In vitro and in vivo comparative study of a novel 68Ga-labeled PSMA-targeted inhibitor and 68Ga-PSMA-11

68Ga-radiolabeled small molecules that specifically target prostate-specific membrane antigen (PSMA) have been extensively investigated, and some of these tracers have been used in the diagnosis of prostate cancer via 68Ga-positron emission tomography (68Ga-PET). Nevertheless, current 68Ga-labeled radiotracers show only fair detection rates for metastatic prostate cancer lesions, especially those with lower levels of prostate specific antigen (PSA), which often occurs in the biochemical recurrence of prostate cancer. The goal of this study was to design and synthesize a new PSMA-targeted radiotracer, 68Ga-SC691, with high affinity for prostate cancer cells and excellent pharmacokinetics. To this end, structural optimization was carried out on the bifunctional group, target motif, and linker while the high affinity targeting scaffold remained. To explore its potential in the clinic, a comparative study was further performed in vitro and in vivo between 68Ga-SC691 and 68Ga-PSMA-11, a clinically approved tracer for PSMA-positive prostate cancer. SC691 was radiolabeled to provide 68Ga-SC691 in 99% radiolabeling yield under mild conditions. High uptake and a high internalization ratio into LNCaP cells were observed in in vitro studies. In vivo studies showed that 68Ga-SC691 had favorable biodistribution properties and could specifically accumulate on PSMA-positive LNCaP xenografts visualized by micro-PET/CT. This radiotracer showed excellent PET imaging quality and comparable, if not higher, uptake in LNCaP xenografts than 68Ga-PSMA-11.

Even though macromolecules such as antibodies were introduced a few decades ago 15 , due to the outstanding merits of small molecules, such as their existence as a pure substance, definitive structure, and clear structure-activity relationships, small molecule PSMA inhibitors have mostly been investigated 16 . According to targeting scaffolds, small molecule PSMA inhibitors fall into one of three major families: (1) thiols 17 ; (2) phosphonate-, phosphate-18 , and phosphoramidates; and (3) ureas 19 . Among these inhibitors, small molecules with a Lys-urea-Glu scaffold as a pharmacophore were found to be able to accumulate in prostate cancer cells specifically and efficiently. Thus, a series of Lys-urea-Glu-based PSMA-targeted imaging agents with favorable biological properties emerged to visualize PCa lesions, such as 18 F-DCFPyL, 18 F-PSMA-1007, 68 Ga-PSMA-11 20 , and 68 Ga-PSMA-617 [21][22][23][24] (Fig. 1). Until now, no PET agents have been approved for clinical use in PCa diagnosis except for 68 Ga-PSMA-11, which was approved in December 2020 25 . Unlike 18 F-radiolabeled tracers, which need cyclotrons to produce 18 F and complex procedures and facilities for the synthesis of 18 F-radiopharmaceuticals, the ready availability and efficient complexing characteristics of 68 Ga(III), together with its favorable biological properties, have made the use of 68 Ga-based PSMA-targeted PET procedures grow rapidly, as evidenced by an increasing number of clinical trials [26][27][28][29][30] . Among them, 68 Ga-PSMA-11 is the most investigated and the only PCa radiotracer approved by the FDA. Currently, it is routinely used in the diagnosis, treatment evaluation, and staging of PCa.
Herein, we present the synthesis and in vitro and in vivo study of a novel and PSMA-targeted radiotracer, 68 Ga-SC691. We further evaluated its affinity and specificity to PSMA-positive tumors by comparing its pharmacokinetics and PET imaging abilities with those of 68 Ga-PSMA-11. 68 Ga-SC691 displayed favorable pharmacokinetics and excellent uptake in PSMA-positive tumors, indicating that 68 Ga-SC691 may serve as a new PET tracer for prostate cancer.

Results
Chemical, radiochemical synthesis and characterization. We first constructed the urea-based scaffold b-3 bearing protected glutamine and lysine residues followed by hydrogenation to provide b-4 with a combined yield of 51.1%. Converting b-4 into b-6 requires attention to the ratio of reactants; otherwise, a disubstituted compound will be the major product instead of the monosubstituted compound b-6. Compound b-6 was produced in a yield of 77.7%. With b-6 in hand, we further converted the secondary amine into tertiary amine b-8 with a para-iodo benzoyl moiety. After reduction of b-8, conjugation between a and b was performed to provide TM-1. The target molecule SC691 was obtained via deprotection of TM-1 in dilute acid in a yield of 39.1% (Scheme 1, Figure S1, Figure S2). The multistep reactions were conducted with a total yield of 2.8%.
Radiolabeling of SC691 with 68 Ga(III) eluate is straightforward. It may be performed in NaAc/HAc buffer or HEPES buffer (pH 4.0-5.0) at ambient temperature or a temperature as high as 95 °C in high radiolabeling yield (~ 95-99%) and radiochemical purity (> 98%) after purification, as analyzed using analytical reversed-phase high performance liquid chromatography (RP-HPLC, equipped with a γ detector) with a specific activity as high as 18.8 MBq/μg (Fig. 2). The retention time increased from 9.9 to 10.4 min after SC691 was radiolabeled with 68 Ga(III). In the cases of PSMA-11 and 68 Ga-PSMA-11, the retention times were 8.7 min and 9.0 min, respectively. Thus, SC691 displayed a longer retention time on HPLC, and an increase in the retention time was observed for both precursors upon radiolabeling.
Lipophilicity and stability. We further investigated the hydrophilicity and stability of 68 Ga-SC691. The partition coefficient (Log P) between octane and phosphate-buffered saline (PBS) is usually used to measure the hydrophilicity of a compound. 68 Ga-SC691 and 68 Ga-PSMA-11 had Log P values of − 3.530 ± 0.086 and − 2.91 ± 0.06 31 , respectively (Table 1). These data indicated that 68 Ga-SC691 is more hydrophilic than 68 Ga-PSMA-11. A stability study of 68 Ga-SC691 was performed both in PBS and fetal bovine serum (FBS) at 37 °C in triplicate. The radiotracer showed time-dependent stability in both systems, but after 2 h, its excellent stability was revealed, as over 96% of the 68 Ga-SC691 remained unchanged as determined by its retention time on analytical radio-HPLC (Fig. 3).
Another important aspect of a radiopharmaceutical is its radiochemical stability, which determines whether it is worth carrying out further in vivo and in vitro studies on a radioligand. 68 Ga-SC691 showed excellent stability    Cell affinity studies. Uptake and internalization experiments of 68 Ga-SC691 in LNCaP cells revealed high uptake and internalization rates (Fig. 4). Both internalization and uptake of 68 Ga-SC691 displayed a timedependent pattern and maintained an increasing trend for 120 min. Interestingly, uptake of 68 Ga-SC691 nearly Table 1. Analytical data of SC691, nat Ga-SC691, and PSMA-11. 1 Mass spectrometry data detected as [M+H] + 2 Value for radiochemical purity measured by RP-HPLC. An Agilent analytical column (250 × 4.6 mm) was utilized with mobile phases consisting of 0.1% TFA in water (A) and ACN (B). For analytical runs, a linear gradient of solvent A (90-10% in 15 min) in solvent B at a flow rate of 1.0 mL/min was used for a 15 min run. 3 Obtained from 68 Ga-SC691. See the detailed method description in the "Materials and methods" section. 4 This value is for 68 Ga-PSMA-11 from the literature 31 . 5 The purity of PSMA-11 was obtained from the ABX GmBH certificate of this compound.   Biodistribution. The results of the biodistribution study of 68 Ga-SC691 in LNCaP mice bearing prostate cancer (NOD/SCID) were decay-corrected and reported as the percentage of the injected activity per gram of tissue mass (% ID/g) and presented as the average ± standard deviation (SD). As summarized in Fig. 5, organ distribution with 68 Ga-SC691 revealed high specific uptake in LNCaP tumors with values of 43.41 ± 8.39% ID/g at 30 min and 27.59 ± 10.38% ID/g at 60 min. This high accumulation of radioactivity was maintained over 2 h of measurements (observed from micro-PET/CT). A similar trend in uptake was observed for the kidneys. The uptake of 68 Ga-SC691 was shown to be specific by coinjection of 68 Ga-SC691 together with 2-PMPA, demonstrating reduced tumor uptake by 30.55% ID/g at 60 min postinjection (12.86 ± 2.80% ID/g, n = 2). Biodistribution studies of 68 Ga-SC691 also demonstrated pronounced uptake in the kidneys and slight uptake by the liver, probably suggesting both hepatobiliary and kidney excretion pathways. However, the much higher uptake of 68 Ga-SC691 shown by the kidneys than the liver suggests that the kidneys contributed most significantly and are thus the principal excretion organs. Tumor-to-background ratios were determined to be 5.86 (tumor/blood) and 34.49 (tumor/muscle) at 1 h postinjection. Consistent with other PSMA-specific radioligands, no significantly reduced uptake, except for that in the kidneys, occurred in normal organs, as seen in blocking experiments.
Micro-PET/CT imaging. Next, whole-body micro-PET/CT imaging was carried out for 68 Ga-SC691 and 68 Ga-PSMA-11 (as a reference) in intact male NOD/SCID mice (Fig. 6) bearing LNCaP tumors only or bearing both LNCaP and PC-3 tumors in opposite upper flanks (Fig. 7, Figure S4). A static imaging method was utilized for both compounds at the time points of 5 min, 30 min, 60 min, and 120 min. Following the static PET scan, a dynamic PET scan was conducted to understand the time-dependent pharmacokinetics of 68 Ga-SC691 (Fig. 8). As a result of both the static and dynamic scans, these radiotracers were able to visualize PSMA-positive LNCaP tumors as early as 5 min postinjection but not PC-3 tumors. The micro-PET/CT images and dynamic uptake curves clearly showed the fast-targeting properties and high retention rate of 68 Ga-SC691 at the tumor site during the 2 h experiment, which was consistent with the biodistribution results. In addition to tumors, uptake by other organs, such as the heart and kidneys, became evident during the initial 5 min, and the uptake by the heart was diminished at 30 min and thereafter. Comparative PET/CT imaging also clearly showed that 68 Ga-SC691 had a renal clearance similar to that of 68 Ga-PSMA-11. Quantitative data from micro-PET/CT showed that the mean % ID/g at the tumor for both 68 Ga-SC691 and 68 Ga-PSMA-11 continued to increase up to 2 h postinjection, and the rapid elimination of radioactivity occurred from the other organs, muscles, and blood (with a quite clear background). However, the uptake curve became flattened over time, as summarized in Fig. 8A. In comparison to 68 Ga-PSMA-11, 68 Ga-SC691 displayed similar uptake in both the tumors and kidneys.

Discussion
PSMA is strongly overexpressed on prostate cancer cells and therefore has been selected as one of the most important drug targets for prostate cancer diagnosis, staging, and follow-up. PSMA inhibitors, after radiolabeling with 68 Ga, present favorable properties, such as availability and facile complexation chemistry. Here, we report the successful synthesis of 68 Ga-SC691 via multistep chemical reactions and complexation with the 68 Ga (III) method. To date, Lys-urea-Glu has become the most common and important targeting motif in PSMA inhibitor structures due to its high affinity and specificity for PSMA. To manage the pharmaceutical and metabolic properties of the desired tracers, amide or secondary amine linkers produced by Lys are the most popular strategy while keeping   The synthesis of SC691 is quite straightforward except for the conversion of the primary amine into a secondary amine, in which a dimer was formed. To enhance the yield of the monosubstituted product, the molar ratio and temperature should be carefully controlled. Compared with DOTA, DOTAGA has one more carboxylic acid group, providing an extra complexation site for Ga(III). The complexation chemistry of DOTAGA is easier than that of DOTA, so this reaction can be carried out at lower temperatures while achieving high radiochemical yields. This tracer was quite stable in PBS and FBS with over 96% intact compound remaining in both media after 2 h of incubation at 37 °C.
To improve precision with regard to comparison, further purification by HPLC was applied to remove the unlabeled precursor (SC691 or PSMA-11). The specificity of 68 Ga-SC691 was confirmed by its high uptake in PSMA-positive LNCaP cells and by blocking experiments in a LNCaP-inoculated NOD/SCID mouse model ( Figure S3). Accumulated radioactivity was not observed on PC-3 tumors but it was observed on LNCaP tumors. Upon coinjection of the PSMA inhibitor 2-PMPA, uptake of 68 Ga-SC691 was completely blocked, so quite low radioactivity was detected, which indicated that the novel tracer had exceptional specificity for PSMA-positive tumors. Biodistribution also showed that the tumor was the major site and that the kidneys played a critical role as a more important excretion organ than the liver. Tumor retention of 68 Ga-SC691 was maintained at a high level for over 2 h postinjection, which is consistent with the in vitro cell uptake and internalization results. Interestingly, the chelator DOTAGA exerted an evident impact on the properties of 68 Ga-SC691 by changing its lipophilicity or charge. In particular, the pharmacological properties were strongly influenced by the more hydrophilic 68 Ga-SC691.
The results obtained from the micro-PET/CT imaging experiments indicated that 68 Ga-SC691 had a slightly higher absolute uptake than 68 Ga-PSMA-11 in PSMA-positive tumors (Fig. 8A). An unexpected and longer blood clearance was observed for 68 Ga-SC691, considering that 68 Ga-SC691 is much more hydrophilic than 68 Ga-PSMA-11 (Table 1). We proposed that structural modification not only changed the hydrophilicity but also changed the binding affinity for plasma proteins, such as albumin. To confirm this hypothesis, a serum albumin binding experiment was carried out, and it was found that introducing a p-iodo benzoyl moiety clearly enhanced the affinity of the compound for serum albumin, thus extending its circulation time in the blood (data not shown). This property of longer blood circulation is favored for therapeutic agents if no safety issues exist. With regard to diagnostic agents, higher accumulation at the targeted organs could also be beneficial in practice due to extended blood circulation, as the diagnostic dose of radioactivity generally does not yield significant adverse reactions but potentially improves precision prognosis.

Materials and methods
Precursor synthesis. The synthetic route and chemical structures of SC691 and 68 Ga-SC691 are shown in Scheme 1. The synthesis of SC691 is quite straightforward through multistep reactions, and SC691 was purified with preparative high-performance liquid chromatography (prep-HPLC). The detailed synthesis is attached in the supporting information.
68 Ga radiolabeling. Typically, 5-20 μL of SC691 (1 mg/mL in pure water) was added to a volume of 100 μL of sodium acetate/acetic acid (NaAc/HAc) buffer (NaAc/HAc = 0.5 M/0.5 M, pH = 4.45), followed by the addition of 400 μL of the 68 Ga(III) eluate . The reaction mixture was incubated at 80 °C for 10 min and the radiochemical purity was determined by reversed-phase HPLC (RP-HPLC).  Biodistribution and imaging studies. Tumor model. The human prostate cancer cell lines LNCaP and PC-3 (PSMA-negative control) were obtained from the American Type Culture Collection (ATCC). Cells were counted, and 5 × 10 6 LNCaP or PC-3 cells in 100 μL of RPMI-1640 medium were injected subcutaneously into 4to 5-week-old male nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Animals were grown under standard conditions for 3-4 weeks to allow the tumors to become established before biodistribution and imaging.
Biodistribution. Biodistribution studies were performed 3 to 4 weeks after LNCaP tumor cell inoculation when the tumor xenografts reached an average mass of approximately 20 ± 5 g, corresponding to a tumor diameter of 5-8 mm. SC691 was radiolabeled with 68 Ga at a specific activity of ~ 24.5 MBq/nmol and diluted in sterilized saline (0.9% NaCl in DI water) to a final specific activity of approximately 2.8 MBq/nmol. Tumor-bearing mice were intravenously injected with 100 µL of 68 Ga-SC691 (~ 2.8 MBq). The mice were sacrificed at 30 min, 60 min, and 120 min postinjection, the major organs were collected and weighed, and the radioactivity was measured with a γ-counter (CAPRAC-t, Edmonton, Canada). Mice were grouped at specific time points, and 5 mice were included in each group.
Imaging studies. The 68 Ga-radiolabeled compound 68 Ga-SC691 was injected into the tail veins of male mice bearing LNCaP and PC-3 tumors (~ 3.7 MBq; 100 μL). All animals were placed in the prone position for micro-PET imaging at 5 min, 30 min, 60 min and 120 min postinjection. During the imaging process, mice were anaesthetized and maintained under 2% isoflurane in oxygen at a flow rate of 2 L/min. Data analysis was performed using Inveon Research Workplace software. The experiment was repeated at least three times.
Institutional Review Board statement. This study was approved by the Ethics Committee for Animals of Southwest Medical University (2019-06-27). All methods were carried out in accordance with relevant guidelines and regulations, and all methods were reported in accordance with ARRIVE guidelines.

Conclusions
In summary, we report a novel 68 Ga-labeled ligand for the noninvasive detection of prostate cancer. This ligand showed promising binding and internalization properties in vitro as well as high specific uptake and a favorable retention time in vivo. This radiotracer showed excellent PET imaging quality and uptake that was comparable with 68 Ga-PSMA-11 in LNCaP xenografts, if not higher. The feasibility of prostate cancer imaging with 68 Ga-SC691 is under evaluation in humans.