BRIEF COMMUNICATIONS

Effect of Pretreatment With Atenolol and

Nifedipine on ZD6126-Induced Cardiac
Toxicity in Rats
Sarah Gould, F. Russell Westwood, Jon O. Curwen, Susan E. Ashton,
David W. Roberts, Susan C. Lovick, Anderson J. Ryan

J Natl Cancer Inst 2007;99:172428

Vascular disrupting agents selectively

destroy the existing tumor vasculature by
inducing morphologic changes in tumor
endothelial cells that lead to vessel occlusion and massive central tumor necrosis
(13). ZD6126, a soluble prodrug of Nacetylcolchinol, is a vascular disrupting
agent that acts by inhibiting tubulin polymerization, which destabilizes the microtubule network and induces morphologic
changes in immature tumor endothelial
cells (46). Several phase I studies have been
carried out to identify the optimal dosing
schedule of ZD6126 and to define its tolerability and pharmacokinetic profile (79).
Dynamic contrast-enhanced magnetic resonance imaging has demonstrated substantially reduced tumor uptake of gadolinium
contrast agent in ZD6126-treated mice and
patients, a finding that is consistent with the
selective vascular disrupting effects of
ZD6126 in tumors (10). However, the
phase I studies (79) reported that ZD6126
1724 Brief Communications

JNCI

treatment was associated with adverse cardiovascular events that generally occurred
within a few hours after ZD6126 was
administered and comprised one or more of
the following: electrocardiogram abnormalities, an increase in the circulating level
of troponin I (a marker of cardiac damage),
and/or arm or chest pain. ZD6126 treatment is also associated with a transient
increase in blood pressure (9). Similar
adverse cardiac events have been reported
for other microtubule-destabilizing vascular
disrupting agents that are in clinical development (1113). In this study, we used a rat
preclinical model to examine the underlying mechanisms responsible for the adverse
cardiac events associated with ZD6126.
For analyses of ZD6126-induced hemodynamic changes, anesthesia was induced in
female Wistar-derived Alpk:ApfSD rats
(175200 g; n = 9; AstraZeneca, Maccleseld,
Cheshire, U.K.) with 3%4% isourane
(Abbott Laboratories, Chicago, IL) in

Affiliation of authors: AstraZeneca, Mereside,

Alderley Park, Macclesfield, Cheshire, U.K.
Correspondence to: Anderson J. Ryan, PhD,
Department of Cancer Bioscience, AstraZeneca,
Mereside, Alderley Park, Macclesfield SK10
4TG, Cheshire, U.K. (e-mail: anderson.ryan@
astrazeneca.com).
See Notes following References.
DOI: 10.1093/jnci/djm202
The Author 2007. Published by Oxford University
Press. All rights reserved. For Permissions, please
e-mail: journals.permissions@oxfordjournals.org.

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Antivascular agents that act by destabilizing microtubules, such as ZD6126 (Nacetylcolchinol-O-phosphate), are associated with adverse cardiovascular effects,
including transient hypertension, cardiac ischemia, myocardial infarction, and
increases in circulating levels of markers of cardiac damage (e.g., troponins). We
investigated mechanisms underlying these effects of ZD6126 in rats by continuously monitoring their heart rate and blood pressure and by assessing heart
histopathology and plasma troponin T levels. ZD6126 induced acute transient
hemodynamic changes (hypertension and delayed tachycardia), which were associated with statistically significant increases in circulating troponin T levels (median
level 3 hours after treatment with vehicle or 12.5 mg/kg ZD6126: <9 pg/mL and 563
pg/mL, respectively; P <.001 [two-sided Wilcoxon rank sum test]) and in the incidence
of left ventricular myocardial fiber necrosis (incidence 24 hours after treatment with
vehicle or 12.5 mg/kg ZD6126: 0/10 rats and 9/10 rats, respectively; P <.001 [twosided Wilcoxon rank sum test]). Pretreatment of rats with atenolol and nifedipine
ameliorated the acute hemodynamic changes and prevented ZD6126-induced
increases in both troponin T and myocardial necrosis but did not prevent ZD6126induced tumor necrosis in an Hras5 tumor xenograft model in nude rats. Our findings suggest that ZD6126-induced acute hemodynamic changes are a prerequisite
for cardiac damage in rats.

medical-grade air and was maintained with

1.5%2.5% isourane throughout the procedure. The abdominal cavity of each rat
was opened, and the descending abdominal
aorta was located and cleared of connective
tissue. A tie placed underneath the vessel
was lifted to occlude the vessel while the
catheter of a DSI TA11PA-C40 radiotelemetry implant (Data Sciences International,
St Paul, MN) was inserted into the aorta
along the bevel of a bent 21-gauge needle.
The catheter was held in place by suturing
it to the inside of the abdominal wall. The
surgical abdominal wound was then closed
with dissolving sutures, and the outer skin
was closed with autoclips. The rats were
allowed to recover from surgery and acclimatized to the handling procedures required
for dosing, and they were randomly assigned
to one of three groups (n = 3 rats per group).
Group 1 (the control group) received 1%
polysorbate 80 by mouth at 150 minutes
and, 1 hour later (i.e., at 210 minutes), 0.9%
sodium chloride solution (physiological
saline) by intravenous injection. Time zero
(0 minutes) was arbitrarily dened as 6:00
am. Group 2 received 1% polysorbate 80 by
mouth at 150 minutes and ZD6126 (12.5
mg in physiological saline/kg) by intravenous injection at 210 minutes. Group 3
received atenolol (6 mg/kg) and nifedipine
(10 mg/kg) (SigmaAldrich, Poole, U.K.),
both by mouth at 150 minutes, each in 1%
polysorbate 80, and ZD6126 (AstraZeneca)
(12.5 mg/kg) by intravenous injection at
210 minutes. Blood pressure and heart rate
data were collected via the radiotelemetry
implant every 5 minutes beginning at 0
minutes for 24 hours. For each rat, data
were collected as a moving point average
over a 30-second period within each 5minute interval.

Prior knowledge
Antivascular agents selectively destroy the
existing tumor vasculature. However, some
vascular disrupting agents in early clinical
development that act by destabilizing
microtubules, such as ZD6126, are associated with adverse cardiovascular effects.
Study design
A rat preclinical model was used to examine the underlying mechanisms responsible
for the adverse cardiac events associated
with ZD6126.
Contribution
ZD6126 induces acute hemodynamic
changes that are a prerequisite for cardiac
damage in rats.

Limitations
Not all patients who receive ZD6126 experience cardiovascular adverse events,
suggesting that the underlying biologic
mechanisms in patients may differ from
those in rats. The direct relevance of this
rat model to patients treated with ZD6126 is
unclear.

120

Control
ZD6126
Atenolol, Nifedipine, ZD6126

110
100
90
80
70
60
0

120

240

360

jnci.oxfordjournals.org

480

600

720

840

Time (minutes)

550

Control
ZD6126
Atenolol, Nifedipine, ZD6126

Mean heart rate (bpm)

500
450
400
350
300
250
0

120

240

360

480

600

720

840

Saline or ZD6126

Time (minutes)
Atenolol/nifedipine

Administration of ZD6126 to rats produced a rapid increase in diastolic blood

pressure that lasted for 120180 minutes,
which was accompanied by a more prolonged and complex effect on heart rate
(Fig. 1). ZD6126 induced tachycardia that
was delayed with respect to the effects on
diastolic blood pressure, possibly due, in
part, to a reex bradycardia resulting from
normal homeostatic mechanisms acting
to reduce the severity of the concurrent
hypertension. When the acute hypertensive response abated 120180 minutes after
ZD6126 treatment, tachycardia became
apparent and persisted for at least an additional 240 minutes. Preliminary experiments showed that direct vasodilation
induced by an oral dose of the calcium
channel-blocking agent, nifedipine, was
effective in abolishing ZD6126-induced
hypertension, whereas the -adrenoceptor
antagonist atenolol was effective in limiting
ZD6126-induced tachycardia (data not
shown). Figure 1 shows that the combina-

and nifedipine (10 mg/kg) alone; atenolol

(6 mg/kg) and nifedipine (10 mg/kg) with
ZD6126 (5 mg/kg or 12.5 mg/kg). When
the three drugs were administered to the
same rat, ZD6126 was given 1 hour after
atenolol and nifedipine, as described previously. Blood samples (0.8 mL in lithium
heparin) collected from each rat via the tail
at 3, 6, and 24 hours after saline or ZD6126
dosing were subjected to a Troponin T Stat
immunoassay (Roche Diagnostics, Lewes,
U.K.) to determine the concentration

Fig. 1. Effects of atenolol and nifedipine on the hemodynamic changes induced by ZD6126. Heart
rate and blood pressure were monitored continuously by telemetry (beginning at 0 minutes) in
three groups of rats (n = 3 per group). The control group received two vehicles, 1% polysorbate
80 by mouth (at 150 minutes) and, 1 hour later (at 210 minutes), 0.9% physiological saline by
intravenous injection. The ZD6126 group received 1% polysorbate 80 by mouth (at 150 minutes)
and, 1 hour later (at 210 minutes), ZD6126 (12.5 mg/kg) by intravenous injection. The atenolol,
nifedipine, ZD6126 group received atenolol (6 mg/kg) and nifedipine (10 mg/kg), both by mouth
(at 150 minutes), and, 1 hour later (at 210 minutes), ZD6126 (12.5 mg/kg) by intravenous injection. Mean blood pressure (A) and heart rate (B) data are shown.
JNCI

Brief Communications 1725

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Implications
Clinical investigations of approaches that
reduce the acute hemodynamic effects of
microtubule-destabilizing vascular disrupting agents are warranted.

tion of atenolol and nifedipine, given

1 hour before ZD6126 administration, was
effective in abolishing both the hypertensive and tachycardic effects of ZD6126.
We next examined the effects of the
three drugs, alone and in combination, on
rat heart histopathology and plasma troponin T levels. Female rats (n = 60) were randomly assigned to one of the following
six treatment groups (10 rats per group):
physiological saline; ZD6126 (5 mg/kg);
ZD6126 (12.5 mg/kg); atenolol (6 mg/kg)

Mean diastolic blood pressure (mmHg)

CONTEXT AND CAVEATS

Table 1. Heart histopathology and plasma troponin T levels in rats treated with ZD6126 alone or in combination with atenolol and
nifedipine*
Treatment group
Vehicle
control
(n = 10)

Atenolol and
nifedipine
(n = 10)

ZD6126
(5 mg/kg)
(n = 10)

Atenolol and
nifedipine,
ZD6126
(5 mg/kg) (n = 10)

Atenolol and
nifedipine,
ZD6126 (12.5
mg/kg) (n = 10)

ZD6126
(12.5 mg/kg)
(n = 10)

Incidence of left ventricle histopathology 24 h after treatment

10
0
0
0

1
4
5
0

10
0
0
0

1
3
6
0

9
1
0
0

10
0
0

10
0
0

8
1
1

10
0
0

4
3
3

10
0
0

6
1
3
0

10
0
0
0

10
0
0
0

Necrosis
None
Minimal
Mild
Hemorrhage
None
Minimal
Mild
Mixed cell infiltration/
fibroplasia
None
Minimal
Mild
3 h after treatment
6 h after treatment
24 h after treatment

9
1
0
0

10
6
10
0
0
0
0
4
0
0
0
0
Incidence of right ventricle histopathology 24 h after treatment

10
0
0

10
0
0

7
2
1

10
0
0

8
0
2

10
0
0

10
0
0

10
0
0

9
1
0

10
0
0

8
1
1

10
0
0

8
1
1

10
0
0

10
0
0
9 (99)
9 (99)
9 (99)

10
8
10
0
0
0
0
2
0
Median plasma troponin T concentration, pg/mL (IQR)
9 (99)
9 (99)
9 (99)

274 (220690)
120 (78257)
9 (99)

9 (99)
9 (99)
9 (99)

563 (454956)
237 (167398)
9 (99)

9 (99)
9 (99)
9 (99)

* Groups of 10 rats were dosed with vehicle, ZD6126 (5 or 12.5 mg/kg), or a combination of atenolol (6 mg/kg by mouth) and nifedipine (10 mg/kg by mouth) with
or without ZD6126 (5 or 12.5 mg/kg). IQR = interquartile range.
Statistically significantly different from vehicle control (P<.001; two-sided Wilcoxon rank sum test). Tests for statistical significance were not adjusted for multiple
testing.

of troponin T. Samples with values at or

below the lower limit of detection for the
assay (9 pg/mL) were recorded as 9 pg/mL.
Data were analyzed for each time point
compared with vehicle-treated control rats
using a Wilcoxon rank sum test. All rats
were subjected to terminal anesthesia by
halothane inhalation 24 hours after saline or
ZD6126 dosing, and their hearts were
removed, xed by immersion in neutral
phosphate-buffered formalin, and bisected
so that left and right ventricular and atrial
tissues were present in both samples. Both
halves of each heart were processed together
and embedded in wax blocks. Step sections
1726 Brief Communications

JNCI

(5 m) were taken at 300-m intervals. Five

hematoxylin-eosinstained sections were
prepared from each block for examination
by light microscopy. The sections were
examined in a blinded fashion by a pathologist (F. R. Westwood) for myocardial necrosis
and associated histopathologic changes (i.e.,
hemorrhage and mixed inammatory cell
inltration/broplasia), which were graded
semiquantitatively as follows: minimal (a
single or a few small areas); mild (up to 20%
of a region of myocardium affected); moderate (more than 20% up to 50% of the myocardium affected); or severe (more than 50%
of the myocardium affected). For each rat

heart, the most severe change on any of the

ve step sections for each of the right and
left ventricles (10 sections in total) was
recorded for each of the histopathologic
parameters. For statistical analysis of left
ventricle necrosis data, each grade of histopathologic change was assigned a score of
14, where no change = 1, minimal change =
2, mild change = 3, and moderate change = 4.
(No severe changes were seen in these
experiments.) The scores for the hearts from
rats within a treatment group were compared with the scores for vehicle-treated
control rats using a Wilcoxon rank sum test.
All statistical tests were two-sided.
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Necrosis
None
Minimal
Mild
Moderate
Hemorrhage
None
Minimal
Mild
Mixed cell infiltration/
fibroplasia
None
Minimal
Mild
Moderate

Table 2. Effect of atenolol/nifedipine pretreatment on ZD6126-induced tumor necrosis in

the Hras5 tumor xenograft model in nude rats*
Treatment group

Mean tumor necrosis, % (95% CI)

Control (1% polysorbate 80 + physiological saline)

Atenolol/nifedipine
ZD6126 (5 mg/kg)
ZD6126 (5 mg/kg) + atenolol/nifedipine
ZD6126 (12.5 mg/kg)
ZD6126 (12.5 mg/kg) + atenolol/nifedipine

0.7
1.5
32.7
65.8
86.9
94.0

(0.1 to 1.3)
(0.1 to 2.9)
(8.7 to 56.7)
(48.3 to 83.4)
(77.5 to 96.3)
(90.2 to 97.8)

* Groups of four rats bearing established Hras5 tumor xenografts were dosed with vehicle (1% polysorbate
80 and physiological saline), ZD6126 (5 or 12.5 mg/kg), or a combination of atenolol (6 mg/kg) and
nifedipine (10 mg/kg) with or without ZD6126 (5 or 12.5 mg/kg). Twenty-four hours later, tumors were
recovered and the necrotic fraction was determined in hematoxylin-eosinstained sections by light
microscopy and image analysis.

jnci.oxfordjournals.org

ZD6126 doses (2.550 mg/kg) in rats indicated a statistically signicant dose-related

increase in plasma troponin T levels 3
hours after treatment (Supplementary
Table 2, available online). Troponin T levels remained elevated 6 hours after treatment with ZD6126, but by 24 hours after
treatment, the levels decreased to those
seen in plasma from vehicle-treated control
rats (Table 1). However, pretreatment of
rats with atenolol and nifedipine fully prevented the increases in troponin T seen at
3 hours after ZD6126 treatment (Table 1).
At the onset of these studies, we considered
three potential mechanisms for ZD6126induced myocardial necrosis to be feasible:
1) by direct and selective morphologic
and/or cytotoxic effects on cardiac endothelial cells, because the endothelium, at least
in tumors, is a proven target of ZD6126; 2)
by direct cytotoxic effects on the myocardium; or 3) as a consequence of the induced
acute hemodynamic alterations. We ruled
out a direct effect of ZD6126 on cardiac
endothelial cells because detailed histopathologic evaluation of rat hearts isolated
2 hours after treatment with ZD6126 at a
dose of 12.5 mg/kg using either 0.75-m
thick tissue sections stained with toluidine
blue or an antibody against the endothelial
cell marker CD31 showed no evidence of
morphologic or histologic changes in
endothelial cells preceding the myocardial
necrosis (data not shown). This nding is in
sharp contrast to the effect of ZD6126 on
tumor vessels, in which endothelial cell
damage has been found to precede tumor
cell necrosis (6). We also ruled out the possibility that ZD6126 had direct cytotoxic
effects on the myocardium because the
myocardial necrosis we observed was localized rather than diffuse (data not shown).

JNCI

Brief Communications 1727

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Compared with hearts from vehicletreated control rats, hearts from rats 24
hours after dosing with ZD6126 at 5 or 12.5
mg/kg showed a statistically signicantly
higher incidence of mild or moderate left
ventricular myocardial necrosis (median
scores in control, 5 mg/kg ZD6126, and
12.5 mg/kg ZD6126 groups were 1, 2.5, and
3, respectively; P<.001) and a higher incidence of left ventricular hemorrhage and
mixed cell inltration/broplasia (Table 1).
Similar histopathologic changes (i.e., necrosis,
hemorrhage, and mixed cell inltration/
broplasias) were seen in the right ventricle,
although these appeared to be less severe.
Similar data were obtained for hearts evaluated 72 hours after dosing and for rats given
higher doses (up to 50 mg/kg) of ZD6126
(Supplementary Table 1, available online).
Histopathologic examination revealed
that pretreatment of rats with atenolol
and nifedipine before ZD6126 abolished
all myocardial damage at both the 5 and
12.5 mg/kg doses of ZD6126 (Table 1).
The single occurrence of minimal ventricular myocardial necrosis seen following
administration of ZD6126 at 12.5 mg/kg
after atenolol and nifedipine could not be
clearly attributed to the administration of
ZD6126 because it was observed in only
one necrotic ber in only one of 10 sections
assessed. By contrast, the minimal necrosis
we observed in the heart of one control rat
involved multiple bers.
Rats treated with ZD6126 at 5 or 12.5
mg/kg had statistically signicantly higher
median troponin T levels at 3 hours after
ZD6126 treatment compared with vehicletreated control rats (control = 9 pg/mL,
5 mg/kg ZD6126 = 274 pg/mL, and
12.5 mg/kg ZD6126 = 563 pg/mL;
P<.001). Testing of a broad range of

Because pretreatment with atenolol and

nifedipine prevented ZD6126-induced
cardiac necrosis and increased plasma
troponin T levels but had no marked effects
on the plasma levels of ZD6126 phenol (the
active form of ZD6126) (Supplementary
Table 3, available online), we conclude that
the ZD6126-induced myocardial necrosis
in the rat is secondary to ZD6126-induced
hemodynamic changes.
The cardiac necrosis we observed in
ZD6126-treated rats was most commonly
seen in the subendocardial to mid-left ventricular wall areas, which include the papillary muscles. This distribution is similar to
that induced by hypoxia and isoprenalin,
which shortens the diastolic period when
ventricular blood perfusion is maximal
(14). The cardiac changes induced by
ZD6126 could be due to an imbalance
between myocardial oxygen delivery and
oxygen demand that results in inadequate
myocardial perfusion, which is analogous
to the situation in which diastolic lling
time, during which the blood irrigates
myocardial tissue, is shortened due to
tachycardia and coronary artery perfusion
pressure is reduced due to systemic hypotension (15,16). We hypothesize that acute
vasoconstriction that is accompanied by
tachycardia, as we have seen in rats following ZD6126 administration, may produce
a similar inadequacy in myocardial perfusion, whereby tachycardia exacerbates
hemodynamic changes associated with
vasoconstriction.
The mechanism of cardiac toxicity suggested by this study (i.e., acute vasoconstriction/hypertension accompanied by
tachycardia) is unlikely to be unique to
ZD6126 because other tubulin-binding vascular disrupting agents (i.e., combretastatin
A4 phosphate and AVE8062) are reported
to have similar cardiac effects in the clinic
(79) and because we have observed a similar pattern of cardiac necrosis after treating
rats with combretastatin A4 phosphate (data
not shown). Although the underlying mechanisms responsible for ZD6126-induced
acute hypertension and tachycardia remain
unknown, other microtubule-destabilizing
agents (i.e., colchicine, nocodazole, and
demecolcine) have been shown to induce
vasoconstriction through their effects on
vascular smooth muscle tone (17).
To investigate the effects of pretreatment with atenolol and nifedipine on the

1728 Brief Communications

JNCI

This small study has several limitations.

In the clinic, only a minority of patients
experience cardiovascular adverse events
following ZD6126 treatment. By contrast,
in the rat models used here the response to
ZD6126 dosing was more homogeneous,
and almost all animals displayed both
increases in plasma troponin T level and
histopathologically conrmed cardiac damage at ZD6126 dose levels of 5.0 mg/kg
or higher. The factors that differentiate
patients at risk for cardiovascular adverse
events following ZD6126 dosing are not
known, but extending the current studies to
different strains of rat may help identify the
underlying biologic mechanisms. In addition, the mechanism of ZD6126-induced
tachycardia seen in rats is unclear but is
likely to be indirect because no effect of
ZD6126 on heart rate has been observed
in isolated perfused rat hearts (data not
shown). Further work is required to understand whether tachycardia observed in this
rat model is relevant to patients treated
with ZD6126.
In conclusion, our data in rats suggest
that the adverse cardiac events observed in
the clinic following ZD6126 administration
may result, at least in part, from acute
hemodynamic changes. This conclusion has
potential therapeutic implications because
our studies also suggest that these acute
hemodynamic changes, and the subsequent
cardiac side effects, may be ameliorated by
antihypertensive therapy without overtly
affecting the antitumor efcacy of ZD6126.

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Notes
The authors are employees of AstraZeneca, which
funded this work. No external funding was received.
Manuscript received January 16, 2007; revised
August 29, 2007; accepted September 11, 2007.
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antitumor efcacy of ZD6126, we measured tumor necrosis in a tumor xenograft

model in nude rats. Athymic female rats
(HsdHan:Rnu-rnu; 100160 g; n = 24;
AstraZeneca, Alderley Park, U.K.) were
implanted subcutaneously with Hrastransformed mouse 3T3 broblasts (Hras5
cells; 2 105 cells per rat) (6). When
Hras5 xenograft tumors reached a size of
approximately 0.5 cm3, the rats were
randomly assigned to one of six treatment
groups (n = 4 rats per group): physiological
saline and 1% polysorbate 80; atenolol
(6 mg/kg) and nifedipine (10 mg/kg);
ZD6126 (5 or 12.5 mg/kg); atenolol (6
mg/kg) and nifedipine (10 mg/kg) plus
ZD6126 (5 or 12.5 mg/kg). When all
three agents were administered to the
same rat, atenolol and nifedipine were
given 1 hour before ZD6126. Rats were
killed 24 hours after ZD6126 or vehicle
dosing and their tumors excised, xed in
10% neutral-buffered formalin, and
processed for embedding in wax blocks.
Necrosis was assessed by light microscopy
in sections (4-m thick) that were stained
with hematoxylineosin. For each tumor,
necrosis was assessed in a single section
cut from the widest part of the tumor with
the use of operator-guided image analysis
software (KS400 version 3.0 image analysis system; Carl Zeiss Vission GmbH,
Hallbergmoos, Germany). For each treatment group, the mean percentage of
necrotic cells and the 95% condence
interval (CI) was calculated.
ZD6126 induced extensive tumor necrosis in Hras5 xenografts 24 hours after dosing (necrotic fraction of cross-sectional
tumor area at 5 and 12.5 mg/kg = 32% and
87%, respectively) (Table 2), consistent
with results from a previous study (4).
Pretreatment with atenolol and nifedipine
did not prevent tumor necrosis induced
24 hours after treatment with ZD6126 at
5 mg/kg (proportion of necrotic cells with
versus without atenolol/nifedipine: 65.8%
versus 32.7%, difference = 33.1%, 95%
CI = 3.9% to 69.9%, P = .07 [two-sided t
test]) or 12.5 mg/kg (proportion of necrotic
cells with versus without atenolol/nifedipine: 94.0% versus 86.9%, difference =
7.1%, 95% CI = 5.2% to 19.5%, P = .208
[two-sided t test]) (Table 2) even though
the same pretreatment schedule with
atenolol and nifedipine ameliorated the
cardiotoxic effects of ZD6126.