity of regression onset and in
duration of regrowth suppression. In contrast, inhibition by ER-076349
was less pronounced at these dosing levels; no measurable effects were
seen at 0.125 mg/kg, and inhibition at 0.5 mg/kg was less than that
with 20 mg/kg paclitaxel. As in the MDA-MB-435 study, there was no
evidence of toxicity based on body weights or water consumption (data
not shown).
Evaluation of 0.1–1.0 mg/kg ER-076349 and 0.05–0.5 mg/kg ER-086526 in
the LOX melanoma model also showed potent antitumor effects of both
compounds (Fig. 7, E and F)
. With ER-076349, all
of the doses led to >90% tumor suppression by day 17, three days
after cessation of dosing. The three top doses, 0.25, 0.5, and 1 mg/kg,
led to virtually complete tumor suppression up to day 17, with
inhibition of regrowth persisting well beyond that seen with 12.5 mg/kg
paclitaxel, its empirically determined MTD with this regimen. Again,
complete tumor suppression by 0.25–1.0 mg/kg ER-076349 in the LOX
model represents an unusually wide 4-fold therapeutic window. In
contrast, doubling the paclitaxel dose to 25 mg/kg in this model is
lethal, whereas halving it to 6.25 mg/kg leads to only minor inhibition
(data not shown); the therapeutic window for paclitaxel with this
dosing regimen is thus <2-fold.
Inhibition of LOX tumor growth by ER-086526 was similar to that with ER-076349 but with about 2-fold greater potency. Thus, 0.05 mg/kg ER-086526 inhibited tumor growth by 78% at day 17, with higher doses of 0.1, 0.25, and 0.5 mg/kg ER-086526 leading to complete tumor suppression. Again, this represents an unusually wide 5-fold therapeutic window. Tumor regrowth rates in the 0.5 mg/kg ER-086526 group were delayed significantly beyond the 12.5 mg/kg paclitaxel group; regrowth in the 0.25 mg/kg ER-086526 group was slightly delayed relative to paclitaxel. Significantly, one and three mice in the 0.25 and 0.5 mg/kg ER-086526 groups (n = 10), respectively, became tumor-free by day 17; all of the mice had measurable tumors on day 10. These cured mice remained completely tumor-free for an additional 7 months. Other than a 2% decrease in body weights in the 0.5 mg/kg ER-086526 group during the first five daily injections, there were no other indications of toxicity in any group with either compound, as assessed by body weights or water consumption (data not shown). The slight decrease in body weights in the latter group resolved itself before the beginning of the second cycle of five daily injections and was not observed again.
Finally, effects of 0.125–1.0 mg/kg ER-076349 and ER-086526 on
NIH:OVCAR-3 ovarian cancer xenograft growth were examined (Fig. 7, G and H)
. During actual dosing periods, both
compounds inhibited tumor growth to about the same degree, with 0.5 and
1 mg/kg leading to inhibition during dosing equivalent to paclitaxel at
its empirically determined MTD of 20 mg/kg. Two of six mice each in the
1 mg/kg ER-076349 and ER-086526 groups and six of six mice in the
paclitaxel group became tumor-free by the end of dosing; these mice
remained tumor-free through the end of the experiment (day 89). After
cessation of dosing, increased efficacy of ER-086526 over ER-076349
became evident in the 0.5 and 1.0 mg/kg groups; no dose of ER-076349
achieved the long-term complete regrowth suppression seen with
paclitaxel, whereas 1 mg/kg ER-086526 achieved complete suppression out
to day 89, similar to paclitaxel. Significant body weight losses during
dosing were only seen in the 20 mg/kg paclitaxel and 1 mg/kg ER-086526
groups (12 and 18% decreases, respectively); body weights in these
groups recovered within about 10 days after cessation of dosing (data
not shown).
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DISCUSSION |
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ER-076349 and ER-086526 differ only in their C35 alcohol and amine
substituents, respectively. Both showed sub-nM in
vitro growth inhibition against a variety of human cancer cell
lines (Table 1)
, with the alcohol ER-076349 being about 2-fold more
potent than the amine ER-086526 in most lines. Both were roughly
5–10-fold more potent than vinblastine and paclitaxel, two
tubulin-based antimitotic agents run as internal standards in each
experiment.
Direct comparisons of ER-076349 and ER-086526 with halichondrin B were not performed in the current study because of the lack of availability of either synthetic or natural halichondrin B. However, the low- to sub-nM in vitro potencies measured for the two macrocyclic analogues were similar to historical halichondrin B data from our laboratory and others, indicating that the in vitro potency of halichondrin B had been retained during structural simplification. Thus, in an early study with synthetic halichondrin B, we measured a mean IC50 of 0.4 nM against 17 human tumor cell lines representing eight different cancer types (24) , whereas other laboratories using natural halichondrin B have reported IC50 values of 0.08 nM, 0.3 nM, and 5 nM against murine B16 melanoma, murine L1210 leukemia, and PtK1 normal kangaroo rat kidney cells, respectively (2 , 6 , 8) . Moreover, performance of ER-076349 and ER-086526 in the 60-cell line screen of NCI was essentially identical to that of natural halichondrin B5 (data not shown). We conclude that virtually all of the growth inhibitory potency was retained during structural simplification, including removing the entire C36-C54 polyether "left half" of halichondrin B and replacing the macrocyclic lactone ester of the latter with a nonhydrolyzable ketone bridge.
Our results indicate that the growth inhibitory mechanisms of ER-076349
and ER-086526 are probably the same as those of halichondrin B. Thus,
ER-076349 and ER-086526 induce G2-M cell cycle
arrest and disrupt mitotic spindles, similar to previous results with
synthetic halichondrin B (24)
and consistent with the
tubulin-based antimitotic mechanism of the latter (6, 7, 8, 9)
.
Moreover, our studies with the biotinylated macrocyclic lactone
analogue ER-040798 showed direct binding to tubulin under affinity
chromatography conditions (Fig. 5)
. Although ER-040798 was prepared
before discovery of the macrocyclic ketone series, direct tubulin
binding by ER-040798 is probably representative of this entire class of
drugs because simplification from halichondrin B to the macrocyclic
ketones involved numerous incremental modifications made without
significant changes in growth inhibitory potency,
G2-M arrest characteristics, or mitotic spindle
disruption. Indeed, ER-076349 and ER-086526 directly inhibit tubulin
polymerization in vitro with IC50
values (5.7 µM and 6.9
µM, respectively) almost identical to the 7.2
µM value reported for halichondrin B
(6)
. We conclude that the anticancer activities of
ER-076349 and ER-086526 derive from a tubulin-depolymerizing
antimitotic mechanism similar or identical to that reported for
halichondrin B.
ER-076349 and ER-086526 showed significant in vivo anticancer efficacy at doses well below 1 mg/kg in several human tumor xenograft models. Interestingly, despite somewhat lower in vitro potency in cell growth and tubulin polymerization studies, ER-086526 showed greater in vivo efficacy, particularly in the MDA-MB-435 breast and COLO 205 colon cancer models. One explanation for this might be that ER-076349 is metabolized in vivo more readily than ER-086526; pharmacokinetic studies are under way to investigate this possibility. Another explanation might relate to our finding that mitotic blocks induced by ER-086526 are much less reversible after drug washout than those induced by ER-076349 (data not shown). This could result in greater in vivo tumor cell killing under the intermittent dosing schedules used in our studies. This observation, which correlates with potential for in vivo efficacy within the halichondrin B macrocyclic analogue series, will be described in a separate manuscript.6
In all of the four in vivo models, ER-076349 and ER-086526 were effective at much lower doses compared with paclitaxel run at empirically determined MTD levels. For instance, potency differentials for ER-086526 versus paclitaxel based on complete tumor suppression ranged from 20-fold in the NIH:OVCAR-3 model, to 40-, 50-, and 100-fold in the COLO 205, LOX, and MDA-MB-435 models, respectively. More importantly, ER-076349 and ER-086526 showed significantly wider in vivo therapeutic windows. For paclitaxel, empirically determined therapeutic windows were relatively small, 1.7 in the MDA-MB-435 model and <2.0 in the LOX model. In marked contrast, therapeutic windows for the macrocyclic ketone analogues were much wider. In the MDA-MB-435 model, ER-086526 showed a 4-fold therapeutic window, whereas in the LOX model, therapeutic windows of 4- and 5-fold were observed for ER-076349 and ER-086526, respectively. We speculate that the wide therapeutic windows seen with ER-076349 and ER-086526 contribute to their substantial in vivo efficacy, in that the ability to increase doses 4–5-fold above fully tumor-suppressive doses probably leads to more complete eradication of residual tumor cells. This might explain the superior in vivo efficacy of ER-086526 over paclitaxel MTD dosing in three of the four models tested and the existence of complete cures in the LOX model by ER-086526. The reasons for the unusually wide therapeutic windows of ER-076349 and ER-086526 are not currently known.
In summary, we have demonstrated highly potent in vitro and in vivo anticancer activities of two fully synthetic, macrocyclic ketone analogues of halichondrin B, ER-076349, and ER-086526. These agents exert their anticancer effects by mechanisms currently indistinguishable from the microtubule-destabilizing effects of halichondrin B. An unexpected but exciting finding was the existence of unusually wide in vivo therapeutic windows, which may contribute to the remarkable in vivo efficacy of these new agents. Our encouraging early results with ER-076349 and ER-086526 strongly support their continued development as novel anticancer agents for human use.
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FOOTNOTES |
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1 These data were presented in part (abstract
1370) at the 91st Annual Meeting of the AACR held April
1–5, 2000 in San Francisco, CA. 
2 To whom requests for reprints should be
addressed, at Department of Anticancer Research, Eisai Research
Institute, 4 Corporate Drive, Andover, MA 01810-2441. Phone:
(978) 837-4638; Fax: (978) 794-4910; E-mail: bruce_littlefield{at}eri.eisai.com
3 M. J. Towle, Y. Kishi, and B. A. Littlefield,
unpublished observations, 1992.
4 The abbreviations used are: NCI, United States
National Cancer Institute; DAPI, 4',6-diamidino-2-phenylindole; MTD,
maximum tolerated dose; PEM, 80 mM PIPES (pH 6.9), 1
mM EGTA, 1 mM MgCl2.
5 D. J. Newman, personal communication.
6 M. J. Towle, Y. Kishi, M. J. Yu, and B. A.
Littlefield, manuscript in preparation.
Received 4/12/00. Accepted 11/30/00.
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), 3
µM (
), 5 µM (
), 7.5 µM
(
), or 15 µM (x) concentrations of ER-076349
(A), ER-086526 (B), or vinblastine
(C) was assessed as described in "Materials and
Methods." Results are plotted as the extent of tubulin polymerization
(A340 nm) as a function of time and
represent means of triplicate determinations at each time point. For
graphic clarity, only data points from odd-numbered minutes are
presented.