In Vivo Imaging of Folate Receptor Positive Tumor Xenografts Using

Apr 12, 2012 - Department of Nuclear Medicine, University Hospital Freiburg, 79106 Freiburg, ... Biodistribution and small-animal PET imaging studies ...
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In Vivo Imaging of Folate Receptor Positive Tumor Xenografts Using Novel 68Ga-NODAGA-Folate Conjugates Melpomeni Fani,*,†,‡ Maria-Luisa Tamma,† Guillaume P. Nicolas,† Elisabeth Lasri,§ Christelle Medina,§ Isabelle Raynal,§ Marc Port,§ Wolfgang A. Weber,‡ and Helmut R. Maecke†,‡ †

Division of Radiological Chemistry and Department of Nuclear Medicine, University Hospital Basel, 4031 Basel, Switzerland Department of Nuclear Medicine, University Hospital Freiburg, 79106 Freiburg, Germany § Research Department, Guerbet, 93600 Aulnay sous Bois, France ‡

S Supporting Information *

ABSTRACT: The overexpression of the folate receptor (FR) in a variety of malignant tumors, along with its limited expression in healthy tissues, makes it an attractive tumor-specific molecular target. Noninvasive imaging of FR using radiolabeled folate derivatives is therefore highly desirable. Given the advantages of positron emission tomography (PET) and the convenience of 68 Ga production, the aim of our study was to develop a new 68 Ga-folate-based radiotracer for clinical application. The chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) was conjugated to folic acid and to 5,8-dideazafolic acid using 1,2-diaminoethane as a spacer, resulting in two novel conjugates, namely, P3246 and P3238, respectively. Both conjugates were labeled with 68/67Ga. In vitro internalization, efflux, and saturation binding studies were performed using the FR-positive KB cell line. Biodistribution and small-animal PET imaging studies were performed in nude mice bearing subcutaneous KB xenografts. Both conjugates were labeled with 68Ga at room temperature within 10 min in labeling yields >95% and specific activity ∼30 GBq/μmol. The Kd values of 68/67Ga-P3246 (5.61 ± 0.96 nM) and 68/67Ga-P3238 (7.21 ± 2.46 nM) showed high affinity for the FR. 68/67Ga-P3246 showed higher cell-associated uptake in vitro than 68/67Ga-P3238 (approximately 72 and 60% at 4 h, respectively, P < 0.01), while both radiotracers exhibited similar cellular retention up to 4 h (approximately 76 and 71%, respectively). Their biodistribution profile is characterized by high tumor uptake, fast blood clearance, low hepatobiliary excretion, and almost negligible background. Tumor uptake was already high at 1 h for both 68Ga-P3246 and 68Ga-P3238 (16.56 ± 3.67 and 10.95 ± 2.12% IA/g, respectively, P > 0.05) and remained at about the same level up to 4 h. Radioactivity also accumulated in the FR-positive organs, such as kidneys (91.52 ± 21.05 and 62.26 ± 14.32% IA/g, respectively, 1 h pi) and salivary glands (9.05 ± 2.03 and 10.39 ± 1.19% IA/g, respectively, 1 h pi). The specificity of the radiotracers for the FR was confirmed by blocking experiments where tumor uptake was reduced by more than 85%, while the uptake in the kidneys and the salivary glands was reduced by more than 90%. Reduction of the kidney uptake was achieved by administration of the antifolate pemetrexed 1 h prior to the injection of the radiotracers, which resulted in an improvement of tumor-to-kidney ratios by more than a factor of 3. In line with the biodistribution results, small-animal PET images showed high uptake in the kidneys, clear visualization of the tumor, accumulation of radioactivity in the salivary glands, and no uptake in the gastrointestinal tract. 68Ga-P3246 and 68Ga-P3238 showed very high tumor-to-background contrast in PET images; however, the tumor-to-kidney ratio remained low. The new radiotracers, especially 68Ga-P3246, are promising as PET imaging probes for clinical application due to their facile preparation and improved in vivo profile as compared to the other folate-based PET radiotracers. KEYWORDS: folate receptor, folic acid, NODAGA-folate conjugates, gallium-68, PET imaging

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INTRODUCTION Folates are vitamins required for the survival and proliferation of eukaryotic cells since they are involved as coenzymes in key biosynthetic and epigenetic processes.1 The uptake of exogenous folates by cells is mediated by three transport systems:2,3 (i) the reduced folate carrier (RFC) with relativity high affinity for reduced folates and low affinity for folic acid, (ii) the proton-coupled folate transporter that transports folates preferentially at low pH, and (iii) the folate receptor (FR) with © 2012 American Chemical Society

high affinity for folic acid (KD = 0.1 nmol/L) and lower affinity for the reduced folates. Although the RFC is ubiquitously expressed in normal tissues, the FR displays a more restricted range of tissue expression, for example, kidneys, lungs, placenta, Received: Revised: Accepted: Published: 1136

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Müller et al. showed that this application significantly reduces kidney uptake of other folate-based radiotracers.23,24

and choroid plexus, while it is highly expressed in a wide variety of human epithelial cancers, such as breast, cervical, colorectal, renal, nasopharyngeal, and especially ovarian and endometrial carcinomas.4−6 FR targeting has been successfully used for the selective delivery of imaging and therapeutic agents to FR-positive tumors.6−9 Similar to folic acid, folic acid conjugates bearing these agents are bound to the FR, and they are transported into the cell by endocytosis.10,11 Several folate-based conjugates labeled with γ-emitters, such as 111In [t1/2 = 2.8 days, Eγ = 171 keV (91%), 245 keV (94%)] and 99mTc [t1/2 = 6 h, Eγ = 140 keV (87%)] have been developed for single photon emission computed tomography (SPECT) imaging of FRpositive tumors,12,13 and two of them have been clinically tested. 111In-DTPA-folate (DTPA: diethylenetriaminepentaacetic acid) has been evaluated in ovarian cancer patients in the United States in a phase I/II study14 and 99mTc-EC20 (99mTc-ethylenedicysteine-folic acid) has been clinically tested in patients with different solid tumors.15,16 Nowadays, positron emitters such as 18F [t1/2 = 110 min, E̅β+ = 250 keV (97%)] and 68 Ga [t1/2 = 67.71 min, E̅ β+ = 740 keV (89%)] attract more and more attention for the development of radiotracers for positron emission tomography (PET) imaging, which combines the potential to quantify tracer uptake within lesions with a relatively high resolution and a remarkably high sensitivity of up to 10−12 mol/L. 68Ga is of major interest as it can be produced from a long-lived 68Ge/68Ga generator, which makes its availability easy, inexpensive, and independent of an on-site cyclotron, rendering 68Ga radiopharmacy possible in every hospital.17,18 We are interested in the development of folate-based PET radiotracers for in vivo imaging of FR. In our previous work, we developed two 68Ga-DOTA-folate conjugates (DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), named 68Ga-P3026 and 68Ga-P1254, which showed similar in vitro and in vivo behavior to the reference radiotracer 111 In-DTPA-folate.19 These encouraging results motivated us to continue our research focusing on the development of a 68 Ga-folate conjugate for clinical application. In the present work, we report the development and preclinical evaluation of two novel folate-based conjugates using the chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7acetic acid (NODAGA) for labeling with 68Ga. Folic acid was attached to NODAGA via 1,2-diaminoethane, as a spacer between the chelator and the pharmacophore, resulting in the conjugate P3246. As an alternative to folic acid, the 5,8dideazafolic acid was used as the pharmacophore based on the structure of the first clinically evaluated folate-based thymidylate synthase inhibitor CB3717, which is transported via the FR, while it is a low affinity substrate for the RFC.20 NODAGA was attached to 5,8-dideazafolic acid using the same spacer, resulting in the conjugate P3238. Both conjugates were labeled with 68Ga and also with 67Ga (t1/2 = 78.3 h) for investigations at later time points given the short half-life of 68Ga. In vitro evaluation of both radiotracers was performed using the KB cells, a human nasopharyngeal carcinoma cell line overexpressing the FR.21,22 In vivo evaluation was performed in nude mice bearing subcutaneous KB xenografts with anatomic (tissue sampling) and imaging (small-animal PET) techniques. Because of the FR expression in the proximal tubular cells, high renal uptake of the radiotracers was expected. In an attempt to block kidney uptake, we injected the antifolate pemetrexed.

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EXPERIMENTAL SECTION Synthesis of the Conjugates. The molecular formulas of the folate conjugates P3246 and P3238 are shown in Figure 1.

Figure 1. Structures of the new NODAGA-folate conjugates P3246 and P3238.

The prochelator 1-(1-carboxy-3-carbo-tert-butoxypropyl)-4,7(carbo-tert-butoxymethyl)-1,4,7-triazacyclononane, NODAGA(tBu)3 (Chematech, Dijon, France), was dissolved in dichloromethane and preactivated for 30 min with N-hydroxysuccinimide and dicyclohexylcarbodiimide under argon atmosphere, followed by filtration of dicyclohexylurea. The solution of the activated ester was added to a dimethyl sulfoxide solution of (i) 4-(2-amino-ethylcarbamoyl)-2-{4-[(2-amino-4-oxo-3,4-dihydro-pteridin-6-ylmethyl)-amino]-benzoylamino}-butyric acid19,25 (P3246) and (ii) 4-(2-amino-ethylcarbamoyl)-2-{4[(2-amino-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-amino]benzoylamino}-butyric acid26 (P3238), in a 1:1.1 ratio, along with 2 equiv of triethylamine. After incubation for 1 h at room temperature (RT), the reaction mixture was precipitated by the addition of diethyl ether. The precipitate was purified by flash chromatography, and the protecting groups were removed after treatment with trifluoroacetic acid for 6 h at RT, followed by evaporation and precipitation in diethyl ether. The final products (P3246 and P3238) were purified by preparative highperformance liquid chromatography (HPLC), lyophilized, and characterized by electrospray ionization−mass spectrometry (ESI-MS) and HPLC using Waters Symmetry C18 column (4.6 mm × 250 mm) and a mixture of water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile (solvent B), in the following gradient: 0−20 min, 95−80% A; 25 min, 80% A; 30 min, 50% A; 32 min, 95% A; and 35 min, 95% A, at a flow rate of 0.75 mL/min. Radiolabeling. The 68Ge/68Ga generator IGG100 and the Modular-Lab PharmTracer cassette-based module (Eckert & Ziegler, Berlin, Germany) were used for labeling based on a slightly modified published method for the processing of the 68 Ga eluate.27 Briefly, the generator was eluted with 7 mL of 0.1 N HCl, and the eluate was loaded onto a cation exchange column (Strata-XC, Phenomenex). 68Ga was eluted with 800 μL of a mixture of acetone/HCl (97.6%/0.02 N) directly into the reaction vial containing 2 mL of 0.2 mol/L sodium acetate buffer, pH 4.0, and 10 μg of P3246 or P3238. The reaction mixture was incubated at RT for 10 min. 1137

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Labeling with 67Ga was performed after incubation of 10 μg of each conjugate with 37 MBq 67GaCl3 in 300 μL of 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (0.4 mol/L, pH 3.9). natGa(NO3)3 × 9H2O was used for the formation of the metal complexes natGa-P3246 and natGa-P3238, following the same protocol. Quality control was performed by HPLC, as described above. The radiotracer solutions were prepared by dilution with 0.9% NaCl for the following studies. For the saturation binding studies, 67/natGa-P3026 and 67/natGaP1254, with tracer amounts of 67Ga, were used to afford structurally characterized homogeneous compounds. Cell Culture. KB cells, a human nasopharyngeal carcinoma cell line overexpressing the FR, were obtained from American Type Culture Collection (CCL-17). The cells were cultured continuously as monolayers at 37 °C in a humidified atmosphere containing 5% CO2, in folate-free RPMI 1640 medium (BioConcept, Switzerland). The medium was supplemented with 10% fetal calf serum (FCS), L-glutamine, and antibiotics (penicillin, 100 IU/mL; streptomycin, 100 μg/mL). For in vitro and in vivo experiments, subconfluent cells were harvested by treatment with trypsin (0.05%) in phosphate-buffered saline (PBS) with ethylenediaminetetraacetic acid (EDTA, 0.02%). In Vitro Studies. The cells were seeded into six-well plates (1 × 106 cells/well) and incubated at 37 °C/5% CO2 to form subconfluent monolayers overnight. The day of the experiment, the medium was removed, and the cells were washed twice with pure RPMI medium (without FCS and antibiotics). Eight-hundred microliters of pure medium was added to each well, and the plates were incubated at 37 °C/5% CO2 for 1 h. Experiments were performed in triplicate for each time point. Cell-Uptake and Internalization Studies. Each radiotracer was added to the cells (2.5 pmol/100 μL/well) and incubated at 37 °C/5% CO2 for preselected time points of 30 min and 1, 2, and 4 h, in a final concentration of 0.25 pmol/mL. An excess of folic acid (1000-fold) was added to determine nonspecific binding and internalization. At preselected time points, the internalization was stopped by removal of the medium followed by washing the cells twice with 1 mL of ice-cold PBS. Cells were treated 2 × 5 min with 1 mL of glycine solution (0.05 mol/L, pH 2.8) on ice to distinguish between cell surfacebound (acid releasable) and internalized (acid resistant) radiotracer.5 Finally, the cells were treated with 1 mL of NaOH, 1 mol/L at 37 °C for 10 min, and washed twice (2 × 1 mL) with the same solution.19,28 The radioactivity of all fractions, the culture medium, the receptor-bound, and the internalized fraction were measured in a γ-counter (Packard, Cobra II). Receptor specific internalization was calculated by subtracting the value found for nonspecific internalization from the total value and expressed as a percentage of the applied radioactivity. Cellular Retention Studies. The cells were allowed to internalize the radiotracer for a period of 2 h at 37 °C and were then exposed to an acid wash, as described in the previous section, to dissociate cell surface-bound radiotracer. Prewarmed pure RPMI medium (1 mL, 37 °C) was then added to each well, and the cells were incubated at 37 °C. At different time points (15, 30, 60, 90, 120, and 240 min), the external medium was removed (followed by two washes with PBS) for quantification of radioactivity in a γ-counter and replaced with fresh 37 °C pure RPMI medium. Internalized radiotracer was extracted in 1 mol/L NaOH. The recycled fraction was expressed as a percentage of the total internalized amount. Saturation Binding Studies. The cells were incubated for 2 h at 4 °C in the presence of 67/natGa-P3246 or 67/natGa-P3238

at different concentrations ranging from 1 to 100 nmol/L. Folic acid at a concentration of 100 μmol/L was used to quantify the nonspecific binding. The medium was removed, and the cells were washed 2 × 1 mL PBS (free fraction), and then, they were collected with 1 mol/L NaOH (bound fraction). The radioactivity of both fractions was measured in a γ-counter. Dissociation constant (Kd) values were calculated from the analysis of the data using GraphPad Prism 5.01 software (GraphPad Software Inc.). Biodistribution Studies. Procedures were approved by the authorities in accordance to the Swiss regulations for animal treatment (approval no. 789) and the guidelines of the German Law for the use of living animals in scientific studies. Athymic female nude mice (4−5 weeks old, 18−20 g) were subcutaneously inoculated in the right flank with 1 × 106 KB cells. Tumors were allowed to grow for 10−15 days (tumor weight, 80−100 mg). The animals were kept under folate low diet (semisynthetic product with 50 μg/kg of folate, S.A.F.E., France) 1 week before the implantation of the tumors until the end of the studies to reduce their serum folate level near that of human serum.29 For the biodistribution studies, the mice were grouped in 3−5 mice/group, and they were injected with 0.4 nmol/10−12 MBq/100 μL of each 68Ga-NODAGA-folate conjugate into the tail vein or with 0.4 nmol/1.2 MBq/100 μL of each 67 Ga-NODAGA-folate conjugate. At preselected time points of 1, 2, 4, and 24 h postinjection (pi), the mice were sacrificed. The organs of interest were collected, blotted dry, weighed, and counted in a γ-counter. The results are expressed as the percentage of injected activity per gram (% IA/g ± SD) for each organ. Nonspecific uptake in tumor and FR-positive organs was determined by 5 min of preinjection of 40 nmol folic acid/100 μL in PBS pH 7.4. Reduction of kidney uptake was attempted by injection of 400 μg/100 μL of the antifolate pemetrexed (Alimta, Eli Lilly, Germany) in 0.9% NaCl, 1 h prior to the injection of the radiotracers. The effect of pemetrexed was studied at 1 and 4 h after injection of 68/67Ga-P3246 and at 1 h after injection of 68/67Ga-P3238. Small-Animal PET Images. PET scans were performed using a dedicated small-animal PET scanner (Focus 120 microPET scanner, Concorde Microsystems Inc.). The mice were intravenously injected with 0.4 nmol/10−12 MBq/100 μL of 68Ga-P3246 into the tail vein. Dynamic scans were performed for 1 h under anesthesia (1.5% isoflurane, 0.5 L/min). Static scans for 20−30 min were acquired in mice sacrificed 1 h pi. Dynamic and static scans were also performed in mice preinjected with pemetrexed as described above. PET images were reconstructed by filtered back projection and were displayed using Advantage Workstation 4.4 from General Electric (GE-Healthcare corp., Milwaukee, WI) and the Volume Viewer version 3.1 software package. No correction was applied for attenuation. The images [maximum intensity projections (MIP) and coronal sections] were set in the same color scale to allow for head-to-head qualitative comparison. For data analysis, regions of interest (ROIs) were drawn on multiple frame dynamic PET images and placed over the tumor, kidneys, salivary glands, and muscles. Maximum standardized uptake values (SUVmax) derived from the ROIs for each time frame were used to generate time−radioactivity curves. Statistical Analysis. Statistical analysis was performed by unpaired two-tailed t test using Prism software (GraphPad Software Inc.). P values of 95% without the need of a postlabeling purification step. The specific activity of both radiotracers was 30 GBq/μmol. In Vitro Studies. Each radiotracer evaluated in cell culture showed high cell-associated activity (cell surface-bound and internalized fraction) with no significant differences from 30 min up to 4 h (Figure 2A). 68/67Ga-P3246 had higher cellassociated uptake values as compared to 68/67Ga-P3238, ranging from 60 to 72% versus 50 to 60%, depending on the assessed

Figure 3. Saturation binding study on intact KB cells using increased concentrations of 67/natGa-P3246 (A) and 67/natGa-P3238 (B), ranging from 1 to 100 nmol/L. The dissociation constant (Kd) was calculated from nonlinear regression analysis using GraphPad Prism. All data results are from two independent experiments with triplicates in each experiment.

the radiotracers to the FR but to avoid internalization. Both 67/nat Ga-NODAGA-folates exhibited similar affinity for the FR, with Kd values of 5.61 ± 0.96 nM for 67/natGa-P3246 and 7.21 ± 2.46 nM for 67/natGa-P3238. Biodistribution Studies. The biodistribution results of 68/67 Ga-NODAGA-folate conjugates in KB xenografts are presented in Tables 1 and 2. The biodistribution profile of both radiotracers is characterized by efficient clearance from the blood, high and receptor-mediated tumor uptake, and high radioactivity accumulation in the kidneys and salivary glands. More specifically, the pharmacokinetics of 68/67Ga-P3246 was studied from 1 h up to 24 h pi (Table 1). Tumor uptake

Figure 2. Time-dependent cell uptake and retention of 68/67Ga-P3246 and 68/67Ga-P3238 into KB cells at 37 °C. (A) Cell uptake (cell surface-bound and internalized fraction), calculated as a percentage of total added radioactivity (mean ± SD), (B) receptor specific internalization expressed as percentage of the applied radioactivity (mean ± SD), and (C) cellular radioactivity retention, expressed as the percentage remaining in the cells from the total amount internalized (100%) 2 h after incubation at 37 °C (mean ± SD). 1139

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Table 1. Biodistribution Results and Tumor-to-Nontumor Ratios of Xenograftsa organ

1h

2h

blood heart liver spleen lung kidney stomach intestine adrenal pancreas muscle bone salivary glands KB tumor

0.24 1.92 2.58 0.80 1.83 91.52 1.68 0.58 2.89 2.32 1.63 0.65 9.05 16.56

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.19 0.26 0.12 0.10 21.05 0.26 0.12 0.52 0.24 0.18 0.14 2.03 3.67

0.09 1.88 1.38 0.48 1.32 133.50 1.58 0.50 3.09 2.91 1.49 1.16 9.10 18.42

tumor:blood tumor:liver tumor:muscle tumor:kidneys

81.32 6.63 9.64 0.18

± ± ± ±

19.94 1.56 1.81 0.02

206.67 14.07 12.40 0.13

68/67

Ga-P3246 in Nude Mice Bearing KB Tumor 4 h of blockingb

4h

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.06 ± 0.11 1.98 ± 0.50 1.07 ± 0.11 0.46 ± 0.15 1.36 ± 5.51 130.31 ± 0.12 1.47 ± 0.12 0.43 ± 0.52 2.80 ± 0.08 2.86 ± 0.18 1.79 ± 0.17 0.65 ± 3.39 8.58 ± 0.74 16.29 ± tumor-to-nontumor ratios ± 38.26 254.03 ± ± 3.40 15.80 ± ± 1.11 7.98 ± ± 0.01 0.12 ±

0.01 0.14 0.18 0.10 0.37 14.65 0.08 0.16 0.10 0.05 0.31 0.18 2.33 4.46

0.01 0.04 0.06 0.04 0.06 6.49 0.16 0.16 0.04 0.05 0.03 0.05 0.18 2.22

± ± ± ± ± ± ± ± ± ± ± ± ± ±

33.94 2.24 1.16 0.02

0.00 0.01 0.01 0.02 0.01 1.03 0.06 0.07 0.01 0.00 0.01 0.01 0.06 0.12

24 h 0.04 1.48 0.87 0.37 0.94 97.62 1.01 0.37 1.60 2.37 1.14 0.89 5.57 14.32

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.23 0.16 0.13 0.31 11.59 0.10 0.13 0.24 0.17 0.01 0.01 0.82 5.80

391.05 16.29 12.61 0.15

± ± ± ±

82.81 2.12 4.22 0.05

Values are expressed as % IA/g ± SD of n = 3−5 mice injected with 0.4 nmol/100 μL 68/67Ga-P3246. bFive minutes of preinjection of 40 nmol folic acid/100 μL in PBS pH 7.4. a

Table 2. Biodistribution Results and Tumor-to-Nontumor Ratios of Xenograftsa organ

1h

68/67

Ga-P3238 in Nude Mice Bearing KB Tumor

2h

blood heart liver spleen lung kidney stomach intestine adrenal pancreas muscle bone salivary glands KB tumor

0.22 1.89 5.82 1.69 1.76 62.26 2.11 0.74 3.03 2.79 1.61 0.79 10.39 10.95

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.11 0.13 1.34 0.58 0.27 14.32 0.58 0.23 0.70 0.91 0.31 0.08 1.19 2.12

tumor:blood tumor:liver tumor:muscle tumor:kidneys

56.71 1.94 6.93 0.18

± ± ± ±

12.85 0.42 1.19 0.03

4 h of blockingb

4h

0.13 ± 0.01 2.26 ± 0.18 3.25 ± 0.37 0.55 ± 0.07 1.53 ± 0.19 92.25 ± 11.94 1.80 ± 0.10 0.57 ± 0.06 2.72 ± 0.45 2.89 ± 0.54 2.11 ± 0.34 0.72 ± 0.18 13.13 ± 3.72 12.89 ± 1.41 tumor-to-nontumor ratios 98.53 ± 8.14 3.97 ± 0.17 6.12 ± 0.26 0.14 ± 0.02

0.10 2.11 2.49 0.71 1.59 111.96 1.69 0.56 3.77 2.79 1.72 1.16 10.91 14.88

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.36 0.21 0.10 0.13 15.33 0.36 0.16 0.27 0.10 0.02 0.08 1.05 2.28

150.48 5.99 7.69 0.13

± ± ± ±

22.34 0.82 1.28 0.00

0.01 0.06 3.34 0.06 0.07 8.58 0.06 0.47 0.06 0.08 0.04 0.03 0.30 2.28

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.00 0.01 0.39 0.02 0.01 1.51 0.01 0.03 0.00 0.01 0.01 0.01 0.04 0.75

a Values are expressed as % IA/g ± SD of n = 3−5 mice injected with 0.4 nmol/100 μL 68/67Ga-P3238. bFive minutes of preinjection of 40 nmol folic acid/100 μL in PBS pH 7.4.

was already high at the initial time point of the investigation (16.56 ± 3.67% IA/g, 1 h pi), showing a very slow washout; more than 85% remained in the tumor after 24 h (14.32 ± 5.80% IA/g). A significant amount of radioactivity was found in FRpositive organs such as the kidneys and salivary glands (91.52 ± 21.05 and 9.05 ± 2.03% IA/g, respectively, 1 h pi), which remained high at all investigated time points. The clearance from the blood pool was fast (e.g., 0.24 ± 0.05 and 0.06 ± 0.01% IA/g at 1 and 4 h pi, respectively), while the concentration of radioactivity in nontargeted organs, such as lungs, spleen, stomach, and muscles was low. The specificity of the radiotracer

for the FR in vivo was confirmed by blocking experiments at 4 h pi using an excess of folic acid. Tumor uptake was reduced by more than 85%, while the uptake in the kidneys and the salivary glands was reduced by more than 90% (Table 1). Similar biodistribution data were obtained with 68/67GaP3238 (Table 2). However, lower tumor uptake was found for 68/67Ga-P3238 than for 68/67Ga-P3246 (10.95 ± 2.12 and 16.56 ± 3.67% IA/g, respectively, at 1 h pi and 14.88 ± 2.28 and 16.29 ± 4.46% IA/g, respectively, at 4 h pi), but this was statistically not significant (P > 0.05). 68/67Ga-P3238 showed lower kidney uptake than 68/67Ga-P3246 (62.26 ± 14.32% IA/g 1140

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kidney uptake was reduced by more than 80% (from 130.31 ± 14.65 to 22.78 ± 2.95% IA/g, Figure 4A), which resulted in a further improvement of tumor-to-kidney ratio (from 0.12 ± 0.02 to 0.76 ± 0.05, ***P < 0.001). The same observation was also made in the case of 68/67Ga-P3238 at 1 h pi where kidney reduction by more than 60% was found when pemetrexed was injected before the radiotracer, while the uptake in the tumor, salivary glands, and the rest of the organs remained essentially unaffected (Figure 4B). Small-Animal PET Images. PET images were performed with 68Ga-P3246 at 1 h pi with and without preinjection of pemetrexed. The PET images (Figure 5) clearly showed high

and 91.52 ± 21.05, respectively, 1 h pi, P > 0.05), while liver uptake was significantly higher (5.82 ± 1.34 and 2.58 ± 0.26% IA/g, respectively, 1 h pi, **P < 0.01). Radioactivity in the liver was washed out by more than 60% from 1 to 4 h pi for both radiotracers. The specificity of 68/67Ga-P3238 was also confirmed by blocking experiments at 4 h pi using an excess of folic acid, where tumor uptake was reduced by 85% and the uptake in the kidneys and the salivary glands by more than 90% (Table 2). The tumor-to-background ratio was high for both radiotracers as early as 1 h. 68/67Ga-P3246 had higher values, especially with regard to tumor-to-blood and tumor-to-liver ratios, while the tumor-to-kidney ratio was low (e.g., tumor-to-kidney = 0.18, 1 h pi) and remained low at all investigated time points. No significant improvement was observed in the tumor-to-background ratios from 2 to 4 h or even 24 h (in the case of 68/67GaP3246) with the exception of the tumor-to-blood ratio, which increased over time. Preinjection of pemetrexed 1 h prior to injection of 68/67 Ga-P3246 significantly reduced the kidney uptake of the radiotracer by 70% at 1 h pi (from 91.52 ± 21.05 to 27.19 ± 4.63% IA/g, Figure 4A) while no significant influence was

Figure 4. Effect of the antifolate pemetrexed (400 μg/100 μL) injected 1 h prior to the injection of 68/67Ga-P3246 (0.4 nmol/100 μL) and 68/67 Ga-P3238 (0.4 nmol/100 μL) in KB xenografts. The biodistribution of 68/67Ga-P3246 was studied at 1 and 4 h pi (A) and of 68/67 Ga-P3246 at 1 h pi. (B) The results are expressed as % IA/g ± SD (n = 3-5). Please note the difference in the scales for the uptake in the tumor, salivary glands, and kidneys as compared to all other organs.

Figure 5. MIP images of 68Ga-P3246 1 h pi without (A) and with (B) preinjection of pemetrexed. Coronal images of 68Ga-P3246 without (C) and with preinjection of pemetrexed (D) 1 h pi. Time− radioactivity curves of 68Ga-P3246 from 10 up to 60 min pi derived from dynamic scans (E).

uptake in the tumor, salivary glands, and kidneys. The background activity, with the exception of kidneys, was negligible, and a very good tumor-to-background contrast was demonstrated, especially in the coronal sections. The PET images of mice preinjected with pemetrexed confirmed the biodistribution results as the accumulation of radioactivity in the kidneys

observed in the accumulation of radioactivity in the tumor, salivary glands, and the rest of the organs. As a consequence, the use of pemetrexed increased the tumor-to-kidney ratio by more than 3-fold (from 0.18 ± 0.02 to 0.60 ± 0.16, ***P < 0.001). This effect was more dramatic at 4 h pi, where the 1141

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limited to discontinuation of folic acid supplements for at least 2 days before scheduled imaging with radiofolates.14,15 Biodistribution studies of 68/67Ga-NODAGA-folates in KB xenografts showed fast blood clearance, receptor-mediated uptake in the FR-positive tumors and organs, such as the kidneys and salivary glands, and very low uptake in nontargeted organs. Higher tumor uptake was found for the 68/67Ga-P3246 than for 68/67Ga-P3238, probably reflecting the in vitro results; however, this was found to be statistically not significant at all investigated time points. To our knowledge, folate-based PET radiotracers for in vivo imaging are limited to 66Ga-DF-folate (DF: deferoxamine)32 and a series of 18F-folates,33−36 all of which have been evaluated in KB xenografts. The DF-folate conjugate was initially used for in vivo imaging labeled with 67Ga.29,37 The chelator DF forms stable complexes with Ga(III); however, it was proven not to be suitable for the development of Ga radiotracers possibly due to the dissociation of the metal at concentrations at which radiotracers are used in vivo.38 The 67/66Ga-DF-folate showed good pharmacokinetics with primary excretion via urine and high uptake and clear visualization of FR-positive tumors and kidneys (∼8.5 and 60% IA/g, respectively, 4 h pi).32,37 However, the high accumulation of radioactivity in the abdomen (∼12−30% IA/g is cleared via the intestine at 4 h pi, depending on the injected dose)37 was the main drawback for further development, as this will interfere with imaging of abdominal tumors near the intestine and bladder, such as ovarian carcinoma. In our previous work, we showed that replacement of DF by DOTA results in 68 Ga-DOTA-folate conjugates, such as 68Ga-P3026 and 68GaP1254,19 with improved pharmacokinetics as compared to 67/66 Ga-DF-folate and comparable to 111In-DTPA-folate, which has been used in clinical trials. These radiotracers are eliminated by the kidneys, while no significant amount of radioactivity was found in the gastrointestinal tract. They all have intestinal uptake 90% of the cases. Diagnosis of ovarian cancer at early stages leads to good prognosis, but it is rarely diagnosed before the cancer has spread because the currently available screening tests do not achieve sufficiently high levels of sensitivity and specificity.30 Therefore, noninvasive imaging of FR using highly selective and specific folate conjugates and highly sensitive PET imaging may be of great importance for the management of these patients. In the present study, the two folate-based pharmacophores, folic acid and 5,8-dideazafolic acid, were coupled to the chelator NODAGA. The two conjugates P3246 and P3238, respectively, were labeled with 68Ga within 10 min at RT. The high labeling yields achieved under the conditions described in the Experimental Section eliminate the need of any postlabeling purification step, often necessary in the preparation of other PET imaging probes, such as 18F-radiopharmaceuticals. Straightforward labeling facilitates the preparation of the aforementioned radiopharmaceuticals as compared to any other known folatebased PET radiotracers, including 68Ga-DOTA-folates, where elevated temperature is required.19,28 A human nasopharyngeal carcinoma cell line overexpressing the FR,21,22 namely, KB cells, were used for the evaluation of the 68 Ga-NODAGA-folates, as they are the most often used cell line for in vitro and in vivo studies of FR-targeting agents. 68/67GaP3246 and 68/67Ga-P3238 were rapidly associated to the FR in vitro, with 68/67Ga-P3246 having higher cell-associated uptake values than 68/67Ga-P3238 (about 70 and 60%, respectively, at 4 h). It has been shown that once folate conjugates are bound to the FR, they are transported into the cell through receptormediated endocytosis.10,11 This internalization process was also seen in the case of both 68/67Ga-NODAGA-folates, with 68/67GaP3246 having a higher internalization rate than 68/67Ga-P3238, while both radiotracers remained in the cells at the same level (about 75% after 4 h at 37 °C). Saturation binding studies exhibited high affinity for both the folic acid conjugate and the 5,8-dideazafolic acid conjugate for the FR with Kd values in a low nanomolar range. Blocking experiments demonstrated the specificity of both radiotracers for the FR, while their FRmediated uptake values in vitro are on the same level referred to in the literature for other radiolabeled folate conjugates in KB cells.11,19,28,31 In vivo studies were performed in KB xenografts about 3 weeks after initiation of folate low diet when the folate level in the mouse serum (25 ± 7 nM) is near the human level (9−14 nM).29 So far in clinical studies, dietary modification is 1142

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of kidney uptake of folate-based radiotracers.23,24,28 In line with these findings, preinjection of pemetrexed 1 h before the injection of the 68/67Ga-P3246 reduced kidney uptake of the radiotracer by 70% at 1 h pi and by 80% at 4 h pi, without influencing the tumor uptake. This resulted in a significant increase of tumor-to-kidney ratio by more than a factor of 3 and a better contrast in the PET images. Also, in the case of 68 Ga-P3238, biodistribution data at 1 h pi showed the same effect, meaning significant reduction of kidney uptake (>60%) with no influence on the tumor uptake. Interestingly, in a recently published study, it was shown that when the antifolates pemetrexed and CB3717 (a 5,8-dideaza analogue) were combined with the folate-based radiotracer 99mTc-EC20 pemetrexed had the same effect as found in the present study, while the 5,8-dideaza analogue CB3717 blocked the uptake of the radiotracer not only in the kidneys but also in the FRpositive organs, including the tumor.24 In vitro studies showed that the affinity of the 5,8-dideaza analogue for the FR was comparable to that of folic acid at physiologic conditions (37 °C), while the affinity of pemetrexed was reduced by more than 2 orders of magnitude. This strongly supports our results as we demonstrated that both pharmacophores, folic acid and 5,8-dideazafolic acid coupled to NODAGA and labeled with 68/67 Ga, retain their affinity for the FR in vitro and in vivo while kidney uptake of both can be blocked with pemetrexed without any influence in the tumor uptake. Moreover, the FR-mediated uptake of both radiotracers could be blocked with an excess of folic acid, confirming their specificity for the FR. In summary, the new NODAGA-folate conjugates, P3246 and P3238, can be labeled with 68Ga in high labeling yields and specific activities that allow clinical application. NODAGA is an ideal chelator for 68Ga and possibly outperforms the commonly used chelator DOTA, especially with regard to facile radiolabeling. The 68Ga-NODAGA-folate conjugates are comparable or even better than the 68Ga-DOTA-folate and similar to clinically evaluated 111In-DTPA-folate, while they showed an improved in vivo pattern as compared to any 18F-folates. Between the two conjugates, 68Ga-P3246 seems to be an excellent candidate for clinical translation.

be mentioned that both 68Ga-P3246 and 68Ga-P3238 had low tumor-to-kidney ratios (0.18, 1 h pi), a disadvantage always found with folate-based radiotracers, but nevertheless, it was found to be the same as the clinically tested 111In-DTPA-folate. During the last 5 years, a series of folate-based PET radiotracers have been developed using 18F. The first, 18Ffluorobenzylamine-folate,33 suffers from low radiochemical yield, multistep and time-consuming synthesis, and a prominent hepatobiliary elimination of the radiotracer, resulting in high radioactivity concentration in the gallbladder and intestinal tract. Even though the problem of low radiochemical yields was overcome using click chemistry,34 the 18F-click-folate analogue had a highly lipophilic character and thus very high abdominal background radioactivity.34,35 A direct 18F-labeling strategy has been recently developed by Ross et al.36 avoiding any prosthetic group, leading to acceptable overall yield. This new 18F-labeled folate has an improved image contrast, as compared to all other 18 F-folates, as it showed efficient renal elimination. PET images demonstrated high radioactivity concentration in the tumor, kidneys, gallbladder, and intestines but also moderate uptake in the liver. 68 Ga-folate radiotracers have some advantages over 18Ffolates. First, 68Ga is available from long-lived 68Ge/68Ga generators, giving flexibility in the preparation of the radiopharmaceutical and independence of an on-site cyclotron. The preparation demands very small amounts of precursor (nmol level) as compared to 18F, where the precursor is usually used in micromole level. The labeling procedure is faster and easier than any synthetic route followed for the preparation of 18 F-folates. More importantly, the preparation of 68GaNODAGA-folates demands very mild conditions since no elevated temperatures or acidic conditions, often used for deprotection steps with 18F or postlabeling purification steps, are required. As far as the specific activity of the product is concerned, both radiotracers 68Ga-folate and 18F-folate radiotracers can be obtained with similar specific activities.33,36 The small-animal PET images of 68Ga-P3246 in KB xenografts showed high and specific uptake of the radiotracer in the tumor, kidneys, and salivary glands. The maximumintensity projection images clearly demonstrated the elimination of the radiotracers via the kidneys, while no gallbladder or intestinal uptake was seen, confirming the ex vivo biodistribution data. The PET images acquired after injection of 68 Ga-P3246 show the superiority of these folate-based PET radiotracers as compared to the 18F-folate radiotracers.33−36 From the time−activity curves, it was seen that radioactivity in the FR-positive organs, such as the kidneys and salivary glands but also the tumor, increases over a 60 min time period. The biodistribution data showed that while tumor and salivary glands remained at the same level from 1 to 4 h pi, kidney uptake is constantly increasing up to 2 h pi. These results indicate that the NODAGA-folate conjugate matches well with a short-lived radionuclide such as 68Ga, since very good image contrast can be achieved at early time points, with the tumor-to-kidney ratio, the main limiting factor, remaining essentially the same up to 1 h pi and not improving over time. Even though the kidney uptake is the main limitation of the folate-based radiotracers, this is mainly of concern if therapeutic applications are planned (radionuclide therapy), rather than for diagnostic purposes, as this will lead to concentration of high amounts of radioactivity in the kidneys with the risk of nephrotoxicity. Müller et al. have shown that the use of the antifolate pemetrexed has a significant impact in the regulation

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ASSOCIATED CONTENT

S Supporting Information *

Detailed comparison between the in vivo results obtained in the present study from the 68/67Ga-NODAGA-folate conjugates and those obtained previously from 68/67Ga-DOTA-folate conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Division of Radiological Chemistry and Department of Nuclear Medicine, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland. Tel: ++41(0)61 556 5891. Fax: + +41(0)61 265 4925. E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s):This work was supported by a research grant from Guerbet (Aulnay sous Bois, France). E. Lasri, C. Medina, I. Raynal and M. Port are employees of Guerbet. M. Fani, M.L. Tamma, G.P. Nicolas, W.A. Weber and H.R. Maecke declare that they have no conflict of interest. 1143

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ACKNOWLEDGMENTS We thank Roswitha Tönnesmann for her expert technical assistance with the 68Ga labelings.

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