Hepatic Hypoxia-Activated Intraarterial Therapy: Effect of Selective Targeting of Hypoxia in
a Rabbit Liver Tumor Model
Rafael Duran¹, Sahar Mirpour¹, Vasily Pekurovsky¹, Shanmugasundaram Ganapathy-Kanniappan¹, Cory F. Brayton¹, Toby C. Cornish¹, Boris Gorodetski¹,
Julius Chapiro¹, Rüdiger Schernthaner¹, Constantine Frangakis², MingDe Lin³, Jessica D. Sun⁴, Charles P. Hart⁴, Jean-François H. Geschwind¹.
• A hypoxia-activated prodrug (HAP)
• Shows efficacy in both preclinical and clinical settings
• Is currently tested in multiple clinical trials including two Phase III
trials for the treatment of pancreatic cancer (NCT01746979) and
sarcoma (NCT01440088 )
Mechanism of Action
Combination Therapy
Anticancer effect of Evo by hypoxia targeting
Evo
Control
•
A
- Quantitative IHC: Color deconvolution image analysis algorithm
(Color Deconvolution v9 and ImageScope software; Aperio®)
cTACE
cTACE + Evo
• Morphometrics
• Loco-regional delivery of high-doses of chemotherapy that have
the potential to reach distal tumor regions which is hypoxic
• Embolic effect increases the hypoxic microenvironment of solid
tumors
• Hypoxia caused by embolization has been shown to trigger
•
•
detrimental effects with the activation of hypoxia-inducible factor 1
alpha (HIF-1α)-dependent pathways
Hypoxia resulting from embolization attributes to the emergence
of chemoresistance after TACE
Induced hypoxia provides a rationale to combine TACE with a
hypoxia-activated prodrug (e.g. evofosfamide) as a strategy
- Hypoxic Fraction (HF): % of hypoxic area in the viable tumor
- Hypoxic Compartment (HC): % of hypoxic area in the whole tumor
- Necrotic Fraction (NF): % of necrotic area in the whole tumor
Hypoxic Fraction
Hypoxic
Compartment
Necrotic Fraction
RESULTS
• Animals tolerated Evo well with no apparent drug-induced toxicity
determined by daily clinical assessment or body weight change.
• Alanine aminotransferase, a liver enzyme, was significantly higher
in cTACE + Evo vs. cTACE at Day 7, but recovered by Day 14.
• cTACE + Evo and cTACE exhibited similar hepatocellular damage,
assessed by semiquantitative scoring system.
Anticancer effect of Evo by quantitative imaging
Quantitative analysis of tumor hypoxia microenvironment changes by
pimonidazole staining. 48 hrs after the treatment, hypoxic fraction
(HF) and hypoxic compartment (HC) were significantly reduced as
compared to control, Evo and cTACE groups. A significant negative
correlation was found across the treatment groups between the HF
and the magnitude of the necrotic fraction (NF), Pearson’s r = -0.753;
P = 0.004.
Evo induces DNA damage, promotes apoptosis
and decreases cellular proliferation
To evaluate the feasibility, safety and efficacy of hepatic hypoxiaactivated intraarterial therapy in a rabbit VX2 liver tumor model
γH2AX
Annexin V
Caspase-3
TUNEL
Ki67
Control
A
METHODS
LDH
VEGF
GAPDH
cTACE + Evo
γH2AX
Annexin V
Caspase-3
48 hrs after treatment
14 days after treatment
whole tumor
Pharmacodynamic biomarker changes
• Animals were sacrificed 48 hrs after saline, Evo, cTACE or
cTACE + Evo treatment (n = 3 per group)
• Histology, Immunohistochemistry (IHC) and Immunofluorescence
Ki67
Ki67-positive nuclei [%]
TUNEL
Anticancer effect of Evo by quantitative pathology
Expression of biomarkers of DNA damage, apoptosis and cellular
proliferation and semi-quantitative analysis apoptosis using TUNEL
performed 48 hrs post intraarterial interventional treatment. ρ P < 0.05
vs. control group. t P < 0.05 vs. Evo group. ψ P < 0.05 vs. cTACE
group.
DISCLOSURES
*, P < 0.05 vs. Control group; # P < 0.05 vs. Evo group;
δ P < 0.05 between TACE groups
(Evo) as a single agent and more importantly in combination with
conventional TACE (cTACE) achieved strong anticancer effects in
the rabbit tumor liver model with minimal toxicity.
Significant correlation between hypoxia-targeting and antitumor
activity further supported the anticancer potential of the
locoregional delivery of the HAP technology.
CONCLUSIONS
• Daily clinical assessment and body weight change
• Liver enzyme by blood chemistry assay
• Pathologically hepatocellular damage
Anticancer efficacy
LDH
Expression of biomarkers of HIF-1α signaling and tumor metabolism
performed 48 hrs post intraarterial interventional treatment. ρ P < 0.05
vs. control group. t P < 0.05 vs. Evo group.
•
Safety profile
www.PosterPresentations.com
MCT-4
• Hepatic hypoxia-activated intraarterial therapy with evofosfamide
cTACE
•
•
•
•
RESEARCH POSTER PRESENTATION DESIGN © 2012
HIF-1α
MCT-4
Evo
VX2 liver tumor-bearing rabbits were assigned to 4 intraarterial
therapy (IAT) groups (n=7):
Saline (control group; 5 mL of 0.9% NaCl)
Evo group (7.7 mg/kg)
cTACE group: Doxorubicin (3-4 mg)-Lipiodol emulsion followed by
bland embolization with 100-300μm beads
cTACE + Evo: A combination of Doxorubicin (3-4 mg)-Lipiodol
emulsion and Evo followed by bland embolization with 100300μm beads
Necrosis: H & E
Hypoxia: pimonidazole
DNA damage: γH2AX
Apoptosis: Annexin V, cleaved caspase-3 and TUNEL
GAPDH
SUMMARY
Animal model and treatment
-
VEGF
Safety Profile
OBJECTIVES
• Quantitative volumetric image analysis (MDCT at baseline and 7
and 14 days after therapy).
• Pathologic tumor necrosis: slide‐by‐slide histosegmentation of the
HIF-1α
cTACE
Evofosfamide
Transarterial chemoembolization (TACE)
Increase in tumor hypoxia by the embolization
validates TACE as an ideal setting for HAPs
Control
Evofosfamide (TH-302, Evo)
- Proliferation: Ki67
- HIF1α signaling: HIF1α and VEGF
- Metabolic markers: GAPDH, MCT4 and LDH
Image analysis
Evo
BACKGROUND
cTACE + Evo
Abstract # 5271
1. The Johns Hopkins Hospital, Baltimore, MD; 2. The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 3. Philips Research North America,
Ultrasound Imaging and Interventions (UII), Briarcliff Manor, NY; 4. Threshold Pharmaceuticals, South San Francisco, CA
This research was funded by Threshold Pharmaceuticals, Inc and Merck KGaA. C Hart and J Sun
are employees of Threshold Pharmaceuticals, Inc. and hold either stock or stock options in the
company. Evofosfamide is currently in clinical trials and not approved for any indication.
• TACE in combination with Evo achieved stronger anticancer
efficacy with limited added toxicity compared to TACE alone.
• The demonstrated correlation between magnitude of hypoxia and
•
Evo efficacy established the in vivo proof-of-concept of selective
hypoxia-activated intraarterial locoregional therapy for liver
cancer.
The results of this study pave the way towards clinical trials in
patients with liver cancer.
REFERENCES
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Sun JD, Liu Q, Wang J, Ahluwalia D, Ferraro D, Wang Y, et al. Selective tumor hypoxia targeting by
hypoxia-activated prodrug TH-302 inhibits tumor growth in preclinical models of cancer. Clin Cancer
Res. 2012;18:758-70.
Saggar JK, Tannock IF. Activity of the hypoxia-activated pro-drug TH-302 in hypoxic and perivascular
regions of solid tumors and its potential to enhance therapeutic effects of chemotherapy. Int J Cancer.
2014;134:2726-34.
Tredan O, Galmarini CM, Patel K, Tannock IF. Drug resistance and the solid tumor microenvironment.
J Natl Cancer Inst. 2007;99:1441-54.
Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nature reviews Cancer. 2006;6:583-92.
Shim JH, Park JW, Kim JH, An M, Kong SY, Nam BH, et al. Association between increment of serum
VEGF level and prognosis after transcatheter arterial chemoembolization in hepatocellular carcinoma
patients. Cancer science. 2008;99:2037-44.
Sergio A, Cristofori C, Cardin R, Pivetta G, Ragazzi R, Baldan A, et al. Transcatheter arterial
chemoembolization (TACE) in hepatocellular carcinoma (HCC): the role of angiogenesis and
invasiveness. The American journal of gastroenterology. 2008;103:914-21.