Article

Antivascular and antitumor evaluation of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes, a novel series of anticancer agents

Shire BioChem, Inc., Laval, Quebec, Canada.
Molecular Cancer Therapeutics (Impact Factor: 5.68). 12/2004; 3(11):1375-84.
Source: PubMed

ABSTRACT

A novel series of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes was identified as potent apoptosis inducers through a cell-based high throughput screening assay. Six compounds from this series, MX-58151, MX-58276, MX-76747, MX-116214, MX-116407, and MX-126303, were further profiled and shown to have potent in vitro cytotoxic activity toward proliferating cells only and to interact with tubulin at the colchicine-binding site, thereby inhibiting tubulin polymerization and leading to cell cycle arrest and apoptosis. Furthermore, these compounds were shown to disrupt newly formed capillary tubes in vitro at low nanomolar concentrations. These data suggested that the compounds might have vascular targeting activity. In this study, we have evaluated the ability of these compounds to disrupt tumor vasculature and to induce tumor necrosis. We investigated the pharmacokinetic and toxicity profiles of all six compounds and examined their ability to induce tumor necrosis. We next examined the antitumor efficacy of a subset of compounds in three different human solid tumor xenografts. In the human lung tumor xenograft (Calu-6), MX-116407 was highly active, producing tumor regressions in all 10 animals. Moreover, MX-116407 significantly enhanced the antitumor activity of cisplatin, resulting in 40% tumor-free animals at time of sacrifice. Our results identify MX-116407 as the lead candidate and strongly support its continued development as a novel anticancer agent for human use.

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Available from: Sui Xiong Cai, May 22, 2014
Antivascular and antitumor evaluation of 2-amino-4-(3-bromo-4,
5-dimethoxy-phenyl)-3-cyano-4H-chromenes,
a novel series of anticancer agents
Henriette Gourdeau,
1
Lorraine Leblond,
1
Bettina Hamelin,
1
Clemence Desputeau,
1
Kelly Dong,
1
Irenej Kianicka,
1
Dominique Custeau,
1
Chantal Boudreau,
1
Lilianne Geerts,
1
Sui-Xiong Cai,
2
John Drewe,
2
Denis Labrecque,
1
Shailaja Kasibhatla,
2
and Ben Tseng
2
1
Shire BioChem, Inc., Laval, Quebec, Canada and
2
Maxim
Pharmaceuticals, Inc., San Diego, California
Abstract
A novel series of 2-amino-4-(3-bromo-4,5-dimethoxy-
phenyl)-3-cyano-4H-chromenes was identified as potent
apoptosis inducers through a cell-based high throughput
screening assay. Six compounds from this series, MX-
58151, MX-58276, MX-76747, MX-116214, MX-
116407, and MX-126303, were further profiled and
shown to have potent in vitro cytotoxic activity toward
proliferating cells only and to interact with tubulin at the
colchicine-binding site, thereby inhibiting tubulin polymer-
ization and leading to cell cycle arrest and apoptosis.
Furthermore, these compounds were shown to disrupt
newly formed capillary tubes in vitro at low nanomolar
concentrations. These data suggested that the com-
pounds might have vascular targeting activity. In this
study, we have evaluated the ability of these compounds
to disrupt tumor vasculature and to induce tumor necrosis.
We investigated the pharmacokinetic and toxicity profiles
of all six compounds and examined their ability to induce
tumor necrosis. We next examined the antitumor efficacy
of a subset of compounds in three different human solid
tumor xenografts. In the human lung tumor xenograft
(Calu-6), MX-116407 was highly active, producing tumor
regressions in all 10 animals. Moreover, MX-116407
significantly enhanced the antitumor activity of cisplatin,
resulting in 40% tumor-free animals at time of sacrifice.
Our results identify MX-116407 as the lead candidate and
strongly support its continued development as a novel
anticancer agent for human use. [Mol Cancer Ther
2004;3(11):1375 83]
Introduction
The initial hit compound of a novel series of 2-amino-4-(3-
bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes was
identified in a cell-based high throughput screening assay
by measuring the induction of apoptosis using our pro-
fluorescent caspase substrate (1 3). Analogues were made
of the hit compound and activity and stability optimization
studies identified six analogues synthesized in this series
(MX-58151, MX-58276, MX-76747, MX-116214, MX-116407,
and MX-126303) for further characterization. Compounds
from this series were shown to have potent in vitro
cytotoxic activity toward proliferating cells only and to
interact with tubulin at the colchicine-binding site, thereby
inhibiting tubulin polymerization and leading to cell cycle
arrest and apoptosis (4). Furthermore, these compounds
were shown to disrupt newly formed capillary tubes
in vitro at low nanomolar concentrations. This activity
suggested that the compounds might have vascular
targeting activity.
Microtubules are highly dynamic assemblies of the
protein tubulin and are major structural components in
cells. They are important in the maintenance of cell shape,
cellular movement, and cell division (5 7). Tubulin
represents one of the best cancer targets identified to date
given the large and diverse group of tubulin targeting
anticancer drugs and their success in the clinic (812).
These drugs, by interfering with the dynamic instability of
tubulin, arrest dividing cells in the M phase of the cell cycle
leading to apoptotic cell death. However, emerging
resistance to antimitotic agents has limited their ultimate
effectiveness and there is a renewed interest in the
discovery and development of new agents that are active
in multidrug-resistant cells and that interact with tubulin at
sites different from those of the taxanes and Vinca alkaloids
(13). This field has exploded in the last few years and led to
the discovery of small molecular weight inhibitors that
have shown promising antitumor activity even in tumors
expressing multidrug-resistant phenotypes (14).
In addition to their tubulin-mediated cytotoxicity, Vinca
alkaloids and colchicine have also been reported to induce
hemorrhagic necrosis of solid tumors (15). However,
because these additive antitumor effects were only ob-
served at doses approaching or exceeding their maximum
tolerated dose (MTD), this attribute has not been fully
explored. This has led to the development of a second
generation of tubulin-binding agents that destabilize the
tubulin cytoskeleton by interacting at the colchicine-
binding site at doses that are well tolerated (16). Due to
their short half-lives, these compounds preferentially target
tumor endothelial cells while sparing normal vasculature
(17, 18). These agents, exemplified by combretastatin A-4
Received 5/7/04; revised 7/16/04; accepted 9/15/04.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Shailaja Kasibhatla, Maxim Pharmaceuticals, Inc.,
6650 Nancy Ridge Drive, San Diego, CA 92121. Phone: 858-202-4042;
Fax: 858-202-4000. E-mail: skasibhatla@maxim.com
Copyright C 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics 1375
Mol Cancer Ther 2004;3(11). November 2004
Page 1
phosphate (CA4P) prodrug and ZD6126, both of which are
analogues of colchicines, are called vascular targeting
agents. Unlike antiangiogenesis drugs, which attempt to
prevent the formation of new tumor blood vessels, vascular
targeting agents starve existing solid tumors by depriving
them of blood flow, causing tumor cell death.
In this study, we have evaluated the ability of 2-amino-4-
(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes
compounds to induce tumor necrosis and their potential to
act as antitumor agents. To this end, we investigated the
pharmacokinetic and toxicity profiles of these six com-
pounds in the series and examined their ability to induce
tumor necrosis. We also examined the antitumor efficacy of
a subset of these compounds in three different human solid
tumor xenografts and addressed the chemoenhancing
potential of the optimal analogue.
Materials and Methods
Compounds
ZD6126 was synthesized at Shire BioChem (Laval,
Quebec, Canada) according to published procedures (19).
MX-58151, MX-58276, MX-76747, MX-116214, MX-116407,
and MX-126303 were synthesized at Shire BioChem and
Maxim Pharmaceuticals according to methods described
previously (4).
3
MX compounds were dissolved in a
mixture of Cremophor EL (5-10%) and ethanol (5-10%) in
saline; ZD6126 was dissolved in water and 5% aqueous
sodium carbonate solution was added drop-wise until a
clear solution was obtained; cisplatin (0.4 mg/mL) was
dissolved in 0.9% saline. Drugs were given at constant
injection volumes of 10 mL/kg of mouse body weight i.p.
(ZD6126 and cisplatin) or i.v. (MX compounds).
Cell Culture
The human lung carcinoma Calu-6 cell line was
purchased from the American Type Culture Collection
(Manassas, VA); the human breast carcinoma MDA-MB-
435 cell line was obtained from the Frederick Research
Tumor Repository (Frederick, MD). Cell lines were main-
tained in either MEM (Calu-6) or RPMI 1640 (MDA-MB-
435) supplemented with fetal bovine serum (10%, Life
Technologies, Burlington, Ontario, Canada) and supple-
ments (nonessential amino acids, glutamine, sodium
pyruvate, vitamins, and glucose; Cellgro Mediatech, Inc.,
Herndon, VA) according to the cell supplier’s instructions.
All cell lines were maintained at 37jC in a humidified
atmosphere of 95% air and 5% CO
2
. Cells were grown in
absence of antibiotics and periodically checked for Myco-
plasma contamination by Hoechst 33258 staining (20).
Tu m o r F r a g m e n t s
The MX-1 breast carcinoma tumor fragments were
originally obtained from the Frederick Research Tumor
Repository and maintained by passage in NCr mice.
Animals
Athymic (Crl:CD-1-nuBr) and severe combined immu-
nodeficient [Crl:Icr:Ha(ICR)-scid] mice 6 to 8 weeks old
were purchased from Charles Rivers Laboratories (St-
Constant, Quebec, Canada). Tac:Cr;(NCr)-Hfh11nu (NCr)
mice 6 to 8 weeks old were purchased from Taconic Farms
(Germantown, NY). Animals were maintained under
specific pathogen-free conditions and had access to sterile
food and water ad libitum. All animal studies were done in
the animal facility at Shire BioChem with the prior
approval of the local Institutional Animal Care Committee
and in agreement with the guidelines provided by the
Canadian Council for Animal Care.
Pharmacokinetic Profiling
The six compounds under evaluation were given i.v. at
a dose of 10 mg/kg to CD-1 male mice (n = 30 mice, per
compound). Blood was collected into EDTA (final concen-
tration, 2 mg/mL) containing tubes by cardiac puncture at
2, 5, 15, 30, 45, 60, 90, 120, 180, and 240 minutes following
compound administration. Samples were centrifuged at
10,000
g for 10 minutes and plasma samples were
collected and stored at 20C until use. Mouse plasma (100
AL) was mixed with 50 AL internal standard and diluted
with deionized water (900 AL). The mixture (
f
1 mL) was
loaded onto Oasis HLB 30-mg 96-well solid-phase extrac-
tion plate (Waters Corp., Milford, PA). After sequential
washes with deionized water (1 mL) and 20% methanol
(1 mL), the sample and the internal standard were eluted
with acetonitrile (1 mL) and then evaporated to dryness
under a gentle stream of nitrogen. The residues were
reconstituted with 50% methanol (100 AL) in water and
aliquots (10 AL) were analyzed by tandem liquid chroma-
tography/mass spectrometry. The chromatography was
achieved on a Luna C18 column (50
2 mm, 5 Am,
Phenomenix, Torrance, CA) with a 5-minute elution
gradient of acetonitrile (20 80%) in ammonium formate
(10 mmol/L). The flow rate was 0.25 mL/min and total run
time was at 12 minutes. Quantitation was done on a
tandem liquid chromatography/mass spectrometry
(TSQ7000, ThermoFinnigan, San Jose, CA) equipped with
APCI source. Standard curve in mouse plasma ranged from
5 to 5,000 ng/mL, with minimum seven calibration points
and three levels of quality controls.
Plasma concentration-time data were analyzed by non-
compartmental linear pharmacokinetic methods using
Kinetica (Innaphase, Philadelphia, PA). Maximum serum
concentrations (C
max
) were read directly from the experi-
mental data, with t
max
defined as the time of the first
measurement (2 minutes). The terminal phase rate con-
stants (k) were estimated using least squares regression
analysis of the serum concentration-time data obtained
during the terminal log-linear phase. The terminal half-life
(t
1
/
2
) was calculated by the equation 0.693/k. The area
under the serum concentration-time curve from time 0 to
240 minutes was estimated using linear trapezoidal
approximation and was extrapolated to infinity according
to the formula: AUC
(0 !1)
= C
last
/k, where C
last
is the
3
W. Kemnitzer et al. Discovery of 4-aryl-4H-chromenes as a new series of apoptosis
inducers using a cell- and caspase-based high throughput screening assay; structure-
activity relationships of the 4-aryl group. J Med Chem. In press 2004.
Novel Vascular-Targeting Agents1376
Mol Cancer Ther 2004;3(11). November 2004
Page 2
estimated concentration at time t
last
=240minutes.
Clearance was calculated as dose/AUC
(0 !1)
and Vss
was estimated as dose
AUMC/AUC
2
, where AUMC is
the area under the first moment curve (21).
To x i c i t y S t u d i e s
Toxicity profile of each tested compound was assessed
after a single dose treatment or either a single or a twice
daily treatment repeated for 5 consecutive days. A 14-day
observation period followed the treatment period. CD-1
male mice (610 mice per dose) were given by a slow bolus
injection at a constant volume of 10 mL/kg through the
caudal vein while restrained in a plexiglass retainer tube.
To monitor the general health condition and drug-
associated toxicity, mice were weighed at least twice
weekly and inspected daily for clinical abnormalities. The
animals were euthanized under isoflurane anesthesia and
blood was collected by cardiac puncture. The collected
blood was immediately distributed into EDTA-containing
tubes (hematology samples) or clotting gel containing
tubes (clinical chemistry samples) and sent for analysis at
Cirion Biopharma, Inc. (Laval, Quebec, Canada). Macro-
scopic necropsies were done at 24 hours post-treatment
(single dose) or after the last treatment (5-day repeat
treatment) and again at the end of the 14-day observation
period. Right kidney, liver, spleen, and thymus were
weighed.
The MTD was estimated as the dose that resulted in
V20% of body weight loss compared with the animal
weight at the beginning of the treatment, <10% death rate,
reversible observed clinical signs and hematology/clinical
chemistry changes, and no visible macroscopic tissue
changes at the end of the observation period. A drug
dose was considered toxic if animals lost z20% of their
initial body weight or if there was >10% lethality when
applying humanized end points as described by the
Canadian Council for Animal Care. The mice were
prematurely euthanized when the MTD criteria were
crossed or the established humanized end points as
described by the Canadian Council for Animal Care were
reached.
Tumor Necrosis and Vascular Dysfunction
Necrosis was assessed by light microscopy at 40
magnification. MDA-MB-435 tumor-bearing mice were
injected with single doses of compounds at different
concentrations as indicated. Tumors were excised 24 hours
later and rapidly snap frozen in CryoMatrix (Cryomatrix,
Pittsburgh, PA). Sections (6 Am) were prepared and stained
with Gomori (Sigma Chemical Co., St. Louis, MO). The
level of necrosis was scored visually according to the
following scale: 1, 0-10% necrosis; 2, 11-20% necrosis; ...;
10, 91-100% necrosis.
Functional vasculature was assessed as described previ-
ously (22). Hoechst 33342 (10 mg/kg) was injected into the
tail vein of MDA-MB-435 tumor-bearing mice followed by
a single bolus injection of vehicle, MX-116407 (10, 20, or 40
mg/kg), or CA4P (150 mg/kg). Four hours later, a second
dye, DiOC
7
(3), was injected; 2 minutes after injection, the
mice were euthanized and tumors were rapidly removed,
embedded in a tissue holder, and immediately frozen in
liquid N
2
for sectioning. Perfused blood vessels in the
tumor could be visualized by the surrounding halo of
fluorescent Hoechst 33342 and/or DiOC
7
(3)-labeled en-
dothelial cells. The two dyes have different excitation and
emission spectra, which allow separate detection. This
method provides an estimate of the relative degree of
perfused tumor vasculature. For each tumor, an average
percentage image area was calculated. A total of three
mice were imaged in each group.
Antitumor Efficacy Studies
Female severe combined immunodeficient mice were
injected s.c. with 2
10
6
MDA-MB-435 cells. Tumor-
bearing animals were randomized (10 mice per group) and
treatment was started when tumor volumes reached 50
to 100 mm
3
(day 20). Doses and administration schedules
are described in Table 1.
Female NCr mice were implanted s.c. by trocar in the
right axillary area with MX1 tumor fragments (average, 2
mm
3
). Tumor-bearing animals were randomized (9 mice
per group) and treatment was started when the average
tumor volumes reached
f
150 mm
3
. MX-76747, MX-116214,
and MX-116407 were given i.v. at 10, 15, or 45 mg/kg,
respectively, daily for 5 consecutive days (days 13-17). Mice
were allowed a 2-day rest period and treated for another
sequence (days 20-24). ZD6126 was given i.p. at 100 mg/kg
following the same schedule.
Female athymic CD-1 (nu/nu) mice were injected s.c. with
2
10
6
Calu-6 cells in 50% Matrigel. Administration of
compounds or vehicle began at day 24 when the average
tumor size reached
f
250 mm
3
. Tumor-bearing animals
were randomized (10 per group) prior to treatment. MX-
76747, MX-116214, and MX-116407 were given i.v. at 10, 15,
or 45 mg/kg, respectively, daily for 5 consecutive days
Table 1. Pharmacokinetic variables of selected compounds in mice after a single i. v. dose of 10 mg/kg
Compounds MX-58151 MX-58276 MX-76747 MX-126303 MX-116214 MX-116407
C
max
(Ag/mL) at 2 min 26.5 28.9 26.3 12.3 19.4 10.0
AUC (Ag
min/mL) 341.5 581.4 271.6 159.6 998.4 421.5
t
1
/
2
(min) 42.1 43.2 33.3 70.8 69.9 39.3
Clearance (mL/min) 0.69 0.43 0.81 1.4 0.25 0.62
Vss (mL) 20.3 11.7 11.5 50.8 20.6 34.9
Molecular Cancer Therapeutics
1377
Mol Cancer Ther 2004;3(11). November 2004
Page 3
(days 24 28). ZD6126 was given i.p. at 100 mg/kg on days
24 to 28. In the combination studies, a single dose of 4
mg/kg cisplatin was given i.p. 24 hours prior to treatment
with MX-116407.
Tumor measurements taken by electronic calipers twice
weekly were converted to tumor volumes (in mm
3
) using
the standard formula: width
2
length
0.52 (23).
Compound efficacy was assessed once the tumors in the
control group had reached at least five times their original
volume (500% growth) by percentage of treated versus
control (% T/C) defined as the (mean treated tumor
volume) / (mean control tumor volume)
100. A % T/C
value of <42 was considered effective (24, 25). Tumor
growth inhibition (TGI) was calculated by subtracting the
% T/C from 100%. Tumor growth delay (TGD) was
determined as the time delay necessary to double the
mean tumor volume (starting from day 1 of treatment) in
treated versus control animals. Statistical analysis was done
by ANOVA or by Student’s t test. Differences were
considered to be significant at P < 0.05.
Results
Pharmacokinetics
The plasma concentration-time profiles and pharmaco-
kinetic variables of MX-58151, MX-58276, MX-76747, MX-
116214, MX-116407, and MX-126303 after single i.v. doses of
10 mg/kg are shown in Fig. 1 and Table 1, respectively. The
clearance of the six compounds varied
f
5-fold. MX-126303
was cleared at a rate close to the mouse liver blood flow (1.4
versus 1.8 mL/min; ref. 26), which resulted in one of the
lowest measured C
max
at 2 minutes (12.3 Ag/mL) and
lowest exposure (159.6 Ag
min/mL) compared with the
other five drug candidates. In contrast, MX-116214 was
characterized by the slowest clearance of this series (0.25
mL/min) and had the greatest exposure (998.4 Ag
min/
mL), resulting in plasma concentrations in the micromolar
range up to 4 hours. Despite their differences in clearance,
these two compounds had the longest terminal half-lives
(
f
70 minutes), suggesting that the tissue distribution is
greater for MX-126303 than for MX-116214. The other
compounds were characterized by intermediate plasma
clearances and terminal elimination half-lives of
f
40
minutes. Highest initial concentrations were achieved by
MX-58151, MX-58276 and MX-76747 (26 29 Ag/mL).
Induction of Tumor Necrosis and Tumor Vascular
Disruption
Table 2 summarizes the toxicity profiles as well as the
dose-response data of the six lead compounds for induction
of tumor necrosis after i.v. administration to mice bearing
the human breast (MDA-MB-435) tumor xenograft. MX-
58151 and MX-58276 induced extensive necrosis (80 90%)
after a single administration of 25 and 110 mg/kg,
respectively. These doses were well tolerated when given
as a single injection but resulted in weight loss and
reversible toxicity when given for 5 consecutive days. For
MX-76747, MX-116214, MX-116407, and MX-126303, MTD
was determined following a single bolus injection and a 2-
to 6-fold toxicity difference was observed between acute
and repeated dosing. MX-116214 and MX-126303 induced
extensive tumor necrosis (80 90%) but only at doses close
to their respective MTD. On the other hand, MX-76747 and
MX-116407 were more effective, causing substantial tumor
necrosis (60 80%) at doses of one-fourth to one-sixth of
their respective MTDs. This compares well with ZD6126 in
this tumor model, where we observed significant tumor
necrosis (6080%) at doses approaching one-third to one-
fourth of its MTD (Table 1). In our hands, ZD6126 was
found to be toxic following a bolus injection of 300 mg/kg
(causing hypothermia, respiratory difficulties, and eventu-
ally death) but was well tolerated at 150 mg/kg (mild loss
of body weight, reduction of spleen weight, decrease in
activity, and matted fur).
MX-116407 was further evaluated by examining the
regrowth of the MDA-MB-435 tumor following a single
bolus administration. Twenty-four hours after treatment,
Figure 1. Plasma concentration-time
curves of selected compounds. The pharma-
cokinetics of MX-58151 (5), MX-58276 (o),
MX-76747 (
y), MX-116214 (4), MX-
116407 (E), and MX-126303 (
.
)were
evaluated in male CD-1 mice (n = 3 per time
point). A single dose of each different com-
pound was given i.v. at 10 mg/kg. Plasma
was coll ected by cardiac puncture at 2, 5,
15, 30, 60, 90, 120, 180, and 240 minutes
following compound administration.
Novel Vascular-Targeting Agents1378
Mol Cancer Ther 2004;3(11). November 2004
Page 4
although 90% of the tumor were necrotic (Fig. 2B; Table 2),
we did not measure tumor reduction (Fig. 2A). This is
likely to be due to edema resulting from an inflammatory
reaction related to the extensive tumor necrosis. The tumor
volume started to decrease thereafter, and by 7 days after
treatment, we observed a 25% growth reduction (Fig. 2A).
Between days 7 and 14 post-treatment, the tumor volume
gradually increased. By day 14, the tumor had repopulated
itself with marginal necrosis observed at the center of the
tumor (Fig. 2C).
The observed rapid and extensive tumor necrosis is
indicative of vascular disruption in the tumor. We have also
examined the level of functional tumor vasculature after
compound treatment by a double fluorescent dye staining
method (22). The DNA-binding fluorescent Hoechst 33342
is first given to allow staining of tumor blood vessels
followed by compound administration. Four hours later, a
second dye was given and tumors were quickly excised,
Table 2. Induction of tumor necrosis in the human breast (MDA-
MB-435) tumor xenograft 24 hours postdosing
Compounds Dose
(mg/kg)
MTD
*
(mg/kg)
Median
necrosis
score
c
ZD6126 140 Single i.p. treatment
between 150 and 300
8.3
100 8
50 6.3
25 4.7
MX-58151 25 qd
5 = 20 9.8
8
43.3
MX-58276 110 b.i.d.
5 = >90 8.3
75
40 4.6
20
MX-76747 60 Single i.v. dose = 60
b.i.d.
5=10
8.2
40 8.2
20 6.5
10 5
MX-116214 25 Single i.v. dose = 25
qd
5=15
9.3
12.5 5.3
6.3 3.3
2.5 3
MX-116407 100 Single i.v. dose = 100
qd
5 = >40
9
75 7
50 6.7
25 8
MX-126303 2.5 Single i.v. dose = 2.5
b.i.d.
5=1
6.3
0.8 5.7
0.4
*The MTD is the dose at which we observed body weight loss (<20%), pale
livers, pale kidneys, thrombocytopenia, and diarrhea. These symptoms were
observed after the treatment period but were no longer present at the 14-day
observation period. qd, single daily dose repeated over 5 consecutive days;
b.i.d., twice daily dose repeated over 5 consecutive days.
cNecrosis index was determined following Gomori staining and scoring
with 1 representing 10% necrosis and 10 representing complete (100%)
necrosis. Three animals were used per time point.
Figure 2. Regrowth of MDA-MB-435 tumors after a single dose of MX-
116407. Twelve mice bearing MDA-MB-435 tumor xenografts were
treated with a single i.v. bolus dose of MX-116407 (100 mg/kg) when
tumors reached an average volume of 250 mm
3
(day 49 ). A, tumors were
measured twice weekly and on days 1, 4, 7, and 14 after treatment with
MX-116407. Three mice were sacrificed for tumor necrosis evaluation.
B,
Gomori staining 24 hours after treatment (
40). C, Gomori staining 14
days after treatment (
40). Representative necrotic (N) and viable ( V )
regions are identified. Regrowth seems to originate from the tumor rim.
Molecular Cancer Therapeutics 1379
Mol Cancer Ther 2004;3(11). November 2004
Page 5
frozen, and then sectioned for examination. The sections
were examined for the amount of fluorescence from the
two distinguishable dyes. The fluorescent image area for
each dye was determined and the results are expressed as
the percentage of the second dye over the first dye (Fig. 3).
The vehicle showed very little effect on the extent of dye
label between the two dyes. MX-116407 showed a dose
dependence of reducing the amount of the second dye, as
did CA4P which serves as a positive control, demonstrating
that it is acting as a vascular disrupting agent.
Antitumor Efficacy Studies
To determine if the antivascular activity of these
compounds was sufficient to result in TGD, the compounds
were evaluated for antitumor activity in the same tumor
xenograft model. Because the MDA-MB-435 tumor grows
slowly, having a doubling time of 12 days, and the half-life
of the compounds varied from 30 to 70 minutes, the
treatment regimens were daily or twice daily for 5
consecutive days repeated for up to 3 weeks. MX-58151,
MX-58276, and MX-126303 resulted in TGDs of 8, 4, and 6
days and % T/Cs of 65, 75, and 75, respectively (Table 3).
These values indicated marginal antitumor activity for
these compounds in this model. For MX-58151 and MX-
58276, the marginal antitumor activity could be due to the
shorter period of treatment (5 consecutive days instead of
up to 3 weeks). On the other hand, MX-76747, MX-116214,
and MX-116407 were more potent, resulting in TGDs of 12,
15, and 16 days and % T/Cs of 58, 55, and 43, respectively
(Table 3). Because MX-76747, MX-116214, and MX-116407
showed good antitumor activity, they were evaluated in
two other human xenografts and compared with ZD6126
using the same schedule of administration.
In the human breast MX1 tumor xenograft, compounds
were given when the tumor fragments had reached
f
150
mm
3
(effect of compounds on tumor growth and body
weight is shown in Fig. 4). Whereas the average tumor size
increased from 147 to 1,879 mm
3
in the control group
(representing a 12.8-fold increase), administration of MX-
76747, MX-116214, and MX-116407 led to increases of 1.9-,
2.4-, and 0.9-fold, respectively. In the ZD6126-treated
groups, the average tumor volume increased 3-fold. At
Figure 3. Functional vasculature determination following single dose of
MX-116407. Perfused vasculature in the Calu-6 tumor was assessed by a
double fluorescent dye staining procedure as described in Materials and
Methods. Vehicle and CA4P were used as negative and positive controls,
respectively. Columns, mean of three animals per group; bars, SD.
Table 3. Antitumor efficacy in the breast (MDA-MB-435) human
tumor xenograft
Compounds Dose/schedule TGD (d) % T/C
ZD6126 200 mg/kg q6d
3868
MX-58151 10 mg/kg b.i.d.
5d 8 65
MX-58276 40 mg/kg b.i.d.
5d 4 75
MX-76747 10 mg/kg (q1d
5) 3 wk 12 58
MX-116214 15 mg/kg (q1d
5) 3 wk 15 55
MX-116407 45 mg/kg (q1d
5) 3 wk 16 43
MX-126303 1 mg/kg (q1d
5) 3 wk 6 75
Figure 4. Activity of selected compounds against the human breast
(MX1) tumor xenograft. MX1 human breast fragments were implanted s.c.
by trocar in nu/nu female mice. Treatment was initiated when the primary
tumor reached a size of
f
150 mm
3
(day 13 ). MX-76747 (10 mg/kg, y),
MX-116214 (15 mg/kg, !), and MX-116407 (45 mg/kg, E) were given
i.v. on days 13 17 and days 20 24. ZD6126 (100 mg/kg, o) was given
i.p. on the same days. Saline control group (solid line); Cremophor/
ethanol/dextrose control gr oup (dashed line). Solid horizontal bar,
treatment period.
Novel Vascular-Targeting Agents1380
Mol Cancer Ther 2004;3(11). November 2004
Page 6
the time vehicle groups were terminated, we observed TGIs
of 75% to 91% for the MX compounds (P < 0.001) compared
with a TGI of 64% (P = 0.03) for ZD6126. Moreover, tumor
regression was observed with MX-116407. Tumor growth
resumed after the termination of treatment, but TGDs of
over 10 days were observed (Fig. 4, top) at doses that were
well tolerated as indicated by no significant body weight
loss (Fig. 4, bottom).
In the human lung Calu-6 xenograft, compounds were
given when the tumor fragments had reached an average
size of 250 mm
3
(24 days after tumor cell inoculation).
MX-76747, MX-116214, MX-116407, and ZD6126 were given
daily for 5 consecutive days at doses near but below their
respective MTDs (Fig. 5). MX-76747 and MX-116214 did not
show significant antitumor activity in this model resulting
in TGIs of 10% and 28% at day 42, time at which the control
group had to be sacrificed (P > 0.5). On the other hand, MX-
116407 showed highly significant antitumor activity with
tumor regressions occurring from days 28 to 45 and TGI
of 86% at day 42 (P = 0.01). ZD6126 had moderate anti-
tumor activity in this model with tumor stasis occurring
from days 28 to 34 and TGI of 47% at day 42 (P = 0.3).
When MX-116407 was examined in combination with a
moderately active dose of cisplatin (4 mg/kg i.p. given 24
hours before MX-116407), we observed significant tumor
regression occurring from day 28 in all 10 treated mice (Fig.
6A), with four animals remaining tumor free at day 104
when the study was terminated, which was 85 days after
the last drug dose. At day 42, the time at which animals
from the control group had to be sacrificed, the average %
T/C value was 13 (Fig. 6B). Body weight losses in the MX-
116407-treated and MX-116407/cisplatin treated groups
were <10%.
Discussion
We have reported previously that a group of 4-aryl-4H-
chromenes (i.e., MX-58151, MX-58276, MX-76747, MX-
116214, MX-116407, and MX-126303) had potent in vitro
cytotoxic activity, bound to tubulin at the colchicine site,
caused a G
2
-M-phase cell arrest, and induced apoptosis (4).
The compounds also had an effect on endothelial cell
function as shown by their ability to disrupt preformed
capillary tubes in the three-dimensional Matrigel tubule
formation assay (4). This property, suggestive of antiangio-
genic and antivascular activities (17, 27), led us to evaluate
the antitumor activity of this group of compounds.
Tubulin-binding agents are known to have antivascular
activity, but for most compounds, this effect is observed at
or close to the MTD (5). However, the newer generation of
tubulin-interacting agents, such as CA4P and ZD6126, have
shown a selective effect against tumor vasculature at doses
around one-third (28) to one-tenth (29, 30) of their
Figure 5. In vivo antitumor activity of selected compounds against the
human lung (Calu-6) tumor xenograft. BALB/c female nude mice 6 8 weeks
old were injected s.c. with 2
10
6
Calu-6 cells (day 0). Treatment was
initiated when the primary tumor reached a size of
f
250 mm
3
(day 24 ).
MX-76747 (10 mg/kg,
y), MX-116214 (15 mg/kg, !), and MX-116407
(45 mg/kg, E) were given i.v. on days 24 28 . ZD6126 (100 mg/kg, o)
was given i.p. on the same days. Vehicle control (Cremophor/ethanol/
saline) group (5). Solid horizontal bar, treatment period.
Figure 6. Growth of Calu-6 human tumor xenografts after treatment
with MX-116407 in combination with cisplatin. Treatment was initiated
when tumors reached an average size of 250 mm
3
(day 24). Cisplatin was
given daily at 4 mg/kg i.p. (
.
) 1 day prior to MX-116407. MX-116407
was given daily for 5 days at 45 mg/kg alone (
w
) or in combination with
cisplatin (
y
). Vehicle control group (Cremophor/ethanol/saline) was dosed
daily for 5 days (n).
A, tumor growth results are expressed as a
percentage of growth in relation to the size of each tumor on the day of
first treatment (this value was set as 100% and tumor growth is reported
in percentage).
B, relative tumor volumes of all the animals from the
different treatment groups are compared at day 42 , after which time
animals from the control group had to be sacrificed due to tumor burden.
Solid horizontal bar, treatment period for MX-116407 and vehicle.
Molecular Cancer Therapeutics 1381
Mol Cancer Ther 2004;3(11). November 2004
Page 7
respective MTDs. The six compounds profiled in this study
all induced extensive tumor necrosis. Within 24 hours of
administration, the entire center of the tumor was necrotic
with a thin viable rim of tumor cells surviving at the
periphery. This pattern of central necrosis with a thin viable
rim of tumor cells was first reported with CA4P (30) and is
consistent with vasculature targeting agents (18, 3133).
However, when the dose required to induce significant
tumor necrosis was compared with the dose that caused
significant but reversible toxicities, it became evident that
some compounds were more effective than others.
Also observed with this series of compounds was that the
compounds with the most potent antivascular effects were
not the most potent in vitro (4). Indeed, although MX-
126303 had the best in vitro cytotoxic activity, it induced
tumor necrosis at levels close to its MTD, suggesting that its
toxicity is linked with its effect on tubulin inhibition rather
than targeting selectively endothelial cells. The antivascular
activity of MX-126303 is reminiscent of other tubulin-
binding agents, such as colchicine and vinblastine, which
havevascular-damagingactivityonlyattheirMTDs.
However, its pharmacokinetic profile is similar to classic
antivascular agents such as ZD6126 and CA4P, which have
short plasma half-lives (29, 34). On the other hand, MX-
116407, while less cytotoxic than MX-126303 in vitro, had
more tumor-selective antivascular activity. We observed
80% tumor necrosis at 25 mg/kg, corresponding to one-
fourth of its MTD. This may be related to the differences in
pharmacokinetic profile. Although both MX-116407 and
MX-126303 have rapid distribution phases, resulting in
maximum concentrations at the earliest time point evalu-
ated (10 and 12.3 Ag/mL, respectively), their clearance rates
were quite different, varying by 2.4-fold. This results in a
8-fold difference in concentration after 45 minutes (3.5 ver-
sus 0.44 Ag/mL for MX-116407 and MX-126303, respectively;
Fig. 1). A rapid distribution coupled with a slow clearance
is thus likely to be important for good antitumor activity.
A second explanation might relate to our finding that
apoptosis induction following a 3-hour treatment with MX-
126303 is irreversible after drug washout, whereas induc-
tion of apoptosis following MX-116407 treatment is
reversible (4). This may suggest that MX-116407 and MX-
126303 have different tubulin-binding kinetics, leading to
differences in pharmacologic profile. Indeed, the improved
therapeutic index observed with CA4P compared with
colchicine has been correlated to their differences in the
on/off rate of binding to tubulin (11, 17, 35), leading to the
hypothesis that a successful vascular targeting agent would
have reversible binding kinetics and relatively rapid
clearance in vivo (19).
The vascular targeting activity of this novel series of
compounds, suggested by the induction of tumor necrosis,
led us to investigate their antitumor potential. Whereas
treatment of mice bearing MDA-MB-435 tumor xenografts
with MX-58151, MX-58276, and MX-126303 resulted in poor
antitumor activity with % T/Cs of 65, 75, and 75,
respectively, MX-76747, MX-116214, and MX-116407
resulted in moderate antitumor activity with % T/Cs of
58, 55, and 43, respectively. According to National Cancer
Institute criteria, compounds resulting in % T/C values of
V42 are considered to have antitumor activity (24). The
moderate antitumor activity is likely a result of regrowth of
the tumor originating from the viable rim of tumor cells
remaining after the treatment. We have observed that the
MDA-MB-435 tumors have repopulated spontaneously 14
days after the cessation of treatment, although % T/C
values were calculated at day 50, 10 days after cessation of
treatment. We have further evaluated MX-76747, MX-
116214, and MX-116407 in other human tumor xenograft
models. In the human breast (MX-1) tumor xenograft, all
three compounds showed significant antitumor activity
with % T/Cs ranging between 0.7 and 17. In the human
lung (Calu-6) tumor xenograft, MX-116407 was highly
active, producing tumor regressions in all 10 animals.
Moreover, MX-116407 significantly enhanced the antitumor
activity of cisplatin, producing tumor-free animals in a
significant number (4 of 10) of cases at time of sacrifice.
In summary, we have identified 2-amino-4-(3-bromo-4,5-
dimethoxy-phenyl)-3-cyano-4H-chromenes as a novel se-
ries of vascular targeting agents with promising antitumor
activity. Our encouraging results with MX-116407 strongly
support its continued development as a novel anticancer
agent for human use.
Acknowledgments
We thank Louis Vaillancourt for the synthesis of ZD6126 and Johanne
Cadieux for assistance in the preparation of the article.
References
1. Cai SX, Nguyen B, Jia S, et al. Discovery of substituted N -ph enyl
nicotinamides as potent inducers of apoptosis using a cell- and caspase-
based high throughput screening assay. J Med Chem 2003;46:2474 81.
2. Cai SX, Zhang HZ, Guastella J, et al. Design and synthesis of
rhodamine 110 derivative and caspase-3 substrate for enzyme and cell-
based fluorescent assay. Bioorg Med Chem Lett 2001;11:39 42.
3. Zhang HZ, Kasibhatla S, Wang Y, et al. Discovery, characterization and
SAR of gambogic acid as a potent apoptosis inducer by a HTS assay.
Bioorg Med Chem 2004;12:309 17.
4. Kasibhatla S, Gourdeau H, Meerovitch K, et al. Discovery and
mechanism of action of a novel series of apoptosis inducers with potential
vascular targeting activity. Mol Cancer Ther 2004;3:1365 73.
5. Ben-Ze’ev A. Cell shape, the complex cellular networks, and gene
expression. Cytoskeletal protein genes as a model system. Cell Muscle
Motil 1985;6:23 53.
6. Bernal SD, Stahel RA. Cytoskeleton-associated proteins: their role as
cellular integrators in the neoplastic process. Crit Rev Oncol Hematol
1985;3:191 204.
7. Thyberg J, Mo skalewski S. Microtubules and the organization of the
Golgi complex. Exp Cell Res 1985;159:1 16.
8. Rowinsky E, Donehower R, editors. Antimicrotubule agents. Philadel-
phia: Lippincott-Raven Publishers; 1997. p. 467 83.
9. Rowinsky EK. The development and clinical utility of the taxane class of
antimicrotubule chemotherapy agents. Annu Rev Med 1997;48:353 74.
10. Jordan MA, Wilson L. Microtubules and actin filaments: dynamic
targets for cancer chemotherapy. Curr Opin Cell Biol 1998;10:123 30.
11. Jordan MA. Mechanism of action of antitumor drugs that interact
with microtubules an d tubulin. Curr M ed Chem An ti-Canc Agen ts
2002;2:1 17.
12. Hadfield JA, Ducki S, Hirst N, McGown AT. Tubulin and microtubules
as targets for anticancer drugs. Prog Cell Cycle Res 2003;5:309 25.
Novel Vascular-Targeting Agents1382
Mol Cancer Ther 2004;3(11). November 2004
Page 8
13. Dumontet C, Sikic BI. Mechanisms of action of and resistance to
antitubulin agents: microtubule dynamics, drug transport, and cell death.
J Clin Oncol 1999;17:1061 70.
14. Li Q, Sham H. Discovery and development of antimitotic agents that
inhibit tubulin polymerizatio n for the treatment of cancer. J Clin Oncol
2002;17:1061 70.
15. Baguley BC, Holdaway KM, Thomsen LL, Zhuang L, Zwi LJ. Inhibition
of growth of colon 38 adenocarcinoma by vinblastine and colchicine:
evidence for a vascular mechanism. Eur J Cancer 1991;27:482 7.
16. Marx M. Small-molecule, tubulin-binding compounds as vascular
targeting agents. Expert Opin Ther Patents 2002;12:769 76.
17. Griggs J, Metcalfe JC, Hesketh R. Targeting tumor vasculature: the
development of combretastatin A4. Lancet Oncol 2001;2:82 7.
18. Micheletti G, Poli M, Borsotti P, et al. Vascular-targeting activity of
ZD6126, a novel tubulin-binding agent. Cancer Res 2003;63:1534 7.
19. Davis PD, Dougherty GJ, Blakey DC, et al. ZD6126: a novel vascular-
targeting agent that causes selective destruction of tumor vasculature.
Cancer Res 2002;62:7247 53.
20. Chen TR. In situ detection of Mycoplasma contamination in cell
cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 1977;
104:255 62.
21. Rowland M, Tozer TN. In: Rowland M, Tozer TN, editors. Clinical
pharmacokinetics: concepts and application. Philadelphia: Lae & Febiger;
1989. p. 9 100.
22. Tufto I, Rofstad EK. Transient p erfusion in human melanoma
xenografts. Br J Cancer 1995;71:789 93.
23. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size
in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148 54.
24. Plowman J, Dykes D, Hollingshead, M, Simpson-Herren L, Alley MC.
In: Teicher B, editor. Anticancer drug development guide: preclinical
screening, clinical trials, and approval. Totowa (NJ): Humana Press Inc;
1997. p. 101 25.
25. Johnson JI, Decker S, Zaharevitz D, et al. Relationships between drug
activity in NCI preclinical in vitro and in vivo models and early clinical
trials. Br J Cancer 2001;84:1424 31.
26. Davies B, Morris T. Phys iological parameters in laboratory animals
and humans. Pharm Res 1993;10:1093 5.
27. Hotchkiss KA, Ashton AW, Mahmood R, et al. Inhibition of
endothelial cell function in vitro and angiogenesis in vivo by docetaxel
(Taxotere): association with impaired repositioning of the microtubule
organizing center. Mol Cancer Ther 2002;1:1191 200.
28. Grosios K, Holwell SE, McGown AT, Pettit GR, Bibby MC. In vivo and
in vitro evaluation of combretastatin A-4 and its sodium phosphate
prodrug. Br J Cancer 1999;81:1318 27.
29. Blakey DC, Westwood FR, Walker M, et al. Antitumor activity of the
novel vascular targeting agent ZD6126 in a panel of tumor models. Clin
Cancer Res 2002;8:1974 83.
30. Dark GG, Hill SA, Prise VE, et al. Combretastatin A-4, an agent that
displays potent and selective toxicity toward tumor vasculature. Cancer
Res 1997;57:1829 34.
31. Nihei Y, Suzuki M, Okano A, et al. Evaluation of antivascular and
antimitotic effects of tubulin binding agents in solid tumor therapy. Jpn J
Cancer Res 1999;90:1387 95.
32. Otani M, Natsume T, Watanabe JI, et al. TZT-1027, an antimicrotu-
bule agent, attacks tumor vasculature and induces tumor cell death. Jpn
J Cancer Res 2000;91:837 44.
33. Nilsson F, Kosmehl H, Zardi L, Neri D. Targeted delivery of tissue fac-
tor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates
the infarction of solid tumors in mice. Cancer Res 2001;61:711 6.
34. Dowlati A, Robertson K, Cooney M, et al. A phase I pharmacokinetic
and translational study of the novel vascular targeting agent combretas-
tatin A-4 phosphate on a single-dose intravenous schedule in patients with
advanced cancer. Cancer Res 2002;62:3408 16.
35. Lin CM, Ho HH, Pettit GR, Hamel E. Antimitotic natural products
combretastatin A-4 and combretastatin A-2: studies on the mechanism
of their inhibition of the binding of colchicine to tubulin. Biochemistry
1989;28:6984 91.
Molecular Cancer Therapeutics 1383
Mol Cancer Ther 2004;3(11). November 2004
Page 9
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