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

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 P₂S₂coordinating 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

123

524

J Biol Inorg Chem (2013) 18:523–538

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 (t_1/2= 20 min) and ¹⁸F

(t_1/2= 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 (t_1/2= 6.02 h; E_c= 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

123

J Biol Inorg Chem (2013) 18:523–538

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

123

526

J Biol Inorg Chem (2013) 18:523–538

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

N

O

P

S

O

c

T

N

C

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

C

N

C

c

T

C

N

C

N

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]

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

MRP2

–

MK571

Cyclosporin A, MK571

Verapamil, cyclosporin A, MC18

Cyclosporin A, MK571

MK571

[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 Na⁹⁹mTcO₄was 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

123

J Biol Inorg Chem (2013) 18:523–538

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 SnCl₂suspended 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 SiO₂F₂₅₄plates

(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]BF₄was 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 SnCl₂suspended 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 C₁₈precolumn (4.6 mm 9

45 mm, 5 lm) and a reversed-phase Vydac 218TP C₁₈col-

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 R_Tvalues 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 C₁₈cartridge 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 R_Tvalues 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

123

528

J Biol Inorg Chem (2013) 18:523–538

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 % CO₂atmosphere,

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 10⁶cells

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 10⁶cells 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 Na₃PO₄and 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,

123

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

530

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

4

2

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

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

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

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

123

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

0

MCF7/ADR+

A2780/ADR+

2008

C13*

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 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

MCF7/ADR+

A2780/ADR+

2008

C13*

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 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

123

532

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

123

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-

123

534

J Biol Inorg Chem (2013) 18:523–538

MDCK-MPR1 (MRP1)

MDCK

22

20

18

16

14

12

10

8

20

18

16

14

12

10

8

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

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

123

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.

123

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 P₂S₂coordination 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].

References

1. Del Vecchio S, Salvatore M (2004) Eur J Nucl Med Mol Imaging

31:S88–S96

2. Meric-Bernstam F, Mills GB (2012) Nat Rev Clin Oncol

9:542–548

3. Vanneman M, Dranoff G (2012) Nat Rev Cancer 12:237–251

4. Mendes F, Paulo A, Santos I (2011) Dalton Trans 40:5377–5393

5. Colabufo NA (ed) (2008) Multidrug resistance: biological and

pharmaceutical advances in antitumour treatment. Research

Signpost, Kerala

6. Sharma V, Piwnica-Worms

252:155–178

7. Piwnica-Worms D, Kesarwala AH, Pichler A, Prior JL, Sharma V

(2006) Neuroimaging Clin N Am 16:575–589

8. Higgins CF, Gottesman MM (1992) Trends Biochem Sci

17:18–21

Moreover, the effects induced by CCCP on drug-resis-

tant cell lines pretreated with P-gp and/or MRP modulator/

inhibitor, combined with the results obtained at 4 °C,

conﬁrmed that blockade of P-gp/MRP allowed ^99mTc(N)-

DBODC(5) to permeate into mitochondria in a membrane-

potential-dependent manner and provide evidence that

^99mTc(N)-DBODC(5) was only minimally absorbed onto

membrane lipids and hydrophobic compartments.

D (2005) Top Curr Chem

¨

9. Loscher W, Langer O (2010) Curr Top Med Chem 10:1785–1791

10. Kannan P, John C, Zoghbi SS, Halldin C, Gottesman MM, Innis

RB, Hall MD (2009) Clin Pharmacol Ther 86:368–377

11. Colabufo NA, Berardi F, Cantore M, Contino M, Inglese C, Niso

M, Perrone R (2010) J Med Chem 53:1883–1897

12. Colabufo NA, Berardi F, Perrone MG, Capparelli E, Cantore M,

Inglese C, Perrone R (2010) Curr Top Med Chem 10:1703–1714

13. Homolya L, Hollo Z, Germann UA, Pastan I, Gottesman MM,

Sarkadi B (1993) J Biol Chem 268:21493–21496

14. Piwnica-Worms D, Sharma V (2010) Curr Top Med Chem

10:1834–1845

15. Luurtsema G, Verbeek J, Lubberink M, Lammertsma AA, Dier-

ckx RA, Elsinga PH, Windhorst B, van Waarde A (2010) Curr

Top Med Chem 10:1820–1833

Concluding remarks

The results from this preliminary report clearly indicate

that the uptake of ^99mTc(N)-DBODC(5) was correlated to

metabolic activity of the cells. In fact, low temperature

inhibited the cellular uptake of the ^99mTc tracers, indicating

that there was not binding to the cell membranes or to

hydrophobic regions of the cell membrane protein. The

cellular accumulation is correlated to the level of P-gp/

MRP expression, and an enhancement of uptake in resistant

cells was observed after treatment with an MDR inhibitor/

modulator, indicating that the selective blockade of P-gp/

MRP prevents efﬂux of the tracer. From the evaluation and

comparison of the usefulness of the two tracers, it seems

that ^99mTc(N)-DBODC(5) may have an advantage over

¨

16. Syvanen S, Hammarlund-Udenaes M (2010) Curr Top Med

Chem 10:1799–1809

17. Okamura T, Kikuchi T, Irie T (2010) Curr Top Med Chem

10:1810–1819

18. Mariani G, Bruselli L, Duatti A (2008) Eur J Nucl Med Mol

Imaging 35:1560–1565

19. Zimmermann RG (2013) Nucl Med Biol 40:155–166

20. Bartholoma MD, Louie AS, Valliant JF, Zubieta J (2010) Chem

Rev 110:2903–2920

123

538

J Biol Inorg Chem (2013) 18:523–538

21. Sharma V (2004) Bioconjug Chem 15:1464–1474

22. Chiu ML, Kronauge JF, Piwnica-Worms D (1990) J Nucl Med

31:1646–1653

42. Cordobes MD, Starzec A, Delmon-Moingeon L, Blanchot C,

´

Kouyoumdjian JC, Prevost G, Caglar M, Moretti JL (1996) J

Nucl Med 37: 286–289

23. Chen CC, Meadows B, Regis J, Kalafsky G, Fojo AT, Car-

rasquillo JA, Bates SE (1997) Clin Cancer Res 3:545–552

24. Moretti JL, Hauet N, Caglar M, Rebillard O, Burak Z (2005) Eur

J Nucl Med Mol Imaging 32:S36–S842

43. Barbarics E, Kronauge JF, Cohen D, Davison A, Jones AG,

Croop JM (1998) Cancer Res 58:276–282

44. Barbarics E, Kronauge JF, Davison A, Jones AG (1998) Nucl

Med Biol 25:667–673

25. Bolzati C, Refosco F, Cagnolini A, Tisato F, Boschi A, Duatti A,

Uccelli L, Dolmella A, Marotta E, Tubaro M (2004) Eur J Inorg

Chem 9:1902–1913

45. Friebe M, Mahmood A, Bolzati C, Drews A, Johannsen B, Ei-

senhut M, Kraemer D, Davison A, Jones AG (2001) J Med Chem

44:3132–3140

26. Cittanti C, Uccelli L, Pasquali M, Boschi A, Flammia C, Bagatin

E, Casali M, Stabin MG, Feggi L, Giganti M, Duatti A (2008) J

Nucl Med 49:1299–1304

27. Boschi A, Uccelli L, Bolzati C, Duatti A, Sabba N, Moretti E, Di

Domenico G, Zavattini G, Refosco F, Giganti M (2003) J Nucl

Med 44:806–814

28. Hatada K, Riou LM, Ruiz M, Yamamichi Y, Duatti A, Lima RL,

Allen R, Goode AR, Watson DD, Beller GA, Glover DK (2004) J

Nucl Med 45:2095–2101

46. Moretti JL, Cordobes MD, Starzec A, de Beco V, Vergote J,

Benazzouz F, Boissier B, Cohen H, Saﬁ N, Piperno-Neumann S,

Kouyoumdjian JC (1998) J Nucl Med 39:1214–1218

47. Nerini Molteni S, Seregni E, Botti C, Martinetti A, Ferrari L,

Crippa F, Bombardieri E (1999) Anticancer Res 19: 255–260

48. Piwnica-Worms D, Kronauge JF, Chiu ML (1990) Circulation

82:1826–1838

49. Lowry OH, Rosebrough NJ, Farr AL, Randall PJ (1951) J Biol

Chem 193:265–275

29. Hatada K, Ruiz M, Riou LM, Lima RL, Goode AR, Watson DD,

Beller GA, Glover DK (2006) J Nucl Cardiol 6:779–790

30. Bolzati C, Cavazza-Ceccato M, Agostini S, Tokunaga S, Casara

D, Bandoli G (2008) J Nucl Med 49:1336–1344

31. Colabufo NA, Berardi F, Cantore M, Perrone MG, Contino M,

Inglese C, Niso M, Perrone R, Azzariti A, Simone GM, Porcelli

L, Paradiso A (2008) Bioorg Med Chem 16:362–373

32. Colabufo NA, Berardi F, Perrone MG, Cantore M, Contino M,

Inglese C, Niso M, Perrone R (2009) Chem Med Chem

4:188–195

33. Van Waarde A, Ramakrishnan NK, Rybczynska AA, Elsinga PH,

Berardi F, de Jong JR, Kwizera C, Perrone R, Cantore M, Si-

jbesma JWA, Dierckx RA, Colabufo NA (2009) J Med Chem

52:4524–4532

34. Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage

H, Dietel M, Greenberger L, Cole SPC, Doyle LA (1999) J Natl

Cancer Inst 91:429–433

50. Tietze F (1969) Anal Biochem 27:502–522

51. Stoddart MJ (2011) Methods Mol Biol 740:1–6

52. Kannan P, Telu S, Shukla S, Ambudkar SV, Pike VW, Halldin C,

Gottesman MM, Innis RB, Hall MD (2011) ACS Chem Neurosci

2:82–89

53. Andrews PA, Albrigt KD (1992) Cancer Res 52:1895–1901

54. Marverti G, Piccini G, Ghiaroni S, Barbieri D, Quaglino D,

Moruzzi MS (1998) Int J Cancer 78:33–40

55. Safaei R, Larson BJ, Cheng TC, Gibson MA, Otani S, Naerde-

mann W, Howell SB (2005) Mol Cancer Ther 4:15951604

56. Lemasters JJ (1999) Am J Physiol 276:G1–G6

57. Gekeler V, Ise W, Sanders KH, Ulrich WR, Beck JT (1995)

Biochem Biophys Res Commum 208:345–352

58. Vezmar M, Deady LW, Tilley L, Georges E (1997) Biochem

Biophys Res Commun 241:104–111

59. Vezmar M, Georges E (1998) Biochem Pharmacol 56:733–742

60. Bolzati C, Cavazza-Ceccato M, Agostini S, Refosco F, Yama-

michi Y, Tokunaga S, Carta D, Salvarese N, Bernardini D,

Bandoli G (2010) Bioconjug Chem 21:928–939

61. Liu S, He Z, Hsieh WY, Kim YS (2006) Nucl Med Biol

33:419–432

35. Benderraa Z, Trussardib A, Morjania H, Villac AM, Dogliac SM,

Manfaita M (2000) Eur J Cancer 36:428–434

36. Azzariti A, Colabufo NA, Berardi F, Porcelli L, Niso M, Simone

GM, Perrone R, Paradiso

5:1807–1816

A (2006) Mol Cancer Ther

62. Kim YS, He Z, Hsieh WY, Liu S (2007) Bioconjug Chem

18:929–936

63. Kim YS, Wang JJ, Broisat A, Glover DK, Liu S (2008) J Nucl

Cardiol 15:535–546

64. Kim YS, Shi JY, Zhai SZ, Hou GH, Liu S (2009) J Nucl Cardiol

16:571–579

65. Kim YS, He Z, Hsieh WY, Liu S (2006) Bioconjug Chem

17:473–484

37. Kool M, de Haas M, Scheffer GL, Scheper RJ, van Eijk MJT,

Juijn JA (1997) Cancer Res 57:3537–3547

38. Evers R, Cnubben NH, Wijnholds J, van Deemter L, van Blad-

eren PJ, Borst P (1997) FEBS Lett 419:112–116

39. Evers R, Kool M, Van Deemter L, Jansen H, Calafat J, Oomen

LCJM, Paulusma CC, Oude Elferink RPJ, Baas F, Schinkel AH,

Borst P (1998) J Clin Invest 101:1310–1319

´

40. Bakos E, Evers R, Szakacs G, Tusnady GE, Welker E (1998)

Szabo, de Haas M, van Deemter L, Borst P, Varadi A, Sarkadi B.

´

66. He Z, Hsieh WY, Kim YS, Liu S (2006) Nucl Med Biol

33:1045–1053

´

J Biol Chem 273:32167–32175

67. Chen WS, Luker KE, Dahlheimer JL, Pica CM, Luker GD,

Piwnica-Worms D (2000) Biochem Pharmacol 60:413–426

41. Tisato F, Refosco F, Bolzati C, Agostini S, Porchia M, Cavazza-

Ceccato M, Tokunaga S (2006) Intermediate compound of

technetium nitride complex for radiodiagnostic imaging. PCT/

JP2006/301260

123

Article Doi

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

DOI: 10.1007/s00775-013-0997-1

Source and publish data:

Authors:

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

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

Products guided by the article

R&D Labs maybe for 113720-90-4

Relevant to this article

Hot Product