JPET #196873

JPET #196873

1

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.

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 7, 2021

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET #196873

2

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

3

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

4

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).


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

5

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

6

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

7

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).


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

8

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

9

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

10

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

11

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

12

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

13

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

14

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

15

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

16

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

17

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

18

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

19

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

20

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

21

References

Beerepoot LV, Radema SA, Witteveen EO, Thomas T, Wheeler C, Kempin S and Voest EE (2006) Phase

I clinical evaluation of weekly administration of the novel vascular-targeting agent, ZD6126, in

patients with solid tumors. J Clin Oncol 24:1491-1498.

Burns CJ, Fantino E, Phillips ID, Su S, Harte MF, Bukczynska PE, Frazzetto M, Joffe M, Kruszelnicki I,

Wang B, Wang Y, Wilson N, Dilley RJ, Wan SS, Charman SA, Shackleford DM, Fida R,

Malcontenti-Wilson C and Wilks AF (2009) CYT997: a novel orally active tubulin polymerization

inhibitor with potent cytotoxic and vascular disrupting activity in vitro and in vivo. Mol Cancer Ther

8:3036-3045.

Cooney MM, Radivoyevitch T, Dowlati A, Overmoyer B, Levitan N, Robertson K, Levine SL, DeCaro K,

Buchter C, Taylor A, Stambler BS and Remick SC (2004) Cardiovascular safety profile of

combretastatin a4 phosphate in a single-dose phase I study in patients with advanced cancer.

Clin Cancer Res 10:96-100.

Dowlati A, Robertson K, Cooney M, Petros WP, Stratford M, Jesberger J, Rafie N, Overmoyer B, Makkar

V, Stambler B, Taylor A, Waas J, Lewin JS, McCrae KR and Remick SC (2002) A phase I

pharmacokinetic and translational study of the novel vascular targeting agent combretastatin a-4

phosphate on a single-dose intravenous schedule in patients with advanced cancer. Cancer Res

62:3408-3416.

Dvorak HF, Nagy JA, Dvorak JT and Dvorak AM (1988) Identification and characterization of the blood

vessels of solid tumors that are leaky to circulating macromolecules. Am J Pathol 133:95-109.

Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH and Augustin HG (2000) Heterogeneity of

angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor

therapies. Cancer Res 60:1388-1393.

Eichholz A, Merchant S and Gaya AM (2010) Anti-angiogenesis therapies: their potential in cancer

management. Onco Targets Ther 3:69-82.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

22

Folkes LK, Christlieb M, Madej E, Stratford MR and Wardman P (2007) Oxidative metabolism of

combretastatin A-1 produces quinone intermediates with the potential to bind to nucleophiles and

to enhance oxidative stress via free radicals. Chem Res Toxicol 20:1885-1894.

Gaya AM and Rustin GJ (2005) Vascular disrupting agents: a new class of drug in cancer therapy. Clin

Oncol (R Coll Radiol) 17:277-290.

Grothey A and Galanis E (2009) Targeting angiogenesis: progress with anti-VEGF treatment with large

molecules. Nat Rev Clin Oncol 6:507-518.

Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, Jain RK and McDonald DM

(2000) Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol

156:1363-1380.

Hida T, Tamiya M, Nishio M, Yamamoto N, Hirashima T, Horai T, Tanii H, Shi MM, Kobayashi K and

Horio Y (2011) Phase I study of intravenous ASA404 (vadimezan) administered in combination

with paclitaxel and carboplatin in Japanese patients with non-small cell lung cancer. Cancer Sci

102:845-851.

Hill SA, Chaplin DJ, Lewis G and Tozer GM (2002a) Schedule dependence of combretastatin A4

phosphate in transplanted and spontaneous tumour models. Int J Cancer 102:70-74.

Hill SA, Toze GM, Pettit GR and Chaplin DJ (2002b) Preclinical evaluation of the antitumour activity of the

novel vascular targeting agent Oxi 4503. Anticancer Res 22:1453-1458.

Holwell SE, Cooper PA, Grosios K, Lippert JW, 3rd, Pettit GR, Shnyder SD and Bibby MC (2002)

Combretastatin A-1 phosphate a novel tubulin-binding agent with in vivo anti vascular effects in

experimental tumours. Anticancer Res 22:707-711.

Hua J, Sheng Y, Pinney KG, Garner CM, Kane RR, Prezioso JA, Pettit GR, Chaplin DJ and Edvardsen K

(2003) Oxi4503, a novel vascular targeting agent: effects on blood flow and antitumor activity in

comparison to combretastatin A-4 phosphate. Anticancer Res 23:1433-1440.

Kanthou C and Tozer GM (2009) Microtubule depolymerizing vascular disrupting agents: novel

therapeutic agents for oncology and other pathologies. Int J Exp Pathol 90:284-294.

Lara PN, Jr., Douillard JY, Nakagawa K, von Pawel J, McKeage MJ, Albert I, Losonczy G, Reck M, Heo

DS, Fan X, Fandi A and Scagliotti G (2011) Randomized phase III placebo-controlled trial of


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

23

carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (ASA404) in

advanced non-small-cell lung cancer. J Clin Oncol 29:2965-2971.

Lorusso PM, Boerner SA and Hunsberger S (2011) Clinical development of vascular disrupting agents:

what lessons can we learn from ASA404? J Clin Oncol 29:2952-2955.

LoRusso PM, Gadgeel SM, Wozniak A, Barge AJ, Jones HK, DelProposto ZS, DeLuca PA, Evelhoch JL,

Boerner SA and Wheeler C (2008) Phase I clinical evaluation of ZD6126, a novel vascular-

targeting agent, in patients with solid tumors. Invest New Drugs 26:159-167.

McKeage MJ and Baguley BC (2010) Disrupting established tumor blood vessels: an emerging

therapeutic strategy for cancer. Cancer 116:1859-1871.

McKeage MJ, Reck M, Jameson MB, Rosenthal MA, Gibbs D, Mainwaring PN, Freitag L, Sullivan R and

Von Pawel J (2009) Phase II study of ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic

acid/DMXAA) 1800mg/m(2) combined with carboplatin and paclitaxel in previously untreated

advanced non-small cell lung cancer. Lung Cancer 65:192-197.

McKeage MJ, Von Pawel J, Reck M, Jameson MB, Rosenthal MA, Sullivan R, Gibbs D, Mainwaring PN,

Serke M, Lafitte JJ, Chouaid C, Freitag L and Quoix E (2008) Randomised phase II study of

ASA404 combined with carboplatin and paclitaxel in previously untreated advanced non-small

cell lung cancer. Br J Cancer 99:2006-2012.

Mita MM, Spear MA, Yee LK, Mita AC, Heath EI, Papadopoulos KP, Federico KC, Reich SD, Romero O,

Malburg L, Pilat M, Lloyd GK, Neuteboom ST, Cropp G, Ashton E and LoRusso PM (2010) Phase

1 first-in-human trial of the vascular disrupting agent plinabulin(NPI-2358) in patients with solid

tumors or lymphomas. Clin Cancer Res 16:5892-5899.

Patterson DM, Zweifel M, Middleton MR, Price P, Folkes LK, Stratford MR, Ross P, Halford S, Peters J,

Balkissoon J, Chaplin D, Padhani AR and Rustin GJ (2012) Phase I Clinical and Pharmacokinetic

Evaluation of the Vascular Disrupting Agent OXi4503 in Patients with Advanced Solid Tumors.

Clin Cancer Res. 18:1415-1425.

Pettit GR, Singh SB and Cragg GM (1985) Synthesis of natural (-)-combretastatin. J Org Chem 50:3404-

3406.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

24

Proia DA, Foley KP, Korbut T, Sang J, Smith D, Bates RC, Liu Y, Rosenberg AF, Zhou D, Koya K,

Barsoum J and Blackman RK (2011) Multifaceted intervention by the Hsp90 inhibitor ganetespib

(STA-9090) in cancer cells with activated JAK/STAT signaling. PLoS One 6:e18552.

Rice L, Pampo C, Lepler S, Rojiani AM and Siemann DW (2011) Support of a free radical mechanism for

enhanced antitumor efficacy of the microtubule disruptor OXi4503. Microvasc Res 81:44-51.

Rustin GJ, Galbraith SM, Anderson H, Stratford M, Folkes LK, Sena L, Gumbrell L and Price PM (2003)

Phase I clinical trial of weekly combretastatin A4 phosphate: clinical and pharmacokinetic results.

J Clin Oncol 21:2815-2822.

Rustin GJ, Shreeves G, Nathan PD, Gaya A, Ganesan TS, Wang D, Boxall J, Poupard L, Chaplin DJ,

Stratford MR, Balkissoon J and Zweifel M (2010) A Phase Ib trial of CA4P (combretastatin A-4

phosphate), carboplatin, and paclitaxel in patients with advanced cancer. Br J Cancer 102:1355-

1360.

Shi W and Siemann DW (2005) Preclinical studies of the novel vascular disrupting agent MN-029.

Anticancer Res 25:3899-3904.

Siemann DW (2011) The unique characteristics of tumor vasculature and preclinical evidence for its

selective disruption by Tumor-Vascular Disrupting Agents. Cancer Treat Rev 37:63-74.

Subbiah IM, Lenihan DJ and Tsimberidou AM (2011) Cardiovascular toxicity profiles of vascular-

disrupting agents. Oncologist 16:1120-1130.

Thorpe PE (2004) Vascular targeting agents as cancer therapeutics. Clin Cancer Res 10:415-427.

Tozer GM, Kanthou C and Baguley BC (2005) Disrupting tumour blood vessels. Nat Rev Cancer 5:423-

435.

Van de Water A, Verheyen J, Xhonneux R and Reneman RS (1989) An improved method to correct the

QT interval of the electrocardiogram for changes in heart rate. J Pharmacol Methods 22:207-217.

van Heeckeren WJ, Bhakta S, Ortiz J, Duerk J, Cooney MM, Dowlati A, McCrae K and Remick SC (2006)

Promise of new vascular-disrupting agents balanced with cardiac toxicity: is it time for oncologists

to get to know their cardiologists? J Clin Oncol 24:1485-1488.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

25

van Heeckeren WJ, Sanborn SL, Narayan A, Cooney MM, McCrae KR, Schmaier AH and Remick SC

(2007) Complications from vascular disrupting agents and angiogenesis inhibitors: aberrant

control of hemostasis and thrombosis. Curr Opin Hematol 14:468-480.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

26

Footnotes

Kevin P. Foley and Dan Zhou contributed equally to this work.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

27

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

28

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

29

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

30

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


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


JPET #196873

31

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.


at ASPE

T Journals on N

ovember 7, 2021


ownloaded from



at ASPE

T Journals on N

ovember 7, 2021


ownloaded from



at ASPE

T Journals on N

ovember 7, 2021


ownloaded from



at ASPE

T Journals on N

ovember 7, 2021


ownloaded from



at ASPE

T Journals on N

ovember 7, 2021


ownloaded from



at ASPE

T Journals on N

ovember 7, 2021


ownloaded from



at ASPE

T Journals on N

ovember 7, 2021


ownloaded from


Documents

JPET #196873