Synthesis and Evaluation of ⁶⁸Ga- and ¹⁷⁷Lu-Labeled [diF-Pro¹⁴]Bombesin(6−14) Analogs for Detection and Radioligand Therapy of Gastrin-Releasing Peptide Receptor-Expressing Cancer

Abstract

Background/Objectives: Overexpressed in various solid tumors, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake of the current clinically evaluated GRPR-targeted radiopharmaceuticals limits their applications. In this study, we replaced the Pro¹⁴ residue in our previously reported GRPR-targeted LW02056 and ProBOMB5 with 4,4-difluoroproline (diF-Pro) to obtain an agonist LW02060 (DOTA-Pip-[D-Phe⁶,Tle¹⁰,NMe-His¹²,diF-Pro¹⁴]Bombesin(6–14)) and an antagonist LW02080 (DOTA-Pip-[D-Phe⁶,NMe-Gly¹¹,Leu¹³(ψ)diF-Pro¹⁴]Bombesin(6–14)), respectively. Methods/Results: The binding affinities (K_i) of Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 were measured by in vitro competition binding assays using PC-3 cells and were found to be 5.57 ± 2.47, 21.7 ± 6.69, 8.00 ± 2.61, and 32.1 ± 8.14 nM, respectively. The ⁶⁸Ga- and ¹⁷⁷Lu-labeled ligands were obtained in 36–75% decay-corrected radiochemical yields with >95% radiochemical purity. PET imaging, SPECT imaging, and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice. Both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 enabled clear tumor visualization in PET images at 1 h post-injection (pi). Tumor uptake values of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 at 1 h pi were 16.8 ± 2.70 and 7.36 ± 1.33 %ID/g, respectively, while their pancreas uptake values were 3.12 ± 0.89 and 0.38 ± 0.04 %ID/g, respectively. Compared to [¹⁷⁷Lu]Lu-LW02080, [¹⁷⁷Lu]Lu-LW02060 showed higher tumor uptake at all time points (1, 4, 24, 72, and 120 h pi). However, fast tumor clearance was observed for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080. Conclusions: Our data demonstrate that [⁶⁸Ga]Ga-LW02060 is promising for clinical translation for the detection of GRPR-expressing tumor lesions. However, further optimizations are needed for [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 to prolong tumor retention for therapeutic applications.

1. Introduction

Gastrin-releasing peptide receptor (GRPR) is a member of G-protein coupled receptors and is expressed in the human body, including in the pancreas, gastrointestinal tract, and central nervous system [1,2]. It is involved in regulating many physiological functions, such as hormone secretion, smooth muscle contraction, and synaptic plasticity [1,2]. Furthermore, GRPR is found to be significantly overexpressed in various solid malignancies, including breast, prostate, lung, and colon cancers, where it functions as a tumor growth factor [3,4,5,6,7,8]. Such characteristics of GRPR makes it an attractive target for developing targeted radiopharmaceuticals for diagnosis and radioligand therapy of GRPR-expressing cancer.

Bombesin (BBN) is a natural peptide showing a very potent binding affinity to GRPR. Its C-terminal heptapeptide, bombesin(8–14), has been identified as the sequence needed for binding to GRPR and is widely utilized for the design of GRPR-targeted pharmaceuticals [9,10,11,12,13,14,15,16]. However, most clinically evaluated GRPR-targeted radiopharmaceuticals derived from BBN showed extremely high pancreas uptake, which not only limits the detection of GRPR-expressing tumor lesions in and near the pancreas but also lowers the maximum tolerated dose for radiotherapeutic applications [9,10,11,12,13,14,17]. Moreover, extracellularly enzymatic degradation by neutral endopeptidase 24.11 (NEP, EC 3.4.24.11, neprilysin) results in low in vivo stability for most of the reported GRPR-targeted radiopharmaceuticals derived from the BBN sequence [18,19].

To address these two major limitations, our group previously developed a series of GRPR-targeted radiopharmaceuticals [20,21]. These ligands were derived from RC-3950-II ([D-Phe⁶,Leu¹³(ψ)Thz¹⁴]Bombesin(6–14)), which has a Thz¹⁴ (thiazoline-4-carboxylic acid) substitution and a reduced peptide bond (CH₂-N) between residues 13–14 (Leu¹³(ψ)Thz¹⁴) [15,16]. All the ⁶⁸Ga-labeled GRPR antagonists derived from the [Leu¹³(ψ)Thz¹⁴]Bombesin(6–14) pharmacophore and all the ⁶⁸Ga-labeled GRPR agonists derived from the [Thz¹⁴]Bombesin(6–14) pharmacophore showed low pancreas uptake (0.78–7.26 %ID/g at 1 h pi) [20,21]. Subsequently, our group improved the in vivo stability of the lead candidates by systematically substituting natural amino acids at potential cleavage sites (Gln⁷, Trp⁸, Ala⁹, Val¹⁰, Gly¹¹, and His¹²) with unnatural amino acids. This led to the discoveries of several GRPR-targeted radiopharmaceuticals with significantly improved in vivo stability [22,23]. We discovered that Tle¹⁰ and NMe-His¹² substitutions, either in combination or alone, can significantly enhance the in vivo stability and improve the tumor uptake of ⁶⁸Ga-labeled GRPR agonists derived from the [Thz¹⁴]Bombesin(6–14) pharmacophore [22]. We also identified that NMe-Gly¹¹ substitution can improve the tumor uptake and tumor-to-organ uptake ratios of ⁶⁸Ga-labeled GRPR antagonists derived from the [Leu¹³(ψ)Thz¹⁴]Bombesin(6–14) pharmacophore [20,23]. Recently, we reported two novel GRPR-targeted ligands, LW02056 and ProBOMB5 (Figure 1), by replacing the Thz¹⁴ in our previously identified LW01110 and TacsBOMB5, respectively, with Pro¹⁴ to avoid oxidation of Thz in the product formulation [24]. Though a slight reduction was observed for the tumor uptake of [⁶⁸Ga]Ga-LW02056 (8.93 ± 1.96 %ID/g at 1 h pi), it had very low pancreas uptake (1.31 ± 0.42 %ID/g at 1 h pi) and good tumor-to-background imaging contrast [24]. The Pro¹⁴-derived antagonist, [⁶⁸Ga]Ga-ProBOMB5, showed good tumor uptake (12.4 ± 1.35 %ID/g), low pancreas uptake (1.37 ± 0.40 %ID/g), and excellent tumor-to-background imaging contrast at 1 h pi [24].

Fluorinated proline derivatives play a crucial role in peptide engineering and drug discovery due to their resistance to degradation by NEP and their ability to modulate polarity and lipophilicity [25,26]. A 4,4-difluoroproline (diF-Pro) derivative, 4,4-difluoropyrrolidine-2-carbonitrile, has been successfully introduced in the design of potent fibroblast activation protein (FAP) inhibitors to mimic Pro [27], and now 4,4-difluoropyrrolidine-2-carbonitrile is widely used for the design of FAP-targeted radiopharmaceuticals [28]. In this study, we synthesized two GRPR-targeted ligands, LW02060 and LW02080, by replacing Pro¹⁴ of LW02056 and ProBOMB5, respectively, with diF-Pro¹⁴ (Figure 1). Subsequently, we characterized the antagonist/agonist characteristics of these two ligands and their Ga/Lu-complexed analogs. We also assessed their potential for PET imaging and targeted radiotherapy using a preclinical tumor model derived from GRPR-expressing PC-3 prostate cancer cells. We hypothesized that (1) diF-Pro¹⁴ substitution would retain GRPR agonist/antagonist characteristics and good binding affinity to GRPR; (2) compared with their Pro¹⁴ analogs, the diF-Pro¹⁴-derived radioligands would have comparable or even higher in vivo stability and could be promising for the detection and radioligand therapy of GRPR-expressing malignancies.

2. Results

2.1. Chemistry and Radiochemistry

LW02060 and LW02080 were synthesized on solid phase using Fmoc chemistry, and their isolated yields were 23% and 39%, respectively. The isolated yields for the synthesis of Ga-LW02060 and Ga-LW02080 were 87% and 93%, respectively, and the isolated yields for Lu-LW02060 and Lu-LW02080 were 87% and 90%, respectively (Tables S1 and S2). ⁶⁸Ga-labeled LW02060 and LW02080 were obtained in 41–75% decay-corrected radiochemical yields with >172 GBq/µmol molar activity and >95% radiochemical purity (Table S3). ¹⁷⁷Lu-labeled LW02060 and LW02080 were obtained in 36–64% decay-corrected radiochemical yields with >125 GBq/µmol molar activity and >97% radiochemical purity (Table S3).

2.2. Agonist/Antagonist Characterization, Binding Affinity, and Hydrophilicity

All LW02060 and its Ga/Lu-complexed analogs were confirmed to be GRPR agonists, while all LW02080 and its Ga/Lu-complexed analogs were confirmed to be GRPR antagonists by intracellular calcium release assays using PC-3 cells (Figure 2). A total of 50 nM of LW02060, Ga-LW02060, Lu-LW02060, ATP (positive control), and bombesin (agonist control) induced Ca²⁺ efflux corresponding to 377 ± 41.9, 405 ± 58.3, 397 ± 43.4, 286 ± 9.44, and 371 ± 17.9 RFU (relative fluorescent unit), respectively. By contrast, 50 nM of LW02080, Ga-LW02080, Lu-LW02080, [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) (antagonist control), and Dulbecco’s phosphate-buffered saline (DPBS, blank control) induced Ca²⁺ efflux corresponding to only 11.4 ± 0.32, 12.4 ± 1.76, 10.8 ± 1.22, 16.6 ± 3.84, and 41.2 ± 5.40 RFU, respectively.

The binding affinities of Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 were determined by in vitro competition binding assays using GRPR-expressing PC-3 cells and [¹²⁵I-Tyr⁴ ]Bombesin as the radioligand. The binding of [¹²⁵I-Tyr⁴]Bombesin to PC-3 cells was inhibited by all tested ligands in a dose-dependent manner (Figure 3). The calculated K_i values for Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 were 5.57 ± 2.47, 21.7 ± 6.69, 8.00 ± 2.61, and 32.1 ± 8.14 nM, respectively (n = 3).

The hydrophilicity of [⁶⁸Ga]Ga-LW02060, [⁶⁸Ga]Ga-LW02080, [¹⁷⁷Lu]Lu-LW02060, and [¹⁷⁷Lu]Lu-LW02080 were determined via the shake flask method, and the calculated logD_7.4 values were −2.57 ± 0.04, −2.60 ± 0.03, −2.45 ± 0.03, and −2.33 ± 0.26, respectively (n = 3).

2.3. PET Imaging and Ex Vivo Biodistribution

Both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 were excreted mainly via the renal pathway and enabled clear visualization of the PC-3 tumor xenografts in PET images at 1 h pi as shown in Figure 4. Except tumor xenografts, kidneys, and the urinary bladder, the uptake of both tracers in other organs/tissues were minimal. [⁶⁸Ga]Ga-LW02060 showed a better tumor uptake, and both tracers had excellent tumor-to-background imaging contrast. Co-injection with 100 μg of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) or nonradioactive Ga-LW02080 significantly decreased the uptake of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080, respectively, in PC-3 tumor xenografts. The kidney uptake in both blocked mice were markedly increased.

The ex vivo biodistribution studies for both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 were conducted at 1 h pi in PC-3 tumor-bearing mice, and the results were consistent with the observations from the PET images (Figure 5 and Figure 6 and Table S4). Compared to [⁶⁸Ga]Ga-LW02080, [⁶⁸Ga]Ga-LW02060 had a higher tumor uptake (7.36 ± 1.13 vs. 16.8 ± 2.70 %ID/g, p < 0.001), leading to better tumor-to-bone and tumor-to-kidney uptake ratios (69.8 ± 13.7 vs. 179 ± 46.9 and 3.18 ± 0.75 vs. 4.86 ± 0.76, respectively). The pancreas uptake of [⁶⁸Ga]Ga-LW02060 was 3.12 ± 0.89 %ID/g, significantly higher than that of [⁶⁸Ga]Ga-LW02080 (0.38 ± 0.04 %ID/g, p < 0.05), resulting in a lower tumor-to-pancreas uptake ratio for [⁶⁸Ga]Ga-LW02060 (5.52 ± 0.78 vs. 18.4 ± 2.86, p < 0.001). Furthermore, [⁶⁸Ga]Ga-LW02060 also showed significantly higher uptake in some normal organs than [⁶⁸Ga]Ga-LW02080 such as in small intestine, large intestine, and stomach. Consistent with the observations from PET images, moderate kidney uptake was obtained for [⁶⁸Ga]Ga-LW02060 (3.58 ± 1.14 %ID/g) and [⁶⁸Ga]Ga-LW02080 (2.42 ± 0.81 %ID/g). Except tumor xenografts and kidneys, the uptake values of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 for all other collected organs/tissues were lower than 1 %ID/g.

Co-injection with 100 μg of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) significantly reduced the average uptake of [⁶⁸Ga]Ga-LW02060 in PC-3 tumor xenografts by 64% (from 16.8 ± 2.70 %ID/g to 6.11 ± 0.42 %ID/g, p < 0.001) (Figure 6A and Table S4). Co-injection with 100 μg of nonradioactive Ga-LW02080 reduced the average tumor uptake of [⁶⁸Ga]Ga-LW02080 by 77% (from 7.36 ± 1.13 %ID/g to 1.71 ± 0.39 %ID/g, p < 0.001) (Figure 6B and Table S4). Co-injection with a blocking agent also significantly increased the uptake of both tracers in most of the collected normal organs/tissues, especially in kidneys (from 3.58 ± 1.14 %ID/g to 11.3 ± 0.97 %ID/g for [⁶⁸Ga]Ga-LW02060, p < 0.001; from 2.42 ± 0.81 %ID/g to 11.2 ± 1.02 %ID/g for [⁶⁸Ga]Ga-LW02080, p < 0.01,) (Figure 6 and Table S4).

2.4. In Vivo Stability

The in vivo stability of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 was evaluated in NRG mice (n = 3) (Figure 7 and Figure 8). There was >99% of [⁶⁸Ga]Ga-LW02060 remaining intact in plasma at 15 min pi, which was higher than that of [⁶⁸Ga]Ga-LW02080, with 87.4 ± 5.34% remaining intact (p < 0.01). No intact tracer was detected in urine samples collected at 15 min pi for either [⁶⁸Ga]Ga-LW02060 or [⁶⁸Ga]Ga-LW02080.

2.5. SPECT Imaging and Ex Vivo Biodistribution

The longitudinal SPECT/CT images of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 were acquired at 1, 4, 24, 72, and 120 h pi (Figure 9). [¹⁷⁷Lu]Lu-LW02060 enabled clear visualization of the PC-3 tumor xenograft in the SPECT images up to 24 h pi, while [¹⁷⁷Lu]Lu-LW02080 can be used to clearly visualize the PC-3 tumor xenograft only at 1 h pi. Both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 were excreted mainly via the renal pathway, which resulted in moderate kidney uptake and extremely high accumulation in the urinary bladders and urethras. Co-injection with 100 μg of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) significantly decreased the uptake of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 in PC-3 tumor xenografts at 1 h pi.

The ex vivo biodistribution results of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 were consistent with the observations from the SPECT images (Figure 9 and Figure 10 and Tables S5 and S6). The tumor uptake values of [¹⁷⁷Lu]Lu-LW02060 were 9.59 ± 3.37, 8.38 ± 0.19, and 5.78 ± 0.40 %ID/g at 1, 4, and 24 h pi, respectively, and then dropped to <3 %ID/g after 72 h pi. By contrast, the tumor uptake of [¹⁷⁷Lu]Lu-LW02080 was only 5.67 ± 1.02 %ID/g at 1 h pi and decreased quickly to 2.96 ± 0.37 %ID/g at 4 h pi and <1 %ID/g after 72 h pi (Figure 10A). The pancreas uptake of [¹⁷⁷Lu]Lu-LW02060 were 2.64 ± 0.63, 1.71 ± 0.08, and 1.04 ± 0.19 %ID/g at 1, 4, and 24 h pi, respectively, while [¹⁷⁷Lu]Lu-LW02080 showed markedly lower pancreas uptake (<0.5 %ID/g for all time points) (Figure 10B). The kidney uptake values of [¹⁷⁷Lu]Lu-LW02060 were 3.62 ± 0.86, 2.85 ± 0.29, and 1.06 ± 0.22 %ID/g at 1, 4, and 24 h pi, respectively, and slightly lower kidney uptake values were obtained from [¹⁷⁷Lu]Lu-LW02080 (2.65 ± 0.48, 1.88 ± 0.35, and 0.89 ± 0.28 %ID/g at 1, 4, and 24 h pi, respectively). Uptake values of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 for all other collected organs/tissues were <1 %ID/g at all time points (Tables S5 and S6).

Co-injection with 100 µg of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) reduced the tumor uptake of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 at 1 h pi by 66% and 64%, respectively. For [¹⁷⁷Lu]Lu-LW02060, significant uptake reductions were also observed for pancreas, stomach, and small intestine by 81%, 72%, and 66%, respectively, with co-injection of the blocking agent. By contract, for [¹⁷⁷Lu]Lu-LW02080, no significant uptake change was observed for the pancreas (p > 0.05), but a slightly higher uptake was found in stomach and small intestine with co-injection of the blocking agent. No significant difference in kidney uptake of [¹⁷⁷Lu]Lu-LW02060 was observed without/with co-injection of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14), while a significant increase in the kidney uptake of [¹⁷⁷Lu]Lu-LW02080 (from 2.65 ± 0.48 %ID/g to 4.17 ± 0.95 %ID/g, p < 0.05) was observed.

2.6. Radiation Dosimetry

The calculated radiation doses absorbed by selected mouse organs/tissues from [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 are provided in Figure 11 and Tables S7 and S8. A higher radiation absorbed dose of [¹⁷⁷Lu]Lu-LW02060 was delivered to the PC-3 tumor xenografts (Unit Density Sphere Model) compared with that of [¹⁷⁷Lu]Lu-LW02080 (Figure 11A). The absorbed dose of [¹⁷⁷Lu]Lu-LW02060 in a 1-g PC-3 tumor xenograft was 272 mGy/MBq, which was 5.8-times the tumor absorbed dose of [¹⁷⁷Lu]Lu-LW02080 (47.0 mGy/MBq) (Figure 11A and Table S7). The urinary bladder received the highest absorbed dose for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 among all selected organs/tissues with 1510 and 655 mGy/MBq, respectively, followed by the kidneys, with 148 and 64.4 mGy/MBq, respectively (Figure 11B and Table S7). Compared with [¹⁷⁷Lu]Lu-LW02060, an overall less absorbed dose was observed for [¹⁷⁷Lu]Lu-LW02080 in all selected organs/tissues. The absorbed dose of [¹⁷⁷Lu]Lu-LW02060 in the pancreas was 118 mGy/MBq, which was around 23-times the pancreas absorbed dose of [¹⁷⁷Lu]Lu-LW02080 (5.03 mGy/MBq).

The estimated radiation absorbed doses with the tumor sink effect correction for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 in an average adult male are provided in

Table 1. Estimated radiation absorbed doses (mGy/MBq) in adult human males for [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080.

Target Organ	[177Lu]Lu-LW02060	[177Lu]Lu-LW02080
Adrenals	2.45 × 10−2	4.24 × 10−3
Brain	2.85 × 10−4	1.04 × 10−4
Esophagus	8.10 × 10−4	3.14 × 10−4
Eyes	5.72 × 10−4	2.45 × 10−4
Gallbladder wall	1.20 × 10−3	4.15 × 10−4
Left colon	2.59 × 10−2	5.58 × 10−3
Small intestine	7.60 × 10−3	3.97 × 10−3
Stomach wall	3.70 × 10−3	1.45 × 10−3
Right colon	1.35 × 10−2	2.98 × 10−3
Rectum	1.30 × 10−2	3.00 × 10−3
Heart	2.66 × 10−3	1.17 × 10−3
Kidneys	6.36 × 10−2	2.78 × 10−2
Liver	1.29 × 10−2	3.17 × 10−3
Lungs	5.31 × 10−3	2.11 × 10−3
Pancreas	5.08 × 10−2	1.69 × 10−3
Prostate	1.64 × 10−3	6.94 × 10−4
Salivary glands	5.91 × 10−4	2.52 × 10−4
Red marrow	7.68 × 10−4	3.23 × 10−4
Skeleton	1.04 × 10−3	4.73 × 10−4
Spleen	9.47 × 10−3	2.92 × 10−3
Testes	2.49 × 10−3	1.05 × 10−3
Thymus	6.92 × 10−4	2.85 × 10−4
Thyroid	6.38 × 10−4	2.68 × 10−4
Urinary bladder	1.50 × 10−1	6.51 × 10−2
Total body	2.55 × 10−3	9.77 × 10−4
Effective dose (mSv/MBq)	1.16 × 10−2	4.15 × 10−3

. The highest estimated absorbed dose was observed in the urinary bladder for both ¹⁷⁷Lu-labeled ligands, with 1.50 × 10⁻¹ and 6.51 × 10⁻² mGy/MBq, respectively. The estimated absorbed dose of [¹⁷⁷Lu]Lu-LW02060 in the pancreas (5.08 × 10⁻² mGy/MBq) was around 30 times of the pancreas absorbed dose of [¹⁷⁷Lu]Lu-LW02080 (1.69 × 10⁻³ mGy/MBq). The estimated absorbed doses of [¹⁷⁷Lu]Lu-LW02060 were consistently higher than those of [¹⁷⁷Lu]Lu-LW02080 across other selected organs/tissues, ranging from 1.9-fold to 5.8-fold. The effective whole-body dose per injected radioactivity of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 to an adult human male were 1.16 × 10⁻² and 4.15 × 10⁻³ mSv/MBq, respectively.

3. Discussion

Based on our previously reported LW02056 and ProBOMB5, we designed two novel DOTA-conjugated GRPR-targeted ligands, LW02060 and LW02080, by replacing Pro¹⁴ in LW02056 and ProBOMB5, respectively, with diF-Pro¹⁴ (Figure 1) [24]. The data from intracellular calcium efflux assay confirm the agonist characteristics of LW02060 and its Ga/Lu-complexed analogs, and the antagonist characteristics of LW02080 and its Ga/Lu-complexed analogs (Figure 2). The agonist/antagonist characteristics of both diF-Pro¹⁴ derivatives are aligned with their Pro¹⁴ analogs (LW02056 and ProBOMB5). This validates our hypothesis that diF-Pro¹⁴ substitution on previously reported LW02056 and ProBOMB5 would retain their GRPR agonist/antagonist characteristics

The binding affinities of the Ga/Lu-complexed LW02060 analogs were better than those of LW02080, respectively (Figure 3). Compared to its Pro¹⁴ analog Ga-LW02056, Ga-LW02060 showed a significantly higher binding affinity to GRPR (K_i = 14.7 ± 4.81 vs. 5.57 ± 2.47 nM, p < 0.05) [24]. In contrast, the binding affinities of the GRPR antagonists, Ga-LW02080 and Lu-LW02080, were found to be lower than those of Ga-ProBOMB5 and Lu-ProBOMB5 (K_i = 21.7 ± 6.69 and 32.1 ± 8.14 nM vs. 12.2 ± 1.89 and 13.6 ± 0.25 nM, respectively) [24]. This observation suggests that the diF-Pro¹⁴ substitution enhances the binding affinity of GRPR agonists (Ga-LW02060) while reducing the binding affinity of GRPR antagonists (Ga-LW02080 and Lu-LW02080). Previously, we also observed that NMe-Gly¹¹ substitution was tolerable for antagonists but decreased the binding affinity of agonists [20,23]. Similarly, NMe-His¹² substitution was found to improve binding affinity for only agonists but to decrease the binding affinity for antagonists [22]. Our accumulated data suggest that agonists and antagonists bind to GRPR in different configurations.

The hydrophilic nature of [⁶⁸Ga]Ga-LW02060, [⁶⁸Ga]Ga-LW02080, [¹⁷⁷Lu]Lu-LW02060, and [¹⁷⁷Lu]Lu-LW02080 were confirmed via the logD_7.4 measurements with logD_7.4 values all ≤−2.33. Compared with their Pro¹⁴ analogs ([⁶⁸Ga]Ga-LW02056 and [⁶⁸Ga]Ga-ProBOMB5), both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 had significantly higher logD_7.4 values (−2.93 ± 0.08 and −2.74 ± 0.04 vs. −2.57 ± 0.04 and −2.60 ± 0.03, respectively; p < 0.01). This suggests that replacing Pro¹⁴ with diF-Pro¹⁴ slightly increases the lipophilicity of two tracers.

Both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 enabled clear visualization of the PC-3 tumor xenografts in PET images at 1 h pi (Figure 4). The extremely high accumulation of both tracers in the urinary bladders seen in PET images indicates that their main excretion was via the renal pathway, likely resulting from the highly hydrophilic nature of both tracers. The ex vivo biodistribution data of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 were consistent with the observations from their PET images (Figure 4, Figure 5 and Figure 6 and Table S4). Possibly owing to its higher binding affinity, [⁶⁸Ga]Ga-LW02060 exhibited 2.3-fold uptake in PC-3 tumor xenografts when compared to that of [⁶⁸Ga]Ga-LW02080 (16.8 ± 2.70 vs. 7.36 ± 1.13 %ID/g), and 1.9-fold uptake when compared to the previously reported [⁶⁸Ga]Ga-LW02056 (16.8 ± 2.70 vs. 8.93 ± 1.96 %ID/g) [24]. On the other hand, compared with [⁶⁸Ga]Ga-ProBOMB5, the diF-Pro¹⁴-derived [⁶⁸Ga]Ga-LW02080 showed a significantly lower tumor uptake (7.36 ± 1.13 vs. 12.4 ± 1.35 %ID/g, p < 0.005), likely due to the inferior GRPR binding affinity of Ga-LW02080 [24].

To avoid the potential toxicity caused by injecting an excessive amount of a GRPR agonist, we conducted the blocking study of the agonist tracer, [⁶⁸Ga]Ga-LW02060, by co-injection with 100 μg of a GRPR antagonist, [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) (Figure 4 and Figure 6, and Table S4). For the antagonist tracer, [⁶⁸Ga]Ga-LW02080, the blocking study was conducted with the co-injection of its nonradioactive standard, Ga-LW02080 (100 μg) (Figure 4 and Figure 6 and Table S4). With a significantly reduced tumor uptake in the blocked mice, both blocking studies confirmed the specific uptake of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 in the GRPR-expressing PC-3 tumor xenografts. The increased background uptake especially in the kidneys of blocked mice mainly resulted from the competitive binding of blocking agents to the GRPR in PC-3 tumor xenografts, leading to more unbound [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 in the blood pool and to be excreted through the renal pathway.

The in vivo stability of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 was assessed at 15 min pi (Figure 7 and Figure 8). More than 99% of [⁶⁸Ga]Ga-LW02060 remained intact in mouse plasma, which was significantly higher than that of [⁶⁸Ga]Ga-LW02080 (87.4 ± 5.34%) and our previously reported GRPR-targeted tracers (12.7–92.9%) [20,21,22,23,24]. The excellent in vivo stability of [⁶⁸Ga]Ga-LW02060 likely contributes to its high tumor uptake (16.8 ± 2.70 at 1 h pi). Same as most of the previously reported GRPR-targeted radioligands, no intact tracer was observed in mouse urine samples, likely due to degradation by NEP, which is expressed abundantly in the kidneys [18,19,20,21,22,23,24].

Subsequently, we labeled LW02060 and LW02080 with Lu-177 and evaluated the therapeutic potential of their ¹⁷⁷Lu-labeled analogs via SPECT/CT imaging, ex vivo biodistribution studies, and dosimetry calculations. The longitudinal SPECT/CT images showed that compared with [¹⁷⁷Lu]Lu-LW02060, [¹⁷⁷Lu]Lu-LW02080 had a lower tumor uptake and was cleared faster from the PC-3 tumor xenograft (Figure 9). This is likely due to the combination of its inferior GRPR binding affinity and being an antagonist (lack of internalization upon binding to GRPR) [1,29,30]. The observations from the SPECT/CT images were confirmed by data from ex vivo biodistribution studies (Figure 10 and Tables S5 and S6). The tumor uptake of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 at 1 h pi was lower than that of their ⁶⁸Ga-labeled analogs, [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 (9.59 ± 3.37 and 5.67 ± 1.02 %ID/g vs. 16.8 ± 2.70 and 7.36 ± 1.13 %ID/g, respectively). The inferior tumor uptake for the ¹⁷⁷Lu-labeled analogs could be due to their slightly inferior GRPR binding affinity, possibly resulting from variation in the charge distribution of different DOTA-metal complexes. It has been previously reported by Fani et al. that complexation with a different metal could have a high impact on the binding affinity of a somatostatin receptor 2 (Sstr2)-targeted ligand, DOTA-JR11 [31]. While the IC₅₀ values of Y-DOTA-JR11 and Lu-DOTA-JR11 were 0.47 and 0.73 nM, respectively, the IC₅₀ value of Ga-DOTA-JR11 was only 29 nM. Surprisingly, with a different chelator, Ga-NODAGA-JR11 was much more potent and had an IC₅₀ value at 1.2 nM, close to that of Y-DOTA-JR11 and Lu-DOTA-JR11. Therefore, we are currently investigating the effect of different chelators on the binding affinity of Ga/Lu-complexed GRPR-targeted ligands.

Owing to their dominantly renal excretion, the highest absorbed dose for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 was received by the urinary bladder in the mouse model. The calculated absorbed dose of [¹⁷⁷Lu]Lu-LW02060 in a 1-g PC-3 tumor xenograft was 272 mGy/MBq, which was 5.8-times that of [¹⁷⁷Lu]Lu-LW02080 (47.0 mGy/MBq) and 4.7-time the tumor absorbed dose of our previously reported [¹⁷⁷Lu]Lu-ProBOMB5 (57.3 mGy/MBq) [24]. However, the absorbed dose of the clinical validated GRPR antagonist [¹⁷⁷Lu]Lu-RM2 in the same tumor model was 429 mGy/MBq, which is 1.6-times that of [¹⁷⁷Lu]Lu-LW02060. This is due to the longer tumor retention of [¹⁷⁷Lu]Lu-RM2, likely resulting from its higher GRPR binding affinity (K_i = 1.19 ± 0.16 nM) [24]. The radiation absorbed doses delivered to the pancreas by [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 (118 and 5.03 mGy/MBq) were markedly lower than that of [¹⁷⁷Lu]Lu-RM2 (316 mGy/MBq) [24]. The estimated absorbed doses of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 in the human pancreas were markedly lower than those of [¹⁷⁷Lu]Lu-RM2 (5.08 × 10⁻² and 1.69 × 10⁻³ mGy/MBq, respectively, vs. 1.16 × 10⁻¹ mGy/MBq) [24]. Because the pancreas is the main dose-limiting organ of GRPR-targeted radiopharmaceuticals [9,10], our data suggested that [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 have higher maximum tolerated doses compared with the clinically validated [¹⁷⁷Lu]Lu-RM2 owing to their relatively lower pancreas accumulation. Additionally, the overall lower absorbed doses of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 in most selected organs/tissues compared to those of [¹⁷⁷Lu]Lu-RM2 indicate less off-target binding of [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 in both the mouse model and adult human.

Though compared to [¹⁷⁷Lu]Lu-LW02080, [¹⁷⁷Lu]Lu-LW02060 has a higher tumor uptake and a longer tumor retention, it is still not ideal for therapeutic application due to its insufficient radiation absorbed dose deposited in tumors. Further optimizations are needed to enhance tumor uptake and extend tumor retention, thereby achieving a potentially better therapeutic efficacy.

4. Materials and Methods

4.1. General Methods

All chemicals and solvents used in this study were obtained from commercial suppliers and utilized without additional purification. GRPR-targeting peptides were synthesized using solid phase approach on an AAPPTec (Louisville, KY, USA) Endeavor 90 peptide synthesizer. Purification and quality control of synthesized peptides were performed using Agilent (Santa Clara, CA, USA) HPLC systems equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (220 nm), and a Bioscan (Washington, DC, USA) NaI scintillation detector. The Agilent HPLC systems were operated using the Agilent ChemStation software. The HPLC columns utilized in this study included a semi-preparative column (Luna C18, 5 µm, 250 × 10 mm) and an analytical column (Luna C18, 5 µm, 250 × 4.6 mm), both acquired from Phenomenex (Torrance, CA, USA). The collected HPLC eluates were lyophilized using a Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze-drier. MS spectra were obtained using an AB SCIEX (Framingham, MA, USA) 4000 QTRAP mass spectrometer system equipped with an ESI ion source. C18 Sep-Pak cartridges (1 cm³, 50 mg) were purchased from Waters (Milford, MA, USA). ⁶⁸Ga was eluted from an ITM Medical Isotopes GmbH (Munich, Germany) generator and purified according to the previously published procedures using a DGA resin column from Eichrom Technologies LLC (Lisle, IL, USA) [24]. ¹⁷⁷LuCl₃ was purchased from Isotopia Molecular Imaging Ltd. (Petah Tikva, Israel) and ITM Medical Isotopes GmbH (Munich, Germany). [¹²⁵I-Tyr⁴]Bombesin was purchased from Revvity (Waltham, MA, USA). Radioactivity of ⁶⁸Ga/¹⁷⁷Lu-labeled peptides was measured using a Capintec (Ramsey, NJ, USA) CRC^®-25R/W dose calibrator. The radioactivity of samples collected from biodistribution studies, binding assays, and logD_7.4 measurements was counted on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter. ¹⁹F NMR is externally referenced to CFCl₃ to 0.00 ppm for all NMR samples.

4.2. Synthesis of Fmoc-Leu(ψ)diF-Pro-OH

Fmoc-Leu(ψ)diF-Pro-OH (5) was synthesized following the reaction steps depicted in Scheme S1. Detailed synthesis procedures and characterizations for Fmoc-Leu(ψ)diF-Pro-OH (5) and its intermediates are provided in Supplementary Materials (Figures S1–S16).

4.3. Synthesis of DOTA-Conjugated Peptides

Both LW02060 and LW02080 were synthesized on solid phase using the Fmoc peptide chemistry. For the synthesis of LW02060, Rink Amide MBHA resin (0.1 mmol) was deprotected by treating it with 20% piperidine in N,N-dimethylformamide (DMF) to remove the Fmoc group. After being pre-activated with HATU (5 eq.), HOAt (5 eq.), and N,N-diisopropylethylamine (DIEA, 15 eq.), Fmoc-protected amino acids (5 eq.) and Fmoc-4-amino-(1-carboxymethyl)piperidine (5 eq.) were sequentially coupled to the resin. Sieber resin (0.1 mmol) was used for the synthesis of LW02080 following the same procedure as LW02060. At the end, DOTA(tBu)₃ (5 eq.), pre-activated with HATU (5 eq.) and DIEA (25 eq.), was coupled to the resins for both LW02060 and LW02080, respectively.

A mixture of trifluoroacetic acid (TFA, 81.5%), triisopropylsilane (TIS 1.0%), water (5%), 2,2′-(ethylenedioxy)diethanethiol (DODT, 2.5%), thioanisole (5%), and phenol (5%) was used to deprotect and cleave the crude peptides from the resins respectively. After 4 h incubation at room temperature, the cleaved peptides were filtered and precipitated in cold diethyl ether. The crude peptides were collected after centrifugation and purified with HPLC (semi-preparative column; flow rate: 4.5 mL/min). The fractions containing the target peptides were gathered and lyophilized. The HPLC conditions, retention times, isolated yields and MS confirmations of LW02060 and LW02080 are detailed in Table S1 and Figures S17 and S18.

4.4. Synthesis of Nonradioactive Ga/Lu-Complexed Standards

The nonradioactive Ga-complexed standards of LW02060 and LW02080 were synthesized by incubating a solution of the DOTA-conjugated precursor with excess GaCl₃ (5 eq.) in NaOAc buffer (0.1 M, 500 µL, pH 4.5) at 80 °C for 15 min. The nonradioactive Lu-complexed standards of LW02060 and LW02080 were synthesized by incubating a solution of the precursor with excess LuCl₃ (10 eq.) in NaOAc buffer (0.1 M, 500 µL, pH 4.5) at 90 °C for 30 min. Subsequently, the reaction mixture was purified using HPLC with a semi-preparative column at flow rate of 4.5 mL/min. The HPLC conditions, retention times, isolated yields and MS confirmations of these nonradioactive Ga/Lu-complexed standards are detailed in Table S2 and Figures S19–S22.

4.5. Synthesis of ⁶⁸Ga/¹⁷⁷Lu-Labeled Ligands

The Ga-68 radiolabeling experiments were performed following previously published procedures [24]. Briefly, purified ⁶⁸Ga (158–523 MBq) in 0.5 mL water was added into a 4-mL glass vial containing 0.7 mL of HEPES buffer (2 M, pH 5.0) and 10 μL of precursor solution (1 mM). The radiolabeling reaction was performed by microwave heating for 1 min at 100 °C, followed by purification using HPLC with a semi-preparative column.

The Lu-177 radiolabeling experiments were conducted following literature procedures [24]. ¹⁷⁷LuCl₃ (994–2240 MBq) was added into a 4 mL glass vial preloaded with 0.7 mL of NaOAc buffer (0.1 M, pH 4.5) and 10 μL precursor solution (1 mM) and incubated at 95 °C for 15 min with a heating block. ¹⁷⁷Lu-labeled ligands were purified using HPLC with a semi-preparative column.

The fraction containing the radiolabeled product was collected, diluted with 50 mL of water, and concentrated in a C18 Sep-Pak cartridge, which was pre-washed with ethanol (10 mL) and water (10 mL). The trapped ⁶⁸Ga/¹⁷⁷Lu-labeled product on the cartridge was eluted off with ethanol (0.4 mL) and diluted with PBS for imaging and biodistribution studies. The HPLC conditions and retention times for the purification and quality control of ⁶⁸Ga/¹⁷⁷Lu-labeled ligands are provided in Table S3. The representative analytical radio-HPLC chromatograms of the purified ⁶⁸Ga/¹⁷⁷Lu-labeled ligands are provided in Figures S23–S26.

4.6. The LogD_7.4 Measurement

The logD_7.4 values of ⁶⁸Ga/¹⁷⁷Lu-labeled ligands were measured using the previously reported shake flask method [24]. An aliquot of the ⁶⁸Ga/¹⁷⁷Lu-labeled ligand was added into a 15 mL falcon tube containing a mixture of n-octanol (3 mL) and DPBS (3 mL, pH 7.4). The mixture was mixed by 1 min vortexing, followed by 15 min centrifugation at 3000 rpm. Samples (1 mL each) from the n-octanol and DPBS layers were collected and then measured using a gamma counter (n = 3). The logD_7.4 value was calculated using the following equation:

$${logD}_{7.4}={log}_{10}\left[{\displaystyle \frac{{\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{s}}_{\mathrm{o}\mathrm{c}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{l} \mathrm{p}\mathrm{h}\mathrm{a}\mathrm{s}\mathrm{e}}}{{\mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{s}}_{\mathrm{b}\mathrm{u}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{r} \mathrm{p}\mathrm{h}\mathrm{a}\mathrm{s}\mathrm{e}}}}\right].$$

4.7. Fluorometric Calcium Release Assay

The agonist/antagonist characteristics of LW02060, Ga-LW02060, Lu-LW02060, LW02080, Ga-LW02080, and Lu-LW02080 were determined following published procedures [24]. The PC-3 cells were obtained from ATCC (via Cedarlane, Burlington, ON, Canada). A 96-well clear bottom black plate was seeded with 5 × 10⁴ PC-3 cells in 100 μL growth media per well 24 h before the assay. The loading buffer (100 μL/well) containing a calcium-sensitive dye (FLIPR Calcium 6 assay kit from Molecular Devices, San Jose, CA, USA) was added into the 96-well plate, followed by 1 h incubation at 37 °C. The plate was then transferred into a FlexStation 3 microplate reader (Molecular Devices, San Jose, CA, USA). LW02060 (50 nM), Ga-LW02060 (50 nM), Lu-LW02060 (50 nM), LW02080 (50 nM), Ga-LW02080 (50 nM), Lu-LW02080 (50 nM), [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14) (50 nM, antagonist control), bombesin (50 nM, agonist control), adenosine triphosphate (ATP, 50 nM, positive control), or DPBS (blank control) was added, and the fluorescent signals were measured for 2 min (λ_Ex = 485 nm; λ_Em = 525 nm; n = 4). The relative fluorescent units (RFU = max − min) were calculated to determine the agonistic/antagonistic characteristics of LW02060, Ga-LW02060, Lu-LW02060, LW02080, Ga-LW02080, and Lu-LW02080, respectively.

4.8. In Vitro Competition Binding Assay

Inhibition constants (K_i) of GRPR-targeted ligands to GRPR were measured via in vitro competition binding assay using PC-3 cells and [¹²⁵I-Tyr⁴]Bombesin as the radioligand following the previously reported method [24]. PC-3 cells were seeded in 24-well poly-D-lysine plates at 2 × 10⁵ cells/well 48 h before the assay. The growth medium was removed before adding 400 μL of reaction medium (RPMI 1640 containing 2 mg/mL BSA, and 20 mM HEPES). After incubating at 37 °C for 1 h, Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 in 50 μL of reaction medium with decreasing concentrations (10 μM to 1 pM) and 50 μL of 0.01 nM [¹²⁵I-Tyr⁴]Bombesin were added into the wells then incubated with gentle agitation for 1 h at 37 °C. The cells were carefully washed twice with ice-cold PBS, harvested using trypsinization, and the radioactivity was measured using a Perkin Elmer (Waltham, MA) 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 10 software (Version 10.1.1).

4.9. PET/CT Imaging and Ex Vivo Biodistribution

PET/CT imaging and biodistribution studies were conducted using male NOD.Cg-Rag1^tm1Mom Il2rg^tm1Wjl/SzJ (NRG) mice following previously published procedures [24]. The studies were carried out in compliance with the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia (protocol number A20-0113, approved on 30 September 2022). The mice were anaesthetized and subcutaneously implanted with 5 × 10⁶ PC-3 cells (100 µL; 1:1 PBS/Matrigel) behind the left shoulder. PET/CT imaging and biodistribution studies were performed once the tumors reached a diameter of 5–8 mm, typically after approximately 4 weeks of growth.

PET/CT imaging experiments were conducted using a Siemens (Knoxville, TN, USA) Inveon micro PET/CT scanner. Around 3–5 MBq of the ⁶⁸Ga-labeled tracer was injected into the mice via lateral caudal tail veins under anaesthesia. The mice were then recovered and roam freely in their cages. A 10 min CT scan was conducted initially for localization, attenuation correction, and reconstructing the PET images at 50 min post-injection, followed by a 10 min static PET imaging acquisition.

For ex vivo biodistribution studies, the mice were injected with the ⁶⁸Ga-labeled tracer (2–4 MBq) via a lateral caudal tail vein. For blocking [⁶⁸Ga]Ga-LW02060, the mice were co-injected with 100 μg of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14). For blocking [⁶⁸Ga]Ga-LW02080, the mice were co-injected with 100 μg of its nonradioactive standard Ga-LW02080. At 1 h pi, the mice were euthanized by CO₂ inhalation. Blood and organs/tissues of interest were collected, weighed, and counted using a gamma counter.

4.10. SPECT/CT Imaging and Ex Vivo Biodistribution

SPECT/CT imaging was conducted using an MI Labs (Houten, The Netherlands) U-SPECT-II/CT scanner with a custom-made ultra-high sensitivity big mouse collimator (2 mm pinhole size) following published procedure [24]. The experiments were obtained in accordance with the same protocol established in the PET/CT imaging and ex vivo biodistributions section. The mice were used for SPECT/CT imaging and ex vivo biodistributions studies once the tumors reached a diameter of 5–8 mm. PC-3 tumor-bearing mice were sedated (2.5% isoflurane in O₂) and injected with 31.4–36.9 MBq of the ¹⁷⁷Lu-labeled ligand through a lateral caudal tail vein. The mouse was imaged at 1, 4, 24, 72, and 120 h pi. At each time point, a 5 min CT scan was performed with 615 μA and 60 kV parameters for localization and attenuation, followed by two 30 min static SPECT scans in list mode with an energy window centered around 208 keV. The U-SPECT II software was used to reconstruct data, and the images were decay corrected to the time of injection with PMOD v 3.402 (PMOD Technologies GmbH, Fallanden, Switzerland).

For ex vivo biodistribution studies, the mice were injected with ~3–5 MBq of the ¹⁷⁷Lu-labeled ligand (n = 5). At 1, 4, 24, 72, and 120 h post-injection, the mice were anesthetized with 2% isoflurane, euthanized by CO₂ inhalation, and the tissues/organs of interest were corrected for radioactivity counting. The blocking study was performed at 1 h pi via co-injection of the ¹⁷⁷Lu-labeled ligand with 100 μg of [D-Phe⁶,Leu-NHEt¹³,des-Met¹⁴]Bombesin(6–14).

4.11. In Vivo Stability Study

For in vivo stability studies, [⁶⁸Ga]Ga-LW02060 or [⁶⁸Ga]Ga -LW02080 (5.23–12.9 MBq) was injected into healthy male NRG mice (n = 3) tough a lateral caudal tail vein. At 15 min pi, mice were anaesthetized and euthanized, followed by collection of the urine and blood samples. The plasma was extracted from whole blood by adding an equal volume of CH₃CN, followed by vortexing, centrifugation, and collection of the supernatant. The plasma and urine samples were analyzed via radio-HPLC.

4.12. Dosimetry Analysis

The uptake values (%ID/g) obtained from ex vivo biodistribution studies (n = 5) were decayed corrected to the appropriate time point and fitted to mono- or bi-exponential equations using SciPy library integrated into an in-house Python script (Python Software Foundation v.3.10.12) [32]. The best fit was selected based on maximizing the coefficient of determination (R²) and minimizing the residuals. Time–activity curves calculated from the parameters obtained from the best fit for each organ were then integrated and normalized to injected activity to acquire time-integrated activity coefficients (TIACs) per unit gram, and subsequently multiplied by the mass of model tissue (30-g mouse phantom) [33]. The TIACs were corrected for the tumor sink effect following formula adopted in the report by Cicone et al. [34], as shown below:

$${TIAC}_{\mathrm{m},\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}\left(organ\right)={TIAC}_{\mathrm{m}}\left(organ\right)+⌈{TIAC}_{\mathrm{m}}\left(tumor\right)\times {\displaystyle \frac{{TIAC}_{\mathrm{m}}\left(organ\right)}{{TIAC}_{\mathrm{m}}\left(WB\right){-TIAC}_{\mathrm{m}}\left(tumor\right)}}⌉.$$

The TIAC values were input into OLINDA (Hermes Medical Solutions, v2.2.3) software [35], which has pre-calculated dose factors for mouse models. Mouse biodistribution data were extrapolated to humans using the method published by Kirschner et al. using the following equation [36]:

$${TIAC}_H\left(organ\right)={TIAC}_{\mathrm{M}}\left(organ\right)\times ⌈{\displaystyle \frac{{\mathrm{m}\left(\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\right)}_H/{WB}_H}{{\mathrm{m}\left(\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\right)}_M/{WB}_M}}⌉.$$

m(organ)_H and m(organ)_M are masses of human and mouse organs, respectively, and WB stands for total-body mass. Human TIACs calculated with the above equation were input into OLINDA, and dosimetry results were assessed for ICRP 89 Adult Male Model [37]. The %ID/g value for the blood was assumed to be that for the heart contents of the phantom. Lastly, the TIAC for the tumor was also calculated on the basis of the biodistribution data, and the values were input into the sphere model available in OLINDA [38].

4.13. Statistical Analysis

Statistical analyses were performed by using Student’s t-test with the Microsoft (Redmond, WA, USA) Excel software. The unpaired two-tailed test was used to compare biodistribution data, binding affinities, and logD_7.4 values of two radioligands. The unpaired one-tailed test was used to compare the biodistribution data of unblocked mice with those of blocked mice. A statistically significant difference was determined when the adjusted p value was <0.05.

5. Conclusions

We synthesized two novel GRPR-targeted ligands, LW02060 and LW02080, by replacing Pro¹⁴ in previously reported LW02056 (an agonist) and ProBOMB5 (an antagonist), respectively, with diF-Pro¹⁴. Our study reveals that the diF-Pro¹⁴ substitution retains their agonist/antagonist characteristics. The diF-Pro¹⁴ substitution on the agonist sequence further improves binding affinity and in vivo stability. By contrast, the diF-Pro¹⁴ substitution significantly reduces the binding affinity of the antagonist ligand which has a reduced peptide bond (CH₂-N) between Leu¹³ and AA¹⁴. Consistent with the low pancreas uptake of their previously reported Pro¹⁴ analogs, both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 showed low pancreas uptake compared to the clinically evaluated GRPR-targeted tracers. With high in vivo stability, great tumor uptake, and excellent tumor-to-background imaging contrast, [⁶⁸Ga]Ga-LW02060 is a promising tracer for clinical translation to detect GRPR-expressing cancer.

In addition, we successfully labeled both GRPR-targeted ligands with Lu-177 and evaluated the therapeutic potential of the resulting [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080. However, moderate tumor uptake and rapid clearance from PC-3 tumor xenografts were observed for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080, resulting in low radiation absorbed doses delivered to tumors. Thus, further optimizations are still needed for both ligands to enhance tumor uptake and extend tumor retention for potential therapeutic applications.

6. Patents

The compounds disclosed in this report are covered by a recent patent application (PCT International Application Serial No. PCT/CA2024/051215; filing date: 13 September 2024). Lei Wang, Chao-Cheng Chen, François Bénard, and Kuo-Shyan Lin are listed as inventors of this filed patent.