A Hydrogen Peroxide Activatable Gemcitabine Prodrug for the Selective Treatment of Pancreatic Ductal Adenocarcinoma

Article abstract of DOI:10.1002/cmdc.201900324

The main concern in the use of anticancer chemotherapeutic drugs is host toxicity. Patients need to interrupt or change chemotherapy due to adverse effects. In this study, we aimed to decrease adverse events with gemcitabine (GEM) in the treatment of pancreatic ductal adenocarcinoma and focused on the difference of hydrogen peroxide levels in normal versus cancer cells. We designed and synthesized a novel boronate-ester-caged prodrug that is activated by the high H₂O₂ concentrations found in cancer cells to release GEM. An H₂O₂-activatable GEM (A-GEM) has higher selectivity for H₂O₂ over other reactive oxygen species (ROS) and cytotoxic effects corresponding to the H₂O₂ concentration in vitro. A xenograft model of immunodeficient mice indicated that the effect of A-GEM was not inferior to that of GEM when administered in vivo. In particular, myelosuppression was significantly decreased following A-GEM treatment compared with that following GEM treatment.

Full text of DOI:10.1002/cmdc.201900324

Accepted Article
Title: A Hydrogen Peroxide-Activatable Gemcitabine Prodrug for the
Selective Treatment of Pancreatic Ductal Adenocarcinoma
Authors: Katsunori Matsushita, Takumi Okuda, Shohei Mori,
Masamitsu Konno, Hidetoshi Eguchi, Ayumu Asai, Jun
Koseki, Yoshifumi Iwagami, Daisaku Yamada, Hirofumi
Akita, Tadafumi Asaoka, Takehiro Noda, Koichi Kawamoto,
Kunihito Gotoh, Shogo Kobayashi, Yuuya Kasahara, Kunihiko
Morihiro, Taroh Satoh, Yuichiro Doki, Masaki Mori, Hideshi
Ishii, and Satoshi Obika
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using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900324
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900324
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10.1002/cmdc.201900324
ChemMedChem
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A Hydrogen Peroxide-Activatable Gemcitabine Prodrug for the
Selective Treatment of Pancreatic Ductal Adenocarcinoma
#
#
[b,d]#
Katsunori Matsushita,[a,b]# Takumi Okuda, Shohei Mori, Masamitsu Konno,
Hidetoshi Eguchi,
[c]
[c]
[a]
Ayumu Asai,[b,d] Jun Koseki, Yoshifumi Iwagami, Daisaku Yamada,
Hirofumi Akita, Tadafumi
[b,d]
†1[a]
[a]
[a]
†2[a]
[a]
[a]
[a]
[a]
Asaoka, Takehiro Noda, Koichi Kawamoto,
Kunihito Gotoh, Shogo Kobayashi, Yuuya
Kasahara,[c,e] Kunihiko Morihiro,†3[c,e] Taroh Satoh, Yuichiro Doki, Masaki Mori,
and Satoshi Obika*[c,e]
Hideshi Ishii,
[b]
†4[a]
*
[d]
[a]
[a]
K Matsushita, Dr. H Eguchi, Dr. Y Iwagami, Dr. D Yamada, Dr. H Akita, Dr. T Asaoka, Dr. T Noda, Dr. K Kawamoto, Dr. K Gotoh, Dr. S Kobayashi, Prof. Dr.
Y Doki, Prof. Dr. M Mori
[b]
K Matsushita, Dr. K Konno, Dr. A Asai, Dr. J Koseki, Prof. Dr. T Satoh
Department of Frontier Science for Cancer and Chemotherapy, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871,
Japan.
[c]
[d]
[e]
T Okuda, Dr. S Mori, Dr. Y Kasahara, Dr. K Morihiro, Prof. Dr. S Obika
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
E-mail: obika@phs.osaka-u.ac.jp
Dr. M Konno, Dr. A Asai, Dr. J Koseki, Prof. Dr. H Ishii
Department of Medical Data Science, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
E-mail: hishii@gesurg.med.osaka-u.ac.jp
Dr. Y Kasahara, Dr. K Morihiro, Prof. Dr. S Obika
National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
†1 Present address: Department of Digestive Surgery Osaka International Cancer Institute Osaka Japan.
†2 Present address: Kinki Regional Bureau of Health and Welfare, Ministry of Health, Labour and Welfare
†3 Present address: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo
†4 Present address: Department of Surgery and Science, Graduate Schools of Medical Sciences, Kyusyu University
Supporting information for this article is given via a link at the end of the document.
1
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Abstract: The major concern in the use of anticancer
chemotherapeutic drugs is host toxicity. Patients need to interrupt or
change chemotherapy due to adverse effects. In this study, we
aimed to decrease adverse events on gemcitabine (GEM) in the
treatment of pancreatic ductal adenocarcinoma and focused on the
difference of hydrogen peroxide (H2O2) levels in normal cells and
cancer cells. We have designed and synthesized a novel boronate
ester-caged prodrug that is activated by the high H2O2 concentration
found in cancer cells to release GEM. An H2O2-activatable GEM (A-
GEM) has higher selectivity for H2O2 over other ROS and cytotoxic
effects corresponding to the H2O2 concentration in vitro. A xenograft
model of immunodeficient mice indicated that the effect of A-GEM
was not inferior to that of GEM, when administered in vivo. In
particular, myelosuppression was significantly reduced following A-
GEM treatment compared with that following GEM treatment.
promising anticancer activities and few toxic side effects by
focusing on the differences between normal and cancer
cells.[18,19] Cancer cells have been reported to exhibit
abnormalities of metabolism and signal transduction.[20,21] These
characteristics of cancer cells could be important in the
development of new anticancer strategies.
A higher level of oxidative stress is observed in various cancer
cells and tumors and results in the overproduction of reactive
oxygen species (ROS).[22-25] H2O2 is a well-known ROS and has
recently been shown to function as an important secondary
messenger in biological systems.[26,27] H2O2 has high membrane
permeability and is a relatively stable ROS.[28,29] In particular, the
higher intrinsic H2O2 concentrations in cancer cells induce the
expression of growth factors that lead to migration and
invasion.[30] Relatively high H2O2 concentrations compared to
normal cells are a distinctive feature of cancer cells[31,21] and
several approaches have been implemented to develop H2O2-
activatable prodrugs for targeting tumors.[33-35]
Introduction
In synthetic organic chemistry, alkyl or arylboronic acids and
their esters are easily dissociated by H2O2. The reaction
between boronates and H2O2 is bioorthogonal and
biocompatible. Indeed, boronated H2O2-activatable small
molecules have been used for H2O2 detection,[36] selective gene
activation,[37] and in cancer therapeutics.[33,34] Boronic acids and
their esters do not appear to be intrinsically toxic and their
hydrolysis end product, boric acid, is considered to be non-toxic
to humans.[38] Additionally, we recently reported specific gene
silencing using aryl-boronated antisense oligonucleotides.[39]
In this study, an H2O2-activatable GEM (A-GEM) was
developed. Under specific conditions in vitro, this novel prodrug
demonstrated anti-tumor activity comparable with that of GEM.
Moreover, we found that interestingly dosed A-GEM exhibited a
unique biodistribution and the potential for efficient drug delivery
into cancer cells in vivo.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most
aggressive types of tumor, with a 5-year survival rate of < 5%.[1-
3]
PDAC is predicted to become the second leading cause of
cancer-related death in the United States by 2030.[4] Surgical
resection remains the only curative treatment, but only 20% of
affected patients are suitable for curative resection, mainly due
to rapid growth, high invasiveness, and metastasis.[5] Surgeons
have developed novel and improved ways of managing PDAC.[6]
Recent reports suggest that adjuvant chemotherapy following
curative surgery significantly contributes to prolong the overall
patient survival time after surgery to remove the pancreatic
cancer.[7,8] Gemcitabine (GEM) is the first-line drug currently
available for treating pancreatic cancer in clinics.[9,10] GEM is
intracellularly phosphorylated by deoxycytidine kinase to GEM
5'-diphosphate and GEM 5'-triphosphate, which are incorporated
into DNA, leading to apoptosis.[11] The common adverse events
of GEM include nausea, vomiting, fever, reversible elevation of
liver transaminases and peripheral edema.[12] Myelosuppression
is the major dose-limiting toxicity[13] and the adverse event lead
to therapy discontinuation.[14] GEM is effective against a variety
of cancers and is used alone or in combination with other
drugs.[15,16] When GEM is used in combination with other
chemotherapeutic drugs, reducing the adverse events due to
GEM is crucial to reduce overall adverse events. In this study,
we aimed to decrease the adverse events due to GEM
administration. One reported approach to reducing the toxicities
of crosslinking anticancer agents in normal cells is to selectively
activate the prodrug in cancer cells.[17] Several molecular-
targeted drugs such as imatinib and trastuzumab exhibit
Results
Design and synthesis of H2O2-activatable gemcitabine
prodrug (A-GEM)
To develop a prodrug of GEM, the enzyme recognition site of
GEM must be temporarily masked using a stimulus-responsive
chemical structure. Intracellularly, GEM is converted to
therapeutically
active
GEM
5'-diphosphate
and
5’-
triphosphate[40] metabolites by sequential phosphorylation with
multiple kinases. The initial step, also the rate-limiting step, is
the monophosphorylation of GEM, which is catalyzed by
2
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10.1002/cmdc.201900324
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deoxycytidine kinase.[11] Enzyme-substrate recognition between
deoxycytidine kinase and GEM relies on hydrogen bonding
between the 4-NH2 group of the nucleobase cytosine and Asp
133, located in the active site of the enzyme.[41] Chemically and
enzymatically activatable GEM prodrugs have been developed
by installing a protecting group onto the 4-NH2 group.[42-44] Thus,
A-GEM was also designed by masking the same position in
GEM with a boronate ester-based carbamate protecting group
(Figure 1). This masking group was expected to be promptly
removed under high H2O2 concentrations in tumor cells,
GEM within 20 min (95% yield, Figure 2a). In contrast, no
conversion or degradation was observed when the negative
a
O
NH2
N
HN
O
O
O
N
B
O
N
O
N
F
HO
HO
F
F
O
O
OH
GEM
OH
F
A-GEM
95%
52%
28%
20
5
1
O
O
H2O2
0
HN
O
O
HN
O
O
5
10
15
20
0
O
Retention time (min)
N
N
B
O
b
N
F
N
F
HO
HO
O
O
O
HN
O
O
OH
F
OH
F
N
A-GEM (Inactive)
C-GEM (Inactive)
N
F
HO
O
H2O2
H2O2
OH
F
O
C-GEM
H2O
60
NH2
N
HN
O
O
H2O2
0
O
N
O
B
5
10
Retention time (min)
15
20
0
N
O
N
F
HO
HO
O
F
F
O
O
control C-GEM was exposed to H2O2 under similar conditions
OH
B
OH
OH
F
(Figure 2b).
O
O
GEM (Active)
+
CO2
+
O
Figure 2. HPLC chromatograms of A-GEM (a) and C-GEM (b) after H2O2
addition at different time points.
resulting in the release of the active drug. In addition, 4-N-Cbz-
2'-deoxy-2',2'-difluorocytidine (C-GEM)[44] was also designed as
a negative control (Figure 1).
Since H2O2 is not the only ROS in human biological systems, we
evaluated the activation of A-GEM by other ROS, such as tert-
butylhydroperoxide (TBHP), hypochlorite (ClO-), hydroxyl radical
(HO·), tert-butoxy radical (tBuO·), nitric oxide (NO), and
Figure 1. Schematic representation of H2O2–triggered A-GEM activation.
H2O2–mediated hydrolysis and release of GEM.
-
The prodrug A-GEM was synthesized according to our previous
report[39] (Scheme S1). Briefly, to introduce the H2O2-responsive
boronate ester moiety into the cytosine, a commercially available
4-(hydroxymethyl)phenylboronic acid pinacol ester was used
and the benzyl alcohol derivative was converted to an imidazole
derivative in two steps. The imidazole derivative was exposed to
Meerwein reagent to synthesize a highly reactive imidazolium
salt in situ, which was then reacted with TIPDS-protected GEM.
TBAF treatment degraded the carbamate moiety, and therefore
HF-pyridine for silyl deprotection, to generate A-GEM.
superoxide (O2 ). The activation of A-GEM was highly selective
for H2O2 compared with the aforementioned ROS species
(Figure S1). In addition to aforementioned ROS, we also
evaluated the activation of A-GEM by peroxynitrite (ONOO-),
which is a potent oxidant of boronate structures.[45] However, the
deprotection of A-GEM did not occur in the same reaction
conditions. This may be due to the poor chemical stability of
ONOO- in neutral buffer. An equimolecular amount of ONOO-
was not enough to activate the A-GEM.
H2O2-decaging of A-GEM
The conversion yield of A-GEM to GEM following H2O2
treatment was determined using HPLC analysis. When exposed
to an equimolar equivalent of H2O2 in sodium phosphate buffer
(pH 7.2), virtually all of the A-GEM prodrug was converted to
3
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Figure 3. Evaluation of cytotoxicity effect and alteration in apoptosis of GEM, A-GEM, and C-GEM in each cell line. The mean ± standard deviation (SD) is
depicted. Significant differences were observed between the values under the horizontal lines (*P < 0.05, **P < 0.01). (a) Growth-inhibitory effects of each
GEM are shown. IC50of each GEM, A-GEM, and C-GEM in each cell line as assessed by the MTT assay. (b) The H2O2 level of two PDAC cell lines and NPEC
is shown.(c and d) Alterations in apoptosis in cell lines with each GEM for 72 hours with the IC50 of A-GEM. (c)The fold-change of Caspase3/7 activity is
represented. The Caspase3/7 activity of DMSO was set as the control. (d) The ratio of apoptotic cells was determined using the Annexin V assay.
Cytotoxicity of A-GEM in human pancreatic cancer cell lines
and normal pancreatic epithelial cell line
C-GEM (Fig. 3c and d). Next, the activity of A-GEM was
evaluated in vitro in the presence of H2O2. The proliferation of
PDAC cells alone with H2O2 after 72 h was evaluated to
determine optimal H2O2 concentration required to inhibit this
proliferation. Optimal H2O2 concentrations for PSN1 and BxPC3
were 250 and 125 μM, respectively (Figure S2a). The
cytotoxicity of C-GEM in the presence of optimal H2O2
concentration was similar to that of C-GEM alone. In contrast,
the cytotoxicity of A-GEM in the presence of H2O2 was elevated
compared with that of A-GEM alone and was similar to that of
GEM (Figure S2b).
Cytotoxicity of GEM, A-GEM, and C-GEM was evaluated using
human pancreatic cancer cell lines (PSN1 and BxPC3) and
normal pancreatic epithelial cell line (NPEC) using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay after 72 h of exposure. As shown in Fig. 3a, all three
drugs inhibited cell growth in a dose-dependent manner.
Osmotic pressure and other such factors may have affected the
cytotoxicity exhibited at high concentrations of C-GEM. The
cytotoxicity of C-GEM may also be due to its partial deprotection
in cells independently of H2O2. Compared with two pancreatic
cancer cell lines, NPEC showed little difference in cytotoxic
effect between A-GEM and C-GEM. The H2O2 level of NPEC
was smaller than that of PDAC cell lines (Figure 3b). GEM is
reported to exhibit cytotoxic effects via the induction of apoptosis.
To quantify the level of cellular apoptosis, both caspase-3/7
activation and flow cytometry analysis of annexin V were
performed. PDAC cells were exposed to GEM, A-GEM, and C-
GEM for 72 h at the IC50 concentration of A-GEM. The apoptotic
frequency of A-GEM was reduced compared to that of GEM.
However, A-GEM had an increased cytotoxic effect compared to
4
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Figure 4. The synergistic effect of 2DG at the optimal concentrations with GEM, A-GEM, and C-GEM using MTT and apoptosis assays. The mean ± standard
deviation (SD) is shown. Significant differences were observed between the values under the horizontal lines (*P < 0.05, **P < 0.01, n.s.; not significant). (a)
The 2DG concentration that did not inhibit the proliferation of each cell was determined. (b) Growth inhibitory effects of GEM, A-GEM, and C-GEM plus 2DG at
the optimal concentration are shown. (c and d) Alterations in apoptosis in cell lines with each GEM alone and each GEM plus 2DG for 72 hours with the IC50 of
A-GEM. (c) The fold-change of Caspase3/7 activity is represented. The Caspase3/7 activity of DMSO was set as the control. (d) The ratio of apoptotic cells was
determined using the Annexin V assay.
An attempt was made to elevate H2O2 concentration in
pancreatic cancer cells. Reportedly, glucose deprivation induces
oxidative stress in cancer cells in humans. Oxidant production
and thiol metabolism disruption consistent with metabolic
oxidative stress have also been noted in cancer cells during
glucose deprivation or upon treatment with the glucose analogue
2-deoxy-D-glucose (2DG).[46] Initially, the proliferation of
pancreatic cancer cells in the presence of 2DG alone was
assessed, and subsequently, the 2DG concentration that did not
inhibit the proliferation of pancreatic cancer cells was
determined. The optimal 2DG concentrations for PSN1 and
BxPC3 were 62.5 and 31.2 nM, respectively (Figure S3).
Intracellular H2O2 concentrations were elevated after
administration of 2DG at these optimal concentrations (Figure
4a). We then evaluated the synergistic effect of 2DG at the
optimal concentrations with GEM, A-GEM, and C-GEM using
MTT and apoptosis assays. Cytotoxic effects and apoptotic
frequencies in the presence of 2DG alone were similar to those
in the presence of DMSO as a solvent or mock control. GEM
and C-GEM did not exhibit a synergistic effect with 2DG;
however, A-GEM exhibited a synergistic effect with 2DG, as
identified using both MTT (Figure 4b) and apoptosis assays
(Figure 4c and d).
5
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Figure 5. In vivo antitumor activity and adverse events. The mean ± standard deviation (SD) is shown. Significant differences were observed between the
values under the horizontal lines (*P < 0.05, ns; not significant). (a) The time course of the animal experiment. (b and c) Tumor volume (b) and the actual
tumors (c) are shown. (d) Myelosuppression was evaluated by comparing blood components before and after the treatment. The variability represents mean
value ratio after/before treatment. Differences among white blood cells, neutrophil, monocyte, lymphocyte, hemoglobin, and platelet levels before and after
the treatment are indicated.
In vivo antitumor activity of A-GEM in a murine xenograft
model
lesser than that in the GEM group (Figure S5). Hematopoietic
stem cells are located in niches characterized by low levels of
ROS, unlike cancer cells.[47,48] The difference in ROS levels,
including H2O2, in the tissue influenced selectivity and reactivity
of A-GEM.
Mice were randomly divided into three groups (n = 6 each): GEM,
A-GEM, and vehicle. The molecular weight of A-GEM is twice
the molecular weight of GEM. Thus, to equalize doses, the GEM
group received 50 mg/kg of GEM and the A-GEM group
received 100 mg/kg of A-GEM. The mice were administered
GEM, A-GEM, or vehicle intraperitoneally four times on days 14,
17, 20, and 23 (Figure 5a). There was no difference in body
weight loss between the GEM and A-GEM groups (Figure S4a).
A-GEM had an antitumor effect compared with the vehicle.
There was no difference in tumor volume between the GEM and
A-GEM groups (Figure 5b and c). Histological findings from the
TUNEL assay revealed no differences in apoptotic frequency
between the GEM and A-GEM groups (Figure S4b).
Discussion
This study has two important findings. First, A-GEM selectively
reacted to H2O2 among ROS species used in this study. Second,
there was a difference in reactivity for A-GEM and C-GEM in
PDAC cells and NPEC. Myelosuppression derived from A-GEM
in vivo was less severe than that of original GEM. These results
indicated that A-GEM has the potential to decrease adverse
events in pancreatic ductal adenocarcinoma in the clinical
setting.
Very interestingly, myelosuppression was reduced in the A-
GEM group compared with the GEM group (Figure 5d). To
investing the cause, GEM accumulation in tumor and bone
marrow was evaluated using LC/MS. A similar amount of GEM
was detected in the tumor tissue of both the GEM and A-GEM-
treated groups. This result suggested that the majority of A-GEM
was converted to GEM in the tumor. In contrast, in the bone
marrow the detected amount of GEM in the A-GEM group was
Recent studies suggest that targeting unique biochemical
alterations in cancer cells is a feasible approach to achieve both
therapeutic activity and selectivity.[38,43] Reactive ROS
homeostasis is important for the survival and progression of both
normal and cancerous cells. Although certain amounts of ROS
are required for proper cell function including normal metabolism
and signaling, excessive amounts of them lead to oxidative
6
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stress.[24] The level of ROS in malignant cells is higher than that
in normal cells. Among ROS, H2O2 is comparatively stable and
one of the principal ROS members. Although H2O2-activatable
prodrugs have been reported so far, to the best of our
knowledge, this is the first example for a prodrug of gemcitabine
bearing the phenyl boronate moiety. The protecting group of A-
GEM was expected to be promptly removed under high H2O2
concentrations in tumor cells, resulting in the release of the
active drug.
myelosuppression compared to GEM, suggesting that H2O2-
targeted modifications can improve the selectivity of cytotoxic
chemotherapeutic
reagents.
We
anticipate
numerous
applications of A-GEM, given its unique combination of high
efficacy and low toxicity.
Experimental Section
In this study, we demonstrated the H2O2 level in PDAC
cells was larger than in normal pancreatic epithelial cell.
The difference of H2O2 level may be comparatively small
between PDAC cells and NPEC because NPEC was not
normal completely by being immortalized with Human
Papilloma Virus. We were unable to quantify H2O2 in cancer
cells and normal cells in vivo, but we successfully
demonstrated the cytotoxicity and selectivity of A-GEM by
evaluating apoptosis and cell viability in vitro and in vivo. It
seems that A-GEM was converted to GEM intracellularly
because its cytotoxicity was increased when the amount of
H2O2 in the cancer cell was raised in vitro. A-GEM had an
antitumor effect and induced apoptosis in vivo. In addition,
the amount of A-GEM was equivalent in cancer tissues
compared to GEM, whereas in the bone marrow tissue the
amount was alleviated by using mass spectrometry.
Hematopoietic stem cells are located in niches
General
Purchased reagents and solvents were used without purification unless
otherwise specified. All experiments involving air and/or moisture
sensitive compounds were conducted under an argon atmosphere. All
reactions were monitored using analytical TLC (Merck Kieselgel 60 F254;
Merck, Darmstadt, Germany). Flash column chromatography was carried
out using an EPCLC–W–Prep 2XY (YAMAZEN, Osaka, Japan). NMR
spectra were obtained using a JNM–ECS–400 spectrometer (JEOL,
Tokyo, Japan) using CDCl3 or DMSO–d6 as
tetramethylsilane as an internal standard. IR spectra were obtained using
FT/IR–4200 spectrophotometer (JASCO, Tokyo, Japan). Optical
a
solvent with
a
rotations were obtained on a JASCO P–2200 instrument. FAB mass
spectra were obtained using a JEOL JIM–700 mass spectrometer. ESI
mass spectra were obtained using a Xevo G2-XS QTof (Waters, Milford,
MA, USA).
H2O2-decaging of A-GEM
A reaction solution comprising A-GEM (1 mM) and H2O2 (1 mM) in a
DMSO-containing buffer (10 mM potassium phosphate, pH 7.2, 100 mM
NaCl, and 5% (v/v) DMSO) was incubated at room temperature for the
prescribed duration and immediately subjected to reverse-phase HPLC
analysis using MeCN in 0.1 M triethylammonium acetate buffer (pH 7.0)
as a solvent. The conversion rate of A-GEM was determined from the
corresponding peak area monitored at 260 nm.
47,48
]
characterized by low levels of ROS, unlike cancer cells.[
The difference in ROS levels, including H2O2, in the tissue
influenced selectivity and reactivity of A-GEM This
.
indicated that A-GEM also functions as an active form in
vivo. Although we have not determined the limiting tolerable
dose of A-GEM, it is considered that there is a possibility
that antitumor effect can be enhanced with the same
degree of side effects as GEM by further increasing the
Other ROS-decaging of A-GEM
A reaction solution of A-GEM (1 mM) and reactive oxygen species (1
mM) in a DMSO-containing buffer (10 mM potassium phosphate, pH 7.2,
100 mM NaCl, and 5% (v/v) DMSO) was incubated at room temperature
for 12 h and immediately subjected to reversed-phase HPLC analysis
using MeCN in 0.1 M triethylammonium acetate buffer (pH 7.0). tert-
Butylhydroperoxide (TBHP) and hypochlorite (NaOCl) were delivered
from 70% and 10% aqueous solutions respectively. Hydroxyl radical
(HO·) and tert-butoxy radical (tBuO·) were generated by the reaction of 5
amount of A-GEM
.
Conclusions
A-GEM selectively reacted to H2O2 rather than other ROS. In
vitro experiments showed A-GEM had less cytotoxicity in NPEC
than in PDAC cells by reacting H2O2 level. A-GEM induced
certain apoptosis than C-GEM and DMSO. A-GEM increased
cytotoxicity in PDAC cell lines by H2O2 production induced by
2DG treatment, while C-GEM and DMSO did not increase. A-
GEM also showed anticancer effect equivalent to that of GEM
mM (NH4)2Fe(SO4)2, 10 mM EDTA with
1 mM H2O2 or TBHP,
respectively. Nitric oxide (NO) was generated from PROLI NONOate.
Superoxide (O2 ) was produced by xanthine oxidase (4.5 × 10-3 mg/100
-
µL) in the presence of hypoxanthine (2 mM) and catalase (0.4 mg mL-1).
Peroxynitrite (ONOO-) was delivered from NaOH aqueous solution and
the concentration of ONOO- was determined using the absorption at 300
nm (ε = 1670 M-1cm-1). The solution was diluted with phosphate buffer
and used immediately. The conversion rate of A-GEM was determined
from the corresponding peak area monitored at 260 nm.
when it was administered to
a
xenograft model of
immunodeficient mice. In addition, it exhibited reduced
7
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calculated as follows: (greatest diameter) × (shortest diameter)2 × 0.5.
Treatments with all GEM and vehicle were initiated when the tumor
volume reached 60–100 mm3. Each GEM was dissolved in 20 μL of
ethanol, 80 μL of PEG, and 100 μL of physiological saline. Each mouse
was administered a total of 200 μL of drug solution. Tumor size and body
weight were measured every 3 days. Xenograft tumors were collected
after sacrificing the mice on day 25, and TUNEL assay was performed on
tumor tissue sections. All tumors of mice were fixed in 10%
formaldehyde for 24ꢀh, embedded in paraffin, and sectioned into 4-μm
slices. We stained the tumor tissue sections of mice with hematoxylin
and eosin. Then, the apoptosis assay was performed using a TdT-
mediated dUTP nick end labeling (TUNEL) kit (Cat. No. G3250,
Promega) according to the manufacturer’s instructions. Finally, nuclei
were visualized using DAPI-containing mounting medium (Cat. No. H-
1200, Vector Laboratories, Burlingame, CA, USA). The images were
captured using a BZ-8000 microscope (Keyence, Tokyo, Japan).
Myelosuppression was evaluated by comparing blood components
before and after the treatment. Myelosuppression was evaluated using a
Vetscan HM2 Analyzer (ABAXIS, Union City, CA). The counts of white
blood cells, neutrophils, monocytes, lymphocytes, and platelets and the
level of hemoglobin before and after treatment were evaluated to observe
whether any hazardous effects occurred in the examined conditions. The
results represent mean value ratio after/before treatment.
Cell culture
Pancreatic cancer cell lines used in this study (PSN1 and BxPC3) were
procured from the Japan Cancer Resource Bank (Tokyo, Japan). Normal
pancreatic epithelial cell (NPEC) was procured from Addexbio
Technology (San Diego, USA). Cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum
containing penicillin (100 IU/ml) and streptomycin (100 μg/ml), and
incubated at 37 °C in a humidified incubator with a 5% CO2 atmosphere.
Cell proliferation assay
To assess the proliferation and sensitivity of pancreatic cancer cells to
2DG (FUJIFILM Wako Chemical Corporation, Osaka, Japan), they were
incubated in the presence of several concentrations of 2DG for 72 h in a
96-well plate. Cell viability was evaluated using the Cell Counting Kit-8
(CCK-8; Dojindo Molecular Technologies, Tokyo, Japan).
Growth inhibition assay and determination of cell viability
Growth inhibition was assessed using MTT assay as previously
described.[49 In brief, cells were incubated for 72 h under varying
]
concentrations of GEM and cell viability was then evaluated using MTT
assay. The results were expressed as the percentage of absorbance
relative to that of the untreated controls.
Statistical analysis
Apoptosis assay
Differences were considered statistically significant at P ≤ 0.05. All
statistical analyses were performed using SPSS 20.0 software (IBM
Japan, Tokyo, Japan).
To quantify the level of cellular apoptosis, both caspase-3/7 activation
and flow cytometric analyses of annexin V were performed. PDAC cells
were exposed for 72 h to GEM, A-GEM, or C-GEM at the 50% inhibitory
concentration (IC50) of A-GEM. Caspase-3/7 activity was evaluated using
the caspase-Glo® 3/7 Assay Kit from Promega (Madison, WI, USA) and
relative luminescence (RLU) was measured using the GloMax®
Microplate Luminometer (Promega). Apoptotic cells that were stained
with annexin V-APC (Biolegend Research Products, San Diego, CA,
USA) or propidium iodide (BioVision Research Products, Milpitas, CA,
USA) were enumerated using flow cytometry using a BD FACS CantoTM
II system (BD Biosciences, SanJose, CA, USA).
Sample preparation for LC/MS analysis
Snap-frozen tissues obtained at necropsy were weighed; Deionized
water (100 μL) was added to homogenize the sample using a Violamo
Homogenizer Mixer VH-10 (AS ONE, Osaka, Japan). Deionized water
(200 μL), methanol (100 μL), and chloroform (200 μL) were added to
homogenized tissue (50 mg) and the resultant sample was incubated at
70 °C for 10 min with vigorous shaking. Vortex mixing was followed by
centrifugation at 19000 rpm for 15 min, and the resulting supernatant was
concentrated by freeze-drying. The residue was reconstituted in 200 μL
of water, of which 10-μL samples were used for LC/MS.
H2O2 quantification assay
H2O2 was quantified using a ROS-Glo H2O2 assay kit (Promega) and
relative luminescence (RLU) was measured using a GloMax® Microplate
Luminometer (Promega). The H2O2 quantification of PDAC cell lines and
NPEC was evaluated 12 hours after seeding each cell not to change the
counts of cells.
Acknowledgements
This work received financial support from grants-in-aid for
Scientific Research from the Japan Agency for Medical
Research and Development (AMED) (18ck0106372h0002 (to M.
Mori)); from the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT), and the Japan Society for the
Promotion of Science (JSPS) (KAKENHI grant nos. 17H04282
(to H. Ishii), 17K19698 (to H. Ishii), 16K15615 (to M. Konno),
and 15H05791 (to M. Mori)); from the Platform Project for
Supporting Drug Discovery and Life Science Research (Basis for
In vivo experiments
This study was approved by the Animal Experiments Committee, Osaka
University (approval number: 30-011-008). It was performed in
accordance with the National Institute of Health guidelines for the use of
experimental animals. Eight-week-old, female, congenitally athymic nude
mice (nu/nu) with immunodeficiency were purchased from CLEA Japan
and were maintained in a pathogen-free environment. For xenografting,
PSN1 cells (1 × 106 cells) were subcutaneously transplanted in 100 μL
PBS/Matrigel (BD Biosciences). Subcutaneous tumor volume was
8
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10.1002/cmdc.201900324
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Thayer, C. R. Ferrone, C. Huttenhower, S. N. Bhatia, D. Gevers, J. A.
Wargo, T. R. Golub, R. Straussman, Science 2017, 357(6356), 1156-
1160.
Supporting Innovative Drug Discovery and Life Science Research)
(BINDS) from the AMED (JP18am0101084), and the Basic Science
and Platform Technology Program for Innovative Biological Medicine
from the AMED (JP18am0301004) (to S. Obika). S. Mori is
grateful for a Research Fellowship for Young Scientists from
JSPS. Institutional endowments were received by YD, MM, and
HI from Taiho Pharmaceutical Co. Ltd., Unitech Co. Ltd. (Chiba,
Japan), IDEA Consultants Inc. (Tokyo, Japan), and Kinshu-kai
Medical Corporation (Osaka, Japan), and by YD, MM, TS from
Chugai Co. Ltd., Yakult Honsha Co. Ltd., and Merck Co. Ltd.
Sponsors had no role in the study design or performance, data
collection, management, or interpretation, or article preparation
and approval.
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Author Information
These authors contributed equally: Katsunori Matsushita, Takumi Okuda,
Shohei Mori and Masamitsu Konno.
Contribution
The individual contributions were as follows:
KMab, TOc, HId: evaluation for feasibility and efficacy of the compound in vitro
and in vivo.
TOc, SOce: synthesis and monitoring by mass spectrometry of the compound
SMc, SOce: synthesis of the compound and evaluation of chemical stability
MKab, TOc, HId: evaluation of the compound in mice.
TOc, SMc, SOce: design and synthesis of the compound
KMab, MKbd, HEa, TSa, YDa, MMa, HId, SOce: study conception
KMab, TOc, SMc, MKbd, YKce, KMce, SOce: study design for the compound
KMab, HEa, AAbd, JKbd, YIa, DYa, HAa, TAa, TNa, KKa, KGa, SKa, HId: Study
design for feasibility
KMab, HEa, AAbd, JKbd, YIa, DYa, HAa, TAa, TNa, KKa, KGa, SKa, SOce: Study
design for efficacy
KMab, HId: in vitro study
KMab, TOc, MKbd, HId: In vivo study
KMab, TOc, SMc, MKbd: acquisition of data
All authors: analysis and interpretation of the data
The compound were continuously designed and synthesized with in vitro and
in vivo feedback data, and two authors (HI and SO) were equally
10
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Entry for the Table of Contents
This article reports on the development of a new gemcitabine prodrug (A-GEM), which has an
arylboronate moiety at the N4-position of cytosine nucleobase. A-GEM was activated in the presence
of hydrogen peroxide (H2O2) that is found in cancer cells, and it showed less myelosuppression in an
immunodeficient model mouse compared to gemcitabine while maintaining the anti-tumor activity.
11
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