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Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models OPEN

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Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.
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Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
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Intravenous Formulation of
HET0016 Decreased Human
Glioblastoma Growth and
Implicated Survival Benet in Rat
Xenograft Models
Meenu Jain1, Nipuni-Dhanesha H. Gamage2, Meshal Alsulami1, Adarsh Shankar1,
Bhagelu R. Achyut1, Kartik Angara1, Mohammad H. Rashid1, Asm Iskander1, Thaiz F. Borin1,
Zhi Wenbo3, Roxan Ara1, Meser M. Ali2, Iryna Lebedyeva4, Wilson B. Chwang2, Austin Guo5,
Hassan Bagher-Ebadian2 & Ali S. Arbab1
Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective
CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to
explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016
with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single
IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at
60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered
vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in
syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed
prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016
and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased
neovascularization (laminin and αSMA), in addition to inammation and angiogenesis markers in the
treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is eective in inhibiting tumor
growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016
signicantly prolonged survival in PDX GBM811 model.
Glioblastoma (GBM) is a hypervascular malignant tumor with poor prognosis1,2. Because of hypervascularity,
anti-angiogenic therapies (AATs) targeting the vascular endothelial growth factor (VEGF) and VEGF receptor
(VEGFR) axis have been attempted in clinical trials, but the results have not been encouraging3. Moreover, our pre-
clinical studies in a rat model of human GBM also showed resistance to the treatment of receptor tyrosine kinase
inhibitors (RTKIs) and resulted in paradoxical enhancement of neovascularization and tumor growth4,5. erefore,
we need an agent that will decrease tumor growth and neovascularization with reduced resistance to therapy.
Recently, studies have shown the role of N-hydroxy-N’-(4-butyl-2 methylphenyl) formamidine (HET0016), a
highly selective inhibitor of 20-hydroxy arachidonic acid (20-HETE) synthesis involving enzymes of the CYP4A
and CYP4F families, in inhibiting tumor angiogenesis, proliferation, migration, and regulation of CD133+ /
CD34+ EPCs6–8. HET0016 was able to inhibit angiogenic responses to several growth factors as well as angio-
genesis in gliosarcoma and in the cornea induced by implanted human U251 GBM cells9,10. Our previous study
in breast cancer also showed decreased tumor growth aer treatment with HET00168. In previous studies of
GBM, HET0016 was prepared in cremophor and DMSO that was administered either orally or intraperitoneally
1Tumor Angiogenesis Laboratory, Georgia Cancer Center, Augusta University, Augusta, GA, USA. 2Cellular and
Molecular Imaging Laboratory, Henry Ford Health System, Detroit, MI, USA. 3Center for Biotechnology and Genomic
Medicine, Augusta University, Augusta, GA, USA. 4Department of Chemistry and Physics, Augusta University,
Augusta, GA, USA. 5Department of Pharmacology, New York Medical College, Valhalla, NY, USA. Correspondence
and requests for materials should be addressed to M.J. (email: mjain@augusta.edu)
Received: 22 July 2016
Accepted: 28 December 2016
Published: 31 January 2017
OPEN
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(IP). However, there was limited success in controlling the glioma due to low bioavailability11. In the present
study, we optimized a condition to make IV formulation of HET0016 with 2-Hydroxypropyl Beta Cyclodextrin
(HPßCD) to improve bioavailability and deliver an eective dose of the drug to the tumor site, especially in the
hypervascular and hypoxic areas of GBM. HPßCD is a derivative of β -cyclodextrin that has been extensively used
as a drug delivery vehicle and recently, FDA has approved the use of HPßCD as a treatment for Niemann Pick
Type C disease12,13. e exploitation of HPßCD as delivery vehicle in GBM may be benecial due to the porosity
of the tumor vasculature, an enhanced permeability and retention (EPR) eect and reduced o target eects in
the tumor neovasculature14. Moreover, due to smaller size (< 1 nm) of the HPßCD encased drug, excess drug can
be cleared rapidly through kidneys. erefore, we believe that the use of HPßCD as a drug delivery system for
delivering HET0016 in GBM will not have any detrimental eect.
In the current rat xenogra model of GBM, we have performed magnetic resonance imaging (MRI) studies,
which help in analysis of changes in tumor vascular physiology, tumor size, backward transfer constant (kep),
tumor plasma volume (vp), vascular permeability (forward transfer constant, Ktrans ), extravascular and extracel-
lular space volume interstitial space volume fraction (ve)15. In addition to MRI, we have also evaluated changes in
histological parameters, eect on survival and signaling pathways following HET0016 treatment. ese parame-
ters may allow for a better understanding of the physiological characteristics of the regional tumor environment
and vascularity aer HET0016 treatment.
e main purposes of the study are (1) to determine whether HPßCD can be complexed with HET0016 within
reasonable period of time (practical kitchen chemistry) to obtain a water soluble formulation that can be admin-
istered intravenously, (2) to investigate the eects of IV administration of HET0016 on human and syngeneic
GBM tumor growth, and survival in patient derived GBM animal models, vascular parameters, histology, and (3)
to identify critical signaling pathways inhibited by HPßCD-HET0016, when administered through IV in the rat
GBM xenogra model.
Results
IV formulation of HET0016 with 30% cyclodextrin. IV formulation of HET0016 using 30% HPβ CD
(in solution with water) was stable both at 4 °C and room temperature for an extended time without any precip-
itation of HET0016 (Fig.1a shows the structure of HET0016 with cyclodextrin). IV administration of 5-day old
complex solution did not cause any immediate or late detrimental eect on health of the animals. ere was no
change in the mass-spectrometric prole between the naked and HPβ CD-HET0016 complex alone or mixed with
plasma or cell lysate (Fig.1b). All proles showed the molecular weight of HET0016 (complex or naked) between
206 and 207 kDa with retention and elution time of 2.9 minutes.
Figure 1. Preparation of HPßCD-HET0016 complex (a) Structure of HET0016 and HPßCD is shown. HPßCD
resembles a shell and it can encase any deliverable drug. To prepare the IV formulation of HET0016, it was rst
dissolved in DMSO and added to 30% HPßCD prepared in water. e bucket like structure of HPßCD with
hydrophilic exterior helps encase HET0016 and delivers to the target area. (b) Mass spectrometry of HET0016
in DMSO (le panel), HET0016 complex in plasma (middle panel). HPßCD-HET0016 complex in tissue lysate
(right panel). Plasma and tissue lysate was collected from the rats bearing glioma and injected with single dose
of HPßCD-HET0016 for evaluation of pharmacokinetics.
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HET0016 levels in plasma and tumor tissue. Rats were orthotopically implanted with U251 cells and
allowed to form the tumor. HET0016 was administered to rats IV and IP as a single dose and sacriced for phar-
macokinetics at dierent time points. A total of 12 rats were serially euthanized for the measurement of HET0016
levels in plasma and brain tumor tissue at 0, 5, 30, 60, 180 min and 24 h (1440 min) aer single IV and IP HET0016
administration (10 mg/kg) (n = 2–4 per time point) and analyzed by LC-MS/MS as described in pharmacokinet-
ics section in material and methods. e level of HET0016 was 7-fold higher in plasma in the group of IV route at
5 min and 60 min time point (105042 and 36880.18 ng/ml) as compared to the IP group (15938.1 and 4773.0 ng/
ml) (p < 0.005) (Fig.2a). e plasma concentration suggests there was rapid elimination of HET0016 with a
half-life of approximately 45 min for both the IV and IP groups. e concentration of HET0016 in tumor tissue in
the IV group was much higher than that of the IP group at 60 min (9251 ng/g vs 325 ng/g) and 24 h (42.7 ng/g vs
7.4 ng/g) (p < 0.01) (Fig.2b). ere was no signicant dierence at middle time point at 180 min.
Eect of HET0016 on tumor growth and vascular parameters. Rats were orthotopically implanted
with U251 cells and allowed to form tumor for 8 days. Aer day 8, animals were treated with HET0016 and fol-
lowed up for 21 days. A detailed schema of treatment schedule is shown in Fig.3. Treatments on the same day and
seven-day waiting period were chosen to mimic the post-surgical cases and post diagnosis of GBM, respectively,
as described previously4. Tumor growth was measured by in vivo MRI. IV administration of HET0016 signi-
cantly reduced the tumor growth (in delayed [day 8–21] treatment) compared to that of vehicle treated animals
(p < 0.001) (Fig.4a) and there was reduced growth with early (day 0–21) treatment although signicant dierence
was not achieved. When compared to the IP administered treatment, tumor growth was reduced only with early
IP treatment, although signicant dierence was not achieved (Fig.4a). However, when all groups were com-
pared, signicant (p < 0.005) reduction of tumor volume was found for delayed IV treatment group compared to
the vehicle and delayed IP HET0016 treated groups (Fig.4a).
Vascular parameters were evaluated to gain information about the vascular kinetics of the tumor environment.
Delayed IV HET0016-treated animals showed signicantly increased blood ow, which may indicate normali-
zation of blood vessels causing enhanced ow although there was no signicant dierence in blood ow (rCBF)
among the IP and early IV treated groups (Fig.4b). As expected, late IP or IV HET0016 treated animals showed
signicantly lower vp (blood plasma pool), ve (extracellular space or interistial volume), Ktrans (forward permea-
Figure 2. Pharmacokinetics of HPßCD-HET0016 in plasma and tissue aer IV and IP dose administration
in rat glioblastoma model. e concentrations of HET0016 in plasma or tissue versus time post dose
administration in rat model are shown. Blood samples were obtained for 0–24 hrs aer dose administration.
Plasma and tissue HET0016 concentrations were determined by LC-MS/MS aer liquid phase extraction. Each
data point is presented as the mean concentration ± SEM (n = 2–4). Concentration of HET0016 in plasma
(a) and tumor tissue (b) aer a single HET0016 (10 mg/kg) dose through IV and IP route. Signicant values
from t Student test and Mann-Whitney’s test are represented by *p < 0.05 in comparison to IV and IP doses in
the same time point.
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bility transfer constant) compared to that of corresponding vehicle treated groups (p < 0.01) (Figs4c and 5a–c).
Both early IP and early IV groups also showed signicantly lower vp compared to that of corresponding vehicle
treated groups (Fig.4c). As rCBF analysis showed higher blood ow (Fig.4b), it is expected that normalized ves-
sels will have less Ktrans (Fig.5a) compared to that of neovessels. erefore, the blood plasma pool (vp) would be
reduced in tumors with normalized vessels (Fig.4c). When the extravascular extracellular space volume (ve) was
compared, both early and delayed IV HET0016- treated groups showed signicantly decreased ve compared to
that of vehicle and IP-treated groups (Fig.5c). ere was no dierence in kep (backow transfer) between the IP
and IV group of animals (Fig.5b). We also evaluated the blood chemistry to determine the toxicity following 0–21
days of treatment with either vehicle, IV formulated or IP HET0016 in GBM xenogras. We did not observe sig-
nicant changes on liver function, renal function, important enzymes, pancreas, and lipid prole aer HET0016
treatment in IP and IV groups (SupplementaryTable1).
Proliferation (Ki67), Micro Vessel Density (MVD), Smooth muscle Actin (αSMA), extracellular
and extravascular space (EES). Brains were removed from the rats (U251 xenogra model) following
euthanasia and prepared for immunohistochemistry as described in material and methods. Tumor section in the
early and delayed IV or IP treatment and vehicle groups were stained for Ki67, which is a marker of cellular prolif-
eration. Ki67 positive and negative tumor cells were counted within the tumor areas. Ki67 positive cells along the
endothelial lining were omitted during counting and calculation of the proliferative cells. In IP treatment groups,
only delayed treatment showed reduced proliferation but not signicant (Fig.6a,c). However, in IV treatment
groups, signicant decreased proliferation was observed in both the early and late treatment groups as compared
to corresponding vehicle groups (Fig.6b–d).
Tumor tissues were also stained for laminin to analyze for microvessel density (MVD). Laminin, a basement
membrane glycoprotein, is found under the endothelium, encasing the pericytes and smooth muscle cells in the
vessel wall and has been shown to be an excellent marker for blood vessels in the brain. Tumor sections were
stained with laminin antibody and images from the positive area were taken at 10x and 20x and counted for
a number of vessels. We found no signicant dierence in expression of laminin among the IP-treated groups
(Fig.7a–c, top le panel). However, there was signicant reduced expression of laminin with lower number of
vessels (3–50 fold less) in the IV treatment group especially around the rim of tumor as compared to vehicle
(p < 0.01) (Fig.7d–f top right panel). We also performed α SMA immunostaining to demonstrate the eect on
vessels in pericyte area around tumor periphery and found there was a signicantly smaller number of ves-
sels with reduced expression of α SMA in the IV treatment group as compared to vehicle (p < 0.02) (Fig.7e,f).
However, no such dierences were detected in the early and delayed IP-treatment groups (Fig.7b,c).
Dierences in the area of extracellular and extravascular space were also analyzed by using H&E stained
images from the IP and IV groups using the color thresholding method as described earlier5. ere was no dif-
ference observed among IP treatment groups as compared to vehicle (SupplementaryFigure2a,b top panel).
However, we found a threefold reduction in the extracellular and extravascular space in the IV-treated groups
(both early and delayed) as compared to vehicle (p < 0.01) (SupplementaryFigure2c,d bottom panel).
Reduced expression of cell proliferation, inammatory and angiogenic proteins were detected
in glioma treated with HET0016. A protein array was performed using tissue lysates for angiogenic
related growth factors using a membrane based protein array kit. SupplementaryFigure3 shows the expres-
sion of important proteins related to angiogenesis among the control, early and delayed IV treatment groups.
Pro-angiogenic proteins such as VE-cadherin (vascular endothelial cadherin-vasculogenesis), bFGF (basic
brobalst growth factor), IL-8 (chemokine CXCL8) and MCP-1 (a CCL2 ligand) were signicantly downregu-
lated in the delayed IV treatment group while expression of anti-angiogenic proteins such as Tie-2, angiostatin
and angiopoietin-2 was increased in the delayed treatment group (day 8–21; SupplementaryFigure3a) as com-
pared to control (p < 0.03). In the early treatment group, expression of proteins, such as MCP-1, angiostatin
and angiopoietin-2 followed a similar trend, indicating a signicant eect of the treatment, particularly on the
factors secreted or expressed by endothelial and inammatory cells. Western blot was performed to determine
the expression of proteins related to cellular proliferation (p-ERK), survival (p-AKT), inammation (COX-1/2),
arachidonic acid metabolism (CYP4A11), signal transducers and activators of transcription (p-STAT1). We also
Figure 3. Schematic representation of treatment schedule. U251 glioma cells were implanted orthotopically
in rat’s brain and treated with HPßCD-HET0016 as described in material and methods.
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Figure 4. HPßCD-HET0016 reduces tumor growth and vascular parameters in rat glioblastoma model.
(a) Representative post contrast T1-weighted images from in vivo imaging of rat with glioma show tumor size
from dierent groups of animals. Semi-quantitative analysis shows signicantly reduced tumor volume in
animals that received HET0016 from day 8 and continued for 2 weeks (n = 5 to 8). (b) Relative cerebral blood
ow maps created from arterial spin labeling techniques. Semi-quantitative analysis (bar graphs) indicates
higher ow in IV HET0016-treated groups (started on day 8) compared to that of vehicle-treated group
(n = 5 to 8). (c) Tumor plasma volume maps and semi quantitative analysis are shown. Both IV and IP
treatment with HET0016 caused a signicant decrease in tumor plasma volume (vp) compared to vehicle and
corresponding IP-treated groups. Each group is represented as the mean of the total measurable MRI sections
from animals of each group (n = 5–8 animals but 7–16 sections). Signicant values from ANOVA test followed
by Fisher’s exact test are represented by *p < 0.05 compared to the corresponding vehicle group. An outlier data
point (from a single MRI section) was removed as described in our material and methods.
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Figure 5. HPßCD-HET0016 inuences vascular parameters in rat glioblastoma model. MRI images from
in vivo rat imaging show signicant changes in vessel permeability (Kt rans) (a), and ve (interstitial space volume)
(C) but no changes in kep (backow rate constant) (b) in early and delayed treatment in IV groups. Signicant
values from ANOVA test followed by Fisher’s exact test are represented by * and #p < 0.05 compared to (*)
vehicle 8–21 vs IV HET 8–21 and vehicle 0–21 vs IV HET 0–21, and (#) IP HET 8–21 vs IV HET 8–21 and IP
HET 0–21 vs IV HET 0–21. Each group is represented as the mean of the total measurable MRI sections from
animals of each group (n = 5–8 animals but 7–16 sections), analyzed by MRI at 0.5 μ m thickness in a range up to
7–16 viewed slices of each animal.
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determined the expression of, HIF-1α , EGFR, VEGF, MMP-2 (angiogenesis) and p-NFKB (inammation), to
investigate the eects of IV administered HET0016 on the rat model of GBM. We observed reduced levels of
COX-1, CYP4A11, p-ERK, p-AKT, p-STAT1, HIF1α , EGFR, VEGF, MMP-2 and p-NFKB proteins in tumors
obtained from both IV treatment groups as compared to vehicle (SupplementaryFigure3b), which conrms the
inuence of HPβ CD-HET0016 on cell proliferation, migration, and inammation pathways.
Eect of HET0016 on survival in GBM811 and HF2303. We evaluated the eect of HET0016, TMZ
alone or in combination with radiation on survival in PDX derived tumor model of GBM811 and HF2303. First,
we tested the eect of treatment in vitro 3D culture model (HF2303) and found that the treatment with HET0016
or TMZ alone or combination showed the inhibition of the growth of neurospheres Fig.8a). Similar studies were
planned in vivo model and we observed that HET0016 treatment alone or in combination with TMZ in irradiated
tumor bearing animals resulted in reduced tumor growth and prolonged survival in both GBM811 (p < 0.004)
and HF2303 PDX models (p = 0.18) (Fig.8b). e overall survival in GBM811 model was prolonged to 26 weeks
aer the treatment with HET0016 plus TMZ and radiation, while control animals (supercontrol and irradiated
only control) survived only for 10 weeks similar to the clinical setting (Fig.8c). e cumulative survival was 100%
for HET0016 plus TMZ combination group as compared to 66% for TMZ alone, 33% for HET0016 alone at 26
weeks until the end of the study without demonstrating any evidence of recurrence (Table in Fig.8d). erefore,
the current study provides evidence that combination therapy (HET0016 + TMZ) with irradiation can improve
survival in GBM. In the second PDX model of HF2303, the HET0016 treatment alone and in combination with
TMZ, resulted in reduced tumor volume and prolonged survival of 26 weeks vs. only 17 weeks of irradiated con-
trol (p < 0.10). e cumulative survival was 25% for both HET0016 plus TMZ combination group and HET0016
alone group at 26 weeks as compared to 0% in radiation only group (p < 0.18). us, data indicates combination
treatment with HET0016 plus TMZ and irradiation resulted in better survival benet as compared to radiation
alone. Overall, the response was better in GBM811 model than HF2303 model and may be attributed to dierence
in growth and genetic characteristics. We also measured the presence of 20-HETE and MGMT in dierent glioma
cells (GL261, U251, HF2303 and GBM811) and GBM811 showed comparatively lower amount of 20-HETE and
methylguanine DNA methyltransferase (MGMT) levels (SupplementaryFigure5and6), which may indicate
better treatment results following HET0016, radiation and TMZ in GBM811 PDX model16,17.
Figure 6. HPßCD-HET0016 treatment results in reduced proliferation, migration in rat glioblastoma
model. (a,b) Ki-67 immunohistochemical staining was done as a marker of proliferation in tumor tissues.
Rats treated with HET0016 starting on day 8 showed signicantly fewer proliferative cells in the tumor in the
IV group compared to the IP group. (c,d) Bar graphs represent the proportion of Ki67 positive tumor cells
compared to total number of tumor cells in tumor area of each group. Images were taken in 40x magnication.
e brown nuclei color shows the positive labelling cells. Statistically signicant dierences were veried by
ANOVA followed by Bonferroni’s test. *p < 0.05 in comparison to the respective vehicle group.
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Eect of IV HET0016 on GL261 syngeneic GBM. We also assessed the eect of IV HET0016 on tumor
growth in syngeneic model of GBM using GL261 cell line. All animals underwent MRI and tumor volume
was measured from the MRI images. ere was signicantly reduced tumor volume in animals treated with
IV HET0016 (54.33 ± 12.37 mm3) compared to that of vehicle treated animals (131.70 ± 19.45 mm3) (p < 0.007)
(SupplementaryFigure1).
Discussion
Despite the availability of current chemotherapy and targeted therapies in GBM (e.g. temozolomide and bevaci-
zumab), not many therapeutic benets have been achieved due to target developing drug resistance18. Most ther-
apies have produced a decrease in tumor growth during early stages of treatment followed by aggressive tumor
recurrence. Mu et al. previously developed water-soluble IV formulation of HET0016 which rapidly penetrated in
the rat normal brain and inhibited the formation of 20-HETE aer cerebral ischemia19. Here, we have developed
an improved IV formulation of HPßCD-HET0016 that dissolves in 2–3 min compared to the 48 hrs as reported
by Mu et al. IV administration of HPßCD-HET0016 in a rat model of human GBM signicantly reduced tumor
growth in developing or developed tumors as compared to IP HET0016 treatment. It appears that the IV formu-
lation facilitated increased delivery of HET0016 to the hypervascular and hypoxic tumor sites, due to EPR eects.
Moreover, the eect of IV HET0016 was not cell specic and showed similar reduced tumor growth in syngeneic
Figure 7. HPßCD-HET0016 treatment results in reduced neovascularization in rat glioblastoma
model. Laminin and α SMA immunohistochemistry staining was done in tumor tissues to determine the
neovascularization (a,b,d and e) Representative images from brain tumor tissues are shown at 10x and 20x
from the IP (le panel) and IV (right panel) groups. Red arrows indicate blood vessels. Four areas on the tissue
section were selected and the number of vessels counted. (c and f) Laminin and α SMA quancation. Each
bar represents an average of four areas and was estimated in multiple samples from each group (n = 2–4).
Signicant dierence is indicated by *p < 0.05 compared to the respective vehicle group.
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tumor (GL261) models. e distribution prole of HPßCD-HET0016 formulation showed better bioavailability
and retention in tissue with a higher brain to plasma ratio in the IV treatment group as compared to the treatment
with IP preparation. e ndings suggest that the IV formulation or encapsulation of HET0016 in a non-toxic
delivery system may have the benets of increased half-life (protect the drug from degradation during circulation
and early clearance), lower toxicity, enhanced EPR eect over the IP preparation and improved the therapeutic
index14. Furthermore, enhanced accumulation of macromolecular drugs in tumor tissues can occur as compared
to normal tissue due to impaired lymphatic drainage and porous blood vessels in GBM with abnormal molec-
ular and uid transport dynamics20,21. e eect of enhanced delivery of IV HET0016 is supported by vascular
parametric analysis in MRI studies. Vascular parametric analysis supported the eect of the drugs, where the IV
formulation decreased vascular permeability (Ktrans), tumor blood volume (vp), and extracellular extravascular or
interstitial space volume (ve) but increased the blood ow to the delayed treated tumors in GBM.
TMZ is widely used alkylating agent for the treatment of primary as well as recurrent GBM. Eect of TMZ
requires functional DNA mismatch repair (MMR), low levels of methylguanine DNA methyltransferase (MGMT)
and DNA repair genes16,22,23. Unfortunately, majority of the primary and recurrent GBMs have unmethylated
or active high level of MGMT, which make TMZ treatment unresponsive17,24,25. We evaluated the synergistic
eect of HET0016 alone or in combination with TMZ on the survival of animals treated with 30 Gy irradiation
in patient-derived xenogra models (PDX) (GBM811, HF2303) (treatment schedule shown in Supplementary
scheme). Aer Administration of HET0016 alone and in combination with TMZ and radiation in GBM811 and
HF2303 models for 6 weeks resulted in good response and tumor did not relapse until 6 months (endpoint of
the study). erefore, HET0016 plus TMZ combination may prolong survival and reduce therapy resistance. e
synergistic eect may be due to the role of HET0016 in sensitizing the action of TMZ and irradiation by reducing
DNA repair mechanisms. Our preliminary study indicated that HET0016 administration resulted in down reg-
ulation of DNA repair genes (unpublished preliminary data, SupplementaryFig.4). e continuous exposure of
Figure 8. Treatment with HET0016 and TMZ prolongs survival in PDX models. (a) Treatment with
HET0016 and TMZ inhibit neurospheres growth in vitro: HF2303 was treated with HET0016 and TMZ alone
(100 μ M), in combination with TMZ and followed up for 14 days. (b) HF2303 and (c) GBM811 show the
eect of HET0016 and TMZ on survival rate of the mice in groups 1, 2, 3, 4 and 5 of treatment schedules (as
described in Material and Methods) were evaluated from the rst day of treatment until death. X-axis represents
cumulative survival time in weeks. Table in Fig. 8d summarize the details of duration in weeks and % survival.
Athymic nude rats were implanted orthotopic with HF2303 and GBM811. Six to ten weeks aer implantation,
rats were randomized into ve treatment groups receiving PBS, radiation, HET0016, TMZ, HET0016 plus TMZ
for another 6 wks, as described in Materials and Methods. #e animals were not included due to the technical
diculty. Signicant dierence is indicated by *p < 0.05 vs irradiation and super-control, $p-value was 0.18 vs
irradiation control, achieved by Kaplan Meier analysis and Log-rank test.
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cellular DNA to potentially harmful environmental and internal insults necessitates redundant and overlapping
DNA repair mechanisms26. erefore, successful destruction of GBM tumors may require a combined approach
utilizing standard treatments in combination with inhibition of DNA repair pathways.
e anti-tumor eect of HPßCD-HET0016 was supported by decreased tumor cell proliferation, migration,
and neovascularization. ere was a clear reduction in the number of Ki67 + cells in the IV treatment groups as
compared to IP groups. e reason for observing the signicant reduced proliferation in IV treatment groups
may be that HET0016 is eective in preventing the regrowth of established tumor due to increased bioavailability
following IV administration. We also believe established tumor has developed-neovascularization and leaky ves-
sels, which allow EPR eect to be more pronounced in groups treated with IV formulated HET0016. In addition,
we suggest that the eect of HET0016 on tumor growth may be attributed to the reduced basic broblast growth
factor expression (bFGF or FGF-2) aer early and delayed treatment. bFGF is a potent mitogen that maintains
cancer cell stemness and leads to drug resistance by enhancing the blood-brain-barrier function of endothelial
cells27,28. ese results are supported by an earlier study of Guo et al.29 which showed chronic in vivo admin-
istration of HET0016 (IP, 10 mg/kg/day) reduced the volume of 9 L gliosarcoma, accompanied by mitotic and
vascularization reduction.
GBM tumor vessels are tortuous, disorganized, highly permeable, decreased pericyte coverage and a solid
basement membrane structure30,31. We found overall fewer vessels in both early and delayed IV treatment group,
with reduced EES, laminin, and α SMA staining, especially at the invasive margin of the tumor indicating the role
of HET0016 in inhibiting the growth of new blood vessels. ese observations were supported by a published
report that suggested that the pericyte line around new endothelial cells sprouts from tumor vessels, play a role
in blood vessel growth, and is suggested to be a potential target in AAT therapy32. In addition, this is further
supported by a high expression of angiopoietin-2, Tie-2 molecules and reduced VE-cadherin expression, which
is associated with pericyte endothelium suggesting an eect of HET0016 on vascular development and stabil-
ity33. We suggest that HPßCD-HET0016 has a primary eect on blood vessels, which by normalization increased
the bioavailability of drug to the hypoxic tumor sites. MRI vascular parametric analysis also correlates with
immunohistochemistry; especially the normalization of vessels in the IV delayed treatment groups that resulted
in decreased permeability (Ktrans) and overall reduced plasma volume fraction (vp). e treatment also caused
decreased EES volume indicated by decreased ve, which is also validated in H&E histological analysis. Similarly,
Guo et al also showed normalization of vessels in 9 L tumor following HET0016 treatment10.
In our study, we observed that HET0016 reduced expression of the pro-angiogenic proteins but increased the
expression of anti-angiogenic proteins expression to achieve equilibrium to reduce tumor growth. Expression
of pro-angiogenic factors such as IL-8, MCP-1, VEGF and SDF-1α were decreased, while expression of inhibi-
tors of the angiogenic process such as angiostatin, angiopoietin-2/Tie-1, and Tie-2 proteins were increased aer
HET0016 treatment. Recently, a study in triple negative breast cancer showed a similar eect of IP HET0016 on
pro-angiogenic factors8. We observed that HPßCD-HET0016 reduced IL-8 expression. IL-8 has recently been
shown to be a critical factor in regulating cancer cell stemness and invasion, and higher expression was associated
with poor survival and therapy resistance in glioma34. Moreover, HET0016 treatment reduces the expression of
MCP-1 and SDF-1α , key chemokines responsible for tracking and activation of monocytes/macrophages and
have been involved in inammation and angiogenesis35. MCP-1-induced angiogenesis has been reported to be
mediated through up-regulation of HIF-1α and subsequent activation of VEGF35. erefore, HPßCD-HET0016
may be acting as an inhibitor of inammation and angiogenesis growth responses in GBM tumor cells through
regulating HIF-1α and VEGF. Interestingly, there were less hypoxic areas in HPßCD-HET0016-treated tumors as
shown by reduced HIF-1α protein expression. Reduced HIF-1α expression also resulted in low VEGF expression
aer HPßCD-HET0016 treatment. VEGF signaling and angiogenesis and highly aected by HIF-1α levels and
regulates endothelial cell proliferation, migration, and permeability of blood vessels. Studies have shown high
expression is correlated with increased metastases, vasculature, and tumor recurrence28. ese ndings suggest
that HPßCD-HET0016 regulated the expression of pro-and anti-angiogenic factors to achieve the balance in the
maintenance of the tumor microenvironment8.
In the current study, we studied eect of HPßCD-HET0016 treatment in the arachidonic acid metabolism
pathway. Previously, HET0016 has been shown to have an eect on expression of CYP4A enzymes. In present
study, we found HET0016 was able to reduce the protein expression of CYP4A11 and COX-1, thereby inuencing
inammation, angiogenesis, and MAPK signaling in GBM36,37. Previous studies have also documented a similar
role of HET0016 in glioma, gliosarcoma, lung, and breast cancers10,29,38. A study in colon cancer suggested com-
bination therapy of rofecoxib and HET0016 might be a new treatment that can improve the anti-tumor ecacy of
rofecoxib alone39. In addition, we also investigated role of p-NFκ B as marker of inammation and proliferation.
Previous studies have shown a pro-survival role for p-NFκ B in glioma and an association with chemo resistance40.
We demonstrated reduced levels of p-NFκ B aer HET0016 treatment suggesting an anti-proliferative eect in
glioma.
Previous study have shown that GBM has frequent overexpression of EGFR, which leads to activation of
the P13/Akt pathway, associated with adverse clinical outcome and has been suggested to be a therapeutic tar-
get41–43. We showed that HPßCD-HET0016 repressed the expression of EGFR, ERK, and Akt, a target of MEK.
We also determined expression of p-STAT1, a tumor suppressor protein involved in the p38/MAPK pathway in
response to IFN-α and stress. Higher expression of p-STAT1 was shown to be associated with poor prognosis
in glioma and depletion of IRF1/STAT1 signaling has been reported to increase the ecacy of anti-VEGF (bev-
acizumab) therapy in a glioma xenogra model44–46. We found that HPßCD-HET0016 treatment also reduced
the levels of pSTAT1, indicating HET0016 can improve overall prognosis in glioma. In summary, we have estab-
lished a highly soluble HPßCD-HET0016 complex for IV administration that reduced GBM growth, normal-
ized vasculatures, decreased permeability, and EES volume both in established and growing tumors through
enhanced bioavailability compared to conventional IP HET0016 preparation. Our results showed that HET0016
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in combination with TMZ and radiation enhanced the anti-tumor ecacy and prolonged survival in GBM xen-
ogra models as compared to TMZ alone. Anti-tumor properties of HPßCD-HET0016 resulted in decreasing
proliferation, hypoxia, migration, stemness, and vasculatures in glioma by altering the balance of pro-angiogenic
and anti-angiogenic balance, PI3K/Akt, p38/MAPK and inammation pathways (Summary model in Fig.9).
erefore, HPßCD-HET0016 could be tested in clinics to explore combination therapies with TMZ or radiother-
apy for GBM.
Materials and Methods
Ethics statement. Animal experiments were performed according to the NIH guidelines and the experi-
mental protocol was approved by the Institutional Animal Care and Use Committee of Henry Ford Health System
and Augusta University (formerly Georgia Regents University) (Approval number: 2014–0625). All animals were
kept under pathogen-free conditions at room temperature (21 to 25 °C) with exposure to light for 12 hours and
12 hours in the dark. Food and water were oered ad libitum. Body weight was measured twice weekly as an
indicator of overall animal health. All surgeries were performed under ketamine - xylazine anesthesia, and eorts
were made to minimize suering. Euthanasia for the moribund animals was performed in a CO2 cha mber.
Chemicals. HPßCD (2-hydroxy Propyl-β -Cyclodextrin) was purchased from Sigma-Aldrich (St. Louis, MO),
cell culture media was from ermo Scientic (Waltham, MA), and fetal bovine serum was purchased from
Hyclone (Logan, Utah). HET0016 was obtained from Dr. JR Falck of UT Southwestern University, Texas, and
also synthesized by Dr. Iryna Lebedyeva in the department of Chemistry and Physics, Augusta University with a
purity of more than 97%. Cell culture grade DMSO was purchased from Fischer Scientic (PA). Blood chemistry
proles and electrolytes were determined Using third party vendor (Antech Diagnostic).
Tumor cells. Human glioma U251 cell line was obtained from Dr. Steve Brown of Henr y Ford Health System.
e cell line was authenticated in July 2014 using the STR proling method. GL261 syngeneic (C57BL/6 mouse
derived) GBM cell line was obtained from Dr. Ted Johnson (Augusta University) and was authenticated in 2016.
e cell line, U251 was grown in high glucose (4.5 g/L) Dulbecco’s modied eagles medium (DMEM) and GL261
in RPMI (Roswell Park Memorial Institute) (ermo Scientic), supplemented with 10% fetal bovine serum
(FBS), 2 mM glutamine and 100 U/ml penicillin and streptomycin at 5% CO2 at 37 °C in a humidied incubator.
Figure 9. Summary and hypothetical model. Tumors consist of an abnormal vasculature, composed mainly
of immature vessels with increased permeability. e less densely packed cells allow drugs or complexes
to accumulate in tumor tissue. Treatment with HPßCD-HET0016 leads to reduced expression of α SMA
and increased expression of angiopoietin-2 and Tie-2, which may lead to reduced tumor vasculature that is
inadequate to support tumor growth and may lead to tumor dormancy. Our results suggest that HET0016
reduces cancer cell growth, invasion, and vasculature by reducing the expression of signaling molecules in the
MAPK, PI3K/AKT, and inammation pathways.
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Patient derived GBM cells (HF2303) was obtained from Dr. Tom Mikkelsen’ s lab at Henry Ford Hospital and
was grown in neurosphere medium (NM), composed of DMEM/F-12 supplemented with N2 (Gibco), 0.5 mg/ml
BSA (Sigma), 25 μ g/ml gentamicin (Gibco), 0.5% antibiotic/antimycotic (Invitrogen), 20 ng/ml basic broblast
growth factor, and 20 ng/ml EGF (Peprotech). Cells were maintained in culture for up to passage 10 (low passage).
Patient derived PDX GBM cells, GBM811 was obtained from North Western University and was propagated in
immunocompromised NOD-SCID mouse and tumor was disintegrated into cell suspension at the time of tumor
implantation in nude rats.
Intraperitoneal (IP) formulation of HET0016. For IP formulation, 2 mg of HET0016 was dissolved in
1 ml of solution containing 80% PBS, 10% of dimethyl sulfoxide (DMSO) (Sigma, St. Louise, MO, USA) and 10%
cremophor (Sigma, St. Louise, MO, USA). e mixture was vortexed and sonicated until HET0016 was dissolved.
Each rat received 10 mg/kg/day dose and the dose has been used according to previous publications10,11.
Intravenous (IV) formulation of HET0016 using HPßCD (2-hydroxy Propyl-B-Cyclodextrin).
Previous published methods have used 48 hours incubation with continuous rotation to make a complex of
HET0016 with HPßCD for the IV formulation19. Here, we describe a rapid and safe method to synthesize IV for-
mulation of HET0016 without using a sonicated water bath or long rotation. HPßCD has a bucket-like structure
with hydrophilic outer shell but hydrophobic inner cavity (Fig.1a). However, optimized conditions are needed
to insert the drug into the HPßCD cage since HET0016 is a heat sensitive compound and can be degraded with
long-term rotation at room temperature. HET0016 was dissolved in DMSO (2 mg/50 μ l) and was added slowly
to a 30% HPßCD solution in sterile water (950 μ l) with continuous vortex, and the turbid solution became clear
within 2–3 minutes. e nal concentration of DMSO was 5%. We have tested mass-spectrometric proles of
HET0016 complexed with HPßCD as well as HET0016 dissolved in DMSO (Fig.1b). e solution of HET0016
in DMSO showed a peak at 2.9 min retention elution time, and it did not change when HET0016 was complexed
or encapsulated with HPßCD.
HET0016 pharmacokinetics in plasma and tissue lysate. Twelve nude rats (RNU nu/nu) obtained
from Charles River Laboratory (Frederick, MD) were used for pharmacokinetics studies. A single IV injection of
HET0016 or vehicle (10 mg/kg) in 30% cyclodextrin or IP injection of HET0016 was administered to the animals
in the respective groups. Animals were euthanized at multiple time points, and brain tumor tissue was collected
(60, 180 min, 24 hrs). e details are provided in Supplementarymethods.
Animal model and treatment schedules. Forty-eight nude rats (RNU nu/nu) weighing 140–150 grams
obtained from Charles River Laboratory (Frederick, MD) were used in these experiments. Human glioma U251
cells (400 k in 5 μ L), were implanted orthotopically at 3 mm to the right and 1 mm anterior to bregma according to
published methods47. Following implantation, animals received treatment of HPßCD-HET0016 (IV, 10 mg/kg per
day); an equivalent dose of IV HPßCD alone as a vehicle; HET0016 (IP, 10 mgkg/day), or an equivalent dose of IP
vehicle alone for either two or three weeks. e details of animal model are described in Supplementarymethods.
In vivo MRI and measurement of vascular kinetics. All animals underwent MRI, vascular kinetic,
and tumor volume analyses as described previously on day 225 as depicted in Fig.3. e details on methods are
provided in Supplementarymethods section.
Tumor volume analysis. Post contrast T1-weighted images were used to determine the tumor volume. At
least two investigators, blinded to the various treatment groups, determined the volume by drawing irregular
region of interest (ROIs) for all slices containing tumor. To calculate the exact volume, investigators summed up
the number of slices and multiplied by the slice thickness.
Survival studies in PDX GBM models. Anti-tumor eects and survival studies were conducted in patient
derived xenogra GBM model (PDX) using HET0016 as an adjuvant to the current treatment strategies of GBM
(radiation and TMZ). Two dierent PDX models were developed using HF2303 (n = 11) and GBM811 (n = 13).
Cells were suspended in PBS and 2 × 105 cells in 5 μ l were injected orthotopically into brain of each, 5–7-week-old
nude rats, as described previously47–50. Prior to the start of treatment, animals underwent MRI at 6 weeks for
GBM811 and 10 weeks for HF2303 before the start of treatment to conrm the presence of a 3 mm3 t umo r.
ereaer, animals were randomly assigned to ve treatment groups and details of the treatment are shown
in Supplementary scheme. e ve groups of treatment in the present study were as follows: (1) supercontrol;
tumor bearing animals were treated with PBS (IV) twice weekly. (2) Irradiation control; tumor bearing animals
received a single dose of 30 Gy radiation encompassing the tumor. (3) Irradiation + HET0016; following single
dose of 30 Gy irradiation tumor bearing animals received IV HET0016 (10 mg/kg, 5 days/week, every other week
for 6 weeks. (4) Irradiation + TMZ; following single dose of 30 Gy irradiation tumor bearing animals received
oral TMZ (50 mg/kg, 2 days/week every other week for 6 weeks). (5) Irradiation + HET0016 + TMZ; follow-
ing single dose of 30 Gy irradiation tumor bearing animals received IV HET0016 plus oral TMZ for six weeks.
HET0016 was given on 1st, 3rd and 5th weeks and TMZ was given on 2nd 4th and 6th week following irradiation. All
animals (GBM 811 and HF2303) received 30 Gy of irradiation in a single fraction before the treatment of TMZ
and HET0016 alone or in combination except the super-control group that did not go through irradiation or any
other treatments. A single dose of 30 Gy radiation was given in an area encompassing the tumor using an X-ray
based image-guided micro small animal radiation research platform from Gulmay Medical Inc. (SARRPTM, an
Xstrahl company). All surviving animals underwent 2nd set of MRI following 6 weeks of treatments to determine
the eects on the tumor volume. 3rd set of MRI was obtained from all surviving animals 4–6 weeks aer the end
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of the treatment. 4th set of MRI was also obtained from all surviving animals at the end of the studies (26 weeks
following tumor implantation). All animals were checked for weight gain or loss at least 2 times a week from the
day of tumor implantation. Any sign of morbidity was also noted. Survival was calculated from the day of tumor
implantation until death. One hundred and eighty-three days aer tumor implantation, the experiment was ter-
minated, and the surviving animals were sacriced. All rats were autopsied at death to examine the antitumor
ecacy of each treatment regimen. Our previous experience showed that HF2303 GBM bearing animals die by
16–20 weeks and GBM811 tumor bearing animals die in 12–16 weeks if not treated.
Histopathology. Tissues were stained for proliferation (anti-Ki 67 antibody, Millipore, USA), human spe-
cic MHC-1 marker (anti-MHC-1 antibody, now HLA-A, Abcam), anti-laminin antibody (for blood vessels), and
smooth muscle actin (SMA) for pericytes (anti-α SMA antibody abcam, USA) using standard immunohistochem-
ical procedure as described previously and included in Supplementarymethods8.
Statistical analysis. Comparison between drug and vehicle-treated groups was done by using Student’s
t-test. When more than two groups were analyzed, we used ANOVA followed by Bonferroni test for normal
distribution or Fisher’s exact test. All data were expressed as means ± SEM. P value of < 0.05 was considered sig-
nicant value in all tests. e criteria to exclude an outlier were the value outside of the range of mean ± twice the
standard deviation (if necessary). Body weight, tumor volume, and survival were compared among PBS-treated,
HET0016-treated, TMZ-treated, and HET0016 plus TMZ–treated groups. Kaplan Meier analysis was performed
to determine the survival of the animals bearing PDX derived GBM and log rank was used to compare the sur-
vival between the two groups.
References
1. Stupp, ., Hegi, M. E., Gilbert, M. . & Charavarti, A. Chemoradiotherapy in malignant glioma: standard of care and future
directions. J Clin Oncol 25, 4127–4136 (2007).
2. Olar, A. & Aldape, . D. Using the molecular classication of glioblastoma to inform personalized treatment. e Journal of
pathology 232, 165–177 (2014).
3. Jain, . .Antiangiogenesis Strategies evisited: From Starving Tumors to Alleviating Hypoxia. Canc er Cell 26, 605–622 (2014).
4. Ali, M. M. et al. Eects of tyrosine inase inhibitors and CXC4 ant agonist on tumor growth and angiogenesis in rat glioma model:
MI and protein analysis study. Transl Oncol 6, 660–669 (2013).
5. Ali, M. M. et al. Changes in vascular permeability and expression of dierent angiogenic factors following anti-angiogenic treatment
in rat glioma. PLoS ONE 5, e8727 (2010).
6. Sei, T., Wang, M. H., Miyata, N. & Laniado-Schwartzman, M. Cytochrome P450 4A isoform inhibitory prole of N-hydroxy-N’-
(4-butyl-2-methylphenyl)-formamidine (HET0016), a selective inhibitor of 20-HETE synthesis. Biological & pharmaceutical bulletin
28, 1651–1654 (2005).
7. Guo, A. M. et al. Activation of vascular endothelial growth factor through reactive oxygen species mediates 20-hydroxyeico-
satetraenoic acid-induced endothelial cell proliferation. J Pharmacol Exp er 321, 18–27 (2007).
8. Borin, T. F. et al. HET0016, a Selective Inhibitor of 20-HETE Synthesis, Decreases Pro-Angiogenic Factors and Inhibits Growth of
Triple Negative Breast Cancer in Mice. PLoS One 9, e116247 (2014).
9. Chen, P. et al. Inhibitors of cytochrome P450 4A suppress angiogenic responses. Am J Pathol 166, 615–624 (2005).
10. Guo, M. et al. 9L gliosarcoma cell proliferation and tumor growth in rats are suppressed by N-hydroxy-N’-(4-butyl-2-methylphenol)
formamidine (HET0016), a selective inhibitor of CYP4A. J Pharmacol Exp er 317, 97–108 (2006).
11. Shanar, A. et al. Combination of vatalanib and a 20-HETE synthesis inhibitor results in decreased tumor growth in an animal
model of human glioma. OncoTargets and therapy 9, 1205–1219 (2016).
12. Sharma, N. & Baldi, A. Exploring versatile applications of cyclodextrins: an overview. Dr ug delivery, 1–19 (2015).
13. Ottinger, E. A. et al. Collaborative development of 2-hydroxypropyl-beta-cyclodextrin for the treatment of Niemann-Pic type C1
disease. Current topics in medicinal chemistry 14, 330–339 (2014).
14. Ma, P. & Mumper, . J. Paclitaxel Nano-Delivery Systems: A Comprehensive eview. Journal of nanomedicine & nanotechnology 4,
1000164 (2013).
15. Bagher-Ebadian, H. et al. Model selection for DCE-T1 studies in glioblastoma. Magn es on Med 68, 241–251 (2012).
16. Zhang, J., Stevens, M. F. & Bradshaw, T. D. Temozolomide: mechanisms of action, repair and resistance. Current molecular
pharmacology 5, 102–114 (2012).
17. Qiu, Z. . et al. Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-lie cells. Chinese journal of
cancer 33, 115–122 (2014).
18. Oonogi, N. et al. Topics in chemotherapy, molecular-targeted therapy, and immunotherapy for newly-diagnosed glioblastoma
multiforme. Anticancer research 35, 1229–1235 (2015).
19. Mu, Y. et al. Intravenous formulation of N-hydroxy-N’-(4-n-butyl-2-methylphenyl) formamidine (HET0016) for inhibition of rat
brain 20-hydroxyeicosatetraenoic acid formation. Drug metabolism and disposition: the biological fate of chemicals 36, 2324–2330
(2008).
20. Proop, A. & Davidson, J. M. Nanovehicular intracellular delivery systems. Journal of pharmaceutical sciences 97, 3518–3590 (2008).
21. Jain, . ., Martin, J. D. & Stylianopoulos, T. e role of mechanical forces in tumor growth and therapy. Annual review of biomedical
engineering 16, 321–346 (2014).
22. Margison, G. P. & Santibanez-oref, M. F. O6-alylguanine-DNA alyltransferase: role in carcinogenesis and chemotherapy.
BioEssays : news and reviews in molecular, cellular and developmental biology 24, 255–266 (2002).
23. oos, W., Baumgartner, M. & aina, B. Apoptosis triggered by DNA damage O6-methylguanine in human lymphocytes requires
DNA replication and is mediated by p53 and Fas/CD95/Ap o-1. Oncogene 23, 359–367 (2004).
24. yrtopoulos, S. A. et al. DNA adducts and the mechanism of carcinogenesis and cytotoxicity of methylating agents of environmental
and clinical signicance. Cancer detection and prevention 21, 391–405 (1997).
25. Capdevila, L. et al. Neoadjuvant cisplatin plus temozolomide versus standard treatment in patients with unresectable glioblastoma
or anaplastic astrocytoma: a dierential eect of MGMT methylation. J Neurooncol 117, 77–84 (2014).
26. Johnathan, E. Lawrence, C. E. B., obert, J. Belton Jr., ichard, A. ovin & obert, J. WinnTargeting DNA epair Mechanisms to
Treat Glioblastoma. (2015).
27. Toyoda, . et al. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via
broblast growth factor 2 secretion: a new in vitro blood-brain barrier model. Cellular and molecular neurobiology 33, 489–501 (2013).
28. Codrici, E., Enciu, A. M., Popescu, I. D., Mihai, S. & Tanase, C.Glioma Stem Cells and eir Microenvironments: Providers of
Challenging erapeutic Targets. Stem cells international 2016, 5728438 (2016).
www.nature.com/scientificreports/
14
Scientific RepoRts | 7:41809 | DOI: 10.1038/srep41809
29. Guo, M., oman, . J., Falc, J. ., Edwards, P. A. & Scicli, A. G. Human U251 glioma cell proliferation is suppressed by HET0016
[N-hydroxy-N’-(4-butyl-2-methylphenyl) formamidine], a selective inhibitor of CYP4A. J Pharmacol Exp er 315, 526–533 (2005).
30. Soda, Y., Mysiw, C., ommel, A. & Verma, I. M. Mechanisms of neovascularization and resistance to anti-angiogenic therapies in
glioblastoma multiforme. J Mol Med (Berl) 91, 439–448 (2013).
31. de Vries, N. A., Beijnen, J. H., Boogerd, W. & van Tellingen, O. Blood-brain barrier and chemotherapeutic treatment of brain tumors.
Expert review of neurotherapeutics 6, 1199–1209 (2006).
32. Moriawa, S. et al. Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. e American journal of pathology
160, 985–1000 (2002).
33. Gaengel, ., Genove, G., Armuli, A. & Betsholtz, C. Endothelial-mural cell signaling in vascular development and angiogenesis.
Arteriosclerosis, thrombosis, and vascular biology 29, 630–638 (2009).
34. Zhang, B. et al. Autocrine IL-8 promotes F-actin polymerization and mediate mesenchymal transition via ELMO1-NF-appaB-Snail
signaling in glioma. Cancer biology & therapy 16, 898–911 (2015).
35. Niu, J., Azfer, A., Zhelyabovsa, O., Fatma, S. & olattuudy, P. E. Monocyte chemotactic protein (MCP)-1 promotes angiogenesis
via a novel transcription factor, MCP-1-induced protein (MCPIP). e Journal of biological chemistry 283, 14542–14551 (2008).
36. Panigrahy, D., aipainen, A., Greene, E. . & Huang, S. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer.
Cancer metastasis reviews 29, 723–735 (2010).
37. Fleming, I. Epoxyeicosatrienoic acids, cell signaling and angiogenesis. Prostaglandins & other lipid mediators 82, 60–67 (2007).
38. Yu, W. et al. Cytochrome P450 omega-hydroxylase promotes angiogenesis and metastasis by upregulation of VEGF and MMP-9 in
non-small cell lung cancer. Cancer Chemother Pharmacol 68, 619–629 (2011).
39. Zhang, Y. et al. Combined therapy with COX-2 inhibitor and 20-HETE inhibitor reduces colon tumor growth and the adverse eects
of ischemic stroe associated with COX-2 inhibition. American journal of physiology. egulatory, integrative and comparative
physiology 307, 693–703 (2014).
40. Ding, G. . et al. adiosensitization by inhibition of IappaB-alpha phosphorylation in human glioma cells. adiation research 160,
232–237 (2003).
41. Opel, D., Poremba, C., Simon, T., Debatin, . M. & Fulda, S. Activation of At predicts poor outcome in neuroblastoma. Cancer
research 67, 735–745 (2007).
42. Charavarti, A. et al. e prog nostic signicance of phosphatidylinositol 3-inase pathway activation in human gliomas. Journal of
clinical oncology: ocial journal of the American Society of Clinical Oncology 22, 1926–1933 (2004).
43. Li, X. et al. PI3/At/mTO signaling pathway and targeted therapy for g lioblastoma. Oncotarget (2016).
44. ota, B. et al. STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in
patients with glioblastoma. Journal of neurosurgery 121, 374–383 (2014).
45. Meng, D. et al. High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human
glioblastoma. Oncotarget 6, 4901–4919 (2015).
46. Liang, J., Piao, Y., Henry, V., Tiao, N. & de Groot, J. F. Interferon-regulatory factor-1 (IF1) regulates bevacizumab induced
auto phagy. Oncotarget (2015).
47. umar, S. et al. Development of a novel animal model to dierentiate radiation necrosis from tumor recurrence. Journal of neuro-
oncology 108, 411–420 (2012).
48. Shanar, A. et al. Subcurative radiation signicantly increases cell proliferation, invasion, and migration of primary glioblastoma
multiforme in vivo. Chinese journal of cancer 33, 148–158 (2014).
49. deCarvalho, A. C. et al. Gliosarcoma Stem Cells Undergo Glial and Mesenchymal Dierentiation In Vivo . Stem Cells 28, 181–190 (2010).
50. Arbab, A. S. et al. Magnetically-labeled sensitized splenocytes to identify glioma by MI: a preliminary study. Mag n eson Med 58,
519–526 (2007).
Acknowledgements
e authors thank the sta of the Georgia Cancer Center core facilities for their ecient assistance. e authors
also thank Rhea-Beth Markowitz, PhD for providing editing assistance in the manuscript and Ana de Carvalho
of Dr. Tom Mikkelsen’s group for providing HF2303 cells. is study is supported by the National Institutes of
Health (NIH) grants R01CA160216, R01CA172048 and Georgia Cancer Center startup funds.
Author Contributions
M.J., B.R.A. and A.S.A., designed research, performed the experiments, interpreted data and wrote manuscript.
M.A., A.I., T.F.B. conducted animal orthotopic injections and prepared paran blocks for histology. A.S., M.H.R.
and K.A., performed the immunohistochemistry and H & E staining. Z.W. conducted mass spectroscopy for
pharmacokinetic studies and R.A. conducted MRI and tumor volume analysis. M.M.A., N.H.G., designed the
composition of HET0016. I.L., A.G., performed the synthesis of HET0016. W.B.C. performed the MRI analysis
and H.B. helped in formulation of mathematical model for MRI analysis.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Jain, M. et al. Intravenous Formulation of HET0016 Decreased Human Glioblastoma
Growth and Implicated Survival Benet in Rat Xenogra Models. Sci. Rep. 7, 41809; doi: 10.1038/srep41809
(2017).
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
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© e Author(s) 2017
... When N-hydroxy-N'-(4-butyl-2methylphenyl) formamidine(HET0016), a highly selective inhibitor of 20-HETE synthesis, is used alone in tumor-bearing animals, tumor neovascularization is reduced (61,62). When the level of different pro-and anti-angiogenic factors in tumor lysates is detected, prominent changes occur after HET0016 treatment compared with placebo-treated tumors (63,64). When certain indicators, including extravascular cell space (EES), vascular parameters and tumor angiogenesis, are analyzed, HET0016 treatment reduces EES, tumor blood volume, permeability and tumor angiogenesis (63,64). ...
... When the level of different pro-and anti-angiogenic factors in tumor lysates is detected, prominent changes occur after HET0016 treatment compared with placebo-treated tumors (63,64). When certain indicators, including extravascular cell space (EES), vascular parameters and tumor angiogenesis, are analyzed, HET0016 treatment reduces EES, tumor blood volume, permeability and tumor angiogenesis (63,64). In the field of cancer, triple-negative breast cancer is not sensitive to chemotherapeutics. ...
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