In Vitro and in Vivo Comparative Study of a Novel 68 Ga-Labeled PSMA-Targeted Inhibitor and 68 Ga -PSMA-11

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


Introduction
Prostate cancer (PCa) is the most common cancer form in males in the United States. It is estimated that there will be more than 190,000 new cases of prostate cancer and an estimated 33,000 deaths from this disease in USA only in 2020, according to the National Cancer Institute 1 . Statistically, even though prostate cancer cells in most PCa cases remain locally, about 10% of total number of PCa patients will suffer from metastasis or biochemical recurrence 2 . Advanced techniques such as Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) provide high contrast and resolution, however, imaging outcomes from these methods often suffer from either short of functional information or explicit explanation from corresponding professionals needed [3][4][5] . As well, they nd di culty in detecting microscale lesions, which are often observed in postoperative examinations. Positron Emission Tomography (PET) as a functional imaging method, together with targeting radiotracers, has been widely used in clinical due to its precision and ultrahigh sensitivity 6 . To date, PET/CT and PET/MRI fusing imaging as novel multimodality technology combining bene ts of anatomic information from CT or MRI scan imaging and functional information from PET, have become one of the most powerful imaging methods in disease diagnosis and single cell tracking 7,8 .
PCa possesses unique pathophysiologic and biological characteristics. A great number of studies found that overexpression of transmembrane protein, namely prostate speci c membrane antigen (PSMA), also known as glutamate carboxypeptidase, dominates in over 90% prostate carcinoma and tumor-associated endothelium of various solid cancer tissue 9,10 . Along with other properties, such as over 94% amino acids of the transmembrane protein locating outside of membrane 11 , PSMA has become one of the most extensively studied drug targets and shows prominent promise in the prostate cancer cell detection [12][13][14] .
Even though macromolecules like antibodies were introduced a few decades ago 15 , due to the outstanding merits of small molecules such as pure substance, de nitive structure, and clear structureactivity-relationship, small molecule PSMA inhibitors have been mostly investigated 16 . According to targeting scaffold, small molecule PSMA inhibitors fall into three major families: (1) thiols 17 ; (2) phosphonate-, phosphate-18 , and phosphoramidates; and (3) ureas 19 . Among these inhibitors, small molecules with Glu-urea-Lysine scaffold as a pharmacophore was found to be able to accumulate on prostate cancer cells speci cally and e ciently. Thus, a series of Glu-urea-Lysine-based PSMA-targeted imaging agents with favorable biological properties emerged to visualized 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 cyclotron to produce 18 F and complex procedure and facility to be applied for synthesis of 18 F-radiopharmaceuticals, the readily availability, e cient complexing characteristic of 68 Ga( ), together with its favorable biological properties make 68 Gabased, PSMA-targeted PET procedures grow rapidly evidenced by increasing number of clinical trials [26][27][28][29][30] . Among them, 68 Ga-PSMA-11 is the mostly investigated and the only approved PCa radiotracer by FDA.
And now it is being routinely used in 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 a nity and speci city to PSMA positive tumor by comparing its pharmacokinetics and PET imaging with 68 Ga-PSMA-11. It turned out that 68 Ga-SC691 displayed favorite pharmacokinetics and excellent uptake at PSMA positive tumor, indicating that 68 Ga-SC691 may serve as a new PET tracer for prostate cancer.

Chemical, Radiochemical Synthesis and Characterization
We rstly constructed the urea-based b-3 scaffold bearing protected glutamine and lysine followed by hydrogenation to provide b-4 with a combined yield of 51.1%. Converting b-4 into b-6 must be paid attention to the ratio of reactants, otherwise, a disubstituted compound will dominate in the products instead of the monosubstituted compound b-6. b-6 was produced with a yield of 77.7%. With b-6 in hand, we further substitute secondary amine into tertiary amine b-8 with 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 with 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( ) eluate is straightforward. It may be performed in NaAc/HAc buffer or HEPES buffer (pH 4.0-5.0) under ambient temperature to 95°C in high radiolabeling yield (~ 95-99%) and radiochemical purity (> 98%) after puri cation analyzed using analytic RP-HPLC (equipped with a gamma detector) with speci c activity as high as 18.8 MBq/µg (Fig. 2). The retention time has increased from 9.9 min to 10.4 min after SC691 radiolabeled with 68 Ga ( ). In the cased of PSMA-11 and 68 Ga-PSMA-11, the retention time were 8.7 min and 9.0 min, respectively. Thus, SC691 displayed a longer retention time on HPLC and extended retention time was observed for both precursor upon radiolabeling.

Lipophilicity and stability
We further investigated the hydrophilicity and stability of 68 Ga-SC691. The partition coe cient (Log P) between octane and Phosphate buffered saline (PBS) is usually used to measure hydrophilicity of a compound. It turned out that 68 Ga-SC691 and 68 Ga-PSMA-11 had Log P values being − 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. Stability study of 68 Ga-SC691 were performed both in PBS and in fetal bovine serum (FBS) at 37 ℃. The radiotracer showed time-dependent stability in both systems, but in a 2 h duration, excellent stability was revealed as it was indicated by over 96% of 68 Ga-SC691 remaining unchanged in retention time of analytical radio-HPLC (Fig. 3). Table 1 Analytical data of SC691, nat Ga-SC691, and PSMA-11.  3 Obtained from 68 Ga-SC691. See the detailed description of the method in materials and method. 4 This value is for 68 Ga-PSMA-11 from literature 31 . 5 The purity of PSMA-11 was taken from the ABX GmBH certi cate of this compound.
Another important aspect of a radiopharmaceutical is the radiochemical stability, which determines whether a radioligand is worth carrying out further in vivo and in vitro studies or not. 68 Ga-SC691 showed excellent stability both in PBS and in FBS incubated for 2 hours at 37 ℃. By the end of 2-hour incubation in PBS and FBS, 68 Ga-SC691 still maintained its radiochemical purity 97.9% and 97.5%, respectively.

Cell A nity Studies
Uptake and internalization experiments of 68 Ga-SC691 in LNCaP cells revealed high uptake and internalization rate. Internalization and uptake of 68 Ga-SC691 displayed a time-dependent pattern and maintained increasing trend for 120 min. Interestingly, uptake of 68 Ga-SC691 nearly reached saturation at 60 min, and only a slight increase was observed. On the other hand, its internalization experienced relatively fast increase in the whole experimental period. A blocking experiment with 2-PMPA was conducted to demonstrate the speci city of 68 Ga-SC691 toward PSMA in vitro. It turned out that cell uptake of 68 Ga-SC691 can be nearly completely blocked with 2-PMPA (data not shown).

Biodistribution
The results of biodistribution study of 68 Ga-SC691 in LNCaP mice bearing prostate cancer (NOD/SCID) was decay-corrected and listed as 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 a high speci c uptake in LNCaP tumor being 43.41 ± 8.39 %ID/g at 30 min and 27.59 ± 10.38 %ID/g at 60 min. High accumulation of radioactivity maintained over 2 hours measurements (observed from micro-PET/CT). Similar trend in uptake was observed for the kidneys.
The uptake of 68 Ga-SC691 was shown to be speci c by the co-injection of 68 Ga-SC691 together with 2-PMPA, demonstrating reduced tumor uptake by 30.55 %ID/g at 60 min post injection (12.86 ± 2.80 %ID/g, n = 2). Biodistribution studies of 68 Ga-SC691 also demonstrated pronounced uptake in kidneys and slight uptake in liver, probably suggesting both hepatobiliary and kidney excretion pathways, but kidneys show much higher uptake of 68 Ga-SC691 over liver suggesting that kidneys contributed most signi cantly and thus the principal excretion organs. Tumor-to-background ratios were determined as 5.86 (tumor / blood) and 34.49 (tumor / muscle), respectively, 1 h post injection. It is consistent with other PSMA-speci c radioligands that no signi cantly reduced uptake except for kidneys occurred in normal organs as it was seen in blocking experiments.
Micro-PET/CT imaging Next, whole body micro-PET/CT imaging was studied for 68 Ga-SC691 and 68 Ga-PSMA-11 (as a reference) in intact male NOD/SCID mice (Fig. 6) bearing LNCaP tumor only or bearing both LNCaP and PC-3 tumors in opposite, upper anks (Fig. 7, Figure S4). Static imaging method was applied to both compounds with time points at 5 min, 30 min, 60 min, and 120 min. Following the static PET scan, a dynamic PET scan was conducted to understand time-dependent pharmacokinetic of 68 Ga-SC691 (Fig. 8). As a result of both static and dynamic scan, these radiotracers were able to visualize the PSMApositive LNCaP tumor as early as 5 min post injection but not the PC-3 tumor. Micro-PET/CT images and dynamic uptake curve clearly showed the fast-targeting property and high retention rate of 68 Ga-SC691 at the tumor site during the 2-hour experiment, which was consistent with the biodistribution results. Besides tumor, uptake of other organs such as heart and kidneys became evident during the initial 5 minutes, and uptake in heart was diminished at 30 minutes and thereafter. The comparative PET/CT imaging also clearly showed that 68 Ga-SC691 had a similar renal clearance with 68 Ga-PSMA-11. Quantitative data from micro-PET/CT showed that the mean %ID/g at tumor for both 68 Ga-SC691 and 68 Ga-PSMA-11 kept increasing up to 2 hours post injection and a rapid elimination of radioactivity occurred from other organs, muscle, and blood (with a quite clear background). But the uptake curve became atter with time as summarized in Fig. 8A. In comparison to 68 Ga-PSMA-11, 68 Ga-SC691 displayed similar uptake both in tumor and in kidneys.

Discussion
PSMA is strongly overexpressed on prostate cancer cells and therefore is selected as one of the most important drug targets for prostate cancer diagnosis, staging, and follow-up. PSMA inhibitors, after radiolabeled with 68 Ga presents favorable properties as availability and facile complexation chemistry.
Here we report the successful synthesis of 68 Ga-SC691 via multistep chemical reactions and complexation with 68 Ga ( ) method. To date, Lys-Urea-Glu has become the most common and important targeting motif of PSMA inhibitor structure due to its high a nity and speci city to PSMA. To manage the pharmaceutical and metabolic properties of desired tracers, amide or secondary amine linker produced by Lys is the most popular strategy while keeping Lys-Urea-Glu untouched. Until 2019, Banerjee and coworkers reported the rst case of the tertiary amination modi cation strategy of the amino group on Lysine side chain modi ed by p-halo benzyl moiety and obtained several PSMA inhibitors with high binding a nity and excellent in vivo properties, which opened a precedent for tertiary amination modi cation strategy 32,33 . Inspired by quite low Ki value of the compound iodo-DCFPyL ( Fig. 1) (Ki = 0.01 nmol), we decided to introduce p-iodo benzoyl moiety into Lysine on the Lys-Urea-Glu scaffold to verify the possibility to obtain a PSMA inhibitor with a high a nity through the tertiary amination strategy. On the other hand, the macrocyclic chelating agent with four acetate donor arms was demonstrated in vitro and in vivo a higher tumor uptake and retention, thus DOTAGA other than DOTA was selected as the chelating agent due to its favorable property. A shorter or longer linker with number of methylene groups > 4 was reported to yield no signi cant impact on tumor uptake and retention. Together with chelating agent and p-iodo benzoyl moiety, SC691 was nalized to be expected excellent in vitro and in vivo properties.
The synthesis of SC691 is quite straight forward except for the step involving the conversion of primary amine into secondary amine, in which dimer was resulted. To enhance the yield of monosubstituted product, molar ratio and temperature should be carefully controlled. In contrast to DOTA, DOTAGA has one more carboxylic acid group providing an extra complexation site to Ga( ). The complexation chemistry of DOTAGA is more facile than DOTA, so that it can be carried out at lower temperature while achieving high radiochemical yield. This tracer was quite stable in PBS buffer and FBS with over 96% intact compound in both media after 2 hours incubation at 37 ℃.
To improve precision with regard to comparison, a further puri cation with HPLC to remove unlabeled precursor (SC691 or PSMA-11) was applied. Speci city for 68 Ga-SC691 was con rmed by its high uptake in PSMA-positive LNCaP cells and by blocking experiments in a LNCaP-inoculated NOD/SCID mouse model ( Figure S3). The accumulated radioactivity was not seen on PC-3 tumor but on LNCaP tumor. Upon co-injection of the PSMA inhibitor 2-PMPA, uptake of 68 Ga-SC691 was completely blocked, so that quite low radioactivity was detected, which indicated that the novel tracer had an exceptional speci city for PSMA-positive tumor. Biodistribution also showed that the tumor is the major site and kidneys played a critical role as the more important excretion organ compared with liver. Tumor retention of 68 Ga-SC691 maintained at a high level over 2 hours post injection, which is consistent with in vitro cell uptake and internalization. Interestingly, chelator DOTAGA exerted an evident impact on the properties of 68 Ga-SC691 by changing its lipophilicity or charges. In particular, the pharmacological property was strong in uenced by the more hydrophilic 68 Ga-SC691.
The results obtained from micro-PET/CT imaging experiments indicated 68 Ga-SC691 had a slightly higher absolute uptake than 68 Ga-PSMA-11 in PSMA-positive tumor (Fig. 8A). To be noticed, 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 modi cation not only changed its hydrophilicity but also changed its binding a nity with plasma protein, such as albumin. To con rm that, serum albumin binding experiment was carried out and demonstrated that introducing piodo benzoyl moiety clearly enhanced its a nity to serum albumin thus extended its circulation time in blood (data not shown). This property of longer blood circulation is favored for therapeutic agents if no safety issue exists. With regard to diagnostic agents, higher accumulation at targeted organs could also be bene cial in practice due to extended blood circulation as diagnostic dose of radioactivity generally does not yield signi cant adverse reactions but potentially improve precision prognosis.

Precursor Synthesis
Synthetic route and chemical structures of SC691 and 68 Ga-SC691 were shown in scheme 1. Synthesis of SC691 is quite straightforward through multi-step reactions and SC691 was puri ed with preparative high performance liquid chromatography (prep-HPLC). The detailed synthesis is attached in the supporting information.

Imaging studies
The 68 Ga-radiolabeled compounds 68 Ga-SC691 were injected into the tail vein of the male mice bearing LNCaP and PC-3 tumors (~3.7 MBq; 100 μL). All animals were performed in prone position for micro-PET imaging at 5 min, 30 min, 60 min and 120 min post injection. During the imaging process, mice were anaesthetized and maintained under 2% iso urane in oxygen at a ow rate of 2 L/min. The data analysis was used the Inveon Research Workplace software.

Conclusions
In summary, we report a novel and 68 Ga-labeled ligand for noninvasive detection of prostate cancer. This ligand showed a promising binding and internalization properties in vitro as well as high speci c uptake and favorite retention time in vivo. This radiotracer showed excellent PET imaging quality and comparable uptake with 68 Ga-PSMA-11 at LNCaP xenografts if not higher. The feasibility of prostate cancer imaging with 68 Ga-SC691 is under evaluation on human.  The uptake and internalization of 68Ga-SC691 in LNCaP cells (~200,000 cells/well, normalized to 106 cells) at 10 min, 30 min, 60 min, and 120 min.

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