Distribution and metabolism of 14C-sulfoquinovosylacylpropanediol (14C-SQAP) after a single intravenous administration in tumor-bearing mice

Article abstract of DOI:10.1080/00498254.2018.1448949

Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65.For the metabolism analysis, a 2?mg (2.98?MBq)/kg of [glucose-U-¹⁴C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body.The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-¹⁴C]-SQAP (CP-3635) was administered through intravenous injection of a 1?mg (3.48?MBq)/kg dose followed by whole body autoradiography of the mice.The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney.Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.

Full text of DOI:10.1080/00498254.2018.1448949

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Distribution and metabolism of 14C-
Sulfoquinovosylacylpropanediol (14C-SQAP) after
a single intravenous administration in tumor-
bearing mice
Tatsushi Ruike, Yoshihiro Kanai, Kazuki Iwabata, Yuki Matsumoto, Hiroshi
Murata, Masahiro Ishima, Keisuke Ohta, Masahiko Oshige, Shinji Katsura,
Koji Kuramochi, Shinji Kamisuki, Hiroeki Sahara, Masahiko Miura, Fumio
Sugawara & Kengo Sakaguchi
To cite this article: Tatsushi Ruike, Yoshihiro Kanai, Kazuki Iwabata, Yuki Matsumoto,
Hiroshi Murata, Masahiro Ishima, Keisuke Ohta, Masahiko Oshige, Shinji Katsura, Koji
Kuramochi, Shinji Kamisuki, Hiroeki Sahara, Masahiko Miura, Fumio Sugawara & Kengo
Sakaguchi (2018): Distribution and metabolism of 14C-Sulfoquinovosylacylpropanediol (14C-
SQAP) after a single intravenous administration in tumor-bearing mice, Xenobiotica, DOI:
10.1080/00498254.2018.1448949
To link to this article: https://doi.org/10.1080/00498254.2018.1448949
Accepted author version posted online: 15
Mar 2018.
View supplementary material
Submit your article to this journal
View Crossmark data
View related articles
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ixen20

14
Distribution and metabolism of C-Sulfoquinovosylacylpropanediol
(14C-SQAP) after a single intravenous administration in tumor-bearing
mice
Tatsushi Ruike1, Yoshihiro Kanai1, Kazuki Iwabata1, Yuki Matsumoto1, Hiroshi Murata1, Masahiro
Ishima1, Keisuke Ohta1, Masahiko Oshige2, Shinji Katsura2, Koji Kuramochi1, Shinji Kamisuki1,
Hiroeki Sahara3, Masahiko Miura4, FumioSugawara1,*, and KengoSakaguchi1,*
1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of
Science, 2641Yamazaki, Noda, Chiba 278-8510, Japan
2 Department of Environmental EngineeringScience, Graduate School of Science and Technology,
Gunma University, 1-5-1Tenjin-cho, Kiryu, Gunma376-8515, Japan
3 Laboratory of Biology, Azabu University School of Veterinary Medicine, 1-17-71 Fuchinobe,
Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan
4 Oral Radiation Oncology, Department of Oral Restitution, Graduate School, Tokyo Medical and
Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
* CorrespondingAuthors:
KengoSakaguchi: kengo@rs.noda.tus.ac.jp
Fumio Sugawara: sugawara@rs.noda.tus.ac.jp
Department ofApplied Biological Science, Facultyof Science andTechnology,
Tokyo Universityof Science,
2641 Yamazaki, Noda, Chiba 278-8510, Japan

ABSTRACT
Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits
angiogenesis in vivo and results in increased hypoxia and reduced tumor volume. We investigated
the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a
human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of
[glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was
decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The
degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635)
was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole
body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly
distributed throughout the whole body and then quickly decreased within 4 hours except the tumor
and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in
other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the
tumor had also completelydisappeared within 72 hours.
Running title: Distribution, metabolism and excretion of 14C-SQAP
Keywords: sulfoquinovosylacylpropanediol (SQAP), 14C-SQAP, radiosensitizer, KSN-nude mice,
ADME, human pulmonary carcinoma, tumorxenograft, whole-bodyautoradiography

INTRODUCTION
There is a need for cancer therapies that cause minimal suffering in cancer patients. One
such therapy could be a nontoxic radiosensitizer, which would enable the use of lower doses and
provide a wider scope for the irradiation of tumors, including metastatic areas, in patients with
terminal disease [Herman et al., 1990; Yuan et al., 2006; Sakaguchi and Sugawara, 2008;
Sakaguchi, 2015]. We previouslydesigned and synthesized an agent that we hopedwould suit such
a purpose. This novel potent radiosensitizer is sulfoquinovosylacylglycerol (SQAG). We
subsequently developed a chemically improved successor model, sulfoquinovosylacylpropanediol
(SQAP) [Ohta et al., 2001; Aoki et al., 2005; Aoki et al., 2006; Sakimoto et al., 2006; Miura et al.,
2007; Sakaguchi and Sugawara, 2008; Takakusagi et al., 2010; Izaguirre-Carbonell et al., 2014;
Iwamoto et al., 2015; Sakaguchi, 2015; Sawada et al., 2015; Takakusagi et al., 2015]. By applying
the concept of radiosensitization using SQAG or SQAP, a new class of anti-angiogenic treatments
has become available. SQAG was originally isolated as a natural product extracted from a red alga,
Gigartina tenella, and was then chemically synthesized [Hanashima et al., 2000a, 2000b and 2001].
SQAP is a synthetic chemical substance refined from SQAG (see Supplement). Both SQAG and
SQAP have great potential for radiosensitization, including the ability to remodel the tumor
microenvironment (such as vascular normalization) and reduce adverse effects [Ohta et al., 2001;
Sakimoto et al., 2006; Miura et al., 2007; Sakaguchi and Sugawara, 2008; Takakusagi et al., 2010;
Sakaguchi, 2015]. To attempt to understand how these compounds affect tumor
microenvironments, the whole-body distribution of the successor compound, SQAP, was
investigated in mice.
The radiosensitization effect of SQAP is promoted by vascular normalization in the
center of a solid tumor under hypoxic conditions (low-oxygen atmosphere), which occurred
immediately after administration [Ohta et al., 2001; Takakusagi et al., 2015]. These studies
indicated that SQAP could be not only a potent anti-angiogenic agent but also a re-oxidizing agent
immediatelyafter administration [Ohta et al., 2001; Takakusagi et al., 2015]. At the radiosensitizing

dose, which differed from the doses of various anti-angiogenic agents developed in the past, SQAP
did not cause any remarkable adverse effects and, thus, the limination due to the adverse effects
often associated with antitumor therapies [Ohta et al., 2001; Sakimoto et al., 2006; Miura et al.,
2007; Sakaguchi and Sugawara, 2008; Takakusagi et al., 2010; Sakaguchi, 2015; Sawada et al.,
2015]. To avoid the adverse effects and the limitations caused by anti-angiogenic agents, a new
radiotherapy with SQAP used as a radiosensitizer was tested for potential clinical use [Ohta et al.,
2001; Takakusagi et al., 2015]. Weak radiation exposure (irradiation) in the presence of low-dose
SQAP [Ohta et al., 2001; Takakusagi et al., 2015] was administered to perform the new
radiotherapy [Herman et al., Yuan et al., 2006]. However, the optimal combinations of the ionizing
radiation and SQAP dose that would provide the most efficient outcomes remained to be
determined beforeSQAP could be clinicallyimplemented.
As shown in Figure 1, SQAP is a novel synthetic derivative of SQAG generated by an
organic synthesis that highly sensitizes tumor parts under ionizing radiation [Sakimoto et al., 2006].
The radiosensitization ability of SQAP is the same as that of SQAG [Sakaguchi, 2015]. SQAG is a
monoacyl derivative of a sulfolipid (Fig. 1) that has been shown to be an extremely potent
radiosensitizer [Hanashima et al., 2000a, 2000b and 2001; Aoki et al., 2005; Ohta et al., 2010;
Takakusagi et al., 2011; Izagurrie-Carbonell et al., 2015]. In our laboratory, SQAG was chemically
modified to SQAP to eliminate the ambiguous diastereomeric mixture generated from the presence
of glycerol in the chemically synthesized SQAG (see Supplement). The diastereomeric mixture is
composed
of
1-O-acyl-3-O-(α-D-sulfoquinovosyl)
glycerol
and
2-O-acyl-3-O-(α-D-sulfoquinovosyl) glycerol. The mixture occurs via chemical resonance, and
separation of the components is very difficult and industrially impossible. This problem made it
unlikely that the compound could be officially licensed as a clinical drug. Therefore, we
synthesized SQAP by using propanediol without a 2-OH group instead of using glycerol (see
Supplement) and confirmed that the biological behavior was the same as that of SQAG [Sakimoto
et al., 2006; Ohta et al., 2010; Sawada et al., 2015; Takakusagi et al., 2015].

Sulfolipids are common secondary metabolites that are widely found in photosynthetic
bacteria, algae, and higher plants and are mostly food components. Investigations have shown that
both SQAG and SQAP have almost no toxic effects following oral administration. This was
expected since SQAG is also a component of food plants and SQAP is a derivative. When directly
injected in vivo, both SQAG and SQAP bound to vascular endothelial growth factor (VEGF) and
then reduced the focal adhesion kinase phosphorylation originated from its interaction with VEGF
[Izagurrie-Carbonell et al., 2015]. Subsequently, anti-angiogenesis caused by both SQAG and
SQAP occurred.
To elucidate the radiosensitization mechanisms of SQAG and SQAP, in vivo imaging
studies were also conducted to evaluate the SQAP mechanism of action for radiosensitization in
tumor parts [Sakimoto et al., 2006; Miura et al., 2007; Sakaguchi and Sugawara, 2008; Takakusagi
et al., 2010; Sakaguchi, 2015]. Electron paramagnetic resonance, oxygen imaging revealed that
intravenously administered SQAP transiently increased tumor oxygenation in murine SCCVII and
human A549 tumor xenografts [Sakimoto et al., 2006; Miura et al., 2007; Sakaguchi and Sugawara,
2008; Takakusagi et al., 2010; Sakaguchi, 2015]. Thus, it was concluded that SQAP transiently
increases tumor oxygenation by both pumping O2 molecules from erythrocytes and improving
tumor perfusion, which sensitizes tumors to radiotherapy [Sakimoto et al., 2006; Miura et al., 2007;
Sakaguchi and Sugawara, 2008; Takakusagi et al., 2010; Sakaguchi, 2015]. Radiation treatment
combined with SQAP significantly delayed angiogenesis-mediated tumor growth and induced
remodeling of the tumor microenvironments [Sakimoto et al., 2006; Miura et al., 2007; Sakaguchi
and Sugawara, 2008; Takakusagi et al., 2010; Sakaguchi, 2015]. In studies using SQAG, SQAP
was found to inhibit angiogenesis in vivo, which resulted in increased hypoxia and reduced tumor
volume [Sakaguchi, 2015; Sakimoto et al., 2006; Miura et al., 2007; Sakaguchi and Sugawara,
2008; Ohta et al., 2010; Takakusagi et al., 2010].
In the present study, we investigated the absorption, distribution, metabolism, and
excretion (ADME) of 14C-labeled SQAP and performed autoradiography in a human Lu65 tumor

xenograft.
MATERIALSAND METHODS
Reagents and Equipment
Scintillator Hionic-Fluor, Scintillator Flo-Scint II and Tissue solubilizer Soluene-350
were obtained from PerkinElmer (Waltham, MA, USA). Saline was obtained from Otsuka
Pharmaceutical Factory (Tokushima, Japan). Distilled water for LC/MS and acetonitrile for
High-performance liquid chromatography (HPLC) were obtained from Kanto Chemical (Tokyo,
Japan). Acetonitrile for LC/MS was obtained from Wako Pure Chemical Industries (Osaka, Japan).
Other reagents used in this study were of analytical grade and purchased from Wako Pure
Chemical Industries, Nacalai Tesque (Kyoto, Japan), Kanto Chemical or Sigma-Aldrich (St. Louis,
MO, USA).
Liquid scintillation counters (LSC) (2500TR, 2700TR, 3100TR or 1900CA,
PerkinElmer, MA, USA) were used for the measurement of radioactivity. HPLC system composed
of System controller (SCL-10Avp, Shimadzu, Kyoto, Japan), Pump (LC-20AD and LC-10ADvp,
Shimadzu), UV detection (SPD-20A and SPD-10Avp, Shimadzu, Kyoto, Japan), Autoinjector
(SIL-10ADvp, Shimadzu, Kyoto, Japan), Column oven (CTO-20AC and CTO-10ASvp,
Shimadzu, Kyoto, Japan), Online degasser (DGU-20A3 and DGU-14A, Shimadzu, Kyoto, Japan),
Manual injector (Rheodyne Model 7725i, Shimadzu, Kyoto, Japan) and Radioactive detector
(RAD) (525TR or 625TR, PerkinElmer). LC/MS/MS system composed of MS (LTQ Orbitrap XL,
Thermo Fisher Scientific, CA, USA), Pump (LC-10ADvp, Shimadzu, Kyoto, Japan), Autosampler
(SIL-HTC, Shimadzu), Column oven (CTO-10ASvp, Shimadzu, Kyoto, Japan), Online degasser
(DGU-14A, Shimadzu, Kyoto, Japan) and Radiomatic flow scintillation analyzer (525TR, Packard
BioScience, CT, USA). Eighteenof the Lu65transplanted mice were used for each experiment.

Synthesis of SQAP
Compound 1-O-stearoyl-3-O-(α-D-sulfoquinovosyl)-1,3-propanediol (SQAP, C18:0;
Fig. 1A; R=H) was prepared by organic synthesis as described in the Supplement. The lot number
of non-radioactiveSQAP was CG-0321 (Lot No. CG-0321).
For autoradiography, radioactive SQAPwas prepared from a sulfoquinovose composed
of 1,3-propanediol[1-14C] (Fig. 1B). 1,3-propanediol[1-14C]-SQAP (C18:0) was synthesized from
3-benzyloxy-1,3-propanediol[1-14C] in the Korea RadioChemicals Center (KRCC), Gyeonggi
Bio-Center, 12FL(864-1, Iui-dong, Yeongtong-gu, Suwon, Korea), by way of Sekisui Medical Co.
Ltd., Gyeonggi-do 443-270, Korea) (see Supplement). The chemically synthetic pathway
described in our Supplement section was followed by the synthetic method for non-radioactive
SQAP [Hanashima et. al., 2000a, 2000b and 2001]. The specific radioactivity of the
1,3-propanediol[1-14C]-SQAP (C18:0) (Lot No. CP-3635) was 3.48 MBq/mg, and the
radiochemical puritywas 97.5%. The synthetic quantity was 165 MBq.
For the pharmacokinetics study, [glucose-U-14C]-SQAP (C18:0) (Fig. 1C) was
prepared by using [glucose-U-14C] as a starting material followed by the synthetic method of
non-radioactive SQAP in KRCC [Hanashima et. al., 2000a, 2000b and 2001]. The specific
radioactivity of the [glucose-U-14C]-SQAP (C18:0) (Lot No. CP-3839) was 1.49 MBq/mg for
the metabolic analysis. The synthetic quantitywas 137 MBq.
14C-SQAP
expresses
14C-labeled
SQAP,
including
both
the
1,3-propanediol[1-14C]-SQAP (Lot No. CP-3635) and the [glucose-U-14C]-SQAP (Lot No.
CP-3839). In this paper, 1,3-propanediol[1-14C]-SQAP (CP-3635) denotes the
1,3-propanediol[1-14C]-SQAP (C18:0) (Lot No. CP-3635), and [glucose-U-14C]-SQAP (CP-3839)
denotes the[glucose-U-14C]-SQAP (C18:0) (Lot No. CP-3839).
Figure 1A-C indicates the structures of chemically synthesized SQAG, SQAP, and
14C-SQAP. The radiochemical purity was 96.4% 5 days before administration of
[glucose-U-14C]-SQAP (CP-3839), and the HPLC column recovery of radioactivity was 98.0%.

The radiochemical purity 1 day after administration (August 3, 2011) was 97.1%, and the HPLC
column recovery of radioactivity was 97.5%. 14C-SQAP was administrated intravenously and its
dose level of 14C-SQAP was adjusted to around 3 MBq/kg for all the radioisotope
experiments.14C-SQAP has undergone Good Laboratory Practice (GLP) compliance inspection,
and the GLP-compliant agent is now in a clinical trial for dogs diagnosed withterminal cancer.
Animals
All experimental protocols were approved by the local Laboratory Animal Care and
Use Committee of Tokyo University of Science and Tokyo Medical and Dental University. All
experiments were carried out in accordance with ADME guidelines. Collected samples were urine,
faeces, expired air, carcasses, and cage washes. In addition, collected organs were adrenal gland, bile in gall
bladder, blood, bone marrow, brain, brown fat, epididymis, eyeball, fat, gastriccontents, harderian gland, heart,
intestinal contents, intestine, kidney, liver, lung, mandibular gland, pancreas, pituitary gland, preputial gland,
skeletal muscle, skin, spleen, stomach, testis, thyroid gland, tumor, urinarybladder, andurine in bladder.
For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP
(CP-3839) was intravenously injected to eighteen of mice were used for the experiments of plasma
and tumor(3 mice/group, groups were 5 min, 1 h, 2 h, 4h, 8h and 24 h after [glucose-U-14C]-SQAP
(CP-3839) administration.).
1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous
injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice.
Sixteen mice in eight groups of two mice/dose group were given. After 5 min, 1 h, 2 h, 4 h, 8 h, 24
h, 72 h, or168 h elapsed from the timeof theinjection.
Six to eight weeks old male KSN nude mice (BALB/cAJcl-nu) were obtained from
Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). Tumor transplanting facility was Oral
Radiation Oncology, Department of Oral Restitution in Tokyo Medical and Dental University. The
mice were transplanted with tumor Lu65, a human cell line with giant cell carcinoma of lung

(JCRB0079). The Lu65 cells were provided by the Japanese Cancer Research Resources Bank.
The cells were maintained in RPMI 1640 containing 1 mmol/L pyruvate, 100 units/mL penicillin,
and 100 μg/mL streptomycin, supplemented with 10% fetal bovine serum. Bovine aortic
endothelial cells were maintained in Eagle's MEM supplemented with 5% fetal bovine serum. The
cell lines were maintained at 37ºC in a humidified atmosphere containing5% CO2.
Lu65 cells, which indicated a high radiation sensitizing effect by applying SQAP to
tumor-bearing mice [Sakimoto et al., 2006; Miura et al., 2007; Sakaguchi and Sugawara, 2008;
Takakusagi et al., 2010; Sakaguchi 2015], cultured at the Oral Radiation Oncology, Department of
Oral Restitution, graduate school of Tokyo Medical and Dental University, were transplanted
subcutaneously into male KSN nude mice (tumor xenograft) (2×106/animal). After the tumor
volume reached 100 to 300 mm3, or 750 to 1521 mm3, and the tumor growth was monitored by
palpation. The sizes of the palpable tumors were measured by using calipers every 2 days. The
tumor volume (V, mm3) was estimated on the basis of the length (mm) × width (mm) × height
(mm) × π/6. Lu65 is a human giant cell carcinoma of the lung cell line (JCRB0079; Japan Human
Sciences Foundation).
Quality Control of DosingFormulation forMetabolicAnalysis
The specific radioactivityof the test substance in the dosing formulation was determined
from the 14C-SQAP used at the preparation of the dosing formulation and the collected dosing
formulation volume. Just after preparation of the dosing formulation, each aliquot was randomly
collected (n = 3) from the dosing formulation. The specific radioactivity of the test substance in the
dosing formulation (actual value) was determined from the observed radioactivity concentration in
each sample, collected amount of the test substance, and total volume of the dosing formulation to
verify within the theoretical value ± 10% (observed value: 103%). 14C-SQAP was administrated
14
intravenously and its dose level of C-SQAP was adjusted to around 3 MBq/kg for all the
radioisotope experiments.

HPLCAnalytical Conditions
The sample was analyzed by HPLC system, and the radioactivity was expressed with
RAD that is unit of a physical dose quantity representing the mean energy imported to matter per
unit mass by ionizing radiation. An absorbed dose of 1 RAD means that 1 g of material absorbed
100 ergs of energyas a result of exposure to radiation.
Column: CAPCELL PAK C18 MGII, 3 µm, 3.0 mm (I.D.) × 50 mm (L) (Shiseido, Tokyo,
Japan)
Mobile phase: A:Acetonitrile
B: 10 mmol/LAmmonium acetate solution
Gradient: Time (min)/B (%) = 0/50 → 20/30 (Linear gradient)
Flow rate: 1 mL/min
Columntemperature: 40°C
Run time: 20 min
Injection volume: 8µL
Sample Collection and Preparation Method
Blood was collected from the inferior vena cava of the mouse using a syringe equipped
with a heparin-treated 25-G injection needle and mixed by inversion. The collected blood was
immediately centrifuged (8000×g) for 5 min at 4°C to separate plasma and blood cells. A 100 µL
aliquot of the plasma was placedin a scintillationvial.
All tumor parts were minced with scissors, placed in a container, and weighed. The
minced tumor parts were mixed with 1 mL of saline, weighed, and homogenized using a glass
homogenizer. The homogenates (0.5 mL) were placed in a scintillation vial for radioactivity
measurement, weighed, and solubilized in 2 mL of Soluene-350 tissue solubilizer, under heating
(50−60°C) as described the manufacturerinstruction.

Determination of Radioactivity
The radioactivity was measured using LSC after the addition of 10 mL of a
Hionic-Fluor scintillator to each sample prepared to determine the radioactivity. The remaining
plasma and the tumor homogenates were immediately frozen and stored in a −80ºC deep freezer
until use as samples for analysis of radioactivityinplasma andtumors.
Sample Processing
Each sample (plasma: 100 µL, tumor: 0.5 mL) was added to a 3-fold volume of
methanol, shaken (10 min), centrifuged (1800×g) for 10 min at 4°C, and then the supernatant was
collected. The residue was dissolved in the same volume of methanol as that of the sample
preparation, shaken, centrifuged, and then the supernatant was collected. These extraction
procedures were performed twice.
The combined supernatants from the experiments performed twice were mixed and
made up to the appropriate volume, and an aliquot of the solution was mixed with 10 mL of
Hionic-Fluor scintillator to determine the radioactivity. The rest of the residue was dissolved in 2
mLof Soluene-350 tissue solubilizer, made up to appropriate volume, and an aliquot of the solution
was mixed with 10 mL of Hionic-Fluor scintillator to determine the radioactivity. The rest of the
supernatant was evaporated to dryness and dissolved in HPLC mobile phase, centrifuged (1800×g)
for 5 min at 4°C, and the supernatant was analyzed by HPLC. From the counts of radioactivity, the
recovery through the sample processing was determined. After the condition was confirmed,
column recoverywas determined from the samples byHPLC analysis.
Column recovery (%) = [Radioactivity recovered from HPLC in the run time (dpm) / Radioactivity
injected to HPLC (dpm)] ×100.

LC/MS (MSn) Measurement Condition
Analysis sample: Tumor (1hour after administration, one sample), plasma (1 hour after
administration, onesample)
Column: Devolosil C30-UG-5, 4.6 mm I.D. ×250 mm L
Mobile phase: A: 10 mmol/Lammonium acetate
B:Acetonitrile
Gradient: Time (min)/B (%) = 0/0→ 45/90→ 65/90
Ionization: ESI(positive and negative mode)
The reference standards and each of the biological samples were measured by LC/MS
(MSn) to identify and estimate the metabolites under the conditions above. For the analysis, the
HPLC eluate was divided by a T-union; approximately 1/5 of the eluate was introduced into the
MS, and approximately 4/5 of the eluate was introduced into the Radiomatic flow scintillation
analyzer.
The radioactivity in each sample was counted for 2 min by using LSC. The counting
efficiency was corrected by the external standard source method. The detection limit of
radioactivity was defined as twice the background value and subtracted from the measured
radioactivity. The radioactivity in each eluate from HPLC was measured using Radiomatic flow
scintillation analyzer. Flo-Scint II scintillator was delivered to the HPLC eluate at a flow rate 3-fold
to that of the mobile phase, and the radioactivity (cpm) was integrated by using Radiomatic flow
scintillation analyzer at 6-sec intervals. The detection limit of radioactivitywas defined as two times
the background value andsubtracted from the counted radioactivity.
Background Radioactivity of LSCand Radiomatic flow scintillation analyzer
LSC: For the radioactivity assay in the dosing formulation, the solvent used for dilution
was analyzed. The column recovery of the HPLC system was analyzed with Radiomatic flow
scintillation analyzer. For the radioactivityassayin the biological samples, the plasma was analyzed.

One aliquot of each sample was analyzed.
For the radioactivity assay of the extract, HPLC assay samples and HPLC eluate,
extraction solvent, reconstitution solvent, and mobile phase were analyzed to determine the
background radioactivity, respectively. For the radioactivity assay in the biological samples, the
purified water was analyzed to determine the background radioactivity. The solvent for the
dissolution of the reference standard (dissolved in distilled water, and the reference standard
(dissolved in 50% ethanol) were analyzed by HPLC, and the mean value of the counts was used as
the background radioactivity level.
Radiochemical Purity and Column Recovery
Radiochemical purity (%) was calculated according to the equation shown below from
the radioactivity counts in each sample by using a calculation system built in Radiomatic flow
scintillation analyzer (Flo-One for Windows Analysis [ver. 3.65], PerkinElmer, MA, USA).
Column recovery (%) was calculated according to the equation shown below. The radiochemical
purity and column recovery (n = 1 each) were rounded off at the second decimal place and
expressed to one decimal place.
Radiochemical purity(%) = (S /T) × 100
T:Total counts over runtime (dpm)
S: Counts of peak correspondingto 14C-SQAP (dpm)
The calculating formula of column recovery(%) was shown in Sample Processing.
Dosing Formulation
Specific radioactivity (observed value) of the test substance in the dosing formulation
was determined from the radioactivity counts in the dosing formulations by using a data processing
system based on the equation shown below accordingto the following equation:
Observed specific radioactivity(MBq/mg) =A× (T/ L)

A: Observed radioactivityconcentration (MBq/g)
T:Total volume ofdosing formulation (g)
L:Amount of the labeledtest substance (mg) of14C-SQAP
Radioactivity Concentration
Radioactivityconcentrations in plasma and tumor were calculated from the radioactivity
counts by using a data processing system according to the equation shown below. The radioactivity
14
14
concentration was converted as C-SQAP (ng eq. of C-SQAP/g or mL), rounded off to one
decimal place, and expressed as an integer. The mean value and the standard deviation (SD) were
calculated.
Radioactivityconcentration(ng eq./g ormL) = (D / F) × S
D: Radioactivityin assaysample (dpm)
F: Specific radioactivityin dosing formulation (dpm/ng)
S:Amount ofsample (gormL) of14C-SQAP
Percentage Distribution
The percentage distribution was calculated from the radioactivity counts in tumors by
using a data processing system according to the equation shown below. The percentage distribution
was converted to a rate for each dosing radioactivity (% of dose), rounded off to the third decimal
place, and expressedtotwodecimal places as the meanvalue± SD ofthree animals.
Percentage distribution (%) = [ (D × T) / (A× S) ] ×100
D: Radioactivityin assaysample (dpm)
T: Total weight of assaysample (g)
S:Amount ofsample (g)14C-SQAP
A: Administered radioactivity (dpm) which was determined with the specific
radioactibity

Analysis of Metabolism
Eighteen of mice were used for the experiments of plasma and tumor(3 mice/group,
groups were 5 min, 1 h, 2 h, 4h, 8h and 24 h after [glucose-U-14C]-SQAP (CP-3839)
administration.).
1) The recoverythrough sample processing was rounded off toone decimal place.
2) The radiochromatogram at each analysis was includedin this report.
3) The MS radiochromatogram, MS spectrum, and MSn spectrum at the measurement of
LC/MS (MSn) were included in this report.
4) For the quantitative data of the parent compound and metabolites, the ratio of the countable
value of the parent compound to all of the radioactivity detected by HPLC-Radiomatic flow
scintillation analyzer analysis was calculated (% on HPLC) and expressed as a ratio for each
sample (% in sample) to one decimal place. Moreover, the radioactivity concentrations of the
parent compound and metabolites in plasma and tumor were converted to SQAP (ng eq. of
SQAP/g or mL), rounded off toonedecimal place, and expressed as an integer.
Calculation of Pharmacokinetic (PK) Parameters
Eighteen of mice were used for the experiments of plasma and tumor (3 mice/group,
groups were 5 min, 1 h, 2 h, 4h, 8h and 24 h after [glucose-U-14C]-SQAP (CP-3839)
administration.). The profiles of radioactivity concentrations in the plasma and tumor were
analyzed by non-compartmental analysis in Phoenix WinNonlin Ver. 6.1 (Pharsight), and the
following PK parameters were calculated. Data of PK parameters were calculated from the mean
concentrations of three animals. For the initial value of AUCt-∞, the observed value was used, and
AUCo-t was determined byusingthe trapezoidal rule.
AUC was rounded up in three digit of significant figure and t1/2 was rounded up in two digit of
significant figure.

1) AUC were determined form radioactivity concentration-time curve (AUC0-t and AUC0- ),
∞
1-24 h forPlasma and 4-24h forTumor and roundedup in three digit of significant figure
2) Area under the radioactivity concentration-time curve (AUC0-t and AUC0- ), 1-24 h for Plasma
∞
and 4-24 h for Tumor
Statistical Analysis
Statistical comparisons of mean values were performed by using one-way ANOVA.
Differences with P<0.05 were considered to be statistically significant. Radioactivity
concentrations were detected as shown in the Table 2, and conditions were described in HPLC
analytical conditions inMaterial andMethods.
Whole-body Autoradiographic Methods
To analyze whole-body autoradiographic patterns, 1 mg/kg SQAP with the
radioactivity (containing 3.48 MBq/mg of 1,3-propanediol[1-14C]-SQAP) was intravenously
injected to the tumor-bearing mouse at a time of 5 ml/kg in each of the SQAP group. The same
amount of saline was injected forthe control group.
Sixteen male KSN nude mice in eight groups of 2 mice/dose group (under GLP
conditions) were given. After 5 min, 1 h, 2 h, 4 h, 8 h, 24 h, 72 h, or 168 h elapsed from the time of
the injection, the autoradiographies of the mice were captured as follows. Mice were euthanized by
CO2 inhalation. The hair of mice was shaved and the nasal cavity and anus were blocked with 4%
carboxymethylcellulose (CMC-Na) and the carcasses were immediately frozen into dried ice and
acetone. The frozen blocks were sectioned by using a Leica CM 3600 cryomicrotome maintained
at −18ºC. Each group was composed of 2 mice. Then, the animals were sacrificed and evaluated by
whole-bodyautoradiography.

Sections were collected at four to six levels of interest and dried in the cryomicrotome
chamber. The sections for each mouse were collected on adhesive tape (No. 810, Sumitomo 3M,
Japan) at 30-μm thicknesses and then lyophilized. The lyophilized sections were mounted,
wrapped with Mitsubishi polyester foil (4 μm, Diafoil; Mitsubishi Plastics), and exposed on high
sensitive BAS Storage Phosphor Screens (GE healthcare Life Sciences, NJ, USA) for 24 hours,
which was sufficient for the exposure. The exposure screens were scanned by FujiFilm Photo
Scanner BAS-2500 (FujiFilm, Tokyo, Japan), and the ionization radiation dose of the screen is
expressed withphoto-stimulated luminescence (PSL) per unit area as an arbitraryunit.

RESULTSAND DISCUSSION
Determination of Radioactivity Concentration inTissues forMetabolism
Initially, [glucose-U-14C]-SQAP (CP-3839) was intravenously administered into human
adenocarcinoma Lu65 transplanted male KSN nude mice (dose: 2 mg (2.98 MBq)/kg as SQAP).
The results areshown inTable 1.
After a single intravenous administration of [glucose-U-14C]-SQAP (CP-3839) to
tumor-bearing male mice, the radioactivity concentration in the plasma was 2,229 ng eq./mL at
5 min after administration and decreased time-dependently. The t1/2 was 3.1 h (1 to 24 h) (Table 1).
The AUC0-24h (area under the concentration-time curve 0 to 24 hours) was 4,400 ng eq.∙h/mLand
AUC0-∞ was 4,420 ng eq.∙h/mL. The radioactivity concentration in the tumor was 331 ng eq./g
5 min after administration, maintained a comparable level (307–373 ng eq./g) ≤4h after
administration, and then decreased, with a t1/2 of 10 h (4–24 h). The AUC0-24 was 4,630 ng eq.∙h/g
and AUC0-∞ was 5,730 ng eq.∙h/g (Table 1). After 8 h or later of the administration, the radio
activityin thetumor is significantlylarger than that ofthe plasma (Table 1).
From these results, soon after a single administration of [glucose-U-14C]-SQAP
(CP-3839), the parent compound and/or its metabolites were distributed into the tumor rapidly, and
the radioactivity was maintained at a comparable level between the plasma and the tumor until 4 h
after administration (Table 1). Thereafter, the radioactivity concentration was higher in the tumor
than in the plasma and decreased more slowlythan that in the plasma. The t1/2 of radioactivityin the
tumor was three times as long as that in plasma. However, it was unknown whether the
radioactivity levels were related to [glucose-U-14C]-SQAP (CP-3839) or to its metabolites.
Therefore, we subsequently performed HPLC radiochromatographyof the product.
Analysis of Radioactivityin Plasma andTumor
After a single intravenous administration of [glucose-U-14C]-SQAP (CP-3839) at adose
of 2 mg/kg, the plasma and tumors obtained from tumor-bearing male mice were analyzed by

HPLC. The radioactivities of the plasma and tumor homogenates were analyzed by HPLC-
Radiomatic flow scintillation analyzer, and the ratios of the parent compound and metabolites in
each sample were determined.
The recovery levels of radioactivity after sample processing and from the HPLC
column after HPLC analysis are shown in Table 2 and indicate that each fraction of SQAP was
almost completely recovered.
A
representative HPLC radiochromatogram of
[glucose-U-14C]-SQAP (CP-3839) clearly shows a mono-peak (Fig. 2A). In the plasma (Fig. 2B)
and tumor (Fig. 2C), radioactive peaks were detected in HPLC radiochromatograms from 5
minutes to 24 hours after the SQAP administration, and the peaks tended to split into SQAP and
another group peak population named Peak 1 (Fig. 2 and Table 3). After 8 h in plasma and after 24
h in tumor, SQAP and Peak 1 were the level of background. (Data not shown). Considering the
LC/MS/MS assay results described later, the radioactive peaks detected in the plasma and tumor at
the retention times between 5 and 10 min were assumed to be one substance (SQAP), and
thereafter were assessed as Peak 1 (Fig. 2 and Table 3). One hour after the administration, Peak 1
appeared to separate into several peaks (Fig. 2B and C). The peaks were mostly
sulfoquinovosylpropanediol (SQP) or sulfoquinovose itself (Fig. 1D, E and Fig. S2) in which
SQAP lost the fatty acid (C18-stearic acid) with or without propanediol. The Peak 1 area in the
tumor in Figure 2C also contained mainlySQPor sulfoquinovose at 8 h. However, there seemed to
be manyother compounds in that area as well (Fig. 2).
The results are shown in Figure 2 and Table 3 for the plasma and tumor. In the plasma,
SQAP accounted for 89.6% of the radioactivity in the sample (1,997 ng/mL) at 5 min, 7.7% (53
ng/mL) at 1 h, and 2.5% (14 ng/mL) at 2 h after administration. SQAPwas not detected at 4 h after
administration (Fig. 2). A polar metabolite (Peak 1) in Figure 4 accounted for 6.0% of the sample
radioactivity (134 ng eq./mL) at 5 min, 87.6% (600 ng eq./mL) at 1 h, 91.1% (509 ng eq./mL) at 2
h, 85.2% (239 ng eq./mL) at 4 h, and 64.8% (52ng eq./mL) at 8h after administration.
The results for the tumor are also shown in Figure 2 and Table 3. Five min after

administration, only SQAP was detected in the tumor, which accounted for 83.1% of the sample
radioactivity (275 ng/g). SQAP accounted for 26.0% of the sample radioactivity (92 ng/g) at 1 h,
14.9% (56 ng/g) at 2 h, 12.1% (37 ng/g) at 4 h, and 9.7% (20 ng/g) at 8 h after administration.
SQAP was not detected at 24 h after administration (Fig. 2). A polar metabolite (Peak 1) was not
detected at 5 min after administration but accounted for 60.3% of the sample radioactivity (212 ng
eq./g) at 1 h, 69.8% (260 ng eq./g) at 2 h, 76.5% (235 ng eq./g) at 4 h, 68.8% (141 ng eq./g) at 8 h,
and 46.2% (35 ng eq./g) at 24 h after administration (Table 3).
Interestingly, the SQAP in the tumor did not degrade to the Peak 1 substance over
longer times (Fig. 2 and Table 3). Just 1 h later, SQAP in the plasma remained only 7.7% of the
administration, but a significant amount of SQAP (26%) remained in the tumor (Table 3). At 4 h,
the radioactivity in the tumor (12.1%) was much larger than that in the plasma (Table 3). In both
cases, the degradation product was mostly the Peak 1 substance within 4 h and then gradually
fragmented to sulfoquinovose and the further metabolic products (Fig. 2 andTable3).
In this study, 87.6% of [glucose-U-14C]-SQAP (CP-3839) (Table 3, Peak 1 of Plasma at
1 h) was excreted as water-soluble metabolites, mainly SQP (See Quantification and identification
of Peak1 in Supplement) 1.8% was SQAPitself together with the intestinal contents, and 7.0% was
exhaled from the lung as 14CO2. 95.8% of the total radioactivity administered was recovered as
SQP in this test. The other non-labeled product, C18-stearic acid, appeared to have been hydrolyzed.
LC/MS (MS/MS) Measurement of Plasma andTumor
In LC/MS (MSn) measurement of the reference standard SQAP, the MS chromatogram
and MS and MSn spectra were investigated (Table 4 and Fig. S2). The [M–H]–ion at m/z 567.3199
was found at the retention time of 49 min and resulted in fragment ions at m/z 283 and 225 (Table 4
and Fig. S2). Assignments of the fragment ions are shown in Figure 6. At m/z 283, SQAPappeared
to be separated into SQP(Fig. 1D) and stearic acid and at m/z 225 to be degraded to sulfoquinovose
(Fig. 1E) and additional compounds.

A comparison of the calculated and found mass data of the molecular formula and the
m/z values of the metabolites identified or estimated in the plasma and tumor are shown in Figure 3
and Table 4. The difference between the calculated and found m/z was within ± 5 ppm. This
suggests that themetabolites can be reliablyidentified bythemass measurement.
In the LC/MS (MSn) measurements of the plasma and tumor, radio-chromatograms and
MS chromatograms were investigated in the samemanner (Fig. 3 andTable4).
In the case of the plasma, an ion at m/z 301.0592 was detected from Peak 1, and the
molecular formula was estimated as C9H17O9S (calculated value as [M–H]–ion: 301.0599) (Fig. 3
and Table 4). This mass number corresponded to the mass number of the parent compound (567–
266). Because the fragment ion at m/z 225 was detected, the chemical structure was estimated to be
a hydrolyzed metabolite. Assignment of the fragment ion is shown in Figure S2. An [M–H]–ion at
m/z 567.3199 (calculated value as [M–H]–ion: 567.3208) was detected at the same retention time as
SQAP (Fig. 3 and Table 4). Because the fragment ions were the same as those of the reference
standardSQAP, this peak was identified as the parent compound.
Aradiochromatogram and MS-chromatogram from the LC/MS (MSn) measurement of
the tumor are also shown in Table 4 and Fig. 3, respectively. Because an ion at m/z 301.0588 was
detected from Peak 1 similar to that from Peak 1 in the plasma (Fig. 3 and Table 4), and the
fragmented ions were similar to those of Peak 1 in the plasma, the peak was considered to be an
identical metabolite. An [M–H]–ion at m/z 567.3193 was detected from the peak at the same
retention time as that of SQAP. Because the fragmented ions were the same as those of the
reference standard SQAP, this peak was identified as the parent compound (See Quantification and
identificationof Peak1 inSupplement).
These results indicated that the metabolic process of SQAP in vivo was the same in
either the plasma or tumor, andthespeed inthe plasma was fast.

Whole-body Autoradiography (Tissue Distribution)
In the ADME tests using 1,3-propanediol[1-14C]-SQAP (CP-3635), the radioactivity
concentrations in the plasma and tumor were analyzed after intravenous administration of 1 mg
(3.48 MBq)/kg of 1,3-propanediol[1-14C]-SQAP (CP-3635) to non-fasting Lu65 transplanted male
nude mice. Analysis showed that the 1,3-propanediol[1-14C]-SQAP (CP-3635)-derived
radioactivity accumulated in the tumor (Fig. 4 and Table 5). Some mechanisms can be deduced as
follows. One possibility is that SQAP was metabolized preferentially and more slowly in the tumor
than in the plasma. The other possibilities are enhanced uptake of SQAP or slower excretion of
SQAP. Another possibility can be also deduced. Our results also suggested that SQAP was caught
in necrotic areas of the tumor where blood flow did not allow for the faster clearance of
radioactivity, because SQAP in the tumor continually kept much higher level than those in brain
and testis.The information about ADME of SQAP is clinically important for the development of a
radiosensitizer.
For further study, whole-body autoradiography for total radioactivity derived from
1,3-propanediol[1-14C]-SQAP (CP-3635) was performed. Figure 4 shows an autoradiographic
image taken on the median line along the mouse whole body. The amounts in Table 5 were
measured by autoluminography, which does not allow for correction of background blood or urine
in the tubules. Table 5 and Figure 4 show photo-stimulated luminescence (PSL) per unit area in
tissues at 5 min, 1 hour, 2 h, 4 h, 8 h, 24 h, 72 h, or 168 h after a single intravenous administration
of 1,3-propanediol[1-14C]-SQAP (CP-3635) to non-fasting male KSN-mice (dose: 1 mg/kg as
SQAP), respectively (Fig. 4 and Table 5). For each time point in Table 5, two non-fasting male
KSN-mice were used. The distribution was almost the same in the two mice. Table 5 presents the
mean values ofthetwo mice.
Autoradiographic data indicated that 1,3-propanediol[1-14C]-SQAP (CP-3635)-derived
radioactivity was widely distributed at first throughout the organs and tissues in the male
KSN-mice (Fig. 4 and Table 5). Interestingly, the photo-stimulated luminescence showed that

SQAP was relatively difficult to distribute in the brain and testis and that the radioactivity did not
remain longin anyorgan (Fig. 4 and Table5).
At 5 min post dose (end of infusion), the highest photo-stimulated luminescence
occurred in the liver, with SQAPbeing two-fold higher concentration than that in blood, and it was
almost the same level as that in blood in the adrenal gland and lung (Fig. 4 and Table 5). The
radioactivity levels in the kidney, spleen, heart, and other organs were lower than that in blood
(Table 5 and Fig. 4). The radioactivitylevel was also not high in the tumor (Fig. 4 and Table 5).
At 1 h, the highest radioactivity levels were in the urinary bladder and the second
highest level was in the liver (Fig. 4 and Table 5). The radioactivity levels in the bile of the gall
bladder and kidney were also very high. In contrast, the radioactivity level in blood was much
lower (Fig. 4 and Table 5). In the other organs, the radioactivitylevels were lower than that in blood
(Fig. 4 and Table 5). The values became much higher in the excretory organs and their excreted
liquids, a tendency that continued later (Table 5). SQAP was rapidly accumulated in the liver and
kidney, and was soon metabolized and excreted (Table 5). The only exception was the tumor. The
photo-stimulated luminescence increased although the value was not high (Table 5). The
radioactivity mostly came from the metabolic products (SQPand sulfoquinovose in Fig. 1D and E),
which was alsoobserved inthe plasma (our unpublished data).
At 2 h, the values in the urinary bladder and the liver became extremely high. On the
other hand, SQAPwas mostlydisappeared within 2 hours in the most organs (Fig. 4 and Table 5).
At 4 h, the radioactivity levels had greatly decreased in most organs except in the tumor
and excretory organs, including excreta (Fig. 4 and Table 5). In the excretory organs or the excreta
(e.g., urinary bladder, urine, intestinal contents, and gall bladder bile) the radioactivity increased
dramatically (Fig. 4 and Table 5). In the process, although the luminescence values in the lung,
blood, adrenal gland, and kidney rapidly decreased, those in the kidney and liver remained very
high (Fig. 4 and Table 5). It appeared that SQAP actively gathered from the entire body and broke
down to SQP and other compounds.

At 8 h, only the tumor showed more than a two-fold higher radioactivity level than the
levels in any other organs except in the excretory organs and excreta (Table 5). As suggested in
Figure 4 and Table 3, the radioactivity of SQAP appeared to be relatively larger in the tumor.
Intra-organ differential distribution was apparent in Lu65 tumor transplanted in the back of the
mice, in which the luminescence unevenly increased in the tumor. Generally, the tissue
radioactivity concentrations decreased over time, but elimination was not complete at 72 h post
dose (Fig. 4 and Table 5). At 168 h, the radioactivityhad been completelyeliminated (Fig. 4).

ACKNOWLEDGMENTS
This work was supported by the Program for Promotion of Fundamental Studies in
Health Science of the National Institute of Biomedical Innovation (NiBio), Japan.
Disclosureof Potential Conflicts of Interest
There are no potential conflicts ofinterest relatedtothis work to disclose.
Authors' Contributions
Conception and design: H. Sahara, M. Miura, F. Sugawara, K. Sakaguchi.
Acquisition of biological data (e.g., provided animals, provided facilities): T. Ruike, Y. Kanai, K.
Iwabata, Y. Matsumoto, M. Ishima, K. Ohta, M. Oshige, S. Katsura, H. Sahara, M. Miura, K.
Sakaguchi.
Biological analysis and interpretation of data (e.g., LC-MS data analysis, computational analysis):
M. Ishima, K. Ohta, M. Miura, F. Sugawara, K. Sakaguchi.
Chemical Synthesis (e.g., organic synthesis, NMR and MS data collection, structural analysis): T.
Ruike,Y. Kanai, H. Murata, K. Kuramochi, S. Kamisuki, F. Sugawara.
Writing, review, and/or revision of the manuscript: M. Oshige, K. Katsura, F. Sugawara, K.
Sakaguchi.
Administrative, technical, or material support (e.g., reporting or organizing data, constructing
databases): H. Murata, M. Ishima, K. Ohta, F. Sugawara, K. Sakaguchi.
Studysupervision: M. Miura, F. Sugawara, K. Sakaguchi.

REFERENCES
Aoki S, Ohta K, Yamazaki T, Sugawara F, Sakaguchi K (2005) Mammalian mitotic
centromere-associated
kinesin
(MCAK),
a
new
molecular
target
of
sulfoquinovosylacylglycerols (SQAGs), novel anti-tumor and immunosuppressive agents.
FEBS J 272:2132-2140
Aoki S, Matsumoto K, Ohta K, Sakai H, Abe M, Miura M, Sugawara F, Sakaguchi K (2006) An
emulsion of sulfoquinovosylacylglycerol and long-chain alkane increases its permeability in
tumor cells. J Membrane Biol 213:11-18
Bowsher, JE, Hedlund LW, Wong T, Dewhirst MW (2006) Intertumoral Differences in Hypoxia
Selectivity of the PET Imaging Agent 64Cu(II)-Diacetyl-Bis(N4-Methylthiosemicarbazone). J
Nucl Med 47:989-998
Hanashima S, Mizushina Y, Ohta K, Yamazaki T, Sugawara F, Sakaguchi K (2000a)
Structure-activity relationship of a novel inhibitor group of mammalian DNA polymerases,
syntheticsulfoquinovosylacylglycerols. Jpn J Cancer Res 91:73-83
Hanashima S, Mizushina Y, Yamazaki T, Ohta K, Takahashi S, Koshino H, SaharaH, Sakaguchi K,
Sugawara F (2000b) Structural determination of sulfoquinovosyldiacylglycerol by chiral
synthesis. TetrahedronLett 41:4403-4407
Hanashima S, Mizushina Y, Yamazaki T, Ohta K, Takahashi S, Sahara H, Sakaguchi K, Sugawara
F (2001) Synthesis of sulfoquinovosylacylglycerols, inhibitors of eukaryotic DNA polymerase
α and β. Bioorg MedChem 9:367-376
Herman TS, Teicher BA, Dewhirst MW, Zimmerman D (1990) Rate of infusion: an important
factor in circulatory effects of perfluorochemical emulsions (PFCES). Int J Radiat Oncol Biol
Phys 19:136
Iwamoto H, Nakamura T, Koga H, Izaguirre-Carbonell J, Kamisuki S, Sugawara F, Abe M,
Iwabata K, Ikezono Y, Sakaue T, Masuda A, Yano H, Ohta K, Nakano M, Shimose S, Shirono
T, Torimura T (2015) Inhibition of hypoxia-inducible factor via upregulation of von

Hippel-Lindau protein induces “angiogenic switch off” in a hepatoma mouse model. Mol Ther
Oncolytics 2: 15020
Izaguirre-Carbonell J, Murata H, Ohta K, Kusayanagi T, Tsukuda S, Iwabata K, Kanai Y, Kamisuki
S, Sakaguchi K, Sugawara F (2014) Identification and study of SQAP-binding proteins from a
T7 phage displayscreen. Planta Med80:PH4
Izaguirre-Carbonell J, Kawakubo H, Murata H, Tanabe A, Takeuchi T, Kusayanagi T, Tsukuda S,
Hirakawa T, Iwabata K, Kanai Y, Ohta K, Masahiko M, Sakaguchi K, Matsunaga S, Sahara H,
Kamisuki S, Sugawara F (2015) Novel anticancer agent, SQAP, binds to focal adhesion kinase
and modulate its activity. Nat Commun 5:15136
Miura M, Sakimoto I, Ohta K, Sugawara F, Sakaguchi K (2007) Sulfoglycolipids as candidate
antiangiogenetic radiosensitizers (a review). Anticancer Drugs 18:1-5
Mizushina Y, Yagi H, Tanaka N, Kurosawa T, Seto H, Katsumi K, Onoue M, Ishida H, Iseki A,
Nara T, Morohashi K, Horie T, Onomura Y, Narusawa M, Aoyagi N, Takami K, Yamaoka M,
Inoue Y, Matsukage A, Yoshida S, Sakaguchi K (1996) Screening of inhibitor of eukaryotic
DNApolymerases produced bymicroorganisms. JAntibiot (Tokyo) 49:491-492
Mizushina Y, Watanabe I, Ohta K, Takemura M, Sahara H, Sugawara F, Matsukage A, Yoshida S,
Sakaguchi K (1997) AIDS-antiviral sulfolipids from a pteridophyta, Athyrium niponicum, are
selective inhibitors of Mammalian DNA polymerase α and β, but not of HIV reverse
transcriptase. Biochem Pharmacol 55:537-541
Ohta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K
(1998) Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA
Polymerases and HIV-Reverse Transcriptase 1 from A Marine Red Alga, Gigartina tennella.
Chem Pharm Bull (Tokyo) 46:684-486
Ohta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K
(1999) Studies on action of sulfoquinovosyldiacylglycerol, a new mammalian DNA
polymeraseinhibitor, from a sea alga, Gigartinatenella. Biol Pharm Bull 22:111-116

Ohta K, Hanashima S, Mizushina Y, Yamazaki T, Saneyoshi M, Sugawara F, Sakaguchi K (2000)
Studies on a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerols:
inhibitoryactionon cell proliferation. Mutat Res 467:139-152
Ohta K, Mizushina Y, Yamazaki T, Hanashima S, Sugawara F, Sakaguchi K (2001) Specific
interaction between oligosaccharide on tumor cell surface and novel antitumor agents,
sulfoquinovosylmonoacylglycerols. Biochem Biophys Res Commun 288:893-900
Ohta K, Murata H, Mori Y, Ishima M, Sugawara F, Sakaguchi K, Miura M (2010) Remodeling of
the tumor microenvironment by combined treatment with a novel radiosensitizer,
α-sulfoquinovosylmonoacylglycerol (α-SQMG) and X-irradiation. Anticancer Res
30:4397-4404
Sakimoto I, Ohta K, Yamazaki T, Ohtani S, Sahara H, Sugawara F, Sakaguchi K, Miura M (2006)
α-Sulfoquinovosylmonoacylglycerol (α-SQMG) is a novel potent radiosensitizer targeting
tumor angiogenesis. Cancer Res 66:2287-2295
Sakaguchi K, Sugawara F (2008) New cancer chemotherapy agents: Inhibitors of DNA
polymerase (a review). Current Drug Therapy 3:44-53
Sakaguchi K (2015) A prospect of a new potent radiosensitizer SQAG/SQAP (a review in
Japanese). Radiat Biol Res Commun 50:1-17
Sawada Y, Omoto K, Kohei H, Sakaguchi K, Miura M, Tanabe K (2015)
Sulfoquinovosylacylpropanediol is a novel potent radiosensitizer in prostate cancer. Int J Urol
22:590-595
Takakusagi Y, Takakusagi K, Sugawara F, Sakaguchi K (2010) Use of phage display technology
for the determination of the targets for small-molecule therapeutics (a review). Expert Opin
Drug Discov 5:361-389
Takakusagi Y, Takakusagi K, Ida N, Takami M, Matsumoto Y, Kusayanagi T, Nakabayashi T, Aoki
S, Murata H, Ohta K, Sugawara F, Sakaguchi K (2011) Binding region and interaction
properties of sulfoquinovosylacylglycerol (SQAG) with human vascular endothelial growth

factor165 revealed bybiosensor-based assays. MedChemComm 2:1188-1193
Takakusagi Y, Matsumoto S, Saito K, Matsuo M, Takakusagi K, Kishimoto S, Ishima M, Murata
H, Ohta K, Miura M, Sugawara F, Sakaguchi K, Mitchell JB, Krishna MC (2015) Increase in
tumor oxygenation and radiosensitivitycaused bySQAP (CG-0321): the mechanism of
actions revealed by in vivo imaging studies. ICRR (15th International Congress of
Radiation Research) 3-PS2E-27
Yuan H., Schroeder T, Bowsher JE, Hedlund LW, Wong T, Dewhirst MW (2006) Intertumoral
Differences
in
Hypoxia
Selectivity
of
the
PET
Imaging
Agent
64Cu(II)-Diacetyl-Bis(N4-Methylthiosemicarbazone). JNucl Med 47:989-998