66Ga-PET-imaging of GRPR-expression in prostate cancer: production and characterization of [66Ga]Ga-NOTA-PEG2-RM26

Molecular imaging of the gastrin-releasing peptide receptor (GRPR) could improve patient management in prostate cancer. This study aimed to produce gallium-66 (T½ = 9.5 h) suitable for radiolabeling, and investigate the imaging properties of gallium-66 labeled GRPR-antagonist NOTA-PEG2-RM26 for later-time point PET-imaging of GRPR expression. Gallium-66 was cyclotron-produced using a liquid target, and enriched [66Zn]Zn(NO3)2. In vitro, [66Ga]Ga-NOTA-PEG2-RM26 was characterized in GRPR-expressing PC-3 prostate cancer cells. In vivo, specificity test and biodistribution studies were performed 3 h and 22 h pi in PC-3 xenografted mice. microPET/MR was performed 3 h and 22 h pi. Biodistribution of [66Ga]Ga-NOTA-PEG2-RM26 was compared with [68Ga]Ga-NOTA-PEG2-RM26 3 h pi. [66Ga]Ga-NOTA-PEG2-RM26 was successfully prepared with preserved binding specificity and high affinity towards GRPR. [66Ga]Ga-NOTA-PEG2-RM26 cleared rapidly from blood via kidneys. Tumor uptake was GRPR-specific and exceeded normal organ uptake. Normal tissue clearance was limited, resulting in no improvement of tumor-to-organ ratios with time. Tumors could be clearly visualized using microPET/MR. Gallium-66 was successfully produced and [66Ga]Ga-NOTA-PEG2-RM26 was able to clearly visualize GRPR-expression both shortly after injection and on the next day using PET. However, delayed imaging did not improve contrast for Ga-labeled NOTA-PEG2-RM26.


Results
Ga production and purification. 66 Ga was successfully produced in a liquid target with end-of-bombardment (EOB) yields up to 0.50 GBq, thus corresponding to saturation yields of 0.25 GBq/μA. When combining all fractions, the isolated [ 66 Ga]GaCl 3 activity was ~ 320 to 340 MBq, of which the fractions used for radiolabeling had activity concentrations ranging from 160 to 280 MBq/mL. A coarse spot check was performed on one [ 66 Ga]GaCl 3 production of which Zn content in the two fractions preceding, and two fractions following the fraction used for radiolabeling were all below the lowest positive color scale of 4 μg/mL. Isolated fractions of [ 66 Ga]GaCl 3 were not analyzed for residual Zn due to the small fraction volumes. However, extensive tests have been performed previously during 68 Ga development efforts, whereby, residual Zn was determined to be 0.33 ± 0.23 μg/mL.
The gamma-spectrum of 66 Ga is presented in Figure S1. The only observable gamma-lines were 511, 834 and 1039 keV belonging to 66 Ga. No other gamma-lines were observed. Data concerning half-live measurement of 66 Ga are presented in Figure S2. The measured data were perfectly fitted (R 2 = 1) in a monoexponetial decay with a the half-life of 9.45 ± 0.05 h, which is in an excellent agreement with the half-life of 66 Ga (9.49 h, http://nucle ardat a.nucle ar.lu.se/toi/nucSe arch.asp ; The Lund/LBNL Nuclear Data Search).
Taken together, the data confirm authenticity and high radionuclide purity of 66 Ga.
Binding specificity, cellular processing and analysis of binding kinetics. Blocking of GRPR in both cell lines cells resulted in significantly (p < 0.008) lower cell-associated activity with both concentrations of blocking peptide (Fig. 2a, Figure S3) compared with the non-blocked cells. Uptake in DU145 cells was significantly lower than in PC-3 cells. Binding of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 to cells was rapid and the amount of total cell-associated activity increased constantly with time. Internalized fraction slowly, but steadily increased over 24 h. The fraction of internalized activity was 17% of cell-associated activity at the end of the observation period (Fig. 2b).
Binding kinetics were measured in real-time using LigandTracer and data were fitted using a 1:1 kinetic binding model. A representative Ligand Tracer curve is displayed in Fig. 3. Equilibrium binding constant (K D ) was 189 ± 50 pM with an association rate (k a ) of 1.78 × 10 5 ± 0.05 × 10 5 1/Ms and a dissocation rate (k d ) of 3.3 × 10 -5 ± 0.8 × 10 -5 1/s. Biodistribution and in vivo specificity. Biodistribution of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 was studied 3 h and 22 h pi in Balb/c nu/nu mice bearing PC-3 xenografts. [ 66 Ga]Ga-NOTA-PEG 2 -RM26 cleared rapidly from blood and bound to GRPR-expressing xenografts as well as organs with natural expression of (murine) GRPR (pancreas, stomach, small intestine) (Fig. 4a). In vivo specificity test showed significant decrease in uptake in GRPR expressing xenografts (p < 0.00001) and pancreas (p < 0.002) indicating GRPR specific binding / of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 (Fig. 4b). Tumor uptake 3 h pi was 14 ± 1% ID g but decreased almost two-fold 22 h pi. However, the tumor uptake was higher than the uptake in all normal organs at both time points. Uptake in GRPR-expressing pancreas decreased significantly (p < 0.004) from 3 to 22 h pi, as well as in GI and carcass. Noticeably, the uptake in bone increased during the observation period. No increase in tumor-to-organs ratios was observed form 3 to 22 h pi (Table 1). Tumor-to-organ ratios were generally higher 3 h pi for all organs except pancreas.
Comparison of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 with its [ 68 Ga]Ga-labeled counterpart showed no remarkable differences in uptake in studied organs and tissues ( Table 2).
Distribution of free gallium-66 in NRMI mice 3 h pi ( Figure S4) showed slow excretion of activity with relatively equal activity distribution in healthy organs. Radio-HPLC analyses of blood plasma demonstrated that 5 min pi of [ 68 Ga]Ga-NOTA-PEG 2 -RM26 up to 57% of radiometal is associated with intact peptide ( Figure  S5). Free gallium-66 represented 6% of activity in blood plasma, while the rest of activity was associated with different peptide's fragments. microPET/MR imaging. microPET/MR imaging was performed 3 h and 22 h pi and images are displayed in Fig. 5. GRPR-expressing xenografts could be clearly visualized at both time points. Tumors showed the highest uptake aside from urinary bladder 3 h pi.

Discussion
Molecular imaging in prostate cancer could improve the diagnostic accuracy for both primary and recurrent disease and thus improve patient management. The GRP receptor is overexpressed in mainly earlier stages of prostate cancer, but not in healthy prostate tissue 1,2 , and is thus an attractive target for diagnostic PET-and SPECT-imaging. The GRPR-antagonist PEG 2 -RM26 (PEG 2 -DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 ) is a promising ligand for imaging of GRPR-expression 18 . First steps towards clinical translation using 68 Ga-labeled RM26 have been reported recently assessing the safety, biodistribution, and dosimetry in humans 26 . Many 68 Galabeled tracers are successfully used in clinical routine for imaging shortly after injection. Pre-clinical studies by our group suggested that imaging contrast, and thereby sensitivity, could be improved by next-day imaging 21,22 . However, limited information is available about the long-term fate of gallium-labeled probes and their potential for later time-point imaging, due to the limited half-life of 68 Ga. Gallium-66 is another positron-emitting galliumisotope, which, because of its longer half-life of 9.5 h, could be an interesting addition to the PET-toolbox for later-time point imaging. The aims of the present study were to produce 66 Ga by cyclotron irradiation of enriched 66 Zn-in a liquid target and to investigate the PET-imaging properties of [ 66 Ga]Ga-NOTA-PEG2-RM26 for later time point imaging of GRPR-expression.
Although the 68 Ga used in this study was acquired from a 68 Ge/ 68 Ga generator, this study demonstrated that the alternative technology developed for the direct cyclotron-based production of 68 Ga with a liquid target could be readily adopted to production of 66 Ga by irradiation of a 66 Zn salt solution, with the [ 66 Ga]GaCl 3 of suitable quality for radiolabeling. Although yields afforded by a liquid target are significantly less than those which can be obtained using a solid target, the achieved yields of 66 Ga were nevertheless adequate to enable in vitro and in vivo pre-clinical studies without the need for sophisticated solid-target infrastructure.
The GRPR-antagonist NOTA-PEG 2 -RM26 was labeled with 66 Ga with yield similar to [ 68 Ga]Ga-PEG 2 -RM26 24,37 . Also, the in vitro and in vivo stability tests did indicate good stability of the 66 Ga-NOTA complex. Because isotopes have identical chemical properties, this was expected. In contrast to the cyclotron-produced 66 Ga, 68 Ga is typically eluted from a 68 Ge/ 68 Ga-generator (as was the case for this study) and the different production routes may potentially result in different impurity profiles of the radioisotope solutions, which in return www.nature.com/scientificreports/ www.nature.com/scientificreports/ could affect the radiolabeling efficiency. However, such differences were not observed in this study-this was also confirmed by HPLC analysis of both radiolabeled products. As expected, the GRPR binding specificity of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 was retained after labeling and the difference in uptake for PC-3 and DU145 cells correlated well to the different levels of receptor expression 38 . For the first time, binding affinity of gallium-labeled NOTA-PEG 2 -RM26 towards GRPR was measured directly and in real-time showing a K D value in the low picomolar range similar to the earlier reported K D for [ 111 In] In-NOTA-PEG 2 -RM26 21 . Due to the longer half-life of 66 Ga, we were also able to follow the cellular processing of gallium-labeled NOTA-PEG 2 -RM26 past 4 h. The cellular processing pattern of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 at early time points matched published data for [ 68 Ga]Ga-NOTA-PEG 2 -RM26 37 . Beyond the 4 h the internalized fraction of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 remained low, with only 17% of the total cell associated activity internalized after 24 h. Slow internalization was expected due to the antagonistic properties of RM26. However, the low level of internalized activity resembled the behavior of radiocobalt-labeled NOTA-PEG 2 -RM26 22,39 , and was considerably lower than the level of internalization of its [ 111 In]In-labeled analog 21 . Similar to [ 55/57 Co]Co-PEG 2 -RM26, the radiocobalt label also led to lower amounts of internalized activity with affibody molecules, and it is hypothesized that the radiocobalt label leaks from or is transported out of the cell by specific cobalt-efflux mechanisms 22,[39][40][41] . It could be speculated that similar mechanisms are in place for gallium.
The general biodistribution pattern of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 was comparable with previous studies of RM26 labeled with different radiometals 21,22,24 . [ 66 Ga]Ga-NOTA-PEG 2 -RM26 cleared rapidly from blood mainly via the renal pathway, and specific uptake of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 was observed in GRPR-positive   Data is presented as %IA/g as average ± standard deviation (n = 3-4 animals/group). Data for GI and body is expressed as %IA. * indicates statistical significant difference (p < 0.05). Ga-NOTA-PEG 2 -RM26 cleared from the GRPR positive pancreas more rapidly than from the tumor, which could be attributed to the differences between human and murine GRPR as well as to the different receptor densities in tumors compared to pancreas. Analyses of blood plasma demonstrated that gallium labeled NOTA-PEG 2 -RM26 has metabolic stability similar to other GRPR-targeting peptides; 5 min pi up to 60% of radiometal is associated with peptide. For comparison, when injected without neprilysin -inhibitor phosphoramidon only 25-30% of activity was associated with truncated human endogenous GRP motifs 45  The microPET/MR images showed excellent visualization of the GRPR-expressing xenografts. For imaging of prostate cancer high contrast in the abdominal region is essential. Despite the somewhat unexpected biodistribution of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 without increase in tumor-to-organ ratios over time, the imaging contrast remained excellent even at 22 h pi due to clearance from GI-tract and whole body with time. Gallium-66 has a rather high positron branching ratio (i.e. 57%) compared with other positron emitters for PET imaging with intermediate ( 64 Cu, 18% β + ; 86 Y, 32% β + ) or longer ( 89 Zr, 23% β + ) half-lives. However, the maximum energy of positron emitted from 66 Ga (4.15 meV) is higher than for many other positron-emitters, and could affect image quality. Nevertheless, the results from our study and others investigating 66 Ga -labeled peptides and monoclonal antibodies for PET-imaging of other molecular targets, such as somatostatin receptor 36   www.nature.com/scientificreports/

Conclusion
In conclusion, we successfully produced 66 Ga by cyclotron irradiation of a liquid [ 66 Zn]Zn(NO 3 ) 2 target, followed by subsequent purification, and radiolabeled the bombesin antagonist NOTA-PEG 2 -RM26 with this radionuclide. We further demonstrated the feasibility of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 for PET-imaging of GRPR expression. In contrast to our initial hypothesis, early and late time point imaging provided similar image quality in our case. Nevertheless, prolonged half-life, and the widely explored gallium-chemistry could make 66 Ga an attractive radionuclide for PET-imaging with other targeting agents that have slightly extended biological half-lives such as engineered scaffold proteins.

Material and methods
General.  The irradiated solution was subsequently diluted in the collection vial with water to a total volume of ~ 8 to 9 mL, and automatically processed on a FASTlab Developer platform as outlined and described in Fig. 6. All resins noted below were obtained from Triskem (pre-packed, Britany, France), with additional reagents obtained as follows: HCl (30%; Ultrapure; Merck), HNO 3 (70%; ≥ 99.999% trace metal basis; Sigma-Aldrich), NaCl (99.999% trace metal basis; Sigma Aldrich).
A coarse spot check was performed on one [ 66 Ga]GaCl 3 production using semi-quantitative colorimetric test strips (Merck, MQuant), of which Zn content in the two fractions preceding, and two fractions following the fraction used for radiolabeling. Isolated fractions of [ 66 Ga]GaCl 3 were not analyzed via ICP-MS (inductively coupled plasma mass spectrometry) for residual Zn due to the small fraction volumes, but extensive tests have been performed previously during 68 Ga development efforts, whereby an identical separation scheme was used (albeit a non-fractionated 5 mL product volume (n = 12)).
To confirm authenticity and radionuclide purity of 66 Ga, the half-life and gamma-spectra of the product were measured. The gamma spectra were measured using ultra-pure germanium detector (Mirion Technologies, San Ramon, CA, US) working in line with the DSA-LX multi-channel analyser (Mirion Technologies, San Ramon, CA, US). Analysis was performed using Genie 2000 software (Mirion Technologies). To determine the half-life, two samples were repeatedly measured using 1480 Wizard gamma-spectrometer during 70 h. Count rate was measured in the range from 10 to 2048 keV. Data fitting was performed using GraphPad software.

Labeling of NOTA-PEG 2 -RM26, and in vitro stability and HPLC analysis of [ 66 Ga]
Ga-NOTA-PEG 2 -RM26. For labeling of NOTA-PEG 2 -RM26 with 66 Ga, 3-10 nmol of peptide was buffered with 1.25 M sodium acetate buffer, pH 3.6, and incubated with 4-8 MBq/nmol for 12 min at 85 °C. Radiochemical yield was determined by instant thin-layered chromatography (ITLC). For this, a sample of the reaction mixture was applied to silica gel-impregnated glass microfiber chromatography paper (Agilent Technologies, Santa Clara, CA, USA), which was eluted with 0.2 M citric acid. Free gallium moves with the citric acid to the front of the chromatography paper, whereas the radiolabeled peptide remains at the application point. The activity distribution was measured using ScanRam radio-TLC Scanner and analyzed with complimentary Software, Laura (v6.0.4.92) (LabLogic, Sheffield S10 2QJ, UK) To test stability of the 66 Ga-label, [ 66 Ga]Ga-NOTA-PEG 2 -RM26 was incubated with 1000-fold molar excess of EDTA and PBS for 1 h at room temperature or in human serum for 1 h at 37 °C. Thereafter, ITLC was used to determine the release of 66 Ga. Analytical high performance liquid chromatography (HPLC) was performed (Hitachi Chromaster, with Luna C18 column (5 µm, 100 Å, 150 × 4.6 mm, Phenomenex, Vaerløse, Denmark) using a gradient of 5% to 70% acetonitrile (with 0.1% TFA) in water over 15 min to study the identity of the radiolabeled products. Binding kinetics of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 were measured in real-time using Ligand Tracer (Ridgeview Instruments AB, Uppsala, Sweden). Cells were plated in a dedicated area of 10 cm Petri dish and the measurement was performed according to previously published protocols 49 . In brief, to measure the association, increasing concentrations (in the range 0.5 nM to 10 nM) of [ 66 Ga]Ga-NOTA-PEG 2 -RM26 were stepwise added to the cell Figure 6. Three column approach for isolating [ 66 Ga]GaCl 3 . Process steps described below. (1) The diluted solution was loaded at a rate of ~ 1.5 mL/min over a 2 mL ZR column, whereby 66 Ga is retained on the resin, and the irradiated 66 Zn was passed through to a collection vial for optional recycling. During the irradiation, coproduced 13 N is passed through in this step as well. (2) The ZR resin was rinsed with 14 mL of 0.1 M HNO 3 to remove residual 66 Zn. (3) The 66 Ga was eluted from the ZR resin with 5 mL 1.75 M HNO 3 , passed over a 1 mL AG 1-X8 column to remove residual trace metals, before being trapped on a TK200 column. (4) The TK200 column was rinsed with 3.5 mL 2 M NaCl in 0.13 M HCl to decrease residual acid on the column. (5) Finally, the 66 Ga, as [ 66 Ga]GaCl 3 was eluted from the TK200 resin in nominal 300 μL fractions, the first 3.5 mL of which was water, followed by 2.5 mL 0.18 M HCl. The fraction(s) with the highest radioactivity concentration(s) were used for radiolabeling. . Tumors were collected as well as samples of blood, lung, liver, spleen, pancreas, stomach, small intestine, kidneys, muscle, and bone. Gastrointestinal (GI) tract and carcass were also collected. Samples were weighed, and measured for activity content in the automated gamma counter. Corrections for background, spill-over and decay were performed for all measurements.

In vitro characterization of [ 66
For the in vivo specificity test, mice in the blocking group were co-injected with 10 nmol of non-labeled NOTA-PEG 2 -RM26 and sacrificed 3 h pi and samples were collected according to the protocol described above.
Distribution of 66 Ga in NMRI mice. 66 Ga-chloride solution was incubated with 1.25 M sodium acetate (pH 3.6) mimicking radiolabeling conditions. The solution was diluted in 1% BSA/PBS and female NRMI mice were injected with 40 kBq 66 Ga-solution. Three hours pi mice were sacrificed and tissue samples were taken according to the protocol described above.
Analysis of blood metabolites. NMRI Mice were injected with 1.5 nmol (6.75-7.3 MBq) [ 68 Ga] Ga-NOTA-PEG 2 -RM26 and sacrificed 5 min pi by cervical dislocation. Immediately after blood was collected by heart puncture into a pre-chilled heparinized Eppendorf tube. Blood samples were centrifuged (10000RCF, 10 min at 4 °C), plasma was collected and mixed with equal amount of ice cold acetonitrile. The mixture was centrifuged (15,000 RCF, 10 min, 4 °C), the supernatant was collected and sterile filtered with a 0.2 µm PTFE syringe filter. The filtrate was analyzed on HPLC according to the method described above. MRI was performed immediately after PET acquisition with a 3 T nanoScan PET/MR scanner (Mediso Medical Imaging Systems Ltd., Budapest, Hungary). MRI parameters were as follows: