Article pubs.acs.org/molecularpharmaceutics
PET and SPECT Imaging of a Radiolabeled Minigastrin Analogue Conjugated with DOTA, NOTA, and NODAGA and Labeled with 64Cu, 68 Ga, and 111In S. Roosenburg,†,‡ P. Laverman,*,†,‡ L. Joosten,† M. S. Cooper,§ P. K. Kolenc-Peitl,∥ J. M. Foster,⊥ C. Hudson,⊥ J. Leyton,⊥ J. Burnet,⊥ W. J. G. Oyen,† P. J. Blower,§ S. J. Mather,⊥ O. C. Boerman,† and J. K. Sosabowski⊥ †
Department of Radiology and Nuclear Medicine, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, U.K. § Division of Imaging Sciences and Biomedical Engineering, King’s College London, London SE1 7EH, U.K. ∥ Department of Nuclear Medicine, University Medical Centre Ljubljana, Ljubljana 1000, Slovenia ⊥
ABSTRACT: Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either 68Ga, 64Cu, or 111In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79−1.51 nM). In the biodistribution studies, 68Ga- and 111In-labeled peptides showed higher tumor-to-background ratios than the 64Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for 111In- and 68Ga-labeled peptides. In contrast, the biodistribution of the 64Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of 64Cu from the chelator and subsequent transchelation to proteins. Based on the present study, 68Ga-DOTA−PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer. KEYWORDS: CCK2R peptide, Ga-68, Cu-64, In-111, macrocyclic chelator, DOTA, NOTA, NODAGA, PET imaging
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INTRODUCTION In several human tumor types, such as medullary thyroid carcinomas (MTC) and small cell lung cancers (SCLC), cholecystokinin-2 (CCK-2) receptors are overexpressed.1 Therefore, CCK-2 receptors serve as targets for peptide receptor radionuclide imaging of these tumors by positron emission tomography (PET) or single photon emission computed tomography (SPECT). Clinical PET imaging provides higher resolution images compared to SPECT and superior quantitation. 68Ga is a cost-effective and convenient positron emitting alternative for SPECT radionuclides such as commonly used 111In (γ-emitter, 2.8 d half-life), since it can be eluted from a 68Ge/68Ga generator twice daily. However, the short68 minhalf-life of 68Ga makes rapid PET imaging necessary. For PET imaging at later time points, PET radionuclides such as 64Cu can be used. Its half-life of 12.7 h provides the possibility of imaging at time points up to 1 d © 2014 American Chemical Society
postinjection. To facilitate labeling of peptides with these radiometals, various chelators can be used. The macrocyclic chelators 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), and 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7triazacyclononane (NODAGA) are known to form complexes with various radiometals. Although these chelators differ in coordination chemistry and in net charge, they can all be labeled with radiometals such as 68Ga or 64Cu (both for PET imaging) or 111In for SPECT imaging. These differences in Special Issue: Positron Emission Tomography: State of the Art Received: Revised: Accepted: Published: 3930
April 17, 2014 June 27, 2014 July 3, 2014 July 3, 2014 dx.doi.org/10.1021/mp500283k | Mol. Pharmaceutics 2014, 11, 3930−3937
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gentisic acid, pH 5). The reactions were stopped by the addition of 0.1 M EDTA (final concentration, 5 mM). The radiolabeled peptides were analyzed by reversed-phase HPLC (RP-HPLC) using a Beckman system gold 128 solvent module and a 166 UV detector module (monitoring at 220 nm) combined with an in-line radiodetector (Raytest, Straubenhardt, Germany). Compounds were separated on a 5 μm Jupiter 300 column, 250 × 4.6 mm i.d. (Phenomenex), or an Agilent Eclipse XDB-C18 column (5 μm pore size, 4.6 × 150 mm). The elution gradient used was as follows: solvent A, 0.1% trifluoroacetic acid (TFA) in water; solvent B, 0.1% TFA in ACN; gradient, 0% B for 2 min, changing to 60% B over 20 min, then to 100% B over 5 min (flow rate of 1 mL/min). ITLC was performed on ITLC-SG strips (ITLC-SG, Agilent Technologies, Palo Alto, CA, USA) with mobile phase A (0.1 M NH4OAc/0.1 M EDTA (1/1 v/v) (Rf labeled peptide = 0, Rf 111 In-EDTA = 1)) as well as mobile phase B (MeOH/3.5% NH3 (1/1 v/v) (Rf colloid = 0, Rf 111In-EDTA + labeled peptide = 1)) as eluents. The radiochemical purities of the peptides used in the studies described here were always above 95%. Radiolabeling of the peptides with 68Ga was performed in 2.5 M 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) buffer (Sigma Chemicals, St. Louis, MO) containing 1.6 mg/mL gentisic acid. 68GaCl3 (1.5 mL) was added to 120 μL of HEPES buffer containing 1 μg (0.47−0.67 nmol) of DOTA/NOTA/NODAGA−peptide. After incubation at 95 °C for 15 min, 10 mM EDTA was added to a final concentration of 1 mM. The reaction mixture was purified on an HLB (Waters, Inc., Milford, MA) and eluted with ethanol. Labeling efficiency and radiochemical purity were checked by HPLC (1200 series LC system, Agilent Technologies, Palo Alto, CA, USA, Alltima RP-C18 column 5 μm, 4.6 × 250 mm (Alltech, Deerfield, IL)) with a similar gradient as described above. The HPLC was equipped with an in-line radiodetector (Raytest, Straubenhardt, Germany). In addition, the formation of 68Ga-colloid was checked by ITLC as described above. Radiolabeling of the peptides with 64Cu was performed in 0.1 M acetate buffer containing 4.15 mg/mL gentisic acid to prevent oxidation. 64Cu acetate was added to 0.4−0.5 nmol of DOTA/NOTA/NODAGA−peptide in the gentisic acid/ acetate solution. After incubation at 98 °C for 10 min, 10 mM EDTA was added to give a final concentration of 5 mM. The radiolabeled peptides were analyzed by HPLC as described for the 111In radiolabeled peptides using the 5 μm Jupiter 300 column. ITLC was performed on ITLC-SA strips (Agilent Technologies) with mobile phase of 0.1 M citrate buffer, pH 5. For all in vivo experiments, DOTA-, NOTA-, and NODAGA−PP-F11 were radiolabeled with 111In, 68Ga, and 64 Cu at a SA of 30 MBq/nmol. Plasma Stability of the 64Cu-Labeled Peptides. To check stability, the 64Cu labeled peptides were incubated at 37 °C either in human plasma or in PBS. Aliquots were removed at 1, 4, and 24 h, after which the plasma proteins were precipitated with acetonitrile (1:1). After centrifugation, the supernatants were evaporated to remove acetonitrile and were analyzed via RP-HPLC as above. Cell Culture. In these studies, A431 cells transfected with CCK2R and A431 cells transfected with a mock vector were used. Cells were constructed as described previously12 and were a gift from Dr. Luigi Aloj (Istituto Nazionale Tumori, Fondazione G. Pascale, Naples, Italy). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L
coordination chemistry and charge may affect the in vitro and in vivo properties when coupled to the peptide. A number of CCK and minigastrin analogues have been synthesized and investigated for their potential to deliver peptide receptor targeted imaging with radionuclides to these tumor types.2−4 Radioligands based on MG0 (D-Glu1minigastrin: chelator-DGlu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-GlyTrp-Met-Asp-Phe-NH2) display very high kidney uptake, probably due to the pentaglutamate sequence. This high kidney uptake limited the application in patients.1,5 Reducing the number of glutamic acids resulted in improved tumor-to-kidney ratios, but also lowered the metabolic stability of the tracer.6 Béhé and colleagues, as well as others, showed that kidney uptake of radiolabeled DOTA-MG0 could be reduced by coadministration of polyglutamic acids.7,8 In a comparative biodistribution study of 12 CCK- or gastrin-based peptides, it was found that replacement of the pentaglutamate sequence in MG0 by five D-glutamic acids lowered the kidney uptake by 95% as compared to the uptake of MG0, without greatly affecting the tumor uptake.2 This minigastrin analogue, known as PP-F11 or CP04 (sequence: chelator-DGlu-DGlu-DGlu-DGluDGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), was shown to be a promising peptide for imaging of CCK2R positive tumors.9 In a quest to find the optimal radionuclide−chelator−peptide combination for the imaging of CCK2 receptor-targeting peptides, we compared PP-F11 conjugated with three chelators (DOTA, NOTA, NODAGA) and radiolabeled with three radioisotopes (111In, 68Ga, and 64Cu). The radiolabeled peptides were evaluated for their IC50 values and biodistribution in mice with CCK2R-expressing tumors. Targeting of CCK2R positive tumors was visualized by PET/CT imaging (68Ga- and 64Culabeled peptides) and SPECT/CT imaging (111In-labeled peptides).
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EXPERIMENTAL SECTION Peptides and Radionuclides. The amino acid sequence of PP-F11 is DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-TrpMet-Asp-Phe-NH2, with a molecular weight of 2048.8 g/mol. The DOTA-, NOTA-, and NODAGA-conjugated peptides were synthesized by piChem (Graz, Austria).111InCl3 was obtained from Mallinckrodt Pharmaceuticals (Petten, The Netherlands). 68GaCl3 was eluted from a TiO2-based 1,850 MBq 68Ge/68Ga generator (IGG-100, Eckert & Ziegler, Berlin, Germany) with 6 mL of 0.1 N Ultrapure HCl (J.T. Baker, Deventer, The Netherlands). 68GaCl3 was collected in four fractions of 1.5 mL; the fraction containing the majority of 68Ga activity was used for radiolabeling. 64Cu was prepared by 64 Ni(p,n)64Cu nuclear reaction on a CTI RDS 112 11 MeV cyclotron.10 64Cu was extracted from the gold target by dissolving in concentrated hydrochloric acid and purified by passing down an anion exchange column (BioRad AG1-X8 resin). The 64CuCl2 solution was evaporated to dryness and redissolved in acetate buffer solution pH 4.6 for complexometry (Sigma-Aldrich, U.K.). Radiolabeling. To a low protein binding 1.5 mL polypropylene tube was added 80 μL (65 MBq) of 111InCl3, 6−8 μL (1/5 the volume) of 1 M ammonium acetate containing 8.3 mg/mL gentisic acid, pH 5.5, and 0.6 nmol of peptide (to obtain a specific activity (SA) of 30 MBq/nmol). Alternatively, where this SA was not achievable with using ammonium acetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer was used instead11 (100 μL (65 MBq) of InCl3 with 100 μL 0.2 M MES buffer, containing 1.6 mg/mL 3931
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Figure 1. Chemical structures of DOTA, NOTA, and NODAGA. R = DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2.
Table 1. Summary of IC50 Values of the Labeled DOTA-, NOTA-, and NODAGA-Chelated Peptidea unlabeled DOTA−PP-F11 NOTA−PP-F11 NODAGA−PP-F11 a
1.40 ± 0.05 0.91 ± 0.07 0.88 ± 0.10
nat
In
0.90 ± 0.02 1.18 ± 0.04 1.51 ± 0.11
nat
Ga
0.79 ± 0.03 1.42 ± 0.05 1.19 ± 0.13
nat
Cu
1.19 ± 0.02 1.32 ± 0.15 1.40 ± 0.12
Values in nM are displayed as mean ± SD (n = 3).
welfare committee and carried out according to national regulations. PET/CT Imaging. Mice were scanned under anesthesia (0.5 L/min 1.5% isoflurane in air) on an animal PET/CT scanner (Inveon; Siemens Preclinical Solutions, Knoxville, TN, USA). Mice were placed in prone position on a heating pad to maintain a body temperature of 37 °C. First, a CT scan (spatial resolution 113 μm, 80 kV, 500 μA) was acquired for anatomic reference, followed by a PET emission scan of 20 min (1 h p.i. and 4 h p.i.). Scans were reconstructed using Inveon Acquisition Workplace software (version 1.5; Siemens Preclinical Solutions), using an ordered-set expectation maximization 3-dimensional maximum a posteriori algorithm with the following parameters: matrix, 256 × 256 × 159; pixel size, 0.43 × 0.43 × 0.8 mm3; and β-value of 1.5 with uniform variance. Scans were processed with Inveon Research Workplace software (IRW, version 4.0). Quantification of VOIs (MBq) was carried out using VivoQuant v 1.23 (inviCRO LLC, Boston), and % ID/g was obtained by dividing by the tumor mass obtained from ex vivo biodistribution. SPECT/CT. Whole body SPECT images were obtained at 1 and 4 h p.i. (45 min each) using a NanoSPECT/CT four-head camera (Bioscan Inc., Washington, DC, USA) fitted with 2 mm pinhole collimators in helical scanning mode (20 projections, 45 min scan) and CT images with a 45 kVP X-ray source. Mice were scanned under anesthesia (0.5 L/min 1.5% isoflurane in air) and were placed in prone position on a heating pad to maintain a body temperature of 37 °C. After scanning at 4 h p.i., the animals were sacrificed by cervical dislocation and the tissues and organs dissected out and counted in a gamma counter. Images were reconstructed in a 256 × 256 matrix using proprietary Bioscan software and fused using PMOD software (Mediso). Quantification was carried out as described above. Statistical Analysis. Statistical analyses were performed using GraphPad Prism version 5.03 (San Diego, CA). Differences in uptake were tested for significance using GraphPad Instat 3.10. A p-value below 0.05 was considered significant.
D-glucose
(Gibco, Invitrogen, Breda, The Netherlands) supplemented with 10% fetal calf serum and 250 μg/mL G418 (Geneticin) in a humidified 5% CO2 atmosphere at 37 °C. The cells were harvested by trypsinization with trypsin/ EDTA. IC50 Determination. The apparent 50% inhibitory concentration (IC50) of the peptides for binding the CCK2R was determined on A431-CCK2R cells. Cells were grown to confluency in 6-well plates. Cells were washed with binding buffer (DMEM supplemented with 0.5% w/v bovine serum albumin (BSA)) and incubated at room temperature for 10 min in binding buffer. Subsequently, peptide labeled with natIn, nat Ga, or natCu was added at final concentrations ranging from 0.1 to 1,000 nM. Cold labeled peptides were synthesized according to literature procedures.13,14 Then a trace amount of 111 In-DOTA−PP-F11 (50,000 dpm/well; 0.9 pM, Kd 16 nM15) was added. After incubation at room temperature for 2 h (previously determined as optimal time point15), binding buffer was removed, cells were washed twice with binding buffer, and cell-associated radioactivity was determined using a well-type gamma counter (2470 Wizard2, PerkinElmer, Waltham, MA). The apparent IC50 was defined as the peptide concentration at which 50% of binding without competitor was reached. IC50 values were calculated using GraphPad Prism software (Version 5.03, GraphPad Software, San Diego, CA, USA). Biodistribution Studies of Radiolabeled Peptides. Tumor targeting of radiolabeled PP-F11 was determined in tumor-bearing female SCID/Bg mice (Janvier or Harlan UK). Mice weighed 22−25 g and were housed in IVC cages with 5 mice/cage in a temperature- and humidity-controlled room with 12/12 h light/dark cycle. Animals had unlimited access to water and food. Subcutaneous tumors were induced by inoculation of 2 × 106 A431-CCK2R cells in a volume of 200 μL in the flank. When tumors had reached a weight of approximately 0.2 g, mice were divided into groups (n = 5/ group) and approximately 5−9 MBq of radiolabeled peptide (0.3 nmol, ∼0.6 μg) was injected intravenously in the tail vein. Groups of mice were euthanized by CO2/O2 asphyxiation or cervical dislocation at 1 h (68Ga, 111In, and 64Cu) and 4 h postinjection (p.i.) (111In and 64Cu), a blood sample was drawn, and organs of interest were dissected, weighed, and counted in a gamma counter along with a standard of the injected activity to allow calculation of the injected dose per gram of tissue (% ID/g). Animal experiments were approved by the local animal
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RESULTS The peptide PPF11 was conjugated with either NOTA, DOTA, or NODAGA (Figure 1). Radiolabeling the conjugated peptide with either 68Ga, 111In, or 64Cu resulted in nine radiolabeled tracers, differing in chelator and/or radiolabel. This method allowed us to investigate the influence of the chelator and that 3932
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of the radiolabel on the behavior of the peptide in vitro and in vivo. IC50 values of the tested compounds are summarized in Table 1. IC50 values were in the low nanomolar range for all compounds (0.79−1.51 nM). Although slight differences (not statistically different, P > 0.05) were observed, the chelator as well as the metal did not seem to strongly affect the IC50 of the peptide, nor did the radiolabel. For all in vivo experiments, DOTA-, NOTA-, and NODAGA−PP-F11 were radiolabeled with 111In, 68Ga, and 64 Cu at a SA of 30 MBq/nmol (prepurification) with greater than 95% radiolabeling efficiency (except for 64Cu-NODAGA− PP-F11 which was 88.8% by HPLC, 25 MBq/nmol). Plasma stability studies carried out on the 64Cu-radiolabeled analogues showed no increase in free 64Cu/64Cu-EDTA for any of the chelators after 48 h (>96% remained intact for all three radiolabeled analogues). Tumor targeting of the 68Ga-labeled peptides was studied by PET/CT imaging 1 h after injection (Figure 2). The tumor was clearly visualized by the 68Ga-labeled peptides 1 h after injection. Uptake of the radiolabel was also seen in the kidneys.
Figure 3. Biodistribution of 68Ga-labeled PP-F11, chelated with either NOTA, DOTA, or NODAGA, in SCID mice bearing CCK2R positive tumors. Dissection 1 h p.i., n = 5 mice/group.
blood and other organs was less than 0.5% ID/g for all 68Galabeled compounds. The biodistribution of the 111In-labeled compounds was visualized by SPECT/CT imaging (Figure 4). Subcutaneous
Figure 4. SPECT/CT images of mice with subcutaneous A431CCK2R tumors 4 h after injection of 12−13 MBq of 111In-labeled (A) DOTA−PP-F11, (B) NOTA−PP-F11, and (C) NODAGA−PP-F11. Radiotracer uptake is clearly visible in the CCK2R tumors (arrow) (scale 1−8% ID/g).
Figure 2. PET/CT images of mice with subcutaneous A431-CCK2R tumors 1 h after injection of 5.5−5.8 MBq of 68Ga-labeled (A) DOTA−PP-F11, (B) NOTA−PP-F11, and (C) NODAGA−PP-F11. Radiotracer uptake is clearly visible in the CCK2R tumors (arrow) (scale 1−8% ID/g).
CCK2R positive tumors were clearly visualized 1 and 4 h after injection. In line with the biodistribution results, pronounced uptake of the radiolabel was found in the kidneys. Some uptake of 111In-labeled NOTA−PP-F11 was observed in the intestines (Figure 4B). When labeled with 111In, tumor uptake seemed not to be affected by the chelator (Figure 5), however, tumor uptake was significantly lower as compared to the 68Ga-labeled compounds. The tumor uptake of the three 111In-labeled peptides was similar (DOTA−PP-F11, 1.89 ± 0.74% ID/g, NOTA−PP-F11, 1.84 ± 0.4% ID/g, and NODAGA−PP-F11, 1.03 ± 0.48% ID/ g). Some uptake of 111In-NOTA−PP-F11 in the duodenum was also observed (1.17 ± 0.61% ID/g), suggesting that this tracer was partly cleared via the hepatobiliary route. Uptake in blood and other organs was very low. Figure 6 shows the PET/CT images of mice injected with 64 Cu-labeled peptides. Besides tumor uptake, each of the three
The biodistribution data (after dissection) of the 68Galabeled peptides in SCID mice bearing A431-CCK2R tumors are summarized in Figure 3. The tumor uptake of the peptides labeled with 68Ga was highest for the DOTA-conjugated peptide (5.21 ± 2.19% ID/ g). The NOTA-conjugated peptide had a slightly lower uptake in the tumor (2.56 ± 2.09% ID/g, P < 0.05), most likely due to the fact that injected mass was unintentionally twice as high as for the DOTA and NODAGA peptide. This lower uptake is in line with a previous study in which we demonstrated that coinjection of 30 nmol of unlabeled peptide indeed resulted in a significantly lower tumor uptake of 111In-DOTA−PP-F11.16 Kidney uptake of 68Ga-labeled peptides was lower than 3.9% ID/g for all chelators. 68Ga-NODAGA−PP-F11 showed a significantly higher uptake in the stomach (1.38 ± 0.73% ID/g) than its DOTA and NOTA counterparts (P < 0.05). Uptake in 3933
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Figure 5. Biodistribution of 111In-labeled PP-F11, chelated with either NOTA, DOTA, or NODAGA, in SCID mice bearing CCK2R positive tumors. Dissection 4 h p.i., n = 5 mice/group.
Figure 7. Biodistribution of 64Cu-labeled PP-F11, chelated with either NOTA, DOTA, or NODAGA, in SCID mice bearing CCK2R positive tumors. Dissection 4 h p.i., n = 5 mice/group.
significantly higher (3.69−9.93% ID/g 4 h p.i.) when compared to the 68Ga- and 111In-labeled compounds (0.14−0.45% ID/g (1 h p.i.) and 0.06−0.08% ID/g (4 h p.i.), respectively) (P < 0.005). Tumor-to-muscle and tumor-to-blood ratios of the radiolabeled DOTA-, NOTA-, and NODAGA-conjugated peptides are provided in Table 2. Table 2. Tumor-to-Muscle and Tumor-to-Blood Ratios of the Radiolabeled DOTA-, NOTA-, and NODAGAConjugated Peptides tumor-to-muscle ratio 68
DOTA−PPF11 NOTA−PPF11 NODAGA− PP-F11
Figure 6. PET/CT images of mice with subcutaneous A431-CCK2R tumors 4 h after injection of 7.4−8.1 MBq of 64Cu-labeled (A) DOTA−PP-F11, (B) NOTA−PP-F11, and (C) NODAGA−PP-F11. Radiotracer uptake is clearly visible in the CCK2R tumors (arrow) (scale 1−12% ID/g).
111
64
tumor-to-blood ratio 68
Ga (1 h)
In (4 h)
Cu (4 h)
Ga (1 h)
111 In (4 h)
64 Cu (4 h)
251
199
19
36
211
6
101
113
34
22
246
8
97
113
26
22
77
5
Quantification of Tumor Volumes of Interest (VOI). To assess tumor washout between 1 and 4 h and to enable comparison of the tumor uptake of the 111In- and 64Cu-labeled peptides with the 68Ga-labeled peptides at the 1 h time point, quantitation of the tumor VOIs was carried out (Figure 8). Tumor uptake values (% ID/g) for the 64Cu labeled peptides at 4 h obtained by quantitation of PET images correlated well with their corresponding values obtained by ex vivo biodistribution (Spearman r value of 0.9048, p = 0.0046). Correlation between the quantitation and biodistribution values for the 111In SPECT data was less good (for the NOTA compound in particular). The decay-corrected data showed no tumor washout between 1 and 4 h for any of the 111In- or the 64 Cu-labeled peptides, thus validating the comparison of 68Ga peptides at 1 h with 64Cu and 111In peptides at 4 h.
64
Cu-labeled peptides also showed a high uptake in the liver and the intestines. For 64Cu-NODAGA−PP-F11 the uptake in the liver was less than that of the other two 64Cu-labeled compounds, indicating a better stability of 64Cu complexed with NODAGA than with DOTA and NOTA in vivo. For the 64Cu-labeled compounds, differences between peptides with various chelators were observed (Figure 7). The DOTA− and NOTA−peptides showed tumor uptakes of 5.84 ± 0.74% ID/g and 7.20 ± 0.44% ID/g, respectively, whereas the tumor uptake of NODAGA-conjugated PP-F11 was significantly lower (2.67 ± 0.98% ID/g, p < 0.001). The slightly lower specific activity of the 64Cu-NODAGA peptide may have had a small effect on the tumor uptake. However, uptake in the liver was also higher for the 64Cu-labeled DOTAand NOTA-conjugated peptides. Overall, uptake of the three 64 Cu-labeled peptides was elevated in all tissues examined as compared to 68Ga and 111In compounds. Blood levels were significantly higher than those for the 68Ga- and 111In-labeled peptides. Liver uptake of the 64Cu-labeled peptides was
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DISCUSSION In previous studies we have shown that DOTA−PP-F11 is a good choice for peptide receptor radionuclide therapy (PRRT)2 as it displays high tumor uptake combined with low kidney retention, a necessary combination to optimize therapeutic efficacy while minimizing toxicity. 3934
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vivo experiments was lower (88.8%) and was injected without purification. This indicates that the 64Cu-NODAGA complex may be more stable in vivo than 64Cu-NOTA and -DOTA complexes. This difference in in vivo behavior was also found in a study with RGD-peptides for PET imaging.21 In addition, the differences in charge between the 64Cu (II) and 111In and 68Ga (III) also might play a role in the altered pharmacokinetic profile. However, at physiological pH, the PP-F11 sequence itself is extremely electronegative (−7) and therefore its biodistribution is unlikely to be greatly influenced by the small changes in overall charge that are seen by variation of the chelator/radioisotope combination. In the case of 64CuDOTA−, -NOTA−, and -NODAGA−PP-F11, for instance, the radiolabeled peptide would be expected to have an overall charge of −8 (64Cu-DOTA and 64Cu-NODAGA) or −7 (64CuNOTA) and this change would be less likely to have a measurable effect on the pharmacokinetic profile than for other peptides reported in the literature, where varying the chelator/ isotope combination can render the peptide neutral or double the charge. For instance, Fani et al. saw improved pharmacokinetic profile of a 64Cu-NODAGA labeled somatostatin analogue (overall neutral charge) compared to that of the 64 Cu-CB-TE2A analogue (+2 overall charge on the peptide).22 Uptake of the 111In- and 68Ga-labeled compounds in nontarget organs, except the kidneys, was negligible. 111Inand 68Ga-labeled PP-F11 therefore seem to be more favorable for the imaging of CCK2R expressing tumors than 64Cu-labeled PP-F11. With all tested radiolabeled compounds visualization of the CCK2 receptor-positive tumor was achieved. Radiotracer uptake was seen in kidneys (111In- and 68Ga-labeled peptides) and liver (64Cu-labeled peptides) as well. Malmberg et al. earlier reported a comparative study with anti-HER2 Affibody molecules conjugated with the chelators DOTA, NOTA, and NODAGA, showing equal efficiency of the DOTA- and NODAGA-chelated Affibody molecule in visualizing HER2 expression of soft tissue metastases of prostate cancer, when radiolabeled with 111In.23 This is in line with our results for 111In-labeled DOTA− and NODAGA−PP-F11. In 2002, Eisenwiener and colleagues reported that NODAGAchelated [Tyr3]octreotide (NODAGATOC) showed a higher tumor uptake than its DOTA-chelated counterpart, and also had more favorable biodistribution properties.24 They concluded that NODAGATOC had superior in vivo characteristics as compared to DOTATOC, likely due to the additional spacer group that separates the chelate from the pharmacophoric part of the peptide. More recently, Dumont et al. reported a study on 64Cu- and 68Ga-labeled RGD peptides in which they found that replacement of DOTA by either CB-TE2A or NODAGA resulted in improved biodistribution profiles.21,25 In addition, Fani et al. described similar findings for a somatostatin analogue.22 In our present study, however, the 111InNODAGA−PP-F11 did not show an improved tumor uptake compared to its DOTA counterpart. We found that the chelator did not change the in vivo behavior of 111In- and 68Ga-labeled PP-F11 but only that of the 64 Cu-labeled peptides with 64Cu-NOTA−PP-F11 showing surprisingly high liver uptake. This is in contrast to some literature reports of 64Cu-NOTA-conjugated peptides showing favorable pharmacokinetic profiles. For example, Prasanphanich et al. have shown that the bombesin analogue 64Cu-NOTA−8Aoc-BBN(7−14)NH2 has minimal accumulation in liver (1.58 ± 0.40% ID/g, 1 h p.i.) and rapid renal-urinary excretion which
Figure 8. Quantitation values of tumor VOIs using VivoQuant v 1.23. Decay-corrected values obtained from the images (in MBq) were used to find % ID/g (where tissue mass was obtained from ex vivo biodistribution).
In this study, the PP-F11 peptide was conjugated with DOTA, NOTA, or NODAGA and each was radiolabeled with 111 In, 68Ga, and 64Cu in order to evaluate which chelator/ radioisotope combination shows the best characteristics for clinical imaging studies. All compounds were evaluated for their IC50, biodistribution, and imaging profile. Binding assays showed that the affinity for the CCK2 receptor is in the low nanomolar range for all tested compounds, indicating that the chelator and the radiolabel do not affect the receptor affinity in vitro. In a previous study we also established the receptor specificity of the PP-F11 peptide in vivo.2 Biodistribution studies in SCID mice bearing CCK2Rexpressing tumors revealed differences in tumor uptake of 111 In-, 68Ga-, and 64Cu-labeled peptides, the latter showing significantly higher tumor uptake (up to 3-fold) than the 111Inand 68Ga-labeled compounds. However, the 64Cu-labeled compounds also showed higher background levels, resulting in lower target-to-background ratios. This high uptake in nontarget tissues is most likely due to in vivo instability of the 64 Cu−chelator complex. In addition, the 64Cu-labeled peptides displayed some differences in uptake between the various chelators: although the 64Cu-labeled DOTA− and NOTA−PPF11 had higher tumor uptake than 64Cu-NODAGA−PP-F11, they also displayed higher blood levels, whichat least partlymay have contributed to the higher tumor uptake. These elevated blood levels may have been caused by in vivo instability, which is confirmed by the higher liver accumulation of the 64Cu-labeled DOTA− and NOTA−peptides. It is known that complexes of Cu(II)-DOTA suffer from in vivo instability due to demetalation followed by transchelation to superoxide dismutase in hepatic tissue as well as to ceruloplasmin and serum proteins.17−20 This can result in high uptake of the radiotracer in blood, liver, and other organs, resulting in low tumor-to-blood ratios. Uptake of 64Cu-NODAGA−PP-F11 in the tumor and most other organs is lower than for the other 64 Cu-labeled peptides, but comparable to the tumor uptake levels of all 111In- and 68Ga-labeled peptides. Liver uptake levels of the NODAGA-chelated compound were much lower. Elevated liver uptake for the DOTA and NOTA analogues cannot be accounted for by the presence of 64Cu-EDTA as they were both >95% purity (i.e.,
