PET study on mice bearing human colon adenocarcinoma cells using [11C]GF120918, a dual radioligand for
P-glycoprotein and breast cancer resistance protein Tomoteru Yamasakia, Kazunori Kawamuraa, Akiko Hatoria, Joji Yuia, Kazuhiko Yanamotoa, Yuichiro Yoshidaa,c, Masanao Ogawaa,b, Nobuki Nengakia,c, Hidekatsu Wakisakab, Toshimitsu Fukumuraa and Ming-Rong Zhanga
Objective To evaluate the functions of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) in human colon adenocarcinoma (Caco-2), we carried out an in-vitro study and a small animal positron emission tomography (PET) study using [11C]GF120918 (elacridar).
Methods [11C]GF120918 was synthesized by reacting the desmethyl precursor with [11C]CH3I. An in-vitro study using [11C]GF120918 was carried out in Caco-2 and Madin–Darby canine kidney cells in the presence or absence of a transporter inhibitor (cyclosporine A and unlabeled GF120918). The biodistribution of radioactivity after the injection of [11C]GF120918 was determined in Caco-2-bearing mice using a small animal PET scanner.
Results In Caco-2 cells expressing Pgp and BCRP, coincubation with unlabeled GF120918 caused an approximately two-fold increase in [11C]GF120918 uptake compared with that of the control ([11C]GF120918 only). In Caco-2-bearing mice, PET results indicated that [11C]GF120918 uptake in the tumor was low, but was significantly increased by treatment with unlabeled
GF120918. In metabolite analysis, the radioactive component in the tumor almost corresponded to intact [11C]GF120918.
Conclusion A PET study combining the administration of [11C]GF120918 with unlabeled GF120918 may be a
useful tool for evaluating the functions of Pgp and BCRP in tumors. Nucl Med Commun 31:985–993 cti 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Nuclear Medicine Communications 2010, 31:985–993
Keywords: breast cancer resistance protein, carbon-11, GF120918 (elacridar), human colon adenocarcinoma, multidrug resistance,
P-glycoprotein, positron emission tomography
Departments of aMolecular Probes, bBiophysics, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba and cSHI Accelerator Service, Ltd., Tokyo, Japan
Correspondence to Ming-Rong Zhang, PhD, Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences,
4-9-1 Anagawa, Inage-Ku, Chiba 263-8555, Japan Tel: + 81 43 206 4041; fax: + 81 43 206 3261;
e-mail: [email protected]
Received 16 May 2010 Revised 6 August 2010 Accepted 21 August 2010
Introduction
A major problem in chemotherapy for cancer patients is tumor resistance to chemotherapeutic drugs. P-glycopro- tein (Pgp) and breast cancer resistance protein (BCRP) are mainly involved in multidrug resistance (MDR) in tumors or in limiting penetration of pharmacological agents into the brain through the blood–brain barrier (BBB) in brain capillaries [1]. Pgp is a 170-kDa transmembrane protein encoded by the Mdr1 gene, located on human chromosome 7q21, belonging to a family of ATP-binding cassette (ABC) transporters [Pgp; ABCB1 or MDR phenotype (MDR1)]
[2]. BCRP is a 70-kDa homodimer transmembrane protein encoded by the BCRP gene located on human chromosome 4q22 and belongs to the family of ABC transporters (BCRP; ABCG2) [2].
Many MDR modulators, including inhibitors and competi- tive substrates, were developed to inhibit the function of these transporters. These agents facilitate the penetration of
chemotherapeutic drugs into the tumor and brain. Com- pounds of the first and second generations are widely used as Pgp and/or BCRP modulators, such as cyclosporine A (CsA), verapamil, fumitremorgin C, PSC833 (CsA analog), and Ko143 (fumitremorgin C analog) [3–9]. Recently, third- generation compounds with high inhibitory action against Pgp and BCRP functions were developed, such as GF120918 (elacridar) and XR9576 (tariquidar) [10–15].
GF120918 was synthesized as an acridonecarboxamide derivative [10]. In-vitro affinity of GF120918 for Pgp was much higher than that of CsA or verapamil [16,17]. More- over, this compound showed high inhibitory action against BCRP-mediated transport in vitro [9]. GF120918 inhibited transport function because of Pgp and BCRP, but did not interrupt efflux because of other ABC transporters, such as MDR protein (MRP, ABCC) subfamily [18]. GF120918 has a good safety profile in humans and, unlike CsA, exhibits no immunosuppressive activity in humans [19,20].
0143-3636 cti 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/MNM.0b013e32833fbf87
Fig. 1
[11C]GF120918 was synthesized by methylation of 5- O-desmethyl GF120918 with [11C]methyl iodide as des-
O
cribed earlier [24]. For injection, [11C]GF120918 was
N
H
N
O
O
dissolved in propylene glycol/water (3/2), and was pre- pared with suitable radioactivity (37–74 MBq) and radio- chemical purity (> 99%).
11
O
O
CH3
NH
Cell culture
The Caco-2 cell line and the Madin–Darby canine kidney (MDCK) epithelial cell line were purchased from the
Chemical structure of [11C]GF120918.
Doner et al. [21] reported that 11C-labeled GF120918 (Fig. 1) can be used as a novel positron emission tomo- graphy (PET) probe to evaluate Pgp localization at the BBB. Specific binding of [11C]GF120918 to Pgp was confirmed by displacement with excess unlabeled GF120918 in in-vitro autoradiography using rat brain slices. However, [11C]GF120918 uptake in the brain of wild-type rats and mice was increased after treatment with unlabeled GF120918 in the PET study [21]. Similar results were also observed in in-vitro and in-vivo distribution studies using [11C]/[12C]laniquidar, a third- generation Pgp inhibitor [22,23]. In our earlier PETstudy using [11C]GF120918 in Pgp and/or BCRP-knockout mice, we found that [11C]GF120918 penetration into the brain was limited by interaction between Pgp and BCRP functions [24]. From these observations, we assumed that GF120918 behaves in a substrate-like manner for Pgp and BCRP in vivo when used in trace carrier amounts as a PET ligand.
In this study, to evaluate the function of Pgp and BCRP, we performed in-vitro uptake and PET studies using [11C]GF120918 as a substrate for Pgp and BCRP in human colon adenocarcinoma (Caco-2) cells and Caco-2- bearing mice. We used the Caco-2 cell line as the target tumor cell line. Caco-2 cells, which naturally express Pgp and BCRP without any artificial technique, are frequently used in in-vitro transport studies of MDR [25–27].
Materials and methods
General methods
We produced 11C by 14N(p,a)11C nuclear reaction using a Cypris HM18 cyclotron (Sumitomo Heavy Industries, Tokyo, Japan). All reagents and organic solvents were commercially obtained and used without further purifica- tion. GF120918 hydrochloride salt was prepared in our laboratory as described earlier, with modifications [24]. CsA was purchased from Wako Pure Industries (Osaka, Japan).
The animal experiments were performed according to the recommendations of the Committee for the Care and Use of Laboratory Animals, National Institute of Radiological Sciences (Chiba, Japan).
RIKEN cell bank (Tsukuba, Japan). Caco-2 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, New York, USA) supplemented with 20% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan), 0.1 mmol/l nonessential amino acids (NEAA; Gibco), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Gibco) in a humidified atmosphere of 5% CO2 at 371C. MDCK cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 0.1 mmol/l nonessential amino acids.
Animals
Male nude mice (BALB/cAJcl-nu/nu; CLEA Japan; weight: 25–30 g; age: 7–9 weeks, n = 25) were trans- planted with Caco-2 cells (107 cells/0.1 ml medium). The Caco-2 cells were prepared for xenografting using Extracel-X (Glycosan Biosystems Inc., Salt Lake City, Utah, USA) [28]. Subsequently, the Caco-2 cell suspen- sion was injected subcutaneously into the right hind leg of the mice. Solid tumor nodules of approximately 1 cm diameter were present 21–28 days after transplantation.
Western blotting
Confluent Caco-2 and MDCK cells were harvested in a lysis buffer containing 50mmol/l Tris (pH 7.4), 1% Nonidet P-40, 1 mmol/l EDTA, 150mmol/l NaCl, 0.1mmol/l PMSF, and leupeptin. After vortexing for 10 s, cell lysates were incubated on ice for 10 min. Crude cell lysates were cen- trifuged in a microcentrifuge at 15 000 rpm for 15min, and the supernatants then underwent immunoblot analysis. Total protein (equivalent to 32 mg for each sample) was loaded and resolved by 7.5% SDS-polyacrylamide gel electrophoresis and then electroblotted onto a polyviny- lidene fluoride membrane (Bio-Rad, Hercules, California, USA). Dual-color molecular weight markers (Bio-Rad) were run concurrently. For signal generation, the mem- brane was incubated at room temperature for 1 h with blocking buffer [5% nonfat dried milk in Tris buffered saline with Tween 20 (TBST)] and then for 2 h at room temperature with the monoclonal primary antibody Pgp C219 (mouse monoclonal; Gene Tex Inc., Irvine, California, USA) diluted 1 : 50 in TBST. Next, the mem- brane was washed thrice in TBST and further incubated for 1 h at room temperature with horseradish peroxidase- conjugated antimouse immunoglobulin G (IgG) rabbit secondary antibody (R&D Systems Inc., Minneapolis, Minnesota, USA) diluted 1 : 1000 in TBST. The membrane
was finally washed five times with TBST and then incubated for 5 min at room temperature with ECL detection reagent (GE Healthcare, Fairfield, Connecti- cut, USA). Image acquisition and band detection were carried out using an LAS-4000 UVmini image analyzer with Multi-Gauge software (Fujifilm, Tokyo, Japan). Immuno- blot analyses for BCRP and b-actin were also carried out as described above. An anti-BCRP antibody (rabbit polyclonal; Proteintech Group Inc., Chicago, Illinois, USA) was used as a primary antibody at 1 : 50 dilution and an horsera- dish peroxidase-conjugated antirabbit IgG antibody (R&D Systems Inc.) was used as a secondary antibody at 1 : 1000 dilution. An anti-b-actin antibody (mouse monoclonal; Novus Biologicals Inc., Littleton, Colorado, USA) was used as a primary antibody at 1: 1500 dilution and an antimouse IgG rabbit (R&D Systems Inc.) was used as a secondary antibody at 1: 1000 dilution.
In-vitro uptake study
Caco-2 and MDCK cells were plated on 24-well plates (105 cells/well) and incubated at 371C with complete growth medium in a humidified atmosphere containing 5% CO2. After a monolayer had formed, the cells were incubated for 120 min in a medium containing 7.4 MBq/ml [11C]GF120918 and distilled water, 10 mmol/l CsA, or 10 mmol/l unlabeled GF120918. After removal of the medium, the cells were washed three times with prechilled PBS and dissolved with 0.2 N NaOH. Radio- activities of the cell lysates were measured using an auto-g scintillation counter (Wizard 300 1480; PerkinEl- mer, Waltham, Massachusetts, USA). After the radio- activity had been counted, the protein concentrations of cell lysates were measured with a DC protein assay (Bio-Rad).
Radioactivity was expressed as percentage of incubation dose per milligram protein of cells [29].
Small animal positron emission tomography study Twenty-one to 28 days after the transplantation of Caco-2 cells, the mice were secured in a custom-made chamber and placed in a small animal PETscanner (Inveon; Siemens Medical Solutions, Knoxville, Tennessee, USA). Body temperature was maintained with a water circulation system (T/Pump TP40, Gaymar Industries, Orchard Park, NY, USA) at 401C. The mice were kept under anesthesia with 1.5% isoflurane during scanning. A 29-gauge needle with 12–15 cm Polyethylene-10 tubing was placed into a tail vein for [11C]GF120918 injection. A dynamic emission scan in the three-dimensional acquisition mode was performed for 90 min (1min ti 4 scans, 2 min ti 8 scans, 5 min ti 14 scans). A bolus of 18–37 MBq [11C]GF120918 in 200 ml of 30% propylene glycol was injected through the tail vein catheter. Forty-five minutes after the scan started, 5 mg/kg unlabeled GF120918 in 0.2ml of distilled water (patients, n = 5) or 0.2 ml of saline (control, n = 4) was injected through the tail vein catheter. The GF120918 dose
selected produced significant inhibition of Pgp and BCRP functions, as reported in an earlier study [30].
Data analysis
Region of interests (ROIs) were drawn in the tumor, brain, and abdominal aorta used for blood. ROI analysis and image reconstruction were carried out using the software ASIPro (Siemens Medical Solutions). Visual analysis was carried out by individuals experienced in PET interpretation using coronal, transverse, and sagittal reconstructions. ROIs were manually placed across image planes to generate time–activity curves (TACs).
Radioactivity was decay-corrected to injection time and expressed as a standardized uptake value (SUV), normal- ized for injected radioactivity and body weight: SUV = (radioactivity per milliliter tissue/injected radioactivity) ti body weight in grams.
We calculated the areas under the concentration–time curves (AUCs) for the tumor and brain using the SUV on the TACs from 0 to 45min and from 45 to 90min, respectively.
Metabolite analysis
A bolus of [11C]GF120918 (38–65 MBq/0.75–1.31 nmol) was injected intravenously into the Caco-2-bearing mice with or without coinjection of unlabeled GF120918 (5mg/kg) (age: 12–13 weeks; weight: 28–34g; n = 2 or 3). The mice were killed by cervical dislocation 60 min after the bolus injection. Blood was removed by puncturing the heart using a heparinized syringe and then the tumor was removed. Blood was centrifuged at 13 000g (MX-105; Tomy Seiko, Tokyo, Japan) for 3 min at 41C to obtain plasma (0.1–0.2 ml) and was deproteinized with the same volume of ice-cold acetonitrile. The mixture was vortexed and centrifuged at 20 000g for 2 min and the supernatant was collected. Tumors were homogenized in 1.0 ml of saline. After adding the same volume of acetonitrile to the homogenate, the mixture was vortexed and centrifuged at 20 000g for 2 min and the supernatant was collected. Supernatants were analyzed using high-performance liquid chromatography (HPLC) with a radioactivity detector [31] and an ultraviolet detector at 254 nm on a Novapak C18 column (100 mm internal diameter ti 8 mm length; Waters, Milford, Massachusetts, USA) con- tained within a radial compression module (RCM-100; Waters). Elution was performed with a mixture of aceton- itrile and 50 mmol/l sodium acetate buffer (pH: 4.7) (v/v: 45 : 55) at a flow rate of 2.0 ml/min. The retention times of [11C]GF120918 and [11C]metabolite were 6.2 and 2.2 min, respectively. Radioactivity in the supernatants, residual precipitates after centrifugation, and waste solu- tion from HPLC were measured using an auto-g scintil- lation counter. The percentage of the unchanged form was then determined.
Fig. 2
(a) MDCK Caco-2
Pgp
BCRP
β-actin
(b)
4000
2000
0
P-glycoprotein
1500
750
0
BCRP
MDCK Caco-2 MDCK Caco-2
(a) Expression of P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and b-actin in Madin–Darby canine kidney (MDCK) and human colon adenocarcinoma (Caco-2) cells. (b) Concentrations of Pgp and BCRP in MDCK and Caco-2 cells. Primary antibodies for Pgp, BCRP, and b- actin were used at 1 : 50, 1 : 100, and 1 : 1500 dilutions, respectively. Caco-2 cells expressed Pgp and BCRP at relatively high levels. Level of expression is represented as arbitrary unit (AU) per square centimeter.
Results
Western blotting
Fig. 3
Figure 2 shows the expression of Pgp and BCRP in MDCK and Caco-2 cells. MDCK is known to express high levels of Pgp without MDR1 gene transfection [32] and was used as a positive control for Pgp in this assessment. Pgp expression in the Caco-2 cells was detected at a similar level to that seen in the MDCK cells. In contrast, BCRP expression was five-fold higher in the Caco-2 cells than that in the MDCK cells. This shows that the Caco-2 cell line expresses both Pgp and BCRP at high levels.
In-vitro uptake study
Figure 3 shows [11C]GF120918 uptake in MDCK and Caco-2 cells on coincubation with 10 mmol/l CsA or
250
200
150
100
50
0
MDCK Caco-2
unlabeled GF120918. In the MDCK cells coincubated with CsA, [11C]GF120918 uptake was 1.3-fold higher than that of the control ([11C]GF120918 only). On coincubation with GF120918, [11C]GF120918 uptake was 2.1-fold above the control level. In contrast, [11C]GF120918 uptake in Caco-2 cells was 2.8-fold above the control level on coincubation with GF120918, although this was not changed by coincubation with CsA.
[11C]GF120918 uptake in Madin–Darby canine kidney (MDCK) and human colon adenocarcinoma (Caco-2) cells. Cells were
incubated for 120 min with 7.4 MBq [11C]GF120918 in the presence of cyclosporine A (CsA) (10 mmol/l) or unlabeled GF120918
(10 mmol/l). Radioactivity is expressed as the mean percentage of incubation dose (ICD) per milligram of protein in the cells
( ± standard error of the mean, n = 4). **P < 0.01. Coincubation with unlabeled GF120918 increased [11C]GF120918 uptake in both the cell lines.
Fig. 4
(a)
0.75
(b)
0
Coronal
Transverse
0–45 min 45–90 min
Representative positron emission tomography images from a human colon adenocarcinoma-bearing mouse. (a) Summation image between 0 and 45 min before treatment with unlabeled GF120918. (b) Summation image between 45 and 90 min after GF120918 treatment. The mouse was injected with a bolus of 18 MBq [11C]GF120918 through a tail vein catheter and injected with 5 mg/kg unlabeled GF120918, 45 min later. The position of the tumor xenograft is indicated in the photographs (left), and positron emission tomography images are indicated by red dotted circles and red arrows. The radioactivity of the tumor region on the 45–90 min summation image is relatively high and that on the 0–45 min summation image is relatively low.
Small animal positron emission tomography study Figure 4 shows distribution images of [11C]GF120918 in a Caco-2-bearing mouse using small animal PET. Figure 4a and b show summation images for 0–45 and 45–90 min in coronal and transverse views, respectively. Radio- activity in the tumor region before GF120918 treatment was lower than that in the muscle region around this tumor. Radioactivity in the tumor region increased after GF120918 treatment, whereas that in the muscle was almost unchanged.
Figure 5 shows TACs of [11C]GF120918 in the tumor, brain, and blood. As shown in the control group, [11C]GF120918 uptake in the tumor and brain remained at a low and consistent level from 5 to 90 min after injection. In contrast, radioactivity in the tumor and brain increased immediately after GF120918 treatment, whereas radioactivity in the blood was not significantly changed. In the GF120918-treated mice, the ratios of radioactivity at 90 min to that at 5 min were 1.8 and 4.6 in the tumor and brain, respectively.
Figure 6 shows AUCs of [11C]GF120918 in the tumor and brain. Hereafter, the AUC value for the uptake from 0 to 45 min is denoted as AUC(0–45 min) and that for the uptake from 45 to 90 min is denoted as AUC(45–90 min) after bolus injection of [11C]GF120918. In the control (untreated
group; n = 4), AUC(45–90 min) in the tumor showed a slight increase compared with AUC(0–45 min), whereas the AUC in the brain was unchanged. In the GF120918-treated group (n = 5), AUC(45–90 min) in the tumor showed a signi- ficant increase compared with AUC(0–45 min) in each mouse. In the brain, AUC(45–90min) showed a large increase compared with AUC(0–45 min) in these mice. As shown in Table 1, in the tumor, the ratios of AUC(45–90 min) to AUC(0–45 min) in the control and GF120918-treated groups were 1.20 ± 0.04 and 1.53 ± 0.15, respectively. The corresponding values in the brain were 0.97 ± 0.04 and 3.56 ± 0.92, respectively. The ratios in the tumor and brain in the GF120918-treated group were significantly higher than those in the control group.
Metabolite analysis
The percentage of unchanged [11C]GF120918 in the tumor tissue and plasma of the mice, 60 min after the bolus injection, was investigated (Table 2). In the control mice, the percentages of the unchanged form in the plasma and tumor averaged 89.6 and 99%, respectively (n = 2). In the mice coadministered with unlabeled GF120918, the percentages of the unchanged form in the plasma and tumor tissue were 64.1 ± 11.7 and 98.4 ± 0.5%, respectively (n = 3). A highly polar radio- labeled metabolite was observed in the HPLC chart. In
Fig. 5
(a)
0.5
0.4
0.3
0.2
0.1
0.0
Tumor
i.v.
Recoveries of radioactivity from the tumor tissue and plasma into acetonitrile for deproteinization were 93.6 ± 2.6 (n = 5) and 92.3 ± 2.0% (n = 5), respectively. Recovery of radioactivity from HPLC analysis was essentially quantitative.
Discussion
Earlier, several PET probes were developed for the evaluation of Pgp function in the BBB [30,33]. Of these, [11C]verapamil and [11C]colchicine were used for eval- uating MDR in tumor [34,35]. Verapamil, a calcium channel blocker, is an inhibitor of Pgp, but worked only as a substrate when used for PET assessment [36–38]. In
0 15 30 45 60 75 90
a PET study on rats bearing small cell lung tumors,
(b)
0.6
0.5
0.4
0.3
0.2
0.1
Brain
i.v.
[11C]verapamil was used to evaluate MDR in the tumor by coadministration with CsA [34]. However, [11C]ver- apamil is easily metabolized in vivo, and approximately 40% was converted to metabolites, 10 min after the bolus injection [32]. Thus, a PET probe that is stable against in-vivo metabolism may be favorable for MDR evaluation. [11C]Colchicine, a radiolabeled natural tubulin-binding agent, was also found to correlate with MDR in in-vitro and in-vivo studies [35]. However, the evaluation of MDR using [11C]colchicine in the tumor required com- plicated kinetic analysis because [11C]colchicine not only
0.0
0
15
30
45
60
75
90
acted as a substrate for Pgp but also had pharmacological actions on the tumor [35]. Hence, a desirable PET probe for the evaluation of in-vivo distribution of MDR would
(c)
2.0
1.5
1.0
0.5
0.0
Blood
i.v.
act only as a substrate for the transporters with no other pharmacological action.
Recently, it has been suggested that [11C]GF120918 may behave in a substrate-like manner when present in a trace amount [21,24]. Moreover, GF120918 had high affinity for Pgp and BCRP. Hence, we tried to evaluate the func- tion of Pgp and BCRP in the tumor using [11C]GF120918 for the first time.
We first assessed Pgp and BCRP expressions in Caco-2 cells as a target cell line by western blotting. The expressions of both Pgp and BCRP in the Caco-2 cell line
0
15
30
45
Time (min)
60
75
90
were detected at relatively high concentrations (Fig. 2). We, therefore, carried out an in-vitro uptake study using a
Control GF120918 treatment
Time–activity curves of [11C]GF120918 in the tumor (a), brain (b), and blood (c). Human colon adenocarcinoma-bearing mice were injected with a bolus of 18–37 MBq [11C]GF120918 and injected with 5 mg/kg unlabeled GF120918 (black squares) or with saline (white circles),
45 min later. Radioactivity in the tumor and brain increased rapidly when the mice were injected [intravenously (i.v.), arrow] with GF120918. Radioactivity concentration is expressed as the mean standardized uptake value (SUV) ( ± standard error of the mean, n = 4, 5).
the plasma, the percentage of unchanged [11C]GF120918 in the control mice was approximately 25% higher than that in mice also treated with GF120918.
cell monolayer of these cell lines to evaluate Pgp-mediated and BCRP-mediated transport of [11C]GF120918. In the MDCK cells expressing only Pgp, [11C]GF120918 uptakes on coincubation with CsA and unlabeled GF120918 were approximately 1.2-fold and 2-fold higher, respectively, than those of the control (Fig. 3). This result supports the assumption that GF120918 behaves in a substrate-like manner for Pgp and BCRP when present in trace carrier amounts. The difference in Pgp inhibition between CsA and GF120918 may be because of the different affinities of the two agents for Pgp. In the Caco-2 cells express- ing Pgp and BCRP, GF120918 coincubation increased [11C]GF120918 uptake, but CsA coincubation did not change this uptake (Fig. 3). A significant increase in
Fig. 6
(a) Tumor (b) Brain
20
15
10
5
0
20
15
10
5
0
0–45 45–90 0–45 45–90
Time (min)
Control GF120918
treatment
Area under the tissue concentration–time curve (AUC) in the tumor (a) and brain (b). AUCs were calculated for the intervals from 0 to 45 min and from 45 to 90 min on the time–activity curves in the tumor and brain, respectively. The AUC(45–90 min) in the tumor and brain in the GF120918-treated mice increased considerably in each experimental mouse compared with the AUC(0–45 min) in the corresponding regions.
Table 1 AUC (mean ± standard deviation) and increase ratio (mean ± standard deviation) of [11C]GF120918 in the tumor tissue and brain
AUCtumor AUCbrain
Time after injection (min)
Controla (n = 4)
GF120918b treatment
(n = 5)
Controla (n = 4)
GF120918b treatment
(n = 5)
0–45 8.52 ± 2.42 8.84 ± 3.49 4.2 ± 0.66 4.49 ± 1.81
45–90 10.22 ± 2.88 14.13 ± 4.28 4.06 ± 0.75 15.62 ± 1.81*
Increase ratio (45–90/0–45) 1.2 ± 0.04 1.53 ± 0.15* 0.97 ± 0.04 3.56 ± 0.92*
AUC, area under the concentration–time curves.
aMice were injected with saline at 45 min after injection of [11C]GF120918.
bMice were injected with 5 mg/kg unlabeled GF120918 at 45 min after injection of [11C]GF120918. *P < 0.01 (t-test vs. control group).
Table 2 Percentage of unchanged [11C]GF120918 at 60 min after bolus injection in the plasma and tumor tissue
radioactivity was increased immediately after GF120918 treatment (Fig. 5a). In addition, the AUC in the tumor
Controla (n = 2)
GF120918b administration (n = 3) aAverage of two samples.
bMean ± standard deviation.
Plasma
89.6 64.1 ± 11.7
Tumor
99.4 98.4 ± 0.5
increased after GF120918 treatment and was significantly different from that of the control (untreated group) (Table 1). In contrast, the level of radioactivity in the blood was not changed after GF120918 treatment. This result suggests that the increase in radioactivity in the tumor was not affected by blood flow but was caused by
radioactivity in Caco-2 cells after GF120918 treatment may explain the fact that Pgp and BCRP act together in limiting intracellular [11C]GF120918 penetration.
We measured [11C]GF120918 distribution in Caco-2- bearing mice using small animal PET. In PET images, uptake in the tumor region was increased after GF120918 treatment, although the level of radioactivity was lower than that in tissue around the tumor before GF120918 treatment (Fig. 4). Moreover, in the TACs of the tumor,
the inhibition of Pgp and BCRP functions. It is consi- dered that radioactivity in the tumor, which remained at a low concentration because of the functions of Pgp and BCRP, rose gradually to reach equilibrium with the radio- activity in blood by passive diffusion, resulting in inhibi- tion of their functions. Similar kinetics of [11C]GF120918 were detected in the brain (Fig. 5b). The AUC in the brain after GF120918 treatment was 3.5-fold higher than that in the control (Table 1). Before GF120918 treat- ment, [11C]GF120918 uptakes in the tumor and brain
remained at low concentrations, which may reflect the level of Pgp-mediated and BCRP-mediated drug trans- port. In fact, penetration of [11C]GF120918 in the brain was regulated at a low concentration more strongly and rigidly than that in the tumor before GF120918 treat- ment (Fig. 6). AUCs in the tumor among the experi- mental mice dispersed, but those in the brain converged. This difference in the ability to limit [11C]GF120918 penetration between the tumor and brain may have been because of a difference in the structural properties of the ABC transporter location. ABC transporters are located on the BBB including tight junctions in the brain capillaries and rigorously prevent molecules from entering the brain. However, tumors and peripheral organs do not have tight junctions similar to those in the BBB, and thus incompletely prevent molecules from entering the tumor and peripheral organs [39]. Therefore, the input of [11C]GF120918 depending on passive diffusion during the initial uptake may reflect the strictness with which [11C]GF120918 penetration is limited because of Pgp and BCRP concentrations.
In the metabolite study, the radioactive component in the tumor and plasma was almost unchanged at 60 min after the injection. Earlier, it was reported that approximately 70% of (R)-[11C]verapamil, the first PET ligand used to evaluate MDR in a tumor, was changed to metabolites in the plasma 60 min after the bolus injection [40]. There- fore, we showed that [11C]GF120918 is a more stable PET probe than [11C]verapamil in vivo. However, ap- proximately 35% of [11C]GF120918 in the plasma was metabolized in the mice coadministered with unlabeled GF120918. Pgp shows high levels of expression in the main pharmacological barriers, such as the brush border membrane of intestinal cells, the biliary canalicular mem- brane of hepatocytes, and the luminal membrane of the proximal tubules of the kidney [18]. We considered that metabolites of [11C]GF120918 in liver, kidney, and intes- tine might return to the blood without being excreted when the functioning of Pgp is inhibited after GF120918 treatment. The polar metabolite of [11C]GF120918 may not penetrate into the tumor (Table 1). A similar result was found in the brain using [11C]GF120918 in the Pgp and BCRP double-knockout mice [24]. These results suggest that the radiolabeled metabolite cannot pene- trate through the BBB and other pharmacological barriers. Therefore, in our PET study, reuptake of radioactivity in the tumor and brain after GF120918 treatment was not because of the [11C]GF120918 metabolite, but only because of intact [11C]GF120918.
Conclusion
An in-vitro study using Caco-2 cells showed that [11C]GF120918 uptake was related to the functions of Pgp and BCRP. In a PET study in Caco-2-bearing mice, [11C]GF120918 uptake in the tumor and brain was increased after treatment with unlabeled GF120918,
again indicating that [11C]GF120918 uptake in the tumor and brain was related to Pgp and BCRP functions. A metabolite analysis showed that [11C]GF120918 was stable against in-vivo metabolism for 60 min after the injection. Consequently, we consider that a PET study with [11C]GF120918 in combination with unlabeled GF120918 may be a useful tool for evaluating the levels of Pgp-mediated and BCRP-mediated MDR in tumors, although this needs more experiments and quantitative analyses.
Acknowledgements
The authors are grateful to the staff of the NIRS for their help in cyclotron operation, radioisotope production, and radiosynthesis procedures. They also thank Fujiko Konno (NIRS) for providing GF120918 hydrochloride. This study was supported in part by a Grant-in-Aid for the Molecular Imaging Program from the Ministry of Educa- tion, Culture, Sports, Science, and Technology of the Japanese Government.
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