68Ga-Labeled [Leu13ψThz14]Bombesin(7–14) Derivatives: Promising GRPR-Targeting PET Tracers with Low Pancreas Uptake

The gastrin-releasing peptide receptor (GRPR) is a G-protein-coupled receptor that is overexpressed in many solid cancers and is a promising target for cancer imaging and therapy. However, high pancreas uptake is a major concern in the application of reported GRPR-targeting radiopharmaceuticals, particularly for targeted radioligand therapy. To lower pancreas uptake, we explored Ga-complexed TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5, and TacsBOMB6 derived from a potent GRPR antagonist sequence, [Leu13ψThz14]Bombesin(7–14), and compared their potential for cancer imaging with [68Ga]Ga-RM2. The Ki(GRPR) values of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, and Ga-RM2 were 7.08 ± 0.65, 4.29 ± 0.46, 458 ± 38.6, 6.09 ± 0.95, 5.12 ± 0.57, and 1.51 ± 0.24 nM, respectively. [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 clearly show PC-3 tumor xenografts in positron emission tomography (PET) images, while [68Ga]Ga-TacsBOMB5 shows the highest tumor uptake (15.7 ± 2.17 %ID/g) among them. Most importantly, the pancreas uptake values of [68Ga]Ga-TacsBOMB2 (2.81 ± 0.78 %ID/g), [68Ga]Ga-TacsBOMB3 (7.26 ± 1.00 %ID/g), [68Ga]Ga-TacsBOMB5 (1.98 ± 0.10 %ID/g), and [68Ga]Ga-TacsBOMB6 (6.50 ± 0.36 %ID/g) were much lower than the value of [68Ga]Ga-RM2 (41.9 ± 10.1 %ID/g). Among the tested [Leu13ψThz14]Bombesin(7–14) derivatives, [68Ga]Ga-TacsBOMB5 has the highest tumor uptake and tumor-to-background contrast ratios, which is promising for clinical translation to detect GRPR-expressing tumors. Due to the low pancreas uptake of its derivatives, [Leu13ψThz14]Bombesin(7–14) represents a promising pharmacophore for the design of GRPR-targeting radiopharmaceuticals, especially for targeted radioligand therapy application.


Introduction
As a member of the G-protein-coupled receptors, the gastrin-releasing peptide receptor (GRPR) is expressed and regulates many physiological functions in the central nervous system, gastrointestinal tract, pancreas, and adrenal cortex tissues, and others [1]. Moreover, GRPR is also overexpressed in several malignancies, including melanoma, prostate, breast, and lung cancers [2][3][4][5][6][7][8]. GRPR is coupled with phospholipase C, followed by protein kinase C (PKC) activation, which regulates cell cycle, cell proliferation, and is implicated in the development of malignant neoplasms [1]. GRPR is also associated with the growth of human prostate carcinoma and pancreatic cancer by an autocrine loop with gastrin-releasing

PET Imaging and Ex Vivo Biodistribution
The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h post-injection using [ 68 Ga]Ga- TacsBOMB2   Ga]Ga-RM2: 0.84 ± 0.55 %ID/g). Uptake values of brain, muscle, bone, heart, and spleen were <1% ID/g for all evaluated tracers.  Ga]Ga-RM2: 0.84 ± 0.55 %ID/g). Uptake values of brain, muscle, bone, heart, and spleen were <1% ID/g for all evaluated tracers.      Compared with [ 68 Ga]Ga-RM2, [ 68 Ga]Ga-TacsBOMB5 has a significantly higher tumor uptake but a lower uptake in most major organs, especially in the pancreas, leading to higher tumor-to-organ (bone, muscle, blood, kidney, and pancreas) uptake ratios (Figure 6 and Table S4).
Co-injection of nonradioactive Ga-TacsBOMB5 reduces the average uptake of [ 68 Ga]Ga-TacsBOMB5 in the PC-3 tumor xenografts by 83% (15.7 down to 2.60 %ID/g at 1 h post-injection), confirming its specific uptake in tumors. In addition, a significant reduction in the average uptake of [ 68 Ga]Ga-TacsBOMB5 was also found in the pancreas (1.98 down to 0.78 %ID/g at 1 h post-injection), which indicates its specific uptake in the pancreas. On the contrary, the average uptake values of [ 68 Ga]Ga-TacsBOMB5 in other major organs were increased at 1 h post-injection with the co-injection of nonradioactive Ga-TacsBOMB5 ( Figure 7 and Table S4).
Co-injection of nonradioactive Ga-TacsBOMB5 reduces the average uptake of [ 68 Ga]Ga-TacsBOMB5 in the PC-3 tumor xenografts by 83% (15.7 down to 2.60 %ID/g at 1 h postinjection), confirming its specific uptake in tumors. In addition, a significant reduction in the average uptake of [ 68 Ga]Ga-TacsBOMB5 was also found in the pancreas (1.98 down to 0.78 %ID/g at 1 h post-injection), which indicates its specific uptake in the pancreas. On the contrary, the average uptake values of [ 68 Ga]Ga-TacsBOMB5 in other major organs were increased at 1 h post-injection with the co-injection of nonradioactive Ga-TacsBOMB5 ( Figure 7 and Table S4).
The average K i values of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB5, and Ga-TacsBOMB6 are comparable (4.29-7.08 nM). This suggests that replacing D-Phe in Ga-TacsBOMB2 with D-2-Nal to obtain Ga-TacsBOMB3, replacing Gly 11 in Ga-TacsBOMB2 with NMe-Gly to obtain Ga-TacsBOMB5, or the addition of a cysteic acid between the Pip linker and DOTA chelator of Ga-TacsBOMB3 to obtain Ga-TacsBOMB6 does not have a major effect on their GRPR-binding affinity. However, replacing D-Phe in Ga-TacsBOMB2 with D-Tpi to obtain Ga-TacsBOMB4 results in a significant loss of binding affinity (K i = 7.08 ± 0.65 vs. 458 ± 38.6 nM). This is likely due to the rigidity of the secondary amino group of D-Tpi, which prohibits free rotation of the added Ga-DOTA complex and Pip linker; therefore, this affects its binding to GRPR.
In vivo stability studies were conducted to investigate if the higher tumor uptake of [ 68 Ga]Ga-TacsBOMB5 compared to [ 68 Ga]Ga-RM2 was the result of improved stability from the NMe-Gly replacement. As shown in Figures S2 and S3, [ 68 Ga]Ga-TacsBOMB5 was not more stable in vivo than [ 68 Ga]Ga-RM2 against peptidase degradation, as their intact fractions in plasma at 15 min post-injection were 67.1 ± 4.76 and 71.9 ± 10.4%, respectively. In addition, the GRPR-binding affinity of [ 68 Ga]Ga-TacsBOMB5 was not better than [ 68 Ga]Ga-RM2 either, as their K i values were 5.12 ± 0.57 and 1.51 ± 0.24 nM, respectively. One possible explanation is the much lower uptake of [ 68 Ga]Ga-TacsBOMB5 in the pancreas when compared to [ 68 Ga]Ga-RM2 (1.98 ± 0.10 vs. 41.9 ± 10.1 %ID/g), enabling more chances for the circulating [ 68 Ga]Ga-TacsBOMB5 to bind to GRPR in PC-3 tumors.
The blocking study (Figure 7 and Table S4) shows that the average uptake of [ 68 Ga]Ga-TacsBOMB5 in PC-3 tumors is reduced by 83% with the co-injection of nonradioactive standard, confirming its specific uptake in tumors. In addition, the average uptake of [ 68 Ga]Ga-TacsBOMB5 in the pancreas is also reduced by 60%, suggesting there is also some specific uptake of [ 68 Ga]Ga-TacsBOMB5 in the pancreas. This is in agreement with the observation that the pancreas is probably the highest GRPR-expressing normal organ [1][2][3]. However, compared to the clinically validated [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-NeoBOMB1, [ 68 Ga]Ga-TacsBOMB5 has~50% more uptake in PC-3 tumors (10.5 ± 2.03 and 9.83 ± 1.48 %ID/g, respectively vs. 15.7 ± 2.17 %ID/g), but only a small fraction of uptake in the mouse pancreas (41.9 ± 10.1 and 122 ± 28.4 %ID/g, respectively vs. 1.98 ± 0.10 %ID/g). This suggests that the extremely high uptake of [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-NeoBOMB1 might not be entirely mediated by GRPR but possibly by some other off-targets as well. However, it cannot rule out the possibility that, compared to [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-NeoBOMB1, [ 68 Ga]Ga-TacsBOMB5 might be more selective for the human GRPR expressed in PC-3 tumors compared to the mouse GRPR expressed in the mouse pancreas. Further clinical studies of [ 68 Ga]Ga-TacsBOMB5 are needed to validate if the observations from the mouse model can be translated to humans.

Synthesis of GRPR-Targeting Ligands
Detailed information for the synthesis and purification of TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5, and TacsBOMB6, their nonradioactive Ga-complexed standards, and 68 Ga-labeled derivatives is provided in the supplementary file (Scheme S1 and Tables S1-S3). Ga]Ga-RM2 were measured using the shake-flask method as previously published [30]. Briefly, aliquots (2 µL) of the 68 Ga-labeled peptides were added to a vial containing 3 mL of 1-octanol and 3 mL of 0.1 M phosphate buffer (pH 7.4). The mixture was vortexed for 1 min and then centrifuged at 5000 rpm for 10 min. Samples of the 1-octanol (1 mL) and buffer (1 mL) layers were taken and counted in a gamma counter. LogD 7.4 was calculated using the following equation: LogD 7.4 = log 10 [(counts in 1-octanol phase)/(counts in buffer phase)].

Cell Culture
The PC-3 cells obtained from ATCC (via Cedarlane, Burlington, Canada) were cultured in RPMI 1640 medium (Life Technologies Corporations) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . The IMPACT Rodent Pathogen Test (IDEXX BioAnalytics) verified that the cells were pathogen-free. Cells were washed with sterile phosphate-buffered saline (PBS, pH 7.4) and collected after 1 min trypsinization when grown to 80-90% confluence. The cell concentration was counted in triplicate using a hemocytometer and a manual laboratory counter.

In Vitro Competition Binding Assay
PC-3 cells were seeded at 2 × 10 5 cells/well in 24-well poly-D-lysine plates 24-48 h prior to the experiment. The growth medium was replaced by 400 µL of reaction medium (RPMI 1640 containing 2 mg/mL BSA and 20 mM HEPES). Cells were incubated for 30-60 min at 37 • C. Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, or Ga-RM2 in 50 µL of decreasing concentrations (10 µM to 1 pM) and 50 µL of 0.011 nM [ 125 I-Tyr 4 ]Bombesin (Perkin Elmer, Waltham, MA) were added to wells, followed by incubation with moderate agitation for 1 h at 27 • C. Cells were gently washed with ice-cold PBS twice, harvested by trypsinization, and measured for radioactivity on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter. Data were analyzed using nonlinear regression (one binding site model for competition assay) with GraphPad (San Diego, CA, USA) Prism 8 software.

Ex Vivo Biodistribution, PET/CT Imaging and In Vivo Stability Studies
Imaging, biodistribution, and in vivo stability studies were performed using male NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG) mice (from in-house breeding colonies) following previously published procedures [25,26,30,31]. The experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia. The mice were anesthetized by inhalation of 2.5% isoflurane in oxygen and implanted subcuta-neously with 5 × 10 6 PC-3 cells (100 µL; 1:1 PBS/Matrigel) behind the left shoulder. When the tumor grew to 5-8 mm in diameter over 3-4 weeks, the mice were used for PET/CT imaging and biodistribution studies.
The PET/CT imaging experiments were carried out using a Siemens (Knoxville, TN, USA) Inveon micro-PET/CT scanner. Each tumor-bearing mouse (n = 1-2) was injected with~3-6 MBq (0.05-0.1 nmol) of a 68 Ga-labeled tracer through a lateral caudal tail vein under 2.5% isoflurane in oxygen anesthesia, followed by a recovery period in which it could roam freely in its cage during the uptake period. At 50 min post-injection, a 10 min CT scan was conducted: first for localization and attenuation corrections after segmentation for reconstructing the PET images, followed by a 10 min static PET imaging acquisition.
For biodistribution studies, the mice (n = 4) were injected with the radiotracer (~2-4 MBq, 0.03-0.07 nmol) as described above. For blocking, the mice were co-injected with 100 µg of nonradioactive Ga-TacsBOMB5. At 1 h post-injection, the mice were euthanized by CO 2 inhalation. Blood was withdrawn by cardiac puncture, and organs/tissues of interest were collected, weighed and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.
For in vivo stability studies, [ 68 Ga]Ga-TacsBOMB2 (6.10 ± 0.04 MBq), [ 68 Ga]Ga-TacsBOMB5 (9.60 ± 0.56 MBq), and [ 68 Ga]Ga-RM2 (5.56 ± 0.03 MBq) were injected via the lateral caudal vein into healthy male NRG mice (n = 3). At 15 min post-injection, the mice were sedated and euthanized, and urine and blood were collected. The plasma was extracted from whole blood by the addition of CH 3 CN (500 µL), vortexing, centrifugation, and the separation of the supernatants. The plasma and urine samples were analyzed via radio-HPLC by using the conditions for quality control of these 68 Ga-labeled radioligands (Table S3).

Statistical Analysis
Statistical analyses were performed by Student's t-test using the Microsoft (Redmond, WA, USA) Excel software. The unpaired two-tailed test was used to compare biodistribution data of [ 68 Ga]Ga-TacsBOMB5 and [ 68 Ga]Ga-RM2. The unpaired one-tailed test was used to compare the biodistribution data of [ 68 Ga]Ga-TacsBOMB5 with/without co-injection of nonradioactive Ga-TacsBOMB5. A statistically significant difference was considered when the adjusted p value was <0.05.

Patents
The compounds disclosed in this report are covered by a recent patent application (PCT/CA2019/051620). Zhengxing Zhang, Jutta Zeisler, François Bénard and Kuo-Shyan Lin are listed as inventors of this filed patent.