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JPET #196873
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The vascular disrupting agent STA-9584 exhibits potent antitumor activity by selectively targeting microvasculature at both the center and periphery of tumors
Kevin P. Foley, Dan Zhou, Chris Borella, Yaming Wu, Mei Zhang, Jun Jiang, Hao Li, Jim Sang, Tim
Korbut, Josephine Ye, Xuemei Zhang, James Barsoum, and Andrew J. Sonderfan.
Synta Pharmaceuticals Corp., Lexington, MA
JPET Fast Forward. Published on July 26, 2012 as DOI:10.1124/jpet.112.196873
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title Page
Running Title
Unique bioactivity of the vascular disrupting agent STA-9584
Corresponding Author
Andrew Sonderfan, Synta Pharmaceuticals Corporation, 125 Hartwell Avenue, Lexington, MA 02421. Tel:
781-541-7785; Fax: 781-541-6350; Email: [email protected]
Document Statistics
Number of text pages 29
Number of Tables 2
Number of Figures 6
Number of References 38
Number of words in Abstract 249
Number of words in Introduction 678
Number of words in Discussion 935
List of Abbreviations: VDA, vascular disrupting agent; CA4P, combretastatin A-4 phosphate; ASA404,
5,6-dimethylxanthenone-4 acetic acid; MTD, maximum tolerated dose; NSCLC, non-small cell lung
cancer.
Recommended section assignment
Drug Discovery and Translational Medicine
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Abstract
Vascular disrupting agents (VDAs) are an emerging class of therapeutics targeting the existing vascular
network of solid tumors. However their clinical progression has been hampered due to limited single-
agent efficacy, primarily due to the persistence of surviving cells at the well-perfused ‘viable rim’ of
tumors, which allows rapid tumor regrowth to occur. In addition, off-target adverse events, including
cardiovascular toxicities, underscore a need for compounds with improved safety profiles. Here we
characterize the mechanism of action, antitumor efficacy and cardiovascular safety profile of STA-9584, a
novel tubulin-binding VDA. In vitro, STA-9122 (active metabolite of STA-9584) displayed increased
potency relative to other tubulin-binding agents and was highly cytotoxic to tumor cells. STA-9584
induced significant tumor regressions in prostate and breast xenograft models in vivo and, in an
aggressive syngeneic model, demonstrated superior tumor growth inhibition and a positive therapeutic
index relative to combretastatin A-4 phosphate (CA4P). Importantly, histological analysis revealed that
STA-9584 disrupted microvasculature at both the center and periphery of tumors. Compared to CA4P,
STA-9584 induced a 73% increase in central necrotic area, 77% decrease in microvasculature and 7-fold
increase in tumor cell apoptosis in the remaining viable rim 24 hours post-treatment. Ultrasound imaging
confirmed that STA-9584 rapidly and efficiently blocked blood flow in highly perfused tumor regions.
Moreover, cardiovascular effects were evaluated in the Langendorff assay and telemetered dogs and
cardiovascular toxicity was not predicted to be dose-limiting. This bioactivity profile distinguishes STA-
9584 from the combretastatin class and identifies the compound as a promising new therapeutic VDA
candidate.
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Introduction
Tumor vasculature is essential for the supply of oxygen and nutrients that promote solid tumor growth and
the spread of metastatic disease. Hence targeting this network represents an intuitively attractive and
clinically validated approach for the treatment of human malignancies (Tozer et al., 2005; McKeage and
Baguley, 2010). First generation therapeutics focused on the use of angiogenesis inhibitors, which
prevent the neovascularization process and/or promote vascular normalization within tumors (Grothey
and Galanis, 2009). This resulted in the successful clinical development of a number of new anticancer
drugs, including bevacizumab (Avastin), sorafenib and sunitinib (Eichholz et al., 2010). As an alternative
strategy, vascular disrupting agents (VDAs) represent a relatively new group of agents that exhibit
selective activity against established tumor vascular networks, causing rapid collapse and shutdown of
tumor blood flow, ultimately culminating in extensive necrosis of the tumor mass (Kanthou and Tozer,
2009). The susceptibility of tumor vessels, but not normal vasculature, to damage by VDAs has been
ascribed to structural differences including their immature pericyte-deficient nature, high intrinsic
permeability, and chaotic organization (Dvorak et al., 1988; Eberhard et al., 2000; Hashizume et al.,
2000).
VDAs are categorized into two types, biological (ligand-directed) and small molecule. Biological VDAs
typically combine an endothelium-targeted molecule with a toxin or pro-coagulant. Small molecule VDAs
can be further subdivided into two classes based on their respective mechanisms of action. One class
contains the flavonoid VDAs, such as ASA404, that exert antivascular effects through induction of local
cytokine production (Gaya and Rustin, 2005). However the largest group of small molecule VDAs are
tubulin binding agents, which target tumor endothelial cells by exploiting their dependence on the tubulin
cytoskeleton to maintain cell shape and vascular integrity. All are structurally related to colchicine and
include combretastatin A-4 (CA4), its prodrug combretastatin A4 phosphate (CA4P), the CA4P analog
Oxi4503 (combretastatin A1 phosphate; CA1P) as well as AVE8062, MN-029, ZD6126, NPI-2358 and
CYT997 (Thorpe, 2004; Shi and Siemann, 2005; Tozer et al., 2005; Burns et al., 2009; McKeage and
Baguley, 2010; Mita et al., 2010).
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Despite causing extensive blood flow shutdown in tumors, only moderate tumor growth suppression has
been achieved when VDAs are used as single agents. One proposed explanation for this mechanism of
resistance is that VDA treatment is highly effective in producing extensive necrosis at the tumor core yet,
even in the most responsive tumors, layers of viable cells remain at the tumor periphery. This ‘viable rim’
accounts for the rapid repopulation of tumors following treatment and the consequent failure to achieve
significant or sustained growth inhibition (Hill et al., 2002a). This characteristic has provided a strong
rationale for investigating VDA activity in combination with other treatment modalities and a number of
agents are presently undergoing clinical evaluation, either as monotherapies or as part of combinatorial
approaches with chemotherapy, radiation and antiangiogenic treatments (Eichholz et al., 2010). However,
the prevalence of off-target adverse vascular events, including cardiotoxicity, ischemia, hypotension,
acute coronary syndromes, and thrombosis (Cooney et al., 2004; Beerepoot et al., 2006; van Heeckeren
et al., 2006; van Heeckeren et al., 2007; Subbiah et al., 2011) highlights an unmet medical need for VDAs
with more favorable safety profiles. Moreover, encouraging early phase results have thus far failed to
translate to improved outcomes for patients and no VDAs are currently approved for the treatment of
human disease.
Herein we provide the first description of the antivascular and antitumor activity of a novel VDA, STA-
9584 (the water soluble prodrug of STA-9122), a potent tubulin binding agent currently in preclinical
development. A series of studies were performed to evaluate its in vitro and in vivo activity across a range
of cancer types, as a single agent and also in direct comparison with members of the combretastatin
family of compounds. Significantly, we found that STA-9584, unlike other VDAs, blocks blood flow by
specifically disrupting tumor microvasculature at both the center and periphery of tumors. In addition,
cardiotoxicity studies revealed a favorable safety profile. Taken together, the results demonstrate a
superior VDA bioactivity and thus STA-9584 represents a potential best-in-class VDA that warrants
further evaluation as a cancer therapeutic.
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Materials and Methods
Cell lines, antibodies and reagents
The B16F10, BT-474, BxPC-3, Daudi, DU145, EMT6, HCT-15, HCT-116, HEL92.1.7, HL-60,
HL60/TX1000, HT-1080, LOX, M14, MDA-MB-231, MDA-MB-435S, MES-SA, MES-SA/DX5, NCI-H1703,
PC-3, P388, Ramos, RERF-LC-AI, SK-OV-3 and U937 cell lines that represent tumors of diverse
hematologic and solid tumor origin were all obtained from the American Type Culture Collection (ATCC,
Manassas, VA) and maintained according to the suppliers’ instructions. Human umbilical vein endothelial
cells (HUVECs) were obtained from Lonza Biologics Inc. (Hopkinton, MA). STA-9122 and its pro-drug
STA-9584 were synthesized by Synta Pharmaceuticals Corp. from 3,4,5-trimethoxybenzaldehyde with
structures determined by H1 NMR and mass spectroscopy, and confirmed by combustion analysis. The
combretastatins AVE8063, CA4, and the pro-drug CA4P were synthesized according to published
procedures (e.g. for CA4: Pettit et al., 1985); the H1 NMR and mass spectra of the synthesized materials
conformed to published data.
In vitro HUVEC assays
To examine microtubule depolymerization, HUVEC cells were exposed to 5 nM STA-9122, CA4 or
AVE8063 for 24 h. Cells were fixed with 3% formaldehyde followed by cold methanol treatment to
preserve both microtubule structures and depolymerized tubulin within the cytoplasm. Cells were
immunostained with an anti-tubulin antibody (DM1A; Sigma-Aldrich, St. Louis, MO) and microtubule
structures visualized using fluorescent microscopy.
The comparative effects of STA-9122 and CA4 treatment on HUVEC migration were determined using a
wound healing assay. HUVECs were grown to confluence on 24-well plates before scraping to generate
linear wounds. The cells were then cultured in the presence of 1 nM STA-9122, 1 or 5 nM CA4, or DMSO
(control). Time lapse imaging was performed using the IncuCyte Live-Cell Imaging System (Essen
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Bioscience, Ann Arbor, MI) where the cells were kept at 37oC and 5% CO2 during the imaging process
and plates were photographed at 24, 48 and 72 h. Cells migrating into wound regions were quantified by
manual counting.
Disruption of vascular structures in vitro was investigated using a capillary tube assay. HUVEC cells were
first seeded in Matrigel-coated 24-well plates in growth factor-rich EGM-2 medium (Lonza Biologics)
overnight to permit formation of capillary tube structures. Cells were exposed to increasing concentrations
of STA-9122 or CA4 (1-100 nM) for 1 h and then cultured for an additional 48 h. Disruption of capillary
tubes was assessed using light microscopy at 10X magnification.
Cell viability assays
HUVECs were treated with 10-fold serial dilutions of STA-9122 or CA4 (1000 - 0.1 nM) or vehicle (DMSO;
≤ 0.5%) for 1 h, and viability assessed at 48 h. Tumor cell lines were seeded overnight and then dosed
with graded concentrations of STA-9122 for 72 h. For primary cell assays, primary cultures of rat PBMCs
and hepatocytes were used. Briefly, whole blood was collected from an adult male SD rat and PBMCs
isolated using Ficoll density centrifugation. Freshly isolated PBMCs were cultured in DMEM plus 10%
FBS and seeded into 96-well plates at 1 x 105 cells/well. For hepatocyte isolation, livers were perfused
and harvested from anesthetized adult male SD rats followed by a collagenase digestion. Hepatocytes
were isolated by low speed centrifugation and suspended in serum-free HepatoStim Medium (HSM;
Becton Dickinson, Bedford MA) plus 2% BSA. This method yielded > 85 % viability as determined by
trypan blue exclusion. Hepatocytes were seeded into collagen-coated 96-well plates at 1.5 x 104 cells/well
and allowed to attach for 2 h. Primary cell cultures were then treated with STA-9122 at concentrations of
2, 10, 25 and 100 μM for 20 h. At the end of the respective incubations, WST-8 reagent (Cell Counting
Kit-8, Dojindo Molecular Technologies, Inc., Rockville, MD) was added to the cells, and absorbance
detected at a wavelength of 450 nm. OD readings were used to generate a standard curve from which the
IC50 values of the compounds were determined using XLFit software (ID Business Solutions, Guildford,
UK).
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In vivo xenograft tumor models
Female BALB/c, immunodeficient BALB/c nude and CB-17/Icr-Prkdcscid/Crl (SCID) mice (Charles River
Laboratories, Wilmington, MA) at 7-12 weeks of age were maintained in a pathogen-free environment and
all in vivo procedures were approved by the Synta Institutional Animal Care and Use Committee. PC-3
and MDA-MB231 tumor cells (5 x 106) were subcutaneously implanted into nude and SCID mice,
respectively, and EMT6 cells (1 x 106) into BALB/c mice. Animals bearing established tumors (100-200
mm3; >500 mm3 for MDA-MB-231 xenografts) were randomized into treatment groups of 8-10 and
intravenously (i.v.) dosed via the tail vein with either vehicle or STA-9584 formulated in 5% dextrose in
water (D5W). Studies were conducted at 4.5 mg/kg STA-9584 1X/week; in the syngeneic EMT6 model,
animals were also treated with CA4P at 100 mg/kg on the same dosing schedule. Tumor growth inhibition
was determined as described previously (Proia et al., 2011). Statistical analyses were performed using a
Kruskal-Wallace one-way ANOVA on ranks followed by the Tukey test.
Histological assessment of EMT6 xenografts
EMT6 tumor-bearing mice (n=10) were administered a single dose of STA-9584 (4.5 mg/kg), CA4P (100
mg/kg) or vehicle for 24 h. Paraffin-embedded sections from the maximum radial regions of each tumor
were subject to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining
using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA) and
counterstained with 2% methyl green. Sections were also stained with an anti-CD31 antibody (BD
Pharmingen, San Diego CA)/hematoxyline. Images were acquired using a Nikon E800 microscope and
Leica DC camera linked to Image-Pro plus software (Media Cybernetics, Inc., Bethesda, MA). Under low
magnification, the dense TUNEL-positive areas correlated with apoptotic/necrotic changes in
corresponding H&E stained sections. Thus the percentage of apoptotic/necrotic area was determined by
the average TUNEL-positive area/average total area at 20X magnification. To determine the proportion of
apoptotic cells in viable tumor areas, image analysis was performed using 5 randomly selected areas per
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slide at 200X magnification. For quantitative evaluation of CD31 positive cells, a total of 6 randomly
selected areas were collected from within the viable zones of 2 independent sections per tumor sample
(200X magnification). Data are expressed as the average CD31 positivity per tumor sample as a
percentage of the control. Statistical significance was determined using one-way ANOVA.
High resolution in vivo imaging of blood flow
Evaluation of tumor perfusion was performed in mice bearing EMT6 xenograft tumors and carried out
under anesthesia (2% isofluorane in oxygen). Tumors were imaged using a Vevo 770 Micro-Ultrasound
platform (VisualSonics, Inc., Toronto, Ontario, Canada), the RMV 706 scanhead, Contrast Analysis
Software, and MicroMarkerTM untargeted ultrasound contrast agent. Using the RMV 706 scanhead,
images were collected at a high spatial resolution (lateral and axial resolution of 100 and 40 μm,
respectively; focal length, 6 mm; transmit power, 50%; mechanical index, 0.14; dynamic range, 52 dB).
Tumor perfusion was measured by using Contrast Mode Software in conjunction with MicroMarkerTM
untargeted ultrasound contrast agent. Lyophilized contrast agent was first reconstituted in 0.7 ml of a
sterile sodium chloride solution (0.9% w/v). A pre-dose baseline was collected by injecting a 50 ul bolus
i.v. injection of contrast agent and capturing the resulting perfusion characteristics. STA-9584 was
immediately dosed at 4.5 mg/kg at t = 0 h, and at 4 h contrast agent imaging was repeated and the
resultant perfusion characteristics captured.
Langendorff assay
Briefly, hearts from male New Zealand white rabbits were used to measure the physiological variables of
PQ interval, QRS complex, RR interval, corrected QT interval (QTc), developed left ventricular pressure
(LVPdev) and coronary blood flow following perfusion with escalating doses of STA-9584 or STA-9122 (10-
8 – 10-5 M) (see Supplementary Materials and Methods for complete details). Mean values were
calculated for each concentration, and mean values (±SEM) were plotted against concentration for all
parameters assessed, both for STA9584/STA-9122-exposed and vehicle-treated hearts.
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Cardiovascular study
Three male Beagle dogs previously instrumented with systemic blood pressure and ECG telemetry
devices (Data Sciences International, St. Paul, MN) were used in the experiment. All experiments were
conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US
National Institutes of Health. The study protocols were reviewed and approved by the Institutional Animal
Care and Use Committee of CorDynamics (Chicago, IL), where the studies were performed. STA-9584
(0.1, 0.25 or 0.5 mg/kg) was administered intravenously into a cephalic or saphenous vein through an in-
dwelling catheter for 1 h. Dogs were returned to their home run at the end of the dosing. Each dosing was
conducted with one-week intervals according to a dose-escalating paradigm. Systemic blood pressure,
heart rate and electrocardiogram (ECG) measurements were recorded continuously for 24 h after dosing
via telemetry. The QT interval was corrected for heart rate using Van De Water’s formula (Van de Water
et al., 1989). A 2 ml blood sample for determination of STA-9584 plasma concentrations was collected
immediately prior to and just before the end of infusion.
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Results
STA-9122 exhibits potent microtubule inhibition activity in endothelial cells and cytotoxic effects
on tumor cells in vitro
STA-9122 (Fig.1A) is a novel small molecule VDA that targets the colchicine–binding (MTc) site of tubulin
(Supplemental Fig. S1). Accordingly, STA-9122 treatment of HUVECs resulted in complete disruption of
microtubule structures, and more effectively than equivalent doses of the combretastatins AVE8063 (the
active component of AVE8062) or CA4 (the active metabolite of CA4P) (Fig. 1B). STA-9122 inhibited
HUVEC migration in vitro at a 5-fold lower dose than CA4 (Fig. 1C). STA-9584 was more potent than CA4
in HUVEC cytotoxicity assays (IC50 values of 30 nM vs. 175 nM, respectively) and at disrupting
endothelial cell capillary tube formation (IC50 4 nM vs 10 nM) (Table 1). In a primary cell cytotoxicity
screening, STA-9122 had little effect on cell viability (Table 1) but was broadly cytotoxic with low
nanomolar potency in a panel of 20 human cancer cell lines (Fig. 1D).
STA-9584 exhibits potent single agent efficacy in tumor xenografts in vivo
Due to limited solubility of STA-9122, for in vivo studies we generated a water soluble phenylalanine ester
prodrug, STA-9584 (Fig. 1A), and evaluated its activity in a series of xenograft models. Initially, BALB/c
nude mice bearing established PC-3 human prostate cancer xenografts were i.v. dosed with STA-9584
on a weekly schedule at its maximum tolerated dose (MTD) of 4.5 mg/kg (Fig. 2A). Significant tumor
regression (40%) was induced by STA-9584 compared with the control group. This regimen was well
tolerated with minimal loss of body weight observed during the course of treatment (Fig. 2B).
We then examined the antitumor effects of STA-9584 in mice bearing large established tumors. To do
this, SCID mice were implanted with MDA-MB-231 human breast carcinoma cells and tumors allowed to
reach > 500 mm3 prior to treatment (Fig. 2C). Weekly dosing of STA-9584 was also highly efficacious in
this model, which resulted in 60% tumor regression. To date, STA-9584 has shown substantial antitumor
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efficacy in each of six xenograft models of human and murine tumors of diverse origins (Table 2). STA-
9584 showed dose-dependent effects using regimens below its MTD and, as is the case with other VDAs,
tumor growth could become re-initiated upon removal of the compound (data not shown).
To directly compare the in vivo efficacies of STA-9584 and CA4P, we next conducted a study in wild type,
immunocompetent BALB/c mice subcutaneously implanted with the highly aggressive, syngeneic
(BALB/c-derived) mouse EMT6 breast carcinoma cell line. In non-tumor bearing BALB/c mice, the MTDs
were determined to be 4.5 mg/kg for STA-9584 and 100 mg/kg for CA4P when dosing 1X/week for 2
weeks (data not shown). Weekly administration of STA-9584 (4.5 mg/kg) was significantly more
efficacious than CA4P dosed at its MTD of 100 mg/kg (T/C values of 11% versus 53%, respectively)
(Fig. 2D). In fact, the 100 mg/kg CA4P dose failed to meet the National Cancer Institute’s %T/C ≤ 42
standard for significant efficacy. Interestingly, even a 200 mg/kg dose (which caused cardiac toxicities)
also failed to meet this standard and was still much less efficacious than 4.5 mg/kg STA-9584 (%T/C =
43%; data not shown).
STA-9584 rapidly and completely blocks tumor blood flow in a syngeneic breast cancer model
The vascular disrupting activity of STA-9584 was examined using the EMT6 model. First, the effects of
STA-9584 and CA4P on tumor blood flow were compared using the standard Evan’s blue dye (EBD)
extravasation assay (as described in the Supplemental Methods) on EMT6 tumors ex vivo, where the
compounds inhibited blood flow by 61% and 43% at 4 h after drug treatment, respectively (Supplemental
Fig. S2). Next we used a high-resolution contrast ultrasound imaging technique to examine tumor blood
flow in vivo. Animals were i.v. injected with a microbubble contrast agent and immediately imaged to
obtain a pre-dose measurement of tumor blood flow. STA-9854 was then dosed at its MTD and 4 h later
contrast agent was again injected and animals imaged for a second time. Figure 3A are representative
images pre- and post-treatment with STA-9584, showing that blood flow was completely blocked in highly
perfused tumor subregions within 4 h of drug administration. As shown in Figure 3B, blood flow was
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monitored by changes in the contrast intensity within the tumors, which was completely abrogated
following STA-9584 treatment. Similar results were observed in 3 of 3 animals.
STA-9584 antitumor effects are not confined to the central region of tumors
In light of this potent antivascular activity, and to further account for its superior efficacy in vivo, EMT6
tumor-bearing animals were treated with single doses of STA-9584 (4.5 mg/kg) or CA4P (100 mg/kg) for
24 h, at which point tumors were isolated for histological analysis. Interestingly, a single dose of STA-
9584 completely arrested tumor growth over this period, as measured by changes in tumor volumes, in
contrast to only a slight delay with CA4P (Supplemental Fig. S3). Tumor sections were analyzed using
methyl green staining to detect viable tissue and TUNEL staining to identify regions of apoptosis/necrosis
(Fig. 4A). As expected, tumors from CA4P-treated animals showed central areas of necrosis; however in
every tumor large regions of viable cells persisted, particularly at the periphery (representing the ‘viable
rim’). In contrast, STA-9584-exposed xenografts displayed far more extensive cellular destruction
throughout the entire tumor, including areas of fibrosis and dramatically reduced viable rim or surviving
tumor zones. In fact, many tumor sections from STA-9584-treated animals showed no detectable viable
rim tissue. Overall, the core necrotic/apoptotic region as a percentage of total tumor area increased by
73% following treatment with STA-9584 relative to CA4P (Fig. 4B).
This finding suggested that STA-9584 blocked blood flow by specifically disrupting tumor
microvasculature at both the center and periphery of tumors. To confirm this, anti-CD31
immunohistochemistry was performed and endothelial cells counted in the remaining periphery/viable rim
of tumors (excluding the central necrotic area). Tumors from STA-9584-treated animals, but not CA4P,
contained large numbers of disrupted microvessels with decreased, patchy CD31 expression, loss of
integrity and thrombosis (Fig. 4C). In total, a 77% decrease in CD31+ endothelial cells in the viable rim
was observed following treatment with STA-9584 relative to CA4P (Fig. 4D). Concomitant changes in
levels of apoptosis were seen when TUNEL-positive cells were counted in the same region (Fig. 4C).
There was little change in the number of apoptotic cells in the viable rim following treatment with CA4P
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relative to vehicle alone. However, a 7-fold increase in apoptotic cells was observed following treatment
with STA-9584 relative to CA4P (Fig. 4E). Taken together, these data suggest that the increased in vivo
efficacy of STA-9584 relative to CA4P (Fig. 2D) can be attributed, at least in part, to increased
microvasculature destruction and consequent apoptosis in peripheral regions of tumors.
STA-9584 has a favorable cardiovascular safety profile
Cardiotoxicity has emerged as an important dose-limiting toxicity (DLT) for a number of VDAs that have
entered clinical development, including CA4P (Subbiah et al., 2011). For this reason, cardiovascular
effects of escalating doses of STA-9584 and STA-9122 on electrophysiology (RR, PQ, QRS, QTc) and
mechanical properties (left ventricular developed pressure [LVPdev], coronary blood flow) were evaluated
in isolated rabbit hearts using the Langendorff assay. Importantly, STA-9122 had no significant effect on
any parameter over the concentration range tested (10-8 - 10-5 M) (Supplemental Fig. S4). For the STA-
9584 prodrug, no significant effects on electrophysiological parameters were seen at concentrations ≤ 10-
6 M (Fig. 5A). At the highest 10-5 M dose, PQ interval, QRS duration and QTc interval increased
compared to baseline, and slight decreases in LVPdev and blood flow were observed (Fig. 5A). However, it
is important to note that this concentration is 50-fold higher than the efficacious dose Cmax value (2 x 10-7
M). Expected physiological changes on these same parameters were observed using increasing
concentrations of quinidine (10-8 – 10-4 M) as a positive control (data not shown).
In a rising-dose tolerance study of STA-9584 in Beagle dogs, the MTD was determined to be 0.5 mg/kg
with the major DLTs involving transient clinical signs typical of many tubulin-binding cytotoxic agents
(emesis, diarrhea, bloody stool) and select hematological changes (data not shown). Importantly, no
treatment-related cardiac effects were observed. To extend this finding, we performed a cardiovascular
function study in dogs surgically instrumented with blood pressure/electrocardiogram (BP/ECG) telemetry
devices. Dogs (n=3) were given rising doses of 0.1, 0.25 and 0.5 mg/kg of STA-9584 (1 h infusion) with a
week between doses and BP/ECG measurements monitored for 24 h post-dosing. As shown in Fig. 5B,
no ECG (QTc interval) effects were observed at any dose or any heart rate or pressure changes seen
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with 0.1 and 0.25 mg/kg dosing. Cardiac effects of STA-9584 administration at the MTD (0.5 mg/kg) were
restricted to transient elevations in mean arterial pressure (driven primarily by increases in diastolic blood
pressure) and heart rate between 1 and 4 h (Fig. 5B). No notable changes in electrocardiographic
intervals (PR, QRS, QTc) or arrhythmogenesis related to STA-9584 were seen at any doses (data not
shown). Overall, these data show that cardiovascular toxicity is not predicted to be dose-limiting for STA-
9584.
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Discussion
Functional disruption of the tumor vasculature is an established approach for the development of novel
antineoplastic therapies. VDAs differ in mechanism to antiangiogenic agents, which prevent
neovascularization, by compromising the preexisting network to rapidly shutdown blood flow, thus
preventing the supply of oxygen and nutrients. Here we report the preclinical characterization of STA-
9584, a promising new investigational VDA of the tubulin binding class. Consistent with other microtubulin
destabilizing agents, STA-9584 targeted endothelial cells of established tumor blood vessels, rapidly
blocking blood flow to elicit tumor necrosis. Unlike most other VDAs, however, STA-9584 displayed
potent single agent antitumor activity in vivo; including tumor regressions in xenograft models of prostate
and breast cancer, even in large established tumors. Importantly, we found that the superior bioactivity
exhibited by this compound was related to its capacity to specifically disrupt tumor microvasculature and
induce cell death at both the center and periphery of tumors (Figure 6).
CA4P is the prototypical member of the combretastatin class and was the first VDA to enter clinical trials
(Dowlati et al., 2002). Here we report that STA-9584 was substantially more efficacious than CA4P using
a highly aggressive syngeneic breast cancer xenograft model. Moreover, morphological examination
revealed significantly increased cellular destruction throughout tumors exposed to STA-9584, leading to a
dramatic reduction in surviving tumor zones. This superior therapeutic index of STA-9584 suggests an
activity profile similar to that of the second generation CA4P analog Oxi4503. Compared to CA4P,
Oxi4503 has been shown to exert more potent antivascular activity and tumor growth delays; treatment
also results in the persistence of a relatively smaller viable rim (Hill et al., 2002b; Holwell et al., 2002; Hua
et al., 2003). The enhanced efficacy of Oxi4503 has been ascribed to the in vivo generation of reactive
quinone species that may be cytotoxic to tumor cells through free radical formation and elevation of
oxidative stress (Folkes et al., 2007; Rice et al., 2011). Here we showed that the active STA-9122 moiety,
which itself is stable and does not generate reactive quinones, was also potently cytotoxic to a panel of
tumor cell lines of diverse hematologic and solid tumor origins. Thus it is reasonable to suggest that the
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direct induction of cancer cell death, in addition to vascular disruption, contributes to the robust single-
agent activity of STA-9584.
To date, the clinical experience with VDAs has failed to deliver on the therapeutic potential of this class of
antivascular agents. Tumor regrowth from the viable rim underscores one of the major shortcomings of
VDA monotherapy, contributing to both limited efficacy and treatment resistance. Accordingly,
investigation of VDAs as a complementary approach to standard of care chemotherapeutics or other
targeted agents is a primary focus of ongoing clinical evaluations (Eichholz et al., 2010; McKeage and
Baguley, 2010). One of the most advanced agents, ASA404, showed potential clinical benefit in early
Phase I and II trials in non-small cell lung cancer (NSCLC) in combination with paclitaxel/carboplatin
(McKeage et al., 2008; McKeage et al., 2009; Hida et al., 2011). These promising findings then prompted
examination of the combination as part of first- or second-line therapy for advanced NSCLC in two large-
scale randomized Phase III clinical trials; however both were halted when no overall survival benefit could
be demonstrated (Lara et al., 2011; Lorusso et al., 2011). Overall, intense efforts at developing VDAs with
increased efficacy have been underway in recent years (Siemann, 2011) and, in light of the observations
we are presenting here, STA-9584 exhibits a preclinical activity profile of significant therapeutic potential.
Another factor contributing to the lack of clinical progression is the prevalence of toxicity profiles marked
by off-target VDA effects on the cardiovascular system. The most common effects include hypertension,
ischemia, arrhythmias, atrial fibrillation and myocardial infarction (for review see (Subbiah et al., 2011)).
While some signs/symptoms (e.g. low grade hypertension) may be transient and manageable for many
VDAs, in some cases these toxicities can be significantly dose-limiting and have resulted in termination of
the development of the drug, as seen for ZD6216 (LoRusso et al., 2008). With respect to the
combretastatins, adverse cardiovascular events have been reported for CA4P when administered either
alone or in combination with other cytotoxic agents (Dowlati et al., 2002; Rustin et al., 2003; Rustin et al.,
2010). As an initial step in evaluating the suitability of STA-9584 for development as a clinical candidate,
a comprehensive assessment of the cardiovascular safety profile of the compound was performed. The
evaluation revealed no effects of STA-9122 on any electrophysiological or mechanical parameter in the
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Langendorff assay, and only minor alterations in QRS duration, QTc interval, LVPdev and blood flow were
observed for STA-9584 at the highest concentration examined. Importantly, this concentration was 50-
fold higher than the efficacious dose Cmax value, thus unlikely to be approached in a therapeutic setting. In
addition, no treatment-related cardiac effects were observed in beagle dogs, either as part of a rising-
dose tolerance study or telemetered cardiovascular function study. Taken together, these findings show
that cardiac toxicity is not likely to be dose-limiting for STA-9584. This represents an important
discriminating feature of the compound, since both hypertension (as a common adverse drug reaction)
and atrial fibrillation (as a dose-limiting toxicity) have recently been reported in a Phase I evaluation of
Oxi4503 (Patterson et al., 2012).
In summary, we have developed and characterized a novel small molecule VDA with distinct bioactivity
and potent antitumor efficacy in preclinical models of human cancer. Further, the favorable cardiovascular
profile exhibited by STA-9584 predicts for a superior therapeutic index compared to other classes of
tubulin binding agents. Taken together, the data presented here identify STA-9584 as a promising new
therapeutic VDA candidate.
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Acknowledgments
We thank Richard Bates, Zhenjian Du, Shoujun Chen, Weiwen Ying, Keizo Koya, Elena Kostik, Lijun Su,
Yumiko Wada, Shuzen Qin and Wenping Song for their contributions and dedication to the project. We
also thank Peter Senese at CorDynamics and Ben Deeley, Tonya Coulthard and Fred Roberts at
VisualSonics for their generous assistance conducting ultrasound imaging.
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Authorship Contributions
Participated in research design: Foley, Zhou, Borella, Jiang, M. Zhang, Sang, Korbut, Barsoum, and
Sonderfan.
Conducted experiments: Zhou, Wu, M. Zhang, Jiang, Sang, Korbut, Ye, and X. Zhang.
Contributed new reagents or analytical tools: M. Zhang, Sang, and Li.
Performed data analysis: Foley, Zhou, Borella, Sang, and Korbut.
Wrote or contributed to the writing of the manuscript: Foley, Sonderfan.
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Footnotes
Kevin P. Foley and Dan Zhou contributed equally to this work.
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Figure Legends
Figure 1. In vitro antivascular and cytotoxic activity of STA-9122. A, Chemical structure of STA-9584.
The prodrug is hydrolyzed at the indicated site to form the active metabolite STA-9122. B, HUVEC cells
were treated at 5 nM for 24 h, and then examined using anti-tubulin immunofluorescence. C, HUVEC
migration was evaluated in a wound healing assay. The numbers of migrating cells were measured at 24,
48 and 72 h in the presence of STA-9122 (1 nM), CA4P (1 and 5 nM) or control (DMSO). D, Tumor cell
lines of breast (BT-474), colon (HCT15, HCT-116), leukemic (HEL92.1.7, HL-60, HL-60/TX1000,P388),
lung (NCI-H1703, RERF-LC-AI), lymphoma (Ramos, U937), melanoma (B16F10, LOX, MDA-MB-435S),
ovarian (SK-OV-3), pancreatic (BxPC-3), prostate (DU-145, PC-3) and sarcoma (HT-1080, MES-SA,
MES-SA/DX5) origin were dosed with graded concentrations of STA-9122 for 72 h and IC50 viability
values determined for each.
Figure 2. STA-9584 exhibits potent antitumor efficacy in human xenograft and mouse syngeneic
models of solid malignancies. %T/C values are indicated to the right of each growth curve; error bars
are the SEM (n = 8 mice/group). *, p < 0.05 A, BALB/c nude mice bearing established PC-3 prostate
cancer xenografts (~150 mm3) were i.v. dosed with STA-9584 at 4.5 mg/kg once weekly as indicated
(arrowheads). B, Body weights were measured for PC-3 xenograft-bearing animals 5 times per week.
Mean values are plotted against vehicle controls. C, SCID mice bearing large (> 500 mm3) MDA-MB-231
breast cancer xenografts were dosed weekly with 4.5 mg/kg STA-9584 (arrowheads). D, BALB/c mice
were implanted with the highly aggressive, syngeneic (BALB/c-derived) mouse breast carcinoma cell line,
EMT6. Mice bearing EMT6 tumors were i.v. dosed with STA-9584 (4.5 mg/kg) or CA4P (100 mg/kg)
weekly (arrowheads).
Figure 3. Microultrasound imaging of blood flow in perfused subregions of tumors in the EMT6
model. EMT6 cells were subcutaneously implanted into BALB/c mice and xenograft tumors allowed to
grow for 7 days. Animals were i.v. injected with contrast agent and immediately imaged to obtain a pre-
dose measurement of tumor blood flow. STA-9584 was dosed at 4.5 mg/kg at t = 0 h, and at 4 h contrast
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agent was again injected and the animals imaged a second time. A, Representative tumor imaged pre-
and post-treatment with STA-9584. Blue ovals indicate the outline of the tumor and blood flow is depicted
by microbubbles (green signal), proportional to perfusion within the region of interest B, Changes in
contrast intensity within one highly perfused subregion of each tumor were imaged at 4 h immediately
after injection of the contrast agent pre- and post-treatment with STA-9584 (representative tumor shown).
Figure 4. STA-9584 antivascular and antitumor effects are not confined to the central regions of
tumors. A, Mice bearing EMT6 tumors were treated with a single dose of CA4P (100 mg/kg), STA-9584
(4.5 mg/kg) or vehicle for 24 h. Tumors were excised and paraffin sections prepared at the maximum
tumor radius. Immunohistochemistry was performed using methyl green and TUNEL staining to detect
regions of viable tissue and necrosis/apoptosis, respectively. Magnification, 20X. B, The extent of regional
apoptosis/necrosis in each treatment was calculated and expressed as a percentage of total tumor area.
*, p < 0.05 vs. CA4P. C, Anti-CD31 immunohistochemical staining (upper panel) and TUNEL-positive
apoptotic cells (lower panel) in the viable rim/periphery of tumors. Magnification, 200X. D, Quantitation of
anti-CD31 immunoreactivity in the viable rim/periphery of tumors following treatment. Values presented
from an average of 2-6 images/tumor (Note, many sections of STA-9584-treated tumors contained no
remaining viable rim); *, p < 0.05 vs. CA4P. E, TUNEL-positive cells were counted in viable region of
tumors (excluding the central necrotic area) for each treatment and values expressed as a percentage of
total area. *, p < 0.05 vs. CA4P.
Figure 5. Cardiovascular safety profile of STA-9584. A, Effects of escalating concentrations of STA-
9584 and vehicle on RR, PQ, QRS and QTc(F) intervals, left ventricular developed pressure (LVPdev) and
coronary blood flow in male rabbit hearts. STA-9584 values presented as the mean (± SEM); n=4. Vehicle
values presented from one animal. B, Cardiovascular assessment of STA-9584 in conscious telemetered
male dogs. Animals previously instrumented with systemic blood pressure and ECG telemetry devices
were i.v. administered rising doses of STA-9584 (0.1, 0.25, 0.5 mg/kg; 1 h infusion) on a weekly
schedule. Systemic blood pressure and ECG measurements were recorded for 24 h. Corrected QT
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interval (Van de Water’s correction; QTcVW), heart rate and diastolic arterial pressure data are reported.
Values are presented as means (n=3).
Figure 6. Proposed mechanism for STA-9584 blocking blood flow by specifically disrupting tumor
microvasculature at both the center and periphery of tumors. The persistence of a viable rim
following VDA treatment permits tumor regrowth and limits the single agent efficacy of current VDAs. The
unique capacity of STA-9584 to cause extensive vascular disruption throughout the entire tumor results in
superior bioactivity.
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Table 1. Comparative biological activity of STA-9122 and CA4 (IC50 values, nM).
Compound
Endothelial cell capillary tube
disruption
Endothelial cell
cytotoxicity
Primary cell
cytotoxicity panel
CA4 10 175 ND
STA-9122 4 30 47500 - 84100
ND, not determined
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Table 2. Antitumor efficacy of STA-9584 in vivo in a panel of xenograft models.
Cell Line
Species
Tumor Phenotype
%T/C
EMT6 Mouse Breast carcinoma 11
Daudi Human B-cell lymphoma 39
M14 Human Melanoma 7
RERF-LC-AI Human NSCLC 5
PC-3 Human Prostate carcinoma -40*
MDA-MB-231 Human Breast carcinoma -60*
Mice bearing established xenografts were i.v. dosed with STA-9584 at 4.5 mg/kg once weekly in all cases. *, tumor regression.
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T Journals on N
ovember 17, 2020
jpet.aspetjournals.orgD
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This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 26, 2012 as DOI: 10.1124/jpet.112.196873
at ASPE
T Journals on N
ovember 17, 2020
jpet.aspetjournals.orgD
ownloaded from
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 26, 2012 as DOI: 10.1124/jpet.112.196873
at ASPE
T Journals on N
ovember 17, 2020
jpet.aspetjournals.orgD
ownloaded from