99mTc(N)-DBODC(5), a potential radiolabeled probe for SPECT of multidrug resistance: In vitro study

Article abstract of DOI:10.1007/s00775-013-0997-1

[^99mTc(N)(DBODC)(PNP5)]⁺ [DBODC is bis(N-ethoxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypropylphosphinoethyl) ethoxyethylamine], abbreviated as ^99mTc(N)-DBODC(5), is a lipophilic cationic mixed compound investigated as a myocardial imaging agent. The findings that this tracer accumulates in mitochondrial structures through a mechanism mediated by the negative mitochondrial membrane potential and that the rapid efflux of ^99mTc(N)-DBODC(5) from nontarget tissues seems to be associated with the multidrug resistance (MDR) P-glycoprotein (P-gp) transport function open up the possibility to extend its clinical applications to tumor imaging and noninvasive MDR studies. The rate of uptake at 4 and 37 C of ^99mTc(N)-DBODC(5) was evaluated in vitro in selected human cancer cell lines and in the corresponding sublines before and after P-gp and/or MDR-associated protein (MRP) modulator/inhibitor treatment using ^99mTc-sestamibi as a reference. The results indicated that (1) the uptake of both ^99mTc(N)-DBODC(5) and ^99mTc-sestamibi is correlated to metabolic activity of the cells and (2) the cellular accumulation is connected to the level of P-gp/MRP expression; in fact, an enhancement of uptake in resistant cells was observed after treatment with opportune MDR inhibitor/modulator, indicating that the selective blockade of P-gp/MRP prevented efflux of the tracers. This study provides a preliminary indication of the applicability of ^99mTc(N)-DBODC(5) in tumor imaging and in detecting P-gp/MRP-mediated drug resistance in human cancer. In addition, the possibility to control the hydrophobicity and pharmacological activity of this heterocomplex through the variation of the substituents on the ligands backbone without affecting the P₂S₂ coordinating sphere makes ^99mTc(N)-DBODC(5) a suitable scaffold for the preparation of a molecular probe for single photon emission computed tomography of MDR.

Full text of DOI:10.1007/s00775-013-0997-1

J Biol Inorg Chem (2013) 18:523–538
DOI 10.1007/s00775-013-0997-1
ORIGINAL PAPER
99mTc(N)-DBODC(5), a potential radiolabeled probe for SPECT
of multidrug resistance: in vitro study
•
Cristina Bolzati Davide Carta Valentina Gandin
•
•
•
•
Cristina Marzano Nicolo Morellato Nicola Salvarese
•
`
Mariangela Cantore Nicola Antonio Colabufo
•
Received: 11 December 2012 / Accepted: 10 March 2013 / Published online: 31 March 2013
Ó SBIC 2013
Abstract
[
99mTc(N)(DBODC)(PNP5)]? [DBODC is bis
cellular accumulation is connected to the level of P-gp/
MRP expression; in fact, an enhancement of uptake in
resistant cells was observed after treatment with opportune
MDR inhibitor/modulator, indicating that the selective
blockade of P-gp/MRP prevented efflux of the tracers. This
study provides a preliminary indication of the applicability
of 99mTc(N)-DBODC(5) in tumor imaging and in detecting
P-gp/MRP-mediated drug resistance in human cancer. In
addition, the possibility to control the hydrophobicity and
pharmacological activity of this heterocomplex through the
variation of the substituents on the ligands backbone
without affecting the P2S2 coordinating sphere makes
99mTc(N)-DBODC(5) a suitable scaffold for the prepara-
tion of a molecular probe for single photon emission
computed tomography of MDR.
(N-ethoxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypr-
opylphosphinoethyl)ethoxyethylamine], abbreviated as
99mTc(N)-DBODC(5), is a lipophilic cationic mixed com-
pound investigated as a myocardial imaging agent. The
findings that this tracer accumulates in mitochondrial
structures through a mechanism mediated by the negative
mitochondrial membrane potential and that the rapid efflux
of 99mTc(N)-DBODC(5) from nontarget tissues seems to be
associated with the multidrug resistance (MDR) P-glyco-
protein (P-gp) transport function open up the possibility to
extend its clinical applications to tumor imaging and
noninvasive MDR studies. The rate of uptake at 4 and
37 °C of 99mTc(N)-DBODC(5) was evaluated in vitro in
selected human cancer cell lines and in the corresponding
sublines before and after P-gp and/or MDR-associated
protein (MRP) modulator/inhibitor treatment using 99mTc-
sestamibi as a reference. The results indicated that (1) the
uptake of both 99mTc(N)-DBODC(5) and 99mTc-sestamibi
is correlated to metabolic activity of the cells and (2) the
Keywords Technetium ꢀ P-glycoprotein ꢀ Multidrug
resistance ꢀ MK571 ꢀ Myocardial
Introduction
The in vivo evaluation of complex cellular processes such
as proliferation, apoptosis, receptor–ligand interactions,
transport of substrates, and metabolism of nutrients in
human cancers is a wide and continuing evolving area of
investigation in nuclear medicine. A major goal in this area
is the noninvasive detection of well-known biochemical,
molecular, and histological markers of tumor aggressive-
ness, invasiveness, and resistance to therapy, which may
provide rational criteria for fine-tuning of therapeutic
strategies in individual patients [1].
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-0997-1) contains supplementary
material, which is available to authorized users.
C. Bolzati (&)
ICIS-CNR, Corso Stati Uniti 4, 35127 Padua, Italy
e-mail: bolzati@icis.cnr.it
D. Carta ꢀ V. Gandin ꢀ C. Marzano ꢀ N. Morellato ꢀ
N. Salvarese
Dipartimento di Scienze del Farmaco, University of Padua, Via
Marzolo 5, Padua, Italy
In recent years, increasing knowledge of the cause and
physiological basis of cancer has permitted the identifica-
tion of biomarkers potentially useful for diagnosis and/or
M. Cantore ꢀ N. A. Colabufo
Dipartimento Farmacia-Scienze del Farmaco, University of Bari,
Via Orabona 4, Bari, Italy
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therapy [2, 3]. In this context, nuclear medicine and
radiopharmaceutical science have explored some of these
substances as potential targets for in vivo molecular
imaging and/or targeted radionuclide therapy for cancer,
but despite the efforts, the development of specific tools
and the successful application of new targeted strategies
are still limited [4].
and tissues in which ABC transporters are not only
expressed but also functional.
Over the past years, different radionuclides suitable for
noninvasive single photon emission computed tomography
(SPECT) and positron emission tomography (PET) tech-
niques have been used in the design of molecular probes
for functional imaging of MDR.
The positron emitters 11C and 18F have been used to
label small molecules such as MDR cytotoxic drugs or
classic MDR modulators/inhibitors [15–17]. In spite of the
encouraging preliminary in vitro and in vivo results, they
present some limitations such as modest radiochemical
yields (RCYs) and fast in vivo metabolization. In addition,
owing to the short half-life of 11C (t1/2 = 20 min) and 18F
(t1/2 = 110 min), the preparation of such molecules needs
a cyclotron nearby, limiting their access and widespread
distribution [18, 19].
Conversely radiometals such as 99mTc, 67/68Ga, and
64Cu, owing to their half-life, chemistry, cost, availability,
and in vivo stability of the corresponding compounds,
present advantages over 11C and 18F and have been
investigated for the in vivo evaluation of MDR [4–14].
Nowadays, 99mTc is, because of its optimal nuclear
properties (t1/2 = 6.02 h; Ec = 141 keV), low cost, and
easy availability, the radioisotope of election in nuclear
medicine, being used in different chemical forms in more
than 80 % of clinical SPECT practices. 99mTc is indispens-
able for the estimated 70,000 medical imaging procedures
that take place daily around the world, primarily in myo-
cardial imaging. The current global interruption of 99Mo
supply has made the situation particularly problematic from
a medical standpoint, underlining the importance of 99mTc in
nuclear medicine practices [18, 19], and the need for a
reliable supply network comprising alternative production
options [20]. Nevertheless, the 99Mo shortage should not be
used as an argument to switch large research programs to
other radionuclides [19]. Although the development of PET
and associated fluorinated agents has led to an almost
complete interruption of SPECT research programs and the
development of new Tc-labeled molecules is quite rare, this
can refocus attention on technetium, also in view of the
arrival on the market of new high-quality SPECT cameras
that can compete with PET technology [19].
Consequently, chemotherapy remains one of the major
therapeutic options for treatment of many malignant
tumors and multidrug resistance (MDR) is one of the pri-
mary causes of failure of this treatment [5]. This phe-
nomenon is mediated by plasma membrane ATP-binding
cassette (ABC) transporters such as P-glycoprotein (P-gp)
and MDR-associated protein (MRP) 1 (MRP1) and MRP2
[5–7]. P-gp function is responsible for diminished drug
cellular accumulation. This effect has been, in part,
explained through a ‘‘flippase model’’ [8], in which P-gp
acts as a transporter of drugs from the inner leaflet of the
lipid bilayer to the outer leaflet or the external medium,
allowing the agents to diffuse away, or acts by altering the
membrane permeability, thus reducing the intracellular
concentration of drugs. Beside this, in cells overexpressing
P-gp, effects associated with variation of the intracellular
pH and the membrane potential may alter indirectly the
intracellular distribution of substrates [7] Like P-gp, MRP1
and MRP2 are involved in MDR, but their specific role
needs to be clarified.
P-gp proteins, in addition to their overexpression in
tumors, are normally expressed in normal tissues involved
in excretory function and in barriers such as the blood–
brain barrier, blood cerebrospinal fluid barrier, and blood–
testis barrier. These strategic locations give P-gp a crucial
role exerting control functions in absorption and excretion
of xenobiotics across these barriers. Recent studies repor-
ted a potential correlation between P-gp activity/expression
in the central nervous system and the onset of neurode-
generative disorders such as Alzheimer’s disease, Parkin-
son’s disease, and epilepsy [7–10] Hence, measurement of
P-gp activity and expression is one of the important goals
in planning systemic therapy not only for cancer but also
for neurodegenerative diseases [11, 12].
Expression of MDR P-gp, as detected at the messenger
RNA or protein level, does not always correlate with the
functional assessment of P-gp-mediated transport activity.
This is because P-gp transport activity is affected by spe-
cific mutations as well as the phosphorylation state of the
protein. Altered or less-active forms of P-gp may be
detected by polymerase chain reactions or immunohisto-
chemistry, which do not accurately reflect the status of
tumor cell resistance. Thus, noninvasive methods to func-
tionally interrogate P-gp transport activity in vivo are
required [11–14]. Imaging with radiolabeled transport
substrates, may help to noninvasively identify those tumors
In the last decade, different 99mTc-based agents have
been characterized as transport substrates for MDR P-gp.
Among these, lipophilic cations such as 99mTc-sestamibi
followed by 99mTc-tetrofosmin, originally developed as
myocardial perfusion agents and subsequently used as
tumor-seeking agents in a variety of human neoplasms
[21], emerged as suitable tools to explore specific cellular
processes and functions in malignant tumors [1, 21–24].
From the clinical point of view, the in vivo examination
of the rate of uptake of these 99mTc-based compounds in
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525
tumors is relevant for tumor response to treatment. The
evaluation of the mitochondrial status in untreated tumors
as well as the evaluation of the MDR-P-gp expression and
function are hot topics in clinical oncology. Indeed, these
assessments allow one to select patients likely to benefit
from therapeutic treatments or to distinguish if the onset of
the resistance is P-gp/MRP-mediated or not; hence, to
select patients likely to benefit from treatment with P-gp/
MRP inhibitors [1, 10]. Moreover, functional measures
revealing the MDR P-gp expression, obtained by nonin-
vasive imaging techniques such as SPECT/PET, have the
advantage that they do not require any surgical biopsy in
contrast to the convectional in vitro evaluation techniques.
Although 99mTc-sestamibi was used as an in vivo mar-
ker of P-gp function, it is not the ideal radiolabeled sub-
strate [10], and its diagnostic and prognostic value is often
limited, owing to insufficient tumor localization and high
liver and muscle uptake followed by slow washout. Thus,
compared with the magnitude of the signal produced by
other radiolabeled substrates, that produced by 99mTc-se-
stamibi is smaller. In addition, its substrate activity for both
P-gp and MRP1 decreases its utility to image the function
of only P-gp [7, 10]; nevertheless, this feature allows
99mTc-sestamibi to be a more general probe of MDR
transporters in some cancers.
possible ‘‘scaffold’’ suitable for the design of 99mTc-based
molecular probes capable of imaging noninvasively P-gp
and closely related transporter activities in tissues as well
as tumors.
Basic parameters such as the rate of uptake (time course
5–120 min), at 4 and 37 °C, of 99mTc(N)-DBODC(5) were
evaluated in vitro in selected cell lines by using 99mTc-se-
stamibi as a reference. The rate of uptake of the radiocom-
plexes was assessed in the corresponding resistant cell lines
before and after treatment with various P-gp/and or MRP
modulators/inhibitors, e.g., verapamil (Vrp), a calcium
channel blocker; cyclosporin A (CsA), an immunosuppres-
sive agent with broad-spectrum MDR modulator activity;
butionine sulfoximine (BSO), a glutathione (GSH) synthesis
inhibitor, which especially inhibits the MRP activity indi-
rectly through the depletion of GSH storage; 5-(3-(2-(7-
chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbam-
yl-4,6-dithiaoctanoic acid sodium salt hydrate (MK571), a
selective MRP family inhibitor; and 6,7-dimethoxy-2- {3-
[4-methoxy-3,4-dihydro-2H-naphthalen-(1E)-ylidene]pro-
pyl}-1,2,3,4-tetrahydroisoquinoline (MC18),
in vitro and in vivo P-gp inhibitor [31–33].
a
potent
To determine the contribution of the mitochondrial
membrane potential, the effect of carbonyl cyanide m-
chlorophenyl hydrazone (CCCP), affecting the mitochon-
drial membrane potential, on the cellular uptake of the
tracers was evaluated.
Thus, there is an unmet medical need for radiotracers
able to monitor noninvasively the MDR transport function
in tumors [4, 10, 14]. Such need has promoted research on
chemistry and biology, with the ultimate goal of identify-
ing a good-performing radiopharmaceutical for MDR
functional assessment.
The cell lines, ABC transporters overexpressed in the
different cell sublines, and the corresponding inhibitors
used in our study are reported in Table 1.
To assess the suitability of 99mTc(N)-DBODC5 in
detecting MRP-mediated drug resistance, the rate of uptake
of the complex was studied in the C13* ovarian adeno-
carcinoma cell line expressing MRP1 and MRP2 trans-
porters, in the A2780/ADR ovarian adenocarcinoma cell
line expressing MRP2 and MRP1 transporters, and in
Madin–Darby canine kidney (MDCK) cells transfected
with human MRP1 (MDCK/MRP1) in the presence or
absence of MK571.
[
99mTc(N)(DBODC)(PNP5)]? [DBODC is bis(N-eth-
oxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypropyl-
phosphinoethyl)ethoxyethylamine], abbreviated as
99mTc(N)-DBODC(5), is a lipophilic cationic mixed com-
pound [25] (Fig. 1) currently under investigation as a
potential myocardial imaging agent [26] owing to its
favorable biodistribution profile characterized by rapid
blood clearance, high and persistent myocardial accumu-
lation, and low lung and liver uptakes followed by a rapid
and quantitative washout [27–29].
The findings that this complex, analogously to 99mTc-
sestamibi and 99mTc-tetrofosmin, accumulates in mito-
chondrial structures through a mechanism mediated by the
negative mitochondrial membrane potential and that the
remarkable rapid efflux of 99mTc(N)-DBODC(5) from
nontarget tissues seems strongly correlated to the P-gp/
MRP transport functions open up the possibility to extend
the applications of this agent and its derivatives to tumor
imaging and noninvasive MDR studies [30].
Materials and methods
Materials
All chemicals were purchased from Sigma-Aldrich (Milan,
Italy). Solvents were reagent grade and were used without
further purification. PNP5 was prepared according to pub-
lished procedures [41]. The DBODC ligand was purchased
from Alchemy (Altedo, Italy). 2-Methoxyisobutylisonitrile
(MIBI) as the copper salt was a gift from R. Pasqualini (IBA
Cis Bio) Vrp, CsA, BSO, MK571 and CCCP were pur-
chased from Sigma-Aldrich (Milan, Italy). Technetium-
With this background, this work aimed to validate
through in vitro studies 99mTc(N)-DBODC(5) as a substrate
of the P-gps/MRP proteins and to identify in this complex a
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Fig. 1 Chemical structure of
the 99mTc compounds with their
corresponding log p values and
molecular volume. Raw
estimation of the shape of the
99mTc complexes indicated that
the molecular volume of the
molecules is comparable. Planar
imaging of the complexes
+
O
O
N
O
P
P
S
S
O
c
T
N
C
O
O
N
O
collected 60 min after injection
with a PRISM 3000 XP gamma
camera is also reported
99mTc(N)-DBODC(5)
log P = 1.61; 830.1 Å3
O
+
N
O
O
O
C
N
N
C
C
C
c
T
C
N
N
C
N
O
O
99mTc-Sesta-MIBI
log P = 0.87; 813.2 Å3
Table 1 Cell lines, ATP-binding cassette (ABC) transporters overexpressed in the different cell sublines, and the corresponding inhibitors used
in our experiments
Cell wild type
Cell subline
ABC
transporters
Inhibitors
References
MCF7 (breast cancer)
–
–
[34–36]
[34–36]
MCF7/ADR (breast cancer; doxorubicin-resistant) P-gp
MRP1
Verapamil, cyclosporin A, MC18
Cyclosporin A, MK571
A2780 (ovarian cancer)
2008 (ovarian cancer)
–
–
[37]
A2780/ADR (ovarian cancer; doxorubicin-
resistant)
MRP2
MRP1
P-gp
MRP1
MRP1
MRP2
–
MK571
Cyclosporin A, MK571
Verapamil, cyclosporin A, MC18
Cyclosporin A, MK571
Cyclosporin A, MK571
MK571
[37]
[37]
C13* (ovarian cancer; cisplatin-resistant)
MDCK/MRP1 (MRP1-transfected)
MDCK
–
MRP1
MK571
[38–40]
MC18 6,7-dimethoxy-2- {3-[4-methoxy-3,4-dihydro-2H-naphthalen-(1E)-ylidene]propyl}-1,2,3,4-tetrahydroisoquinoline, MDCK Madin–Darby
canine kidney, MR571 5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid sodium salt hydrate, MRP
multidrug-resistance-associated protein, P-gp P-glycoprotein
99m as Na99mTcO4 was eluted from a Elumatic III
99Mo/99mTc generator purchased from IBA Cis Bio. Sep-
Pak RP-C18 and Sep-Pak CM cation-exchange cartridges
were purchased from Waters (Milford, MA, USA).
A2780 and the corresponding doxorubicin-resistant
A2780/ADR human ovarian cancer cells were kindly pro-
vided by A. Rosato (Istituto Oncologico Veneto, Padua,
Italy). The 2008 and the corresponding cisplatin-resistant
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527
C13* human ovarian cancer cells were kindly provided by G.
Marverti (Department of Biochemical Science, University of
Modena, Italy). MCF-7 human breast cancer cells were pur-
chased from ATCC (Rockville, MD, USA). MCF-7/ADR,
MDCK, and the corresponding transfected MDCK/MRP1
cells were provided by N.A. Colabufo. RPMI-1640 culture
medium was purchased from EuroClone (Milan, Italy).
dihydrazide, 5.0 mg ethylenediaminetetraacetic acid, and
0.1 mg SnCl2 suspended in 0.1 mL saline. The vial was
kept at room temperature for 15 min, giving a mixture of
99mTc-nitrido precursors. Then, 1.0 mg PNP5 ligand (dis-
solved in 0.5 mL saline containing 2 mg/mL c-cyclodex-
trin) and 2.0 mg DBODC (dissolved in 0.2 mL saline)
were simultaneously added to the reaction vial. RCY
determined by TLC and HPLC after 30 min at 80 °C was
greater than 96 %.
Analysis
Thin-layer chromatography (TLC) and/or high-perfor-
mance liquid chromatography (HPLC) analyses were used
to evaluate the radiochemical yield (RCY) and stability of
the compounds as radiochemical purity (RCP) over time.
TLC analysis was performed on SiO2 F254 plates
(Merck) using a mixture of 0.5 M EtOH/CHCl3/toluene/
NH4Ac (5:3:3:1). The activity on the plates was detected
using a Cyclone phosphorous imaging instrument (Packard
Instruments, Meridien, CT, USA). Under these analysis
conditions, the Rf values for 99mTc(N)-DBODC(5) and
99mTc-sestamibi were 0.78 and 0.85, respectively.
99mTc-sestamibi 99mTc-sestamibi was prepared following
the manufacturer’s instructions. A stock solution of
[Cu(MIBI)4]BF4 was prepared by adding 1 mL saline to a
vial containing [Cu(MIBI)4]BF4 (1 mg), cysteine (1 mg),
sodium citrate (2.6 mg), and mannitol (20 mg). Na(99mTcO4)
(1 mL, 200 MBq) was added to a vial containing 0.2 mL of
stock solution and 0.1 mg SnCl2 suspended in 0.1 mL saline.
The vial was heated at 100 °C for 15 min. RCY determined
by TLC and HPLC was greater than 97 %.
Dose preparation for in vitro studies Before the experi-
ments the 99mTc compounds were purified from the
reagents using the following procedure.
HPLC analysis was performed using a Beckman System
Gold instrument equipped with a model 126 programmable
solvent module, a model 210A sample injection valve, a
model 166 scanning detector module, and a Bioscan B-FC-
3200 radioisotope detector. HPLC analysis was done using a
reversed-phase Vydac 218TP C18 precolumn (4.6 mm 9
45 mm, 5 lm) and a reversed-phase Vydac 218TP C18 col-
umn (4.6 mm 9 250 mm, 5 lm). Solvent A was water
(0.1 % trifluoroacetic acid) and solvent B was acetonitrile
(0.1 % trifluoroacetic acid). The gradient was as follows:
0–2 min, 20 % solvent B; 2–38 min, 80 % solvent B;
38–40 min, 20 % solvent B. The flow rate was 1 mL/min for
30 min. UV detection was at 215 nm. Under these analysis
conditions the RT values for 99mTc(N)-DBODC(5) and
99mTc-sestamibi were 23.69 and 24.19, respectively.
A two-step procedure was employed for 99mTc(N)-
DBODC(5) purification. A Sep-Pak C18 cartridge was
activated with EtOH (5 mL) and equalized with deionized
water (5 mL). Then, the reaction solution containing the
99mTc(N)-DBODC(5) complex was diluted with deionized
water (8.0 mL) and loaded on the cartridge. All the activity
was retained. After the cartridge had been washed with
water (20.0 mL) and 35 % EtOH (5.0 mL), the complex
was eluted using a mixture of 90:10 EtOH–saline (1.0 mL).
Ninety percent of the starting activity was collected.
Subsequently a Sep-Pak CM cation-exchange cartridge
was preconditioned with deionized water (10.0 mL). The
99mTc(N)–DBODC(5) solution collected from the first
purification step was diluted with deionized water (8 mL),
withdrawn with a syringe (10.0 mL), and loaded on the
cartridge. All the activity was retained. After the cartridge
had been washed with water (10.0 mL) and 70 % EtOH
(5.0 mL), the complex was eluted using a mixture of 90:10
EtOH–saline (0.250 mL 9 1, 0.750 mL 9 1). Approxi-
mately 90 % of the starting activity was collected in the
second fraction.
Alternatively, a different HPLC analysis can be applied.
This condition used a reversed-phase Beckman octadecylsi-
lane precolumn (4.6 mm 9 45 mm, 5 lm), and a reversed-
phase Beckman octadecylsilane column (4.6 mm 9
250 mm, 5 lm). For isocratic elution the solvents were as
follows: 0.02 M phosphate buffer pH 7.4 (solvent A) and
MeOH (solvent B) (20:80). The flow rate was 1 mL/min for
30 min. UV detection was at 215 nm. Under these analysis
conditions the RT values for 99mTc(N)-DBODC(5) and
99mTc-sestamibi were 18.13 and 9.15, respectively.
99mTc-sestamibi was purified on a Sep-Pak CM cation-
exchange cartridge following the procedure described for
99mTc(N)-DBODC(5).
Methods
In both cases the elution solvents were completely
evaporated under a dinitrogen stream; the complex was
dissolved in dimethyl sulfoxide (DMSO), diluted with
water to obtain an isotonic saline solution containing less
than 0.5 % (v/v) DMSO, and used for in vitro studies. After
dilution, the activity of the 99mTc complex was 6.5 lCi/
Radiopharmaceutical preparation
99mTc(N)-DBODC(5) Na(99mTcO4) (1 mL, 200 MBq)
was added to
a vial containing 5.0 mg succinic
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5 lL. The RCP of the purified compounds determined by
TLC and HPLC was 99 %.
Packard Instruments). The amount of supernatant in the cell
pellet was determined to be less than 1 % in separate
experiments. Uptake of 99mTc complexes expressed as
percentage cellular uptake of the total activity was calcu-
lated according to the literature [42, 43, 45–47] as follows:
½cpmðpelletÞꢁ
Cell studies
Cellular uptake of 99mTc(N)-DBODC(5) was evaluated in
suspension in suitable drug-sensitive cell lines and in the
corresponding resistant sublines (Table 1) before and after
MDR modulator/inhibitor treatment using 99mTc-sestamibi
as a reference. Experiments were conducted according to
the published procedures [42–45].
% Cell uptake =
ꢂ 100
½cpm ðpelletÞ þ cpm ðsupernatantÞꢁ
where cpm is counts per minute.
All assessments were conducted in duplicate for three
experiments.
Stability in RPMI-1640 medium Before the uptake
experiments, stability studies were conducted on the puri-
fied 99mTc complexes. To a glass test tube containing
450 lL of RPMI-1640 cell culture medium was added
50 lL of the purified 99mTc complexes. The mixture was
vortexed and incubated at 37 °C for 2 h. At 30 min, 1 h,
and 2 h, aliquots of the reaction mixture were withdrawn
and analyzed by TLC and HPLC. The RCP of the com-
plexes was greater than 95 %.
Uptake after exposure to MDR modulator/inhibitor To
identify the maximal accumulation of 99mTc agents after
modulator/inhibitor treatment, pilot experiments were done
by preincubating selected cell lines (MCF7/ADR and 2008)
with different concentrations (20, 50, 80 lM) of modula-
tor/inhibitor in order to identify the best concentration. The
concentration of Vrp and MK571 was fixed at 50 lM and
the concentration of CsA and MC18 was set to 80 lM.
All MDR modulators/inhibitors were prepared in
DMSO. The final concentration of DMSO in the experi-
mental buffers was less than 0.5 %, which has been found
to have no effect on the cell viability and on the net uptake
of the 99mTc agents in cultured cells [48]. Before the
addition of the 99mTc tracer, the cells were treated with
3.5 lL of the selected MDR modulator/inhibitor (50 mM/
80 mM) and preincubated at 37 °C for 30 min.
Uptake in baseline conditions Cell lines were maintained
in the logarithmic phase at 37 °C in 5 % CO2 atmosphere,
using RPMI-1640 medium supplemented with 10 % heat-
inactivated fetal bovine serum (FBS; EuroClone), 25 mM
N-(2-hydroxyethyl)piperazine-N0-ethanesulfonic acid buf-
fer, 4 mM L-glutamine, 50 U/mL penicillin, and 50 mg/mL
streptomycin. Cells adhering to the culture flask were
harvested with 0.05 % trypsin–0.53 mM ethylenediami-
netetraacetic acid (EuroClone), washed, and suspended in
the appropriate medium at a concentration of 5 9 106 cells
per milliliter for the uptake studies.
The experiments were conduced as described in the
previous section. The uptake of the 99mTc complexes was
expressed as the percentage cellular uptake of the total
activity. All assessments were conducted in duplicate
for three experiments.
Preliminary studies were performed in order to set the
experimental conditions to avoid cell damage and/or death
during uptake experiments. With use of these results, cell
suspensions were preincubated in sterile glass tubes at 4 and
37 °C for 30 min. MCF7 and MCF7/ADR cells were pre-
incubated in RPMI-1640 medium added to 10 % FBS; all
other cell lines were serum-starved for 30 min and then
resuspended in serum-free RPMI-1640 medium. Afterward,
350 lL of the cell suspension (5 9 106 cells per milliliter)
was incubated with intermittent agitation with 5 lL
(6.5 lCi) 99mTc agents. At select time points (5, 15, 30, 60,
90, and 120 min), the tubes were vortexed and triplicate
samples of the suspension (8 lL) were layered on 350 lL
cold FBS in a 500-lL Eppendorf microcentrifuge tube.
After centrifugation at 14,000g for 2 min, the tubes were
frozen in a dry ice–EtOH bath. The bottom tip containing
the cell pellet was cut off and placed in a counting tube. The
remaining portion of the tube with the supernatant was
placed in a separate counting tube. The activity of both
fractions was determined using a gamma counter (Cobra II,
Determination of intracellular GSH levels Cells
(1 9 106) were washed with cold phosphate-buffered saline,
treated with 6 % metaphosphoric acid, kept on ice for
15 min, and then centrifuged (2,000g for 15 min). The pellet
was dissolved in radioimmunoprecipitation assay buffer and
used for protein determination according to the method of
Lowry et al. [49]. Aliquots of supernatant were neutralized
with Na3PO4 and assayed for total GSH following the pro-
cedure of Tietze [50]. To deplete intracellular GSH, cells
were pretreated with BSO (25 lM) added to the culture
medium for 24 h prior to analysis and uptake experiments.
Cell viability Cell viability was determined by trypan blue
dye exclusion before and at the end of each experiment [51].
Evaluation of CCCP effects After 120 min of incubation
with the radiotracers, in baseline conditions, the cells were
treated with CCCP (10 lM final concentration) and
maintained at 37 °C for an additional 5 min. Then,
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J Biol Inorg Chem (2013) 18:523–538
529
triplicate samples of suspension (8 lL) were layered on
350 lL of cold FBS in a 500-lL Eppendorf microcentri-
fuge tube and treated as described earlier.
complex 99mTc-sestamibi. The experiments were con-
ducted at 37 °C to measure the net accumulation and at
4 °C to measure membrane-potential-independent (non-
specific) uptake; at this temperature, the plasma membrane
potential is depolarized and other energy-dependent pro-
cesses, such as acidification of cell lysosomes, are abro-
gated [52]. Hence, the lysosomal trapping component of
cellular accumulation was eliminated.
Data analysis
The results are presented as the mean the standard devi-
ation. The calculation of the significance was performed by a
Student t test; p \ 0.05 was considered significant.
Figure 3 shows the time-dependent variation in cellular
uptake, in baseline conditions at 4 and 37 °C of 99mTc(N)-
DBODC(5) and 99mTc-sestamibi in human breast and
human ovarian cancer drug-sensitive cell lines and in the
corresponding drug-resistant sublines.
Western blots
Western blot and cytoflorimetric analyses for the detection
of P-gp, MRP1, and MRP2 were performed as previously
described [36–40].
It is noteworthy to note that both 99mTc agents are
retained by human breast and human ovarian cancer cells.
In general, in terms of cellular uptake, the different
lipophilicity and solubility of the two compounds affects
their permeability and relative affinity for the MDR
transporters, resulting in the difference in their initial
accumulations. Variations of percentage cellular uptake of
the complexes into the different cell lines were strictly
correlated to the intrinsic characteristics of the cells, in
particular to the diverse mitochondrial density and over-
expression of ABC transporters. In C13* and 2008 cell
lines, the high level of accumulation of the tracers may be
attributed to their greater intrinsic electron-gradient
potential or may be linked to their high number of mito-
chondria. Thus, the tracer uptake could reflect the high
level of metabolic activity of these cell lines [53–55].
The complexes display an almost similar accumulation
profile. The uptake values of each tracer rapidly increased
within the first 5 min and the plateau level reached was
different for the cell lines and occurred at different times
(30–60 min).
For Western blot experiments, equal amounts of total
proteins were resolved by gel electrophoresis, transferred
to nitrocellulose membranes, and subjected to immuno-
blotting using the following primary antibodies: mouse
anti-MDR, D-11 (Santa Cruz); rat anti-MRP1, MRPr1
(Abcam) [38–40]; mouse anti-MRP2, M2III-5 (Santa
Cruz). Following incubation with the appropriate anti-
mouse horseradish peroxidase linked secondary antibodies,
chemoluminescence images were obtained using a Fujifilm
LAS-3000 intelligent dark box and the LAS-3000 Lite
Image Reader software program.
As examples, Western blot analyses of MCF7/ADR and
transfected MDCK/MRP1 cell lines are reported in Fig. 2.
Results
Each experiment compares the uptake of 99mTc(N)-
DBODC(5) with that of the commercially available
For the drug-sensitive cell lines, the percentage cellular
accumulation of the 99mTc compounds was higher in
human ovarian cancer cell lines than in human breast cell
cancer lines and no variation in cellular efflux between
99mTc(N)-DBODC(5) and 99mTc-sestamibi was found. At
the steady-state level, the percentage cellular uptake in
MCF7 cells was 6.64 0.83 and 8.64 1.44 for
99mTc(N)-DBODC(5) and 99mTc-sestamibi, respectively
(p \ 0.001), and in A2780 cells, the percentage cellular
accumulation was 17.67 1.35 and 18.98 0.99
(p B 0.1). In the 2008 cell line (cisplatin-sensitive), the
percentage cellular accumulation was 10.68 1.50 and
10.53 1.85 for 99mTc(N)-DBODC(5) and 99mTc-se-
stamibi, respectively (p C 0.1).
Fig. 2 Western blot analyses of MCF7, MCF7/ADR, Madin–Darby
canine kidney (MDCK), and MDCK/multidrug-resistance-associated
protein (MRP) 1 cell lines. MDCK and MDCK/MRP1 cell lines were
obtained from Netherlands Cancer Institute-Antoni van Leeuwenhoek
Hospital (NKI-AVL), with an Material Transfer Agreement between
NKI-AVL and N.A. Colabufo. P-gp P-glycoprotein, WT wild type
At 4 °C a significant reduction of cellular accumulation
was observed for both 99mTc agents. In particular, the
nonspecific uptake was assessed as around 2 % after
60 min (p \ 0.001). A slight increase was detected for both
tracers with time (3 % at 120 min). A reduction of the net
123

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J Biol Inorg Chem (2013) 18:523–538
Human Brest Cancer Cell line
MCF7
MCF7/ ADR (Pgp/MDR1 / MRP1)
22
20
18
16
14
12
10
8
22
20
18
16
14
12
10
8
6
6
4
2
4
2
0
0
5 min
15 min
30 min
60 min
90 min
120 min
5 min
15 min
30 min
60 min
90 min
120 min
time
time
DBODC5 4 °C
DBODC5 37 °C
MIBI 4 °C
MIBI 37 °C
DBODC5 4 °C
DBODC5 37 °C
MIBI 4 °C
MIBI 37 °C
Human Ovarian Cancer Cell line
A2780
A2780/ADR (MRP2/MRP1)
22
20
18
16
14
12
10
8
22
20
18
16
14
12
10
8
6
4
6
4
2
0
2
0
5 min
15 min
30 min
60 min
90 min
120 min
5 min
15 min
30 min
60 min
90 min
120 min
time
time
4 °C DBODC5
37 °C DBODC5
4 °C MIBI
37 °C MIBI
4 °C DBODC5
37 °C DBODC5
4 °C MIBI
37 °C MIBI
2008 (Pgp/MDR1; MRP1)
C13* (MRP1/ MRP2)
22
20
18
16
14
12
10
8
6
4
2
0
22
20
18
16
14
12
10
8
6
4
2
0
5 min
15 min
30 min
60 min
90 min
120 min
5 min
15 min
30 min
60 min
90 min
120 min
time
time
DBODC5 4 °C
DBODC5 37 °C
MIBI 4 °C
MIBI 37 °C
DBODC5 4 °C
DBODC5 37 °C
MIBI 4 °C
MIBI 37 °C
Fig. 3 Kinetics of cellular accumulation of 99mTc(N)-DBODC(5) (DBODC5) and 99mTc-sestamibi (MIBI) evaluated in baseline conditions at 4
and 37 °C, in human breast and human ovarian cancer drug-sensitive cell lines and the corresponding drug-resistant sublines
cellular uptake between drug-sensitive cells and drug-
resistant tumor cells was observed [p \ 0.001 for
99mTc(N)-DBODC(5) and 99mTc-sestamibi in all cell lines]
(Fig. 3).
pump activity was found to be higher than that produced by
P-gp.
MDR inhibitor/modulator effects
The ratio of the percentage cellular accumulation of the
99mTc complexes in drug-sensitive and drug-resistant cell
lines was used as a measure of the P-gp, MRP1, and MRP2
activities. Data are reported in Tables S1–S3.
To evaluate the effect of the different MDR reversal
agents, drug-sensitive and drug-resistant cell lines were
preincubated at 4 and 37 °C with various P-gp and/or
MRP modulators/inhibitors before the addition of 99mTc
agents.
Under similar experimental conditions, the change in
99mTc(N)-DBODC(5) cellular uptake produced by MRP1
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J Biol Inorg Chem (2013) 18:523–538
531
As documented by cytofluorimentric [36] and Western
blot analyses, cells express good levels of P-gp, MRP1, and
MRP2 transporters, confirming the literature data [34–40].
Thus, the mediated efflux is expected to result in decreased
cellular accumulation with respect to the corresponding
drug-sensitive cell lines.
cell lines in the presence or absence of the selected MDR
inhibitor/modulator.
Drug effect diagrams were generated for Vrp, CsA,
MC18, BSO, and MK571 and for the combined addition of
MK571 and MC18. The ratio of the percentage cellular
uptake of the 99mTc complexes in drug-resistant cell lines
in the presence of the selected MDR modulator/inhibitor to
the percentage cellular uptake in absence of the drug was
Figures 4 and 5 display the percentage cellular uptake
after 30 min at 37 °C of the complexes in drug-resistant
VRP effects
40
35
30
25
20
15
10
5
0
MCF7/ADR+
A2780/ADR+
2008
C13*
(Pgp/MDR; MRP1) (MRP2; MRP1) (Pgp/MDR; MRP1) (MRP1; MRP2)
cell lines
DBODC5 37°C
MIBI 37°C
DBODC5 VRP 50 uM
MIBI VRP 50uM
CSA effects
MC18 effects
16
14
12
10
8
24
22
20
18
16
14
12
10
8
6
4
6
4
2
2
0
0
MCF7/ADR+
A2780/ADR+
2008
C13*
MCF7/ADR+
A2780/ADR+
2008
C13*
(Pgp/MDR; MRP1) (MRP2; MRP1) (Pgp/MDR; MRP1) (MRP1; MRP2)
(Pgp/MDR; MRP1) (MRP2; MRP1) (Pgp/MDR; MRP1) (MRP1; MRP2)
cell lines
cell lines
DBODC5 37°C
MIBI 37°C
DBODC5 CSA 80 uM
MIBI CSA 80uM
DBODC5 37°C
MIBI 37°C
DBODC5 MC18 80 uM
MIBI MC18 80uM
MK571 effects
MK571 + MC18 effects
55
50
45
40
35
30
25
20
15
10
5
55
50
45
40
35
30
25
20
15
10
5
0
0
MCF7/ADR+
A2780/ADR+
2008
C13*
MCF7/ADR+
A2780/ADR+
2008
C13*
(Pgp/MDR; MRP1) (MRP2; MRP1) (Pgp/MDR; MRP1) (MRP1; MRP2)
(Pgp/MDR; MRP1) (MRP2; MRP1) (Pgp/MDR; MRP1) (MRP1; MRP2)
cell lines
cell lines
DBODC5 37°C
MIBI 37°C
DBODC5 MK571 50 uM
MIBI MK571 50uM
DBODC5 37°C
MIBI 37°C
DBODC5 MK571 50 uM
+ MC18 80 uM
MIBI MK571 50 uM
+ MC18 80 uM
Fig. 4 Percentage cellular uptake of 99mTc(N)-DBODC(5)
(DBODC5) and 99mTc-sestamibi (MIBI) in human drug-resistant cell
lines evaluated at 37 °C after 30 min of incubation in the presence or
absence of the selected multidrug resistance (MDR) inhibitor/
modulator. CSA cyclosporin A, MC18 6,7-dimethoxy-2- {3-[4-
methoxy-3,4-dihydro-2H-naphthalen-(1E)-ylidene]propyl}-1,2,3,4-
tetrahydroisoquinoline, MR571 5-(3-(2-(7-chloroquinolin-2-yl)eth-
enyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid sodium salt
hydrate, VRP verapamil
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J Biol Inorg Chem (2013) 18:523–538
Fig. 5 Comparison of effects
induced by butionine
MK571 and BSO effects
50
45
40
35
30
25
20
15
10
5
sulfoximine (BSO) and MK571
on cellular uptake of 99mTc(N)-
DBODC(5) (DBODC5) and
99mTc-sestamibi (MIBI) in 2008
and C13* cell lines. Data are
expressed as percentage cellular
uptake evaluated at 37 °C after
30 min of incubation
0
2008
C13*
(Pgp/MDR; MRP1)
(MRP1; MRP2)
cell lines
DBODC5 37°C
MIBI 37°C
DBODC5 MK571 50 uM
MIBI MK571 50uM
DBODC5 BSO
MIBI BSO
used as an assessment of the modulator-induced effect;
data are reported in Tables S1–S3.
MRP2 transporters. This may be attributable to the effects of
CsA on the mitochondrial energetics [56].
Vrp is a calcium channel blocker capable of reversing
P-gp function. At the cellular level the effects of Vrp are
responsible for modification of the cell permeability, which
consequently may induce alteration of the uptake ratio of
the 99mTc agents [42]. This could explain the small
increase of the 99mTc cell-associated activity found in the
drug-sensitive cell lines (data not shown).
Modulation by MC18, a P-gp inhibitor which has a
pharmacological profile similar to that of CsA [31, 32],
resulted in an increase of 99mTc-sestamibi accumulation in
cells expressing a high level of P-gp (p \ 0.001), whereas no
variation of the 99mTc(N)-DBODC(5) uptake was observed
in the same cell line (p C 0.1). Minimal or no effects on
cellular accumulation of the radiotracers were found in drug-
resistant cell lines with no expression of P-gp transporters
such as A2780/ADR and C13* cells [0.05 B p \ 0.1 for
99mTc(N)-DBODC(5) and 99mTc-sestamibi].
In general, the addition of Vrp (P-gp substrate) was
responsible for an increase of the cellular uptake of both the
radiotracers in all cell sublines [p \ 0.001 for 99mTc(N)-
DBODC(5) and 99mTc-sestamibi]; however, for 99mTc(N)-
DBODC(5) its effect was much more evident in cells
expressing a high level of P-gp compared with drug-resis-
tant cell lines with no expression of P-gp such as A2780/
ADR and C13* (Fig. 4). Indeed, the 99mTc(N)-DBODC(5)
uptake was increased by a factor of 5 in MCF7/ADR cells
and by a factor of 3/3.5 in 2008 cells, which express high
P-gp levels; conversely, its accumulation was increased
twofold in A2780/ADR and C13* cell lines, which do not
express detectable P-gp levels [35]. These differences were
less marked for uptake of 99mTc-sestamibi: its accumulation
was increased by a factor of 15 in MCF7/ADR cells, by a
factor of 3 in 2008 cells, by a factor of 5 in A2780/ADR
cells, and by a factor of 2.5 in C13* cells.
MRP1 proteins may act as a GSH–drug cotransporter.
To examine the contribution of intracellular GSH to
99mTc(N)-DBODC(5) transport, some uptake experiments
were performed by pretreating cells with BSO (25 lM).
The effect of cellular GSH depletion on net accumulation
of the complex in C13* (MRP1, MRP2) and 2008 (P-gp,
MRP1) cells was evaluated and compared with that of
99mTc-sestamibi (Fig. 5, Table S3). In both cases, BSO
induced a slight enhancement of 99mTc(N)-DBODC(5) and
99mTc-sestamibi uptake [p \ 0.001 for 99mTc(N)-
DBODC(5) and 99mTc-sestamibi in both cell lines], sug-
gesting a possible recognition of 99mTc(N)-DBODC(5) as a
substrate of MRP1-mediated transport. This hypothesis was
strongly supported by the data collected after pretreatment
of cells with MK571. In fact, the addition of MK571, a
potent and selective MRP1 inhibitor, was responsible for a
considerable increase in uptake of complexes in MRP1-
expressing cell lines, which was mostly evident in the C13*
MRP1-overexpressing subline [p \ 0.001 for 99mTc(N)-
DBODC(5) and 99mTc-sestamibi]. After 60 min of incu-
bation, the ratio of the percentage cellular accumulation in
the presence of MK571 to the percentage cellular accu-
mulation in the absence of the drug was 6.76 for 99mTc(N)-
DBODC(5) and 4.40 for 99mTc-sestamibi (Tables S1–S3).
Complete inhibition of the P-gp and MRP1 activity was
obtained by incubating the different drug-resistant cell
The addition of CsA (a broad-spectrum MDR modula-
tor) resulted in an increase of 99mTc(N)-DBODC(5) and
99mTc-sestamibi accumulation in MCF7/ADR cells
[p \ 0.001 for 99mTc(N)-DBODC(5) and 99mTc-sestamibi],
which was quantified to be two times and six times the
basal value, respectively.
A slight enhancement of the 99mTc(N)-DBODC(5) uptake
(1.5-fold) was observed in 2008 cells and A2780/ADR cells
(p \ 0.001), whereas no differences in cell-associated
activity of 99mTc-sestamibi were detected (0.05 B p \ 0.1).
A reduction of the uptake levels of both 99mTc agents was
detected in the C13* cell line, which expresses MRP1 and
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J Biol Inorg Chem (2013) 18:523–538
533
lines simultaneously with MK571 and MC18. In all cases
the drugs induced an enhancement of the tracer uptake with
respect to the basal conditions [p \ 0.001 for 99mTc(N)-
DBODC(5) and 99mTc-sestamibi]. The data showed dif-
ferences in cellular accumulation of the two agents, which
were dependent on P-gp, MRP1, and MRP2 expression
levels in the diverse cell lines (Tables S1–S3).
fected MDCK cell line overexpressing MRP1 transporter
(MDCK/MRP1). This cell line provides a good model
system for analysis of MRP1 [38–40]. The cellular uptake
was compared with that of the corresponding wild type
(MDCK). 99mTc-sestamibi was used as a reference.
The rate of uptake of the two tracers evaluated in
baseline conditions at 4 and 37 ° C in MDCK and MDCK/
MRP1 cell lines is reported in Fig. 6.
Inhibition of ABC transporters in the MCF7/ADR cell
line (P-gp; MRP1) by MK571 and MC18 resulted in a
significant increase of the uptake of radiotracers
(p \ 0.001). In these conditions, the ratio of the percentage
cellular accumulation in the presence of MK571 and MC18
to the percentage cellular accumulation in the absence of
the drugs evaluated at steady-state levels was 4.79 for
99mTc(N)-DBODC(5) and 17.23 for 99mTc-sestamibi. For
both 99mTc complexes, the ratio was comparable with that
observed after treatment with only MK571 (p C 0.1), and
was much greater than that attained after treatment with
only MC18, underlining a preponderant effect of MK571
compared with MC18.
Cell-associated activity increased with time and reached
a plateau after 60 min of incubation. For 99mTc(N)-
DBODC(5) a lower cellular uptake was found with respect
to 99mTc-sestamibi in the MDCK and MDCK/MRP1 cell
lines. Steady-state levels of 99mTc(N)-DBODC(5) were
8.53 0.87 in MDCK cells and 3.21 0.88 in MDCK/
MRP-1 cells versus 11.01 1.28 and 4.68 0.49 for
99mTc-sestamibi in the same cultured cells.
The ratio of the percentage accumulation (at 60 min) for
the 99mTc agents in MDCK and MDCK/MRP-1 cells was
used as a measure of the function of MRP1 proteins. This
value was comparable for the two 99mTc agents [2.65 for
99mTc(N)-DBODC(5); 2.26 for 99mTc-sestamibi], suggest-
ing a similar recognition of the complexes by the MRP1
transporters.
The rate of uptake of 99mTc(N)-DBODC(5) and 99mTc-
sestamibi after MDR modulator/inhibitor treatment is
reported in Table S4. Figure 7 shows the enhancement of
the percentage cellular uptake of the two agents in MDCK/
MRP1 cells induced by the selected MDR modulator/
inhibitor after 60 min of incubation at 37 °C.
In the 2008 cell line expressing a high level of P-gp and
MRP1, for both the complexes the drugs induced a similar
99mTc cellular enrichment which was approximately 4.5-
fold the basal uptake value [p \ 0.001 for 99mTc(N)-
DBODC(5) and 99mTc-sestamibi]. In this cell line the
estimated ratios were twice those found after treatment
with only MK571 and four times those obtained after
addition of MC18 (p \ 0.001), evidencing a putative syn-
ergic action of the two inhibitors.
Inhibition of A2780/ADR or C13* cells, which express
a high level of MRP transporters and no P-gp pump,
resulted in a similar behavior characterized by an increase
of the uptake of the 99mTc agents that at steady-state levels
was determined to be sixfold and 3.5-fold the basal uptake
value for 99mTc(N)-DBODC(5) and 99mTc-sestamibi,
respectively. For these sublines the ratio of the percentage
cellular accumulation in the presence of MK571 and MC18
to the percentage cellular accumulation in the absence of
the drugs was coincident with the data produced by MK571
inhibition [p C 1 for 99mTc(N)-DBODC(5) and 99mTc-
sestamibi].
Incubation of transfected MDCK/MRP1 cells with
MK571 enhanced the cellular accumulation of 99mTc(N)-
DBODC(5) by 3.2 times with respect to the baseline con-
dition. For 99mTc-sestamibi, this improvement was only 1.7
times (p \ 0.001). Treatment with the other MDR modu-
lators/inhibitors which do not possess specific and selective
activity for MRP1 transporters did not result in a significant
effect on the accumulation of 99mTc(N)-DBODC(5) and
99mTc-sestamibi (p C 0.1). Exposure to Vrp induced a
partial increase of cellular accumulation of both 99mTc
agents. Since tranfected MDCK/MRP1 cells do not express
P-gp proteins (Fig. 2) [38–40], this effect can be attributed
to an overall modification of the cell permeability.
Inhibition/modulation experiments performed at 4 °C
generate very low 99mTc cellular accumulations. Also for
these cell lines the percentage uptake of the 99mTc agents
was around 2 %, evidencing that the modulator-induced
tracer enhancement was a temperature-sensitive process.
Cell viability
Cell viability, determined before and during all uptake
experiments by the trypan blue dye exclusion technique,
was greater than 90 % in each the experiments reported.
No differences between preincubation and postincubation
with the 99mTc agents were detected, indicating that incu-
bation with the 99mTc compounds did not affect cell
viability.
Specificity of 99mTc(N)-DBODC(5) for MRP1
transporter
To test the specificity of 99mTc(N)-DBODC(5) for MRP1
transporters, uptake assays were performed on the trans-
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J Biol Inorg Chem (2013) 18:523–538
MDCK-MPR1 (MRP1)
MDCK
22
22
20
18
16
14
12
10
8
20
18
16
14
12
10
8
6
6
4
4
2
0
2
0
5 min
15 min
30 min
60 min
90 min
120 min
5 min
15 min
30 min
60 min
90 min
120 min
time
time
DBODC5 4 °C
DBODC5 37 °C
MIBI 4 °C
MIBI 37 °C
DBODC5 4 °C
DBODC5 37 °C
MIBI 4 °C
MIBI 37 °C
Fig. 6 Kinetics of cellular accumulation of 99mTc(N)-DBODC(5) (DBODC5) and 99mTc-sestamibi (MIBI) evaluated in baseline conditions at 4
and 37 °C in MDCK cells and in the corresponding transfected MDCK/MRP1 subline
Fig. 7 Percentage cellular
uptake of 99mTc(N)-DBODC(5)
(DBODC5) and 99mTc-sestamibi
(MIBI) in the transfected
MDCK/MRP1 cell line
P-gp/ MDR MRP hinibitor/modulator effects
25
20
evaluated at 37 °C after 60 min
of incubation in the presence or
absence of the selected MDR
inhibitor/modulator. Vpr
verapamil
15
10
5
0
Vpr 50 uM
CSA 80 uM
MIBI 37°C
MC18 80 uM
MK571 50 uM
MK571 50 uM+ MC18
80 uM
cell lines
DBODC5 37°C
DBODC5 MDR-modulator
MIBI MDR-modulator
Evaluation of CCCP effects
rapidly emerging target in the progression of neurodegen-
erative disorders, such as Alzheimer’s disease, Parkinson’s
disease, and epilepsy [10, 14]. Thus, molecular probes
capable of imaging noninvasively P-gp and closely related
transporter activities in tissues as well as tumors would be
expected to contribute to personalized medicine. Interro-
gation of P-gp-mediated transport activity in vivo via
noninvasive SPECT could be helpful for stratification of
patient populations likely to benefit from a given thera-
peutic treatment, assist in the management of chemother-
apy, and aid the study of neurodegenerative diseases [14].
In the past decade, 99mTc-sestamibi has been charac-
terized as a transport substrate for MDR P-gp and MRP1
(although to a lesser degree) and used in functional imag-
ing of MDR [7]. Cross-reactivity with MRP1 may reduce
the specificity of the tracer for functional imaging of P-gp
in tumors; nevertheless, SPECT scans with 99mTc-sestam-
ibi have accurately predicted chemotherapy response in a
range of cancers, even though 99mTc-sestamibi is also a
substrate for MRP1 [7, 23]. In spite of this, the extensive
The influence of CCCP, which affects the mitochondrial
membrane potential, on cellular uptake of the 99mTc
complexes was evaluated for drug-sensitive and drug-
resistant cell lines after treatment with P-gp/and or MRP
modulators/inhibitors. The addition of CCCP released
approximately 70 % of the accumulated activity of both
99mTc(N)-DBODC(5) and 99mTc-sestamibi.
For drug-resistant cell lines, these findings suggest that
the enhanced uptake of 99mTc(N)-DBODC(5) generated by
the addition of a modulator/inhibitor was sequestered into
the structure of mitochondria.
Discussion
MDR mediated by overexpression of P-gp is one of the
best characterized transporter-mediated barriers limiting
the success of chemotherapy in cancer patients and is also a
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J Biol Inorg Chem (2013) 18:523–538
535
hepatobiliary excretion of this radiopharmaceutical and its
uptake in heart, liver, kidneys, and gastrointestinal tract
make imaging in the abdomen difficult.
MRP1 transporter; and (5) the reverse effect induced by
MK571 was significantly greater than that generated by
P-gp MDR inhibitor/modulator.
To explore the potential use of 99mTc(N)-DBODC(5) in
tumor imaging and in detecting the P-gp/MRP-mediated
drug resistance in cancer and in neurodegenerative dis-
eases, the rate of uptake of this agent was evaluated in vitro
in selected drug-sensitive human tumor cell lines and in the
corresponding drug-resistant sublines before and after
treatment with a suitable P-gp and/or MRP modulator/
inhibitor. For this purpose some pharmacological proper-
ties and experimental criteria must be fulfilled: (1) low
nonspecific binding to cell membranes and hydrophobic
regions in biological preparations; (2) high distinction in
net uptake levels between drug-sensitive cells and P-gp-
expressing multidrug-resistant cells; (3) high enhancement
of uptake in resistant cells after addition of an MDR
modulator/inhibitor.
Similarly to P-gp, the MRP family of transporters is also
a member of the ABC transporter superfamily [5]. Among
this superfamily, MRP1 transporter is widely expressed in
normal tissues such as lung, testes, kidneys, skeletal tis-
sues, cardiac muscles, and placenta. Overexpression of
MRP1 and sister proteins, such as MRP2, has subsequently
been shown for a number of drug-resistant cancer cell lines,
including lung, gastric, and colorectal cancer cell lines, and
their expression levels are thought to confer resistance to
many antineoplastic agents and chemotherapeutic drugs as
well as vincristine, doxorubicin, and cisplatin [5].
MRP1 transporter is involved in GSH homeostasis and
is responsible for its export. In addition, it is also involved
in inflammatory process mediated by leukotriene D4
(LTD4), which constitutes the highest-affinity substrate for
this transporter [5]. Whereas P-gp is primarily involved in
the active transport of organic cations [5], MRP1 typically
transports compounds that have been GSH-, glucuronide-
or, sulfate-conjugated, or compounds that are amphiphatic
and anionic, such as LTD4 [57]. However, studies have
shown that cytotoxic natural products that are neutral or
cationic in character can be also transported by MRP1.
Furthermore, these compounds appear to be cotransported
with GSH but not conjugated to it. More than one mech-
anism has been proposed to mediate the resistance by
MRP1 [58, 59], the mechanism by which the resistance is
associated with the intracellular GSH levels and entails the
conjugation of the compounds to GSH via glutathione S-
transferases; and the mechanism by which the efflux of
unmodified drug is mediated by direct interaction/binding
of the compound with MRP1.
99mTc(N)-DBODC(5) is a lipophilic compound charac-
terized by a single positive charge. Biological studies
revealed for this compound a remarkable in vitro and
in vivo stability: no evidence of biotransformation and
metabolization was detected after incubation and extraction
of 99mTc(N)-DBODC(5) from biological fluids and tissues
[27–30]. Challenge experiments conducted in presence of
high excess of GSH showed a high inertness of the com-
plex toward transchelation and the formation of GSH
adducts was never observed.
In general, the percentage cell accumulation of
99mTc(N)-DBODC(5) was found to decrease in drug-
resistant cell lines in proportion to the P-gp expression
levels, and an enhancement of the tracer has been observed
upon treatment with an MDR modulator/inhibitor. Addi-
tion of MDR modulator/inhibitor (CsA and MC18), which
blocks the function of P-gp, was responsible for enhanced
cellular accumulation of the 99mTc(N)-DBODC(5) tracer,
indicating a possible recognition of 99mTc(N)-DBODC(5)
by this transporter. This finding supports a possible use of
this agent in the functional assessment of MDR P-gp even
though the data collected for 99mTc-sestamibi show that
this latter compound is better recognized by P-gp pumps
than is 99mTc(N)-DBODC(5). Hence, 99mTc-sestamibi
probably remains the most appropriate compound for the
detection of P-gp activity. It is interesting to note that for
both 99mTc complexes the uptake was significantly lower in
cells expressing MRP1 or MRP2 with respect to those
expressing P-gp/MRP (p \ 0.001).
The data reported here suggest that 99mTc(N)-
DBODC(5) is better recognized by MRP1 than by P-gp.
This finding is supported by the fact that the accumulation
of 99mTc(N)–DBODC(5) in the drug-resistant cell line was
the inverse of the expression of MRP1 and MRP2. Thus,
(1) the percentage cellular accumulation of 99mTc(N)-
DBODC(5) was lower in the A2780/ADR and C13* cell
lines, which are characterized by high expression of MRP1
and MRP2 with respect to the MCF7/ADR and 2008 cell
lines, which express both P-gp and MRP1 transporter; (2)
the net accumulation of 99mTc(N)-DBODC(5) was the
inverse of the expression of MRP1 in the transfected
MDCK/MRP1 cell line; (3) depletion of GSH in MRP1-
expressing cells (2008 and C13*) resulted in enhancement
of the cellular uptake; (4) a potent reverse effect was
observed by inhibition with MK571, which is selective for
To reverse the efflux of 99mTc(N)-DBODC(5) mediated
by MRP1, the effects of BSO (a GSH synthesis inhibitor)
and MK571 (an LTD4 receptor antagonist) were investi-
gated (Fig. 4). GSH depletion resulted in a slight increase
of 99mTc(N)-DBODC(5) accumulation in C13* cells
(1.5 times the basal level at the steady state). This value
was drastically lower compared with that induced by
MK571 inhibition. Under the same conditions, the per-
centage cellular accumulation in MK571-pretreated cells
123

536
J Biol Inorg Chem (2013) 18:523–538
was approximately six times higher than the basal uptake
value [30 min after incubation the percentage cellular
uptake was 49.23 2.23 for 99mTc(N)-DBODC(5) versus
42.45 5.13 for 99mTc-sestamibi].
These results seem to indicate that 99mTc(N)-
DBODC(5) is only partially effluxed as GSH-conjugated
from the cells and that the effects of MK571 are mostly due
to the blocking of 99mTc(N)-DBODC(5) efflux. The data
obtained with MK571 suggest a different mechanism of
recognition of the complex by MRP1, by which the resis-
tance might involve the direct interaction/binding of the
unmodified 99mTc(N)–DBODC(5) with MRP1.
The collected data indicate that 99mTc(N)-DBODC(5)
has almost the same transporter profile as 99mTc-sestamibi.
Actually, 99mTc(N)-DBODC(5) is recognized as a transport
substrate for both P-gp and MRP1, but findings from
inhibition experiments revealed a better recognition of
99mTc(N)-DBODC(5) by MRP1 than by P-gp transporters.
Resembling 99mTc-sestamibi, cross-reactivity with MRP1
may reduce the specificity of the tracer for functional
imaging of P-gp in tumors, but alternatively this property
may cause 99mTc(N)-DBODC(5) to be a more general
probe of transporter-mediated MDR in some cancers and
neurodegenerative diseases [7].
DBODC(5) for myocardial imaging may also be applied to
tumor imaging. For 99mTc(N)-DBODC(5), hydrophilicity
and pharmacological activity can be controlled through the
independent variation of the phosphine substituents (at the
phosphorous and nitrogen atoms) and the dithiocarbamate
ligand without impacting the coordinating sphere and
consequently the remarkable stability properties of this
compound [60]. This fact makes 99mTc(N)-DBODC(5) a
suitable platform for the preparation of a molecular probe
for SPECT of MDR.
Furthermore, different 99mTc mixed compounds charac-
terized by the presence of an alcohoxyalkyl aminodiphos-
phine ligand (PNP) such as
[
99mTc(N)(L)(PNP)]?
(L is a linear-chain, alicylic, or crown-ether-containing
dithiocarbamate or thiomaltol derivative) [27, 60–64],
[
99mTc(NNPh)(L)(PNP)]?
(L is crown-ether-containing
dithiocarbamate) [65], and [99mTc(CO)3(PNP)]? have
recently been reported [66]. The evidence that all these
compounds maintain similar biodistribution patterns, char-
acterized by a high and persistent myocardial uptake and an
extremely rapid elimination of the activity from nontarget
tissues such as lung, liver, kidney, and intestine, regardless of
the presence of the terminal technetium–nitrogen multiple
bond and regardless of the nature of the dithiocarbamate,
malthol, or CO co-ligands, suggests that this behavior may be
mainly correlated to the [Tc(PNP)]n? scaffold and, in par-
ticular, to the chemical structure of the PNP ligand as well as
to the presence of alcohoxyalkyl pendant groups. Animal
studies aimed at elucidating the mechanisms of distribution,
retention, and elimination of some of these [Tc(PNP)]-based
complexes demonstrated that the remarkably rapid efflux of
these compounds from nontarget tissues seems strongly
correlated to the action of P-gp and MRP1 transporters
[30, 64]. This behavior suggests that the [Tc(PNP)]n? and
[Tc(N)(PNP)]2? scaffolds can be viewed as a substrate for
P-gp and sister proteins. Consequently, it should be possible
to conjugate, through a suitable chelating system, these
moieties to conventional cytotoxic compounds and MDR
modulator and/or substrates in the development of more
specific molecular probesfor use in assessingthe activity, the
expression, and the function of P-gp and sister proteins in
cancer and neurodegenerative diseases.
These in vitro studies confirm what we previously
observed from gender-related pharmacokinetic studies, in
which the influence of P-gp and MRP1 on the remarkable
rapid elimination of 99mTc(N)-DBODC(5) from critical
nontarget tissues was investigated by evaluating the effect
of CsA on the biodistribution profile of the complex [30].
When CsA was administered before intravenous injection
of the radiocomplex, a significant reduction of lung, liver,
and kidney washout was observed along with a consider-
able variation in the activity distribution in the intestinal
tract and in myocardial uptake, thus indicating a role of
P-gp and MRP1 transporters in determining the pharma-
cokinetic behavior of 99mTc(N)-DBODC(5).
Therefore, it is realistic to believe that 99mTc(N)-
DBODC(5) is recognized both in vitro and in vivo as a
transport substrate for both P-gp and MRP1. These findings
provide indications concerning the possibility to extend the
diagnostic application of 99mTc(N)-DBODC(5) in tumor
and in noninvasive MDR studies, even if further in vivo
studies need to be performed in more suitable animal
models to evaluate the diagnostic value of 99mTc(N)-
DBODC(5) as well as to identify with respect to 99mTc-
sestamibi the optimal in vivo imaging agent. For in vivo
application, not only is the accumulation of radioactivity in
target organ or tissue critical, also critical is the ratio of
radioactivity in the target versus the background.
99mTc(N)-DBODC(5) has a more favorable pharmacoki-
netic profile than 99mTc-sestamibi [25–27]. Therefore, the
advantages of in vivo pharmacokinetics of 99mTc(N)-
99mTc(N)-DBODC(5) uptake
The application of 99mTc-sestamibi as a tumor-seeking
agent is made possible by irreversible trapping of the tracer
in the mitochondrial structures driven by a passive diffu-
sion process also supported by an electric gradient due to a
high mitochondrial transmembrane potential [21–24]. For
this technetium-based compound, the net cellular content is
a function of both passive potential-dependent influx and
transport-mediated efflux.
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J Biol Inorg Chem (2013) 18:523–538
537
The mechanism of uptake of 99mTc(N)-DBODC(5) is
unknown. Subcellular distribution studies conduced on
isolated rat myocardial tissue indicated that 99mTc(N)-
DBODC(5), analogously to 99mTc-sestamibi and 99mTc-
tetrofosmin, is localized in the mitochondrial fraction
(86.3 %) of the myocardial cells, suggesting for this
monocationic agent a mechanism of myocardial accumu-
lation similar to that observed for 99mTc-sestamibi [30].
To add some information to the mechanism of
99mTc(N)-DBODC(5) cellular incorporation, we studied
the effects of two different temperatures (4 and 37 °C) and
the addition of CCCP on the cellular uptake. We found that
the tracer cellular incorporation was correlated to the
metabolic activity of the cells, with a cellular accumulation
profile that was temperature-dependent. In fact, low tem-
perature restricts the cellular uptake under all conditions
investigated, whereas a considerable increase of the cell-
associated radioactivity was detected at 37 °C.
99mTc-sestamibi in assessing MRP-mediated drug resis-
tance. The collected data provide indications concerning
the possibility to extend the diagnostic application of
99mTc(N)-DBODC(5) in tumor and in noninvasive MDR
studies. In vivo studies on appropriate animal models are in
progress.
For this mixed compound, the pharmacological activity
and the pharmacokinetic profile can be controlled by var-
iation of the substituents at the periphery of the molecule
without affecting the P2S2 coordination sphere. Hence,
99mTc(N)-DBODC(5) might be a suitable ‘‘scaffold’’ for
the design of new 99mTc-based molecular probes capable of
imaging noninvasively P-gp and closely related transporter
activities.
Acknowledgments This work was financially supported by Minis-
della Ricerca (PRIN
`
tero dell’Istruzione, dell’Universita
20097FJHPZ-004).
e
Likewise, the addition of CCCP, which depolarizes the
mitochondrial membrane potential and may affect the cell
membrane potential, caused approximately 70 % release of
accumulated 99mTc(N)-DBODC(5) from the cells, indi-
cating that the percentage released was connected to
mitochondrial accumulation. The residual fraction of 99mTc
activity may be attributed to the effect of cell membrane
potential, which could not have been totally diminished
after addition CCCP, on the passive influx of the tracers
into the cells [67].
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