International Journal of

Molecular Sciences
ArticleAssessment of Amino Acid/Drug Transporters for
Renal Transport of [18F]Fluciclovine (anti-[18F]FACBC)
in Vitro
Masahiro Ono 1,† , Atsumi Baden 1,† , Hiroyuki Okudaira 1 , Masato Kobayashi 2 , Keiichi Kawai 3 ,Shuntaro Oka 1, * and Hirokatsu Yoshimura 1
1

2
3

*
†

Research Center, Nihon Medi-Physics Co., Ltd., Chiba 299-0266, Japan; masahiro_ono@nmp.co.jp (M.O.);
atsumi_baden@nmp.co.jp (A.B.); hiroyuki_okudaira@nmp.co.jp (H.O.);
hirokatsu_yoshimura@nmp.co.jp (H.Y.)
Wellness Promotion Science Center, Institute of Medical, Pharmaceutical and Health Science,
Kanazawa University, Ishikawa 920-0942, Japan; kobayasi@mhs.mp.kanazawa-u.ac.jp
Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University,
Ishikawa 920-0942, Japan; kei@mhs.mp.kanazawa-u.ac.jp
Correspondence: shuntaro_oka@nmp.co.jp; Tel.: +81-438-627-611; Fax: +81-438-626-969
These authors contributed equally to this work.

Academic Editors: Sundaresan Gobalakrishnan and Jamal Zweit
Received: 28 July 2016; Accepted: 8 October 2016; Published: 14 October 2016

Abstract: [18 F]Fluciclovine (trans-1-amino-3-[18 F]fluorocyclobutanecarboxylic acid; anti-[18 F]FACBC),a positron emission tomography tracer used for the diagnosis of recurrent prostate cancer,
is transported via amino acid transporters (AATs) with high affinity (Km : 97–230 µM). However,
the mechanism underlying urinary excretion is unknown. In this study, we investigated the
involvement of AATs and drug transporters in renal [18 F]fluciclovine reuptake. [14 C]Fluciclovine(trans-1-amino-3-fluoro[1-14 C]cyclobutanecarboxylic acid) was used because of its long half-life.
The involvement of AATs in [14 C]fluciclovine transport was measured by apical-to-basal transport
using an LLC-PK1 monolayer as model for renal proximal tubules. The contribution of drugtransporters herein was assessed using vesicles/cells expressing the drug transporters P-glycoprotein(P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4),
organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) , organic cation transporter 2(OCT2), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporting
polypeptide 1B3 (OATP1B3). The apical-to-basal transport of [14 C]fluciclovine was attenuated by
L -threonine, the substrate for system alanine-serine-cysteine (ASC) AATs. [14 C]Fluciclovine uptake
by drug transporter-expressing vesicles/cells was not significantly different from that of control
vesicles/cells. Fluciclovine inhibited P-gp, MRP4, OAT1, OCT2, and OATP1B1 (IC50 > 2.95 mM).
Therefore, system ASC AATs may be partly involved in the renal reuptake of [18 F]fluciclovine. Further,
given that [18 F]fluciclovine is recognized as an inhibitor with millimolar affinity for the tested drugtransporters, slow urinary excretion of [18 F]fluciclovine may be mediated by system ASC AATs,
but not by drug transporters.
Keywords: fluciclovine; anti-FACBC; positron emission tomography; amino acid transporter;
drug transporter

1. Introduction[18 F]Fluciclovine (trans-1-amino-3-[18 F]fluorocyclobutanecarboxylic acid, also known asanti-[18 F]FACBC or Axumin™) is a positron emission tomography (PET) tracer that has been approvedby the U.S. Food and Drug Administration for the detection of recurrent prostate cancer since 2016 [1].

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Recently, phase II clinical trials of [18 F]fluciclovine have been simultaneously conducted in Japan forthe diagnosis of glioma [2] and primary prostate cancer [3].
[18 F]Fluciclovine is a synthetic leucine analog that is developed as a next-generation PET tracer tocompensate for the high accumulation of [18 F]fluorodeoxyglucose ([18 F]FDG) in the brain and urinary
tract under physiological conditions [4]. The low urinary excretion of [18 F]fluciclovine differs fromthat of many other 18 F-labeled amino acids such as O-(2-[18 F]fluoroethyl)-L-tyrosine ([18 F]FET) anda range of tracers that have been preclinically evaluated [5]. Clinical trials have demonstrated that
[18 F]fluciclovine accumulates readily in the liver and pancreas, mildly in the kidneys, and minimally inthe brain after administration as an intravenous bolus infusion; it is excreted slowly in the urine for up
to 24 hours, but not in bile [6,7]. These results suggest that [18 F]fluciclovine-PET may have advantages
over [18 F]FDG and [18 F]FET for imaging tumors in the brain and/or in the pelvis.
Our previous preclinical studies have shown that [18 F]fluciclovine is not incorporated into
proteins [8], and accumulates in cells through amino acid transporters (AATs) that serve as its
primary means of transport [8–10]. We have also shown that two Na+ -dependent AAT subtypes(i.e., alanine-serine-cysteine transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter2 (SNAT2)) and Na+ -independent L-type amino acid transporter 1 (LAT1) have high affinities for[18 F]fluciclovine with Km values of 92.0, 222.0, and 230.4 µM, respectively [11]. This suggests that AATs
may be involved in the whole-body distribution of [18 F]fluciclovine, because these AATs are expressedin the liver, renal proximal tubule, by the blood-brain-barrier (BBB) [12], and/or tumors [13].
Drug transporters expressed in the intestines, liver, kidneys, and/or at the BBB play an importantrole in regulating drug absorption, distribution, metabolism, and excretion [14]. Numerous
studies have shown that drug transporters influence pharmacokinetics and thus affect drug efficacy
and are involved in the occurrence of adverse effects, including those produced by drug-druginteractions. The International Transporter Consortium advocated the assessment of drug-druginteractions in the context of drug transporters in accordance with their significant clinical importance.
In particular, three ATP-binding cassette (ABC) transporters—P-glycoprotein (P-gp; MDR1), breast
cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4)—are important
efflux transporters that regulate intestinal absorption, distribution to the brain, and/or excretion in theliver and kidneys for several therapeutic drugs [14]. Three solute carrier (SLC) transporters, organic
anion transporter 1 (OAT1), organic anion transporter 3 (OAT3), and organic cation transporter 2(OCT2), are thought to be involved in transporting anionic (OAT1/3) and cationic (OCT2) drugs
from the blood to renal tubules. In addition, two other SLC transporters, organic anion transporting
polypeptide 1B1 (OATP1B1) and organic anion transporting polypeptide 1B3 (OATP1B3), are believed
to be critical hepatic transporters of anionic drugs [14].
Recent studies have reported that therapeutic drugs that have both amino and carboxyl
residues are transported via several transporters. For example, melphalan (an anticancer drug)
and gabapentin (antiepilepsy drug) are transported by an AAT (LAT1) [15,16] and drug transporters(P-gp and organic cation/carnitine transporter 1 (OCTN1)) [17,18]. Efflux drug transporters suchas P-gp are overexpressed in several tumors, causing resistance to chemotherapeutic drugs [19,20].
Therefore, the contributions of hepatic and renal drug transporters to [18 F]fluciclovine influx/efflux
merit evaluation, because drug transporters such as those described above may be involved in[18 F]fluciclovine accumulation following [18 F]fluciclovine administration during PET procedures.
As described above, we have previously reported that AATs are the primary transporters involvedin [18 F]fluciclovine uptake into cells. However, the mechanisms underlying hepatic/renal distributionof [18 F]fluciclovine by AATs and drug transporters, especially the slow excretion into urine, has notbeen fully identified. To address this, we assessed the contribution of AATs on renal reabsorptionof [18 F]fluciclovine using the LLC-PK1 monolayer model, which is used to simulate the transport ofnovel drugs to the proximal tubules [21,22]. Furthermore, the contribution of drug transporters to thehepatic/renal distribution of [18 F]fluciclovine was investigated.

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2. Results

2. Results

2.1. Tight Junction Formation in the LLC-PK1 Monolayer Model
2.1. Tight Junction Formation in the LLC-PK1 Monolayer ModelThe
LLC-PK1of renal
renal proximal
proximaltubules
tubulesfor
forthetheThe
LLC-PK1monolayermonolayermodel
modelisis aa well-known
well-known model
model ofestimationofrenal
transport[21,22].
To
validatetheformationoftightjunction
inthe
LLC-PK1
estimation of renal transport [21,22]. To validate the formation of tight junction in the LLC-PK1
3monolayers,penetration,andtransepithelial
electricalresistance(TER)
monolayers,paracellularparacellular [[3H]mannitol
H]mannitol penetration,andtransepithelial
electricalresistance(TER) were
3H]mannitol permeation was3
weremeasured.
As
shown
in
Figure1,
themeanpercentageof[measured. As shown in Figure 1, the mean percentage of [ H]mannitol permeation was significantly
significantly
decreased
19.3%(day(day1) to(day 4),and reachedplateau
at day7 afterdecreased from
19.3% from(day 1)
to 1.23%4),1.23%
and reacheda plateau
at day a7 after
seeding(2.29%).
2 at day 1, 4,
2
seeding(2.29%).
In
addition,
TER
valuesgradually
increased
to
206,
284,
and
413
Ω·cm
In addition, TER values gradually increased to 206, 284, and 413 Ω·cm at day 1, 4, and 7, respectively
and(Figure
7, respectively
These results
suggested
that
tight
junctions formed
were sufficiently
formed1). These(Figure
results 1).
suggestedthat tightjunctions
weresufficiently
in the LLC-PK1
monolayers
day 4 after
seeding.
in the
LLC-PK1frommonolayers
fromday 4 after seeding.

Figure1. 1.The
formationof tight
junctionsin the LLC-PK1
model asFigureThechronologicalchronological formationof tight
junctionsin the LLC-PK1
monolayermonolayer
model as monitored3
3
by the [ byH]mannitol
penetration
rate (left axis,
blackaxis,
bar; blackn = 3–9)electricalpenetration
rate (leftbar;andn =transepithelial
3–9) and transepithelial
monitored
the [ H]mannitol
resistance(TER, right
axis,
white
circle;n =circle;
3–4). Eachdata Eachpoint data
representsmean ±thestandard
electricalresistance
(TER,
right
axis,
white
n = 3–4).
point therepresentsmean ±
deviation
(SD).
*
p
<
0.05
vs.
insert
only,
#
p
<
0.05
vs.
day
1
after
seeding.
standard deviation (SD). * p < 0.05 vs. insert only, # p < 0.05 vs. day 1 after seeding.

Apical-to-Basal
TransportofofAmino
AminoAcid
AcidTracers
Tracers through2.2.2.2.
Apical-to-Basal
Transport
throughthe
theLLC-PK1
LLC-PK1Monolayers
Monolayers
estimatetranscellulartranscellular amino
throughthe LLC-PK1
monolayers
mimicking
renal
ToTo
estimateaminoacid
acidtransporttransport
throughthe LLC-PK1
monolayers
mimicking
14 C-labeled amino acid tracers (10 µM).
proximaltubules,
apical-to-basaltransport
wasmeasuredusing
14
renal proximal tubules, apical-to-basal transport was measured using C-labeled amino acid tracers
shown
Figure in
2a, Figure
the apical-to-basaltransport of eachtracer increased
in a time-dependent(10AsμM).
Asinshown
2a, the apical-to-basaltransportof each tracer
increased in amanner.
The
meanpenetration
amountofeach
tracer
at
120
min
was
145
with
[14 C]fluciclovine
time-dependent manner. The mean penetration amount of each tracer at 120 min
was 145 with(fluciclovine),
85.5
with
L -[14 C]alanine (Ala),
and
149
pmol
with
L -[14 C]leucine (Leu).
In addition,
14
14
14[ C]fluciclovine (fluciclovine), 85.5 14
with L-[ C]alanine (Ala), and 149 pmol with L-[ C]leucine(Leu).
the mean residual radioactivity of [ C]fluciclovine in the cells at 120 min was significantly higher thanIn addition, the mean residual radioactivity of [14C]fluciclovine in the cells at 120 min wasthat of Ala and Leu: 238, 15.2, and 179 pmol, respectively (Figure 2b).

significantly higher than that of Ala and Leu: 238, 15.2, and 179 pmol, respectively (Figure 2b).

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Figure 2. (a) Time course14 of 14C-labeled amino acid (10 μM) transport through the LLC-PK1
Figure2.2. (a)(a) Time courseof C-labeled
aminoacid (10µM)
through the
LLC-PK1
monolayers
14C-labeled
Figurecourseamino(10transportμM)amounttransport
throughtheamino
LLC-PK1
monolayers Timeafter 4–5
daysofculture(n = 3–6);(b) acid
the residualof intracellular
acid
after
4–5
days
culture
(n
=
3–6);
(b)
the
residualamountofintracellular
amino
acid
tracermonolayers(n = 3–6); (b) the
residualof intracellularaminoacidat14C]fluciclovine”,
tracer at after
120 4–5
min days(n =culture3–6); “fluciclovine”,“Ala”,
and amount“Leu”
indicate“[
120 min(n“fluciclovine”,“Ala”, and “Leu”
indicate “Leu”“[14 C]fluciclovine”,“L-[14 C]alanine”,
tracer120= 3–6);
minand(n “L=-[143–6);“fluciclovine”,“Ala”,
indicate the“[14meanC]fluciclovine”,
C]alanine”,
C]leucine”,
respectively.
Each anddata point represents± SD. * p <“L-[14at14
and“L-[ C]leucine”, respectively.
Each data point represents the mean ± SD. * p < 0.05 vs. fluciclovine.
14C]alanine”,
14C]leucine”,
and
“
L
-[
respectively.
Each
data
point
represents
the
mean
±
SD. * p <“L-[0.05
vs. fluciclovine.
0.05 vs. fluciclovine.

2.3. The Inhibition of Apical-to-Basal Transport in the LLC-PK1 Monolayers by Amino Acids
2.3. The Inhibition of Apical-to-Basal Transport in the LLC-PK1 Monolayers by Amino Acids
14
2.3. The
Inhibition of Apical-to-Basal
Transporttransport
in the LLC-PK1
Monolayersby Amino
Acids and synthetic
The(10 μM)µM)
by naturally
naturallyoccurring
Theinhibition
inhibition ofof [ [14C]fluciclovine
C]fluciclovine transport(10
byoccurringand synthetic
amino
acids
(1
mM)
was
assessedusing
the
LLC-PK1
monolayermodel.
As
shown
insynthetic
Figure14
The acids
inhibitionof was[ C]fluciclovine(10 μM)by naturally
occurringamino(1 mM)
assessed usingtransport
the LLC-PK1
monolayermodel. As
shown inand
Figure3, the3,14 C]fluciclovine in Na+ -free buffer was 58.8% of the control in Na+
theapical-to-basaltransport
rate
of
[
14
+
amino
acids (1 mM)
was assessedthe LLC-PK1
monolayermodel.
As shown
Figurein3, Nathe+apical-to-basaltransport
rate of [using
C]fluciclovine
in Na-free buffer
was 58.8%of theincontrol+ -dependent transport was an estimated 41.2% of the total
buffer,indicatingthat
thecontributionofNa
apical-to-basaltransport
ratecontributionof [14C]fluciclovine
in Na+-freetransportbuffer wasof the control
buffer, indicatingthat theof Na+-dependent
was58.8%
an estimated
41.2% inof Na
the+
+ -free buffer” that was subtracted from
penetration
amount(Figure3;
thepercentage“controlinNa++total indicating
penetrationthatamount(Figure 3; theofpercentage“controlin Na -freethat wassubtracted
buffer,the contribution
Na -dependenttransport
wasbuffer”
an estimated41.2%
of the“controlin Na+ buffer”).
Inaddition,significant
inhibitionof inapical-to-basaltransport
wasobservedfrom“controlinNa+ buffer”).
In theaddition,significant
inhibitionof apical-to-basaltransport
wasin
total
penetration
amount(Figure3;
percentage“controlNa+-freebuffer”
that wassubtracted
+
+
+
the
presencea combinationand L-threonine(thesubstratefor
an Na
system+ buffer”).
observed
inofinthe
ofofInaNa
combinationof Na(Thr)andL-threonine(Thr)(the -dependent
substrate
for
an
from
“controlNapresence
addition,significant
inhibitionof apical-to-basaltransport
was+ . In contrast,+-dependent
ASC
AATs,
including
ASCT2)
tothesame
extentasthatobserved
intheabsenceofNa+NasystemASC
AATs,
including
ASCT2)
tothesame
extentasthatobserved
intheobserved in the presence of a combination of Na and L-threonine (Thr) (the substrate for an+. In contrast,
noabsence
significant
inhibition
was observed
when using
2-(methylamino)-isobutyricacid
a substrate+-dependentof Na
no significant
inhibition
wasobserved
when asusing
2-(methylamino)Na
systemASC
AATs,
including
ASCT2)
to thesame extent
that(MeAIB,
observedin the+++forthe Na-dependent
system
A AATs,including
SNAT2)
in the A
presenceof Na2-(methylamino), andwheninusing+. In
isobutyricacid(MeAIB,a substrate
for theNa -dependent
systemAATs,
including
SNAT2)
theabsenceof Na
contrast,
no significant
inhibition
was observed
when using+ -independent AAT system+, and when using 2-aminobicyclo[2,2,1]heptane-2-carboxylic
2-aminobicyclo[2,2,1]heptane-2-carboxylic
acid
(BCH,
a
substrate
for
the
Na
presence
of
Na
acid
(BCH,
a
substrate+
isobutyric acid (MeAIB, a substrate for the Na -dependent system A AATs, including SNAT2) in the+ -dependent+-independent+. ThesetheofNaAAT 2-aminobicyclo[2,2,1]heptane-2-carboxylic
systemincludingtheNaabsenceof Na
results
L,forincluding
LAT1)
the absenceof Na+ . L,
Theseresults LAT1)
suggestinthatsystemASC
AATs+, andinwhen
presence
Na
using
acid
(BCH,
a substrate
14+
14
suggestNa-dependentASCpartlyLAT1)
mediate[ C]fluciclovine
partlyrenal[ C]fluciclovinetransport.
+-independentforthe mediate
Nathat
AATsystem
system
L, AATs
includinginrenalthe absenceof Na+. transport.
These results

suggest that Na+-dependent system ASC AATs partly mediate renal [14C]fluciclovine transport.

Figure3. 3.
Theinhibition
profile[14C]fluciclovine(10 apical-to-basalμM) apical-to-basaltransport
using
theFigureThe
inhibition
profileof [14ofC]fluciclovine(10 µM)
transport
using the
LLC-PK1
LLC-PK1 monolayer
model
under theconditions,indicated conditions,
i.e., temperature
1 mM inhibitorsinmonolayermodel under
the indicatedi.e., temperatureand 1 mMand
inhibitors
in Na+ buffer
14C]fluciclovine+ buffer+-free buffer (black
Figure3. The(white
inhibition
profileof[Na(10 μM)apical-to-basaltransport
using the
+
Na
bar,
n
=
5–14)
or
bar,
n
=
9).
Each
data
point
represents
the(white bar, n = 5–14) or Na -free buffer (black bar, n = 9). Each data point represents the mean ± SD.
+ buffer. conditions, i.e., temperature and 1 mM inhibitors in
LLC-PK1
model±monolayerSD.
* p < 0.05
inthe
Naindicated+ under* pmean< 0.05vs control
in vsNacontrol
buffer.
++Na buffer (white bar, n = 5–14) or Na -free buffer (black bar, n = 9). Each data point represents themean ± SD. * p < 0.05 vs control in Na+ buffer.

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5 of 132.4. [14C]Fluciclovine Transport via ATP-binding Cassette (ABC) Transporters and the Inhibitory Effect of
Fluciclovine
on ABC Transporters
2.4. [14 C]Fluciclovine
Transport via ATP-binding Cassette (ABC) Transporters and the Inhibitory Effect of
Fluciclovine
on ABC Transporters[14C]Fluciclovine
uptake was measured using ABC transporter-expressing vesicles. As shown in
14 C]Fluciclovine
Figure [4,no significantuptake
differencein [14C]fluciclovine
was observed between
theshown
control,
was measured
using ABCuptake
transporter-expressing
vesicles. As
14
14
P-gp,
and MRP4(Figure4a,c).inIn[ contrast,[ C]fluciclovine
by the
BCRPthevesicleswasin Figure4, no vesicles
significant
differenceC]fluciclovine
uptake wasuptake
observed
betweencontrol,
P-gp, andvesicles (Figure
In contrast,
C]fluciclovine
the BCRP vesicles
reducedin MRP4
comparison
to that4a,c).
of thecontrol[14vesicles,
albeituptake
not bysignificantly(Figurewas
4b).
14
reduced in comparison
to that of
the control
vesicles,significantly
Furthermore,
Furthermore,[ C]fluciclovine
uptakeby theBCRPalbeit
and not
MRP4
vesicles (Figure
showed4b).
Michaelis-Menten
14 C]fluciclovine uptake by the BCRP and MRP4 vesicles showed Michaelis-Menten kinetics (Table 1).
[
kinetics (Table 1).

Figure4. 4.
(a–c):
TheTheconcentration
dependency
of of[14C]fluciclovine
in in(a)(a)
P-glycoprotein
Figure(a–c):
concentration
dependency[14 C]fluciclovine
P-glycoprotein(P-gp)-;(P-gp)-;(b)breastcancerresistance
protein(BCRP)-;
and(c)multidrug
resistance-associatedprotein(b) breast cancer resistance protein (BCRP)-; and (c) multidrug resistance-associated protein 4 4(MRP4)-expressing
oftracertraceruptake
uptakeby
byunlabeledunlabeledfluciclovine(MRP4)-expressinginside-out
inside-outvesicles;
vesicles; (d–f):(d–f): thethe inhibition
inhibition of
fluciclovine
P-gp-;(e)(e)BCRP-;
BCRP-;and
and(f)(f)MRP4-expressing
MRP4-expressing inside-out
represents
thethein in(d)(d)
P-gp-;
inside-outvesicles.
vesicles.Each
Eachdata
datapoint
point
represents
33H]estrone-3-sulfate” and “[3 H]estradiol-17β3
mean
±
SD
(n
=
3–6).
“ES”
and
“E2G”
indicate“[
D -Dmean ± SD (n = 3–6). “ES” and “E2G” indicate “[ H]estrone-3-sulfate” and “[ H]estradiol-17βglucuronide”,respectively.
respectively.
glucuronide”,
Table1.1.KKmmand
andIC
IC5050values
valuesof
offluciclovine
fluciclovine forfor specificspecificdrug
drugtransporters.
transporters.
Table

Transporters
Transporters
Efflux Transporters
Efflux Transporters
P-gp
P-gp
1
BCRP
BCRP 1
1
MRP4 1
MRP4
Uptaketransporters
Uptaketransporters2
OAT1
OAT1 23
OAT3
OAT3 3
OCT2 3 3
OCT2
OATP1B1 4 4
OATP1B1OATP1B3 5
OATP1B3 5

m (mM)
Km
K
(mM)

IC50
IC(mM)
50 (mM)

N.D.
N.D.
1.34
± 1.451.34 ±
1.45
15.3 ±± 44.115.3

2.95± 2.562.95± 2.56
N.D.
N.D.
9.28
± 1.909.28± 1.90

258
48.2
258 ±
± 48.2
N.D.
N.D.
275 ± 36.6
275 ± 36.6
401 ± 41.0
401 ± 41.0
238 ± 82.5

3.98
± 1.77
3.98± 1.77238 ± 82.5

8.95 ± 1.96
8.95 ± 1.96
5.41 ± 2.49
5.41 ± 2.49
N.D.
N.D.

All values represent the mean ± SD (n = 3–6). N.D.: Not determined within the concentration range used in thisAll values
mean
SDsignificant(n = 3–6).
Not determinedwithinvesicles,
the concentration
range
study. 1 represent
Values at allthe
pointswere±notin N.D.:
comparison
with those of control
but Km is shown
2–5 The calculated K values (µM) of the mock cells were1
because
these
data
fit
Michaelis–Menten
kinetics;mused in this study. Values at all points were not significant in comparison with those of control
258 ± 127 (n = 6), 340 ± 43.1 (n = 3), 349 ± 40.0 (n = 3), and 340 ± 161 (n = 6). P-gp: P-glycoprotein; BCRP: breast
2–5 The calculated Km
vesicles,Km isprotein;shownMRP4:
becausetheseresistance-associateddata fit Michaelis–Menten
kinetics;
cancerbut
resistancemultidrug
protein 4; OAT1:
organic aniontransporter 1;
OAT3:
organic
anion
transporter
3;
OCT2:
organic
cationtransporter
2;
OATP1B1:
organic
anion
values (μM) of the mock cells were 258 ± 127 (n = 6), 340 ± 43.1 (n = 3), 349 ± 40.0
(n =transporting3), and 340 ±polypeptide 1B1; OATP1B3: organic anion transporting polypeptide 1B3.
161 (n = 6). P-gp: P-glycoprotein; BCRP: breast cancer resistance protein; MRP4: multidrug
resistance-associated protein 4; OAT1: organic anion transporter 1; OAT3: organic anion transporter
3; OCT2: organic cation transporter 2; OATP1B1: organic anion transporting polypeptide 1B1;
OATP1B3: organic anion transporting polypeptide 1B3.

Int. J. Mol. Sci. 2016, 17, 1730

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Int. J. Mol. Sci. 2016, 17, 1730

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Next, the inhibitory effect of fluciclovine on ABC transporters was also determined using
ABC-transporter-expressing vesicles. As shown in Figure 4d–f and Table 1, fluciclovine at millimolar
Next, the inhibitory
effect andof fluciclovinetransporters
wasandalsoTable
determined
concentrations
inhibited P-gp
MRP4, but on
notABCBCRP(Figure 4d–f1). Theusing
ABC
ABC-transporter-expressing
vesicles.
As
shown
in
Figure
4d–f
and
Table
1,
fluciclovineatmillimolar
transporter-mediated transport of specific substrates in the absence of inhibitors was 0.739,
0.523,
concentrations
inhibited
P-gp forandP-gp,
MRP4,
butandnotMRP4,
BCRPrespectively.
(Figure 4d–f and Table 1). The ABC
and 159 pmol/mg
protein/min
BCRP,
transporter-mediated transport of specific substrates in the absence of inhibitors was 0.739, 0.523,
andpmol/mg protein/min
forSoluteP-gp,Carrier
BCRP,(SLC)and MRP4,
respectively.
2.5. 159[14C]Fluciclovine
Transport via
Transporters
and the Inhibition of SLC Transporters
by Fluciclovine
2.5. [14 C]Fluciclovine Transport via Solute Carrier (SLC) Transporters and the Inhibition of SLC Transporters
Figure 5 shows [14C]fluciclovine uptake by SLC drug transporter-expressing cell lines. The
by Fluciclovine
intracellular uptake 14
of [14C]fluciclovine by mock cells was almost equal to that of
Figure 5 shows [ C]fluciclovine uptake by SLC drug transporter-expressing cell lines.
OATP1B3-expressing cells, and was higher than that of OAT1-, OAT3-, OCT2-, and
The intracellular uptake of [14 C]fluciclovine by mock cells was almost equal
to that
OATP1B1-expressing cells (Figure 5a–d). Therefore, the drug transporter-mediated [14C]fluciclovine
of OATP1B3-expressing cells, and was higher thanthatof
OAT1-,
OAT3-,
OCT2-,
anduptake appeared to be negligible. Moreover, [14C]fluciclovine uptake in all cells showed
OATP1B1-expressing cells (Figure 5a–d). Therefore, the drug transporter-mediated [14 C]fluciclovine
Michaelis-Menten kinetics (see Table 1 for the Km values). Similarly, the inhibition of SLC
uptake appeared to be negligible. Moreover, [14 C]fluciclovine uptake in all cells showedtransporters by fluciclovine was assessed using SLC transporter-expressing cell lines. As shown in
Michaelis-Menten kinetics (see Table 1 for the Km values). Similarly, the inhibition of SLC transporters
Figure 5e–i and Table 1, the inhibition by fluciclovine at millimolar concentrations was observed inby fluciclovine was assessed using SLC transporter-expressing cell lines. As shown in Figure 5e–i
OAT1, OCT2, and OATP1B1 cells, but not in OAT3 and OATP1B3 cells (Figure 5e–i). The
and Table 1, the inhibition by fluciclovine at millimolar concentrations was observed in OAT1, OCT2,transporter-mediated uptake using specific substrates in the absence of inhibitors was 38.5, 6.38,
and OATP1B1 cells, but not in OAT3 and OATP1B3 cells (Figure 5e–i). The transporter-mediated uptake
93.5, 3.37, and 0.570 pmol/mg protein/min for OAT1, OAT3, OCT2, OATP1B1, and OATP1B3,
using specific substrates in the absence of inhibitors was 38.5, 6.38, 93.5, 3.37, and 0.570 pmol/mg
respectively.
protein/min for OAT1, OAT3, OCT2, OATP1B1, and OATP1B3, respectively.

FigureFigure5.5.(a–d):(a–d): The
Theconcentration
concentrationdependency
dependencyof
ofthe
the[14[14C]fluciclovine
C]fluciclovine uptake
uptake inin (a)(a)organicorganicanion
anion
transportertransporter11(OAT1)(OAT1)-(b)(b)organicorganicanion
aniontransportertransporter33(OAT3)-/(OAT3)-/organicorganiccation
cation transportertransporter 22 (OCT2)(OCT2)(c)(c) organicorganic anion
anion transportingtransporting polypeptide
polypeptide 1B1
1B1 (OATP1B1)(OATP1B1)-and
and(d)(d)organicorganicanion
aniontransportingtransporting
polypeptide
polypeptide 1B3
1B3 (OATP1B3)-expressing(OATP1B3)-expressing cells;
cells; (e–i):(e–i): thethe inhibition
inhibition ofof tracer
tracer uptake
uptake byby unlabeledunlabeledfluciclovinein(e)
OAT1-;(f)
OAT3-;(g)
OCT2-;(h)OATP1B1-;
and(i)
OATP1B3-expressing
fluciclovine in (e) OAT1-; (f) OAT3-; (g) OCT2-; (h) OATP1B1-; and (i) OATP1B3-expressing cells.
cells.
Each
data
point representsmean± * SD(n =“PAH”,
3–6); *“ES”,
p <“Metformine”
0.05. “PAH”,
Each data
point
represents
the mean ±theSD (n= 3–6);
p < 0.05.
and“ES”,
“E2G”
14
3
14
3H]estrone-3-sulfate”,“Metformine”
and“E2G” indicate “p-[ “[C]aminohippurate”,“[ H]estrone-3-sulfate”,“[ C]metformin”,
C]aminohippurate”,“[14C]metformin”, and“[3H]estradiolindicate “p-[143 H]estradiol-17β- D -glucuronide”, respectively.
andD-glucuronide”,
respectively.
17β-“[

3.3. Discussion
Westudies toto assessassessthetheinvolvement
involvementofofAATs
AATsandanddrug
drugtransporterstransporters
Weconductedconducted aa series of studies
in14C]fluciclovineindistribution[14 C]fluciclovine
in vitro,
therebyspecificallyfocusing
on renal
reuptake.
thethedistributionof [ofin vitro,
therebyspecificallyfocusing
on renal
reuptake.
The

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The apical-to-basal transport of [14 C]fluciclovine in the LLC-PK1 monolayer model was inhibitedby Thr, a substrate representative for system ASC AATs [9,12]. Furthermore, control vesicles/cells
and ABC/SLC drug transporter-expressing vesicles/cells showed no significant differences in drugtransporter-mediated [14 C]fluciclovine uptake. Fluciclovine at millimolar concentrations inhibitedthe uptake of specific substrates by some of the drug transporters tested. Our study suggests thatthe slow urinary excretion of [18 F]fluciclovine is partly mediated by system ASC AATs, which serveas high-affinity transporters for [18 F]fluciclovine uptake/efflux, but that the contribution of drugtransporters to [18 F]fluciclovine distribution is negligible compared to that of AATs.
LLC-PK1 cells acquire cell polarity similar to that of renal proximal tubule cells during monolayer
cultivation [21,22]. Regarding the localization of AATs that mediate [18 F]fluciclovine uptake,
ASCT2 (a subtype of system ASC AATs) is localized in the apical membrane of the renal proximal
tubule cells [23], and SNAT2 (a subtype of system A AATs) is ubiquitously expressed, including in thekidneys [12,24]. The system L AAT LAT1 is localized both in the apical and basolateral membrane, but
its basolateral expression is higher in LLC-PK1 cells [22]. As shown in Figure 3, the contribution of
SNAT2 and LAT1 to [14 C]fluciclovine uptake, which was calculated from the inhibition by MeAIB and
BCH, was low at the apical side. These results reveal that system ASC AATs, including ASCT2, at theapical side are the main transporters mediating [14 C]fluciclovine reuptake by renal proximal tubules.
Furthermore, system L in the basolateral membrane may be involved in [14 C]fluciclovine transport
from the renal proximal tubule cells to the blood [22].
System A and ASC AATs recognize L-alanine as a substrate, but its permeation is significantly
lower than that of [14 C]fluciclovine in the LLC-PK1 monolayer model (Figure 2a). If the activity of
SNAT2 at the apical side is low as described above, the influx and backflux of fluciclovine may be
mediated via system ASC AATs because system ASC AATs function as exchangers [12]. In contrast,
L -leucine is one of the substrates of system L AATs, and its permeability profile is similar to that of[14 C]fluciclovine (Figure 2a). If the involvement of system L is low at the apical side, other AATs
present at the apical side (e.g., system B, may be involved in the reabsorption of L-Leu [23]). LLC-PK1
cells are derived from pig kidney; thus, another possibility is that the localization and/or the affinityof amino acids for AATs may be different in other species.
We have previously reported that p-aminohippurate (PAH), a substrate for OAT1/3, slightly(approximately 5%) inhibited [18 F]fluciclovine uptake [8], suggesting that [18 F]fluciclovine might
be anionic at physiological pH and thus might be recognized by anion transporters such as MRP4,OAT1, OAT3, OATP1B1, and OATP1B3 [14]. As shown in Figures 4 and 5 and Table 1, the uptake
profiles of [14 C]fluciclovine fit the Michaelis-Menten kinetics for some drug transporters. Nevertheless,in the present study, there was no significant difference in the [14 C]fluciclovine uptake between
control vesicles/cells and vesicles/cells expressing MRP4, OAT1, OAT3, OATP1B1, or OATP1B3.
These observations suggest that AATs, but not drug transporters, are high-affinity transporters involvedin [18 F]fluciclovine uptake, because our previous in vitro study showed that a substrate of system L
AATs inhibited [18 F]fluciclovine uptake to a higher degree than that observed for PAH [8].
As shown in Figure 4b, we observed a difference, although nonsignificant, in [14 C]fluciclovine
uptake by control and BCRP vesicles, suggesting that BCRP might be somewhat involved in[14 C]fluciclovine transport. BCRP is an efflux transporter that has an important role in drug dispositionand distribution, and prevents drugs from penetrating not only the brain and intestines but also tumors,and is also involved in biliary and renal excretion of drugs [14]. Consequently, if the observed difference
mentioned above is physiologically relevant, BCRP may play a role in the excretion of [18 F]fluciclovinefrom the liver, kidneys, and other organs. In cancer patients, our tracer is not expected to affect the
distribution of BCRP-mediated anticancer drugs such as mitoxantrone [25], because here we showthat fluciclovine does not inhibit the transport of the BCRP-specific substrate [3 H]estrone-3-sulfate(ES, Figure 4e and Table 1).
Clinical trials have shown that [18 F]fluciclovine accumulates in the liver, is excreted slowly in theurine, and provides high tumor-to-normal contrast in cerebral PET imaging because the distribution of

Int. J. Mol. Sci. 2016, 17, 1730

8 of 13[18 F]fluciclovine to the brain under physiological conditions is restricted [6,26,27]. The major organs
targeted by [18 F]fluciclovine are the liver, kidneys, and brain (the latter via BBB transport) as theyexpress system A (SNAT2), ASC (ASCT2), and L (LAT1) AATs as well as drug transporters [12,14].
However, O’Kane et al. [28] reported that the concentrations of neutral amino acids in the extracellular
cerebral fluid are maintained at <10% of those in the plasma by the action of AATs at the BBB. Thus,[18 F]fluciclovine accumulation in the brain is likely restricted because of neutral amino acid efflux
via AATs at the BBB. [11 C]Methionine, an amino acid PET tracer used in tumor detection, has someproperties similar to those of [18 F]fluciclovine: (1) [11 C]methionine is transported via AATs [10];(2) [11 C]methionine accumulates readily in the liver, moderately in the kidney, but accumulates
little in the brain [29]; and (3) [11 C]methionine accumulation is not affected by P-gp expression [30].
Considering the similarity in the distribution mechanisms underlying [18 F]fluciclovine and[11 C]methionine, the present study suggests that, in contrast to that for most therapeutic drugs [31],AATs are the primary contributors to the hepatic, renal, and cerebral biodistribution of [18 F]fluciclovine.
As shown in Figures 4 and 5 and Table 1, millimolar concentrations of fluciclovine inhibited someof the transporters tested. OAT3-mediated ES uptake increased in the presence of 10 mM fluciclovine,which is in contrast to what was observed for the other transporters tested. A potential explanation forthis finding is that the indirect effects, such as a change in the intracellular dicarboxylate concentration,
may affect ES uptake because OAT3 functions as an exchanger that exchanges its extracellular substrate
and intracellular dicarboxydrate [12]. In contrast, fluciclovine competes directly with the uptakeof ES. However, it is unlikely that such inhibition/stimulation will occur clinically because onlya small amount of tracer (in the picomolar or nanomolar range) is commonly administered in PET
imaging [32]. This, and the fact that [18 F]fluciclovine is distributed throughout the entire body, prevent
the [18 F]fluciclovine plasma concentration from reaching levels required for inhibition. Therefore,
the initial administration of [18 F]fluciclovine to patients with brain or prostate cancer during PET
procedures [2,3] is unlikely to cause drug transporter-mediated drug–drug interactions if generally
accepted initial dosages for PET imaging are used.
The glucose analog [18 F]FDG is a PET tracer that is widely used for cancer diagnosis and, similar18
to [ F]fluciclovine, is not a P-gp substrate [33]. However, treatment guidelines recommend morethan four hours of fasting before conducting [18 F]FDG-PET imaging to avoid drug-food interactions,because high blood glucose levels caused by food ingestion directly affect the tumor-to-normal
contrast [4]. In the case of amino acid-derived PET tracers, there is some controversy whether or not
short-term fasting is needed to enhance contrast by reducing the plasma amino acid concentration
prior to administration of amino acid PET tracers [34,35]. For example, clinical trials of [18 F]fluciclovine
in Japan were conducted with fasted volunteers/patients [2,3,6]. Furthermore, high-protein meals
increase plasma levels of free amino acids, including those of branched-chain amino acids [36],which compete for AAT-mediated [18 F]fluciclovine uptake [9,10]. The present in vitro study indicatesthat drug transporters are not as crucial for [18 F]fluciclovine biodistribution as AATs are. Based ontreatment guidelines and our present study outcomes, the relationship between food intake andimaging contrast in [18 F]fluciclovine-PET may warrant evaluation in future studies, because AATs are
the predominant transporters involved in the [18 F]fluciclovine biodistribution.
4. Materials and Methods
4.1. Reagents and Radioisotope-Labeled Tracers
All reagents were purchased from commercial suppliers (Wako Pure Chemical Industries,
Osaka, Japan; Sigma-Aldrich, Japan, Tokyo, Japan; and Nacalai Tesque, Kyoto, Japan) unlessindicated otherwise.
In all experiments, 3 H- or 14 C-labeled tracers were used because their long half-lives (12.3 and5700 years, respectively) make them more suitable for in vitro experiments than 18 F (110 min).
trans-1-Amino-3-fluoro[1-14 C]cyclobutanecarboxylic acid ([14 C]fluciclovine, 2.09 GBq/mmol) wasInt. J. Mol. Sci. 2016, 17, 1730

9 of 13synthesized [9] by Nemoto Science (Tokyo, Japan). L-[14 C]alanine (5.9 GBq/mmol, for system
A and ASC AATs); L-[14 C]leucine (12.0 GBq/mmol, for system L AATs); D-[1-3 H(N)]mannitol(455.1 GBq/mmol, for the evaluation of paracellular transport on LLC-PK1 monolayer experiment);[3 H]digoxin (1104 GBq/mmol, for P-gp); [3 H]estrone-3-sulfate ([3 H]ES; 1655 GBq/mmol, for BCRP
and OAT3); and p-[14 C]aminohippurate ([14 C]PAH; 2.04 GBq/mmol, for OAT1) were purchasedfrom PerkinElmer (Waltham, MA, USA). [3 H]estradiol-17β-D-glucuronide ([3 H]E2G; 1269 GBq/mmol,for MRP4, OATP1B1, and OATP1B3) and [14 C]metformin (3.7 GBq/mmol, for OCT2) were purchasedfrom American Radiolabeled Chemicals (St. Louis, MO, USA).
4.2. Monolayer Establishment Using LLC-PK1 Cells
The LLC-PK1 cell line was purchased from DS Pharma Biomedical (Osaka, Japan). Cells weremaintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Japan, Tokyo, Japan)
supplemented with 10% (v/v) fetal bovine serum (American Type Culture Collection, Manassas,
VA, USA) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin (Life Technologies
Japan)). Cells were cultured at 37 ◦ C under an atmosphere containing 5% CO2 before the start ofthe experiments.
Cells were seeded at a density of 1.5 × 106 cells/0.25 mL on the membrane of cell culture inserts
fitting 24-well plates (upper compartment, apical side; Becton Dickinson, Franklin Lakes, NJ, USA).
Flat-bottom 24-well tissue culture plates (Becton Dickinson) containing 0.95 mL of culture medium perwell were used as the basolateral side (bottom compartment).
LLC-PK1 monolayer formation was monitored by the measurement of TER using an EVOM-2
and Endohm24 (World Precision Instruments, Inc., Sarasota, FL, USA) according to the manufacturer’s
protocol. After cultivation for 4–5 days, the monolayers with a TER value of >250 Ω·cm2 were used forthe experiment as described in Sections 4.3 and 4.4. [3 H]Mannitol transport was measured as describedin Section 4.3 for the validation of tight junction formation because mannitol passes through the cellmonolayers via the paracellular route.
4.3. Apical-to-Basal Transport Using LLC-PK1 Monolayers
Experimental procedures were performed as described previously [37] with minor modifications.
After cultivation as described above, the culture media in both compartments were replaced with Na+
buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2 HPO4 , 1.5 mM KH2 PO4 , 5.6 mM D-glucose, 0.9 mM
CaCl2 , and 0.5 mM MgCl2 ; pH 7.4 at 37 ◦ C), and the cells were preincubated for 10 min at 37 ◦ Cunder normal air conditions. After the buffer was removed from each compartment, the permeability
experiments were started with the addition of 0.25 mL of Na+ buffer containing radiolabeled tracers(final concentration: 81 nM for [3 H]mannitol and 10 µM for the 14 C-labeled amino acid tracers) in each
upper compartment, and 0.95 mL of Na+ buffer in each bottom compartment. The inserts were placed
into new 24-well culture plates containing 0.95 mL/well of Na+ buffer after 5, 15, 30, 60, and 90 min of
incubation. Subsequently, the cells were incubated up to 120 min at 37 ◦ C under normal air conditions.
The apical-to-basal permeability was measured by sampling an aliquot of the buffer from theupper and bottom compartment at the indicated times. To evaluate intracellular accumulation, the cells
were lysed in 0.5 mL of 0.1N NaOH at room temperature following rapid washing of the cells with
ice-cold Na+ buffer. The radioactivity of each aliquot was measured with an LSC6000SC (Beckman
Coulter, Inc., Brea, CA, USA) or Tri-Carb 2910TR liquid scintillation counter (PerkinElmer) using
Ultima Gold (PerkinElmer).
4.4. Inhibition Study Using the LLC-PK1 Monolayer Model
After having developed the LLC-PK1 monolayer model as described in Section 4.2,
an apical-to-basal transport study was conducted in Na+ buffer (a condition in which all AATs
were active) or Na+ -free buffer (NaCl was replaced with an equivalent concentration of choline
chloride, ensuring that only Na+ -independent AATs were active) containing 10 µM [14 C]fluciclovine

Int. J. Mol. Sci. 2016, 17, 1730

10 of 13for 120 min at 37 ◦ C under normal air conditions in the presence or absence of 1 mM of the inhibitors.
Natural and synthetic amino acids were used as inhibitors [9]: MeAIB (2-(methylamino)-isobutyricacid, for system A in the presence of Na+ ); Thr (L-threonine, for system ASC in the presence ofNa+ ); and BCH (2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, for system L in the absence of Na+ ).
The apical-to-basal permeability of [14 C]fluciclovine in Na+ buffer without inhibitors was normalized
to 100%, and the inhibition of [14 C]fluciclovine permeation by the natural and synthetic amino acids
was calculated as a percentage of the control in Na+ buffer.
4.5. Uptake and Inhibitory Assay Using ABC Transporter-Expressing Vesicles
For ABC transporters, experimental kits containing Sf9-derived vesicles with high expression
levels of human P-gp, MRP4, BCRP, and each ABC transporter (including buffer, ATP/AMP solution,and glutathione) were purchased from GenoMembrane Co., Ltd. (Kanagawa, Japan).
Vesicular transport assays were carried out according to the manufacturer’s protocol. The reaction
buffer (50 mM MOPS-Tris, 70 mM KCl, and 7.5 mM MgCl2 ; pH 7.0) contained 4 mM ATP or AMP,
unlabeled and labeled fluciclovine, and 2 mM glutathione (only for MRP4). The reaction was started
by suspending 50 µg of vesicles (10 µL) in 40 µL of the reaction buffer. After incubation for an
appropriate period of time, the reaction was stopped by the addition of ice-cold stop buffer (40 mM
MOPS-Tris and 70 mM KCl; pH 7.0), after which the solution was filtered through a 0.45 µm HA filter(Millipore Corp., Billerica, MA, USA) using suction, and was washed twice in 5 mL of ice-cold stopbuffer. The radioactivity of the filter was measured with a Tri-Carb 2910TR liquid scintillation counter(PerkinElmer) using Ultima Gold (PerkinElmer). The amount of tracer uptake was normalized to themembrane protein amount described in the product information sheets.
The inhibitory study was performed as described above using appropriate tracers (digoxin: 1 µM,
ES: 0.1 µM, or E2G: 10 µM; as described in “4.1 Reagents and radioisotope-labeled tracers”) in thepresence of 100 µM–12.5 mM of unlabeled fluciclovine. The tracer uptake amount in the absence of
fluciclovine was set as 100%.
4.6. Uptake and Inhibitory Assay Using SLC Transporter-Expressing Cells
For the SLC transporter experiments, Mock Flp-in-293 cells (Flp/mock), OAT1-expressing
Flp-in-293 cells (Flp/OAT1) [38], mock HEK293 cells (HEK/mock), and OATP1B3-expressing HEK293
cells (HEK/OATP1B3) [39] were obtained from Kanazawa University (Ishikawa, Japan). Briefly,
Flp-in-293 and HEK293 cells were transfected with the appropriate plasmid DNA and selectedusing antibiotics (hygromycin or G-418). All cell lines were cultured in DMEM (Life Technologies
Japan) supplemented with 10% (v/v) fetal bovine serum (American Type Culture Collection) andantibiotics (100 U/mL penicillin and 100 µg/mL streptomycin (Life Technologies Japan) for mock cells,
0.2 mg/mL hygromycin for Flp/OAT1 cells, and 1.2 mg/mL G-418 (Roche Diagnostics K.K., Tokyo,
Japan) for HEK/OATP1B3 cells) at 37 ◦ C under an atmosphere containing 5% CO2 . For the
OAT3 and OCT2 experiments, mock HEK293 cells (HEK/mock), OAT3-expressing HEK293 cells(HEK/OAT3), and OCT2-expressing HEK293 cells (HEK/OCT2) were purchased in culture plates
from GenoMembrane, Co., Ltd.. For the OATP1B1 experiments, mock HEK293 cells (HEK/mock) and
OATP1B1-expressing HEK293 cells (HEK/OATP1B1) were purchased in vials from GenoMembrane,Co., Ltd. All cells were cultured according to the manufacturer’s protocol, and were maintained at
37 ◦ C under an atmosphere containing 5% CO2 before the experiments.
The experiments were performed as described previously [39], with minor modifications.
Cells provided by Kanazawa University were seeded at a density of 5 × 104 cells per well inpoly-D-lysine-coated 24-well flat-bottom tissue culture plates (Becton Dickinson). After 3 days ofcultivation, the cells were washed twice with Hank’s balanced salt solution containing Ca2+ and Mg2+
(HBSS; Life Technologies Japan), after which they were incubated with 0.3 mL of HBSS containing
unlabeled and labeled fluciclovine for 5 min at 37 ◦ C under normal air conditions. Tracer uptake
was terminated by removing the tracer solution and rapidly washing the cells twice with ice-cold

Int. J. Mol. Sci. 2016, 17, 1730

11 of 13

HBSS. The cells were lysed in 0.3 mL of 0.1N NaOH at room temperature, after which the radioactivityof each aliquot was measured as described above. The protein concentration of the cell lysate wasdetermined with a VersaMax microplate reader (Molecular Devices Japan K.K., Osaka, Japan) or
SpectraMax i3x (Molecular Devices Japan K.K.; only for OATP1B1) using a BCA Protein Assay Kit(Thermo Fisher Scientific, Kanagawa, Japan). Tracer uptake was expressed as pmol/mg protein.
For the cells purchased from GenoMembrane Co., Ltd., the experiments were carried out as describedabove after cultivation according to the manufacturer’s protocol.
The inhibitory study was performed as described above using appropriate tracers (PAH: 1 µM,
ES: 1 µM, metformin: 10 µM, or E2G: 0.1 µM; as described in “4.1 Reagents and radioisotope-labeledtracers”) in the presence of 100 µM–10 mM unlabeled fluciclovine. The tracer uptake amount in theabsence of fluciclovine was set as 100%.
4.7. Data and Statistical Analysis
All data are presented as the mean ± standard deviation (SD), and are rounded off tothree significant figures. Km and IC50 values were calculated using GraphPad Prism 5.0 (GraphPad
Software, Inc., La Jolla, CA, USA). Statistical analyses were carried out in the SAS software (version 5.0;
SAS Institute Japan, Tokyo, Japan) using two-tailed unpaired t-tests or Tukey’s multiple comparison
tests. The threshold for significance was p < 0.05.
5. Conclusions
We show that [18 F]fluciclovine is recognized as a drug transporter inhibitor with millimolar affinity
to the tested drug transporters in vitro, suggesting that slow urinary excretion of [18 F]fluciclovine ismediated in part by system ASC AATs, but not by drug transporters.
Acknowledgments: The authors express their appreciation to Sachiko Naito (Nihon Medi-Physics Co., Ltd.) for
performing the cell culture, as well as to Soichi Nakamura (Nihon Medi-Physics Co., Ltd.) for providing unlabeled
fluciclovine. The authors also thank Ikumi Tamai and Takeo Nakanishi (Kanazawa University) for providing drugtransporter-expressing cell lines. The authors thank Kodai Nishi (Nagasaki University) for helpful discussions.
Author Contributions: Masahiro Ono, Atsumi Baden, Hiroyuki Okudaira, and Shuntaro Oka conceived
and designed the experiments; Masahiro Ono and Atsumi Baden performed the experiments; Masahiro Ono
and Atsumi Baden analyzed the data; Hiroyuki Okudaira, Masato Kobayashi, Keiichi Kawai, Shuntaro Oka,and Hirokatsu Yoshimura provided reagents/materials/analysis tools; Masahiro Ono, Atsumi Baden,
and Shuntaro Oka wrote the manuscript draft; all authors discussed the acquired data and contributed to therevision of the manuscript.
Conflicts of Interest: Masahiro Ono, Atsumi Baden, Hiroyuki Okudaira, Shuntaro Oka, and Hirokatsu Yoshimura
are employees of Nihon Medi-Physics Co., Ltd. Keiichi Kawai had research collaborations with Nihon
Medi-Physics Co., Ltd. until March 2016. Masato Kobayashi has no conflict of interest to declare.

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