Evaluation of Met-Val-Lys as a Renal Brush Border Enzyme-Cleavable Linker to Reduce Kidney Uptake of 68Ga-Labeled DOTA-Conjugated Peptides and Peptidomimetics

High kidney uptake is a common feature of peptide-based radiopharmaceuticals, leading to reduced detection sensitivity for lesions adjacent to kidneys and lower maximum tolerated therapeutic dose. In this study, we evaluated if the Met-Val-Lys (MVK) linker could be used to lower kidney uptake of 68Ga-labeled DOTA-conjugated peptides and peptidomimetics. A model compound, [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (AmBz: aminomethylbenzoyl), and its derivative, [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH, coupled with the PSMA (prostate-specific membrane antigen)-targeting motif of the previously reported HTK01166 were synthesized and evaluated to determine if they could be recognized and cleaved by the renal brush border enzymes. Additionally, positron emission tomography (PET) imaging, ex vivo biodistribution and in vivo stability studies were conducted in mice to evaluate their pharmacokinetics. [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was effectively cleaved specifically by neutral endopeptidase (NEP) of renal brush border enzymes at the Met-Val amide bond, and the radio-metabolite [68Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted via the renal pathway with minimal kidney retention. [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH retained its PSMA-targeting capability and was also cleaved by NEP, although less effectively when compared to [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH. The kidney uptake of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was 30% less compared to that of [68Ga]Ga-HTK01166. Our data demonstrated that derivatives of [68Ga]Ga-DOTA-AmBz-MVK-OH can be cleaved specifically by NEP, and therefore, MVK can be a promising cleavable linker for use to reduce kidney uptake of radiolabeled DOTA-conjugated peptides and peptidomimetics.


Introduction
The use of low molecular weight radiolabeled peptides and antibody fragments for applications in oncology is rapidly gaining momentum [1][2][3][4][5]. Such a site-directed radiation delivery involves targeting of certain specific receptors overexpressed on the surface of cancer cells for the purpose of targeted imaging and radionuclide therapy [6]. While these oncophilic molecules serve as biological targeting vectors, they commonly exhibit very high and sustained renal uptake [7]. This is caused by either a high We first synthesized the model compound, [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (Figure 2), and confirmed that it could be recognized by the renal brush border enzymes and cleaved at the Met-Val amide bond. We conducted PET imaging and in vivo stability studies in mice and confirmed that the expected radio-metabolite [ 68 Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted through the urinary pathway with minimal uptake in kidneys. We then replaced the acetyl group of [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH with the pharmacophore of HTK01166 (Figure 2), a peptidomimetic targeting the prostate-specific membrane antigen (PSMA), which is highly expressed in prostate cancer [15]. The targeting pharmacophore of [ 68 Ga]Ga-HTK01166 was selected as it was shown previously to have very high renal uptake (147 %ID/g, 1 h p.i.) in mice. Since Met is prone to oxidation to generate methionine sulfoxide (Met(O) or M(O)) [16]   We first synthesized the model compound, [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (Figure 2), and confirmed that it could be recognized by the renal brush border enzymes and cleaved at the Met-Val amide bond. We conducted PET imaging and in vivo stability studies in mice and confirmed that the expected radio-metabolite [ 68 Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted through the urinary pathway with minimal uptake in kidneys. We then replaced the acetyl group of [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH with the pharmacophore of HTK01166 (Figure 2), a peptidomimetic targeting the prostate-specific membrane antigen (PSMA), which is highly expressed in prostate cancer [15]. The targeting pharmacophore of [ 68 Ga]Ga-HTK01166 was selected as it was shown previously to have very high renal uptake (147 %ID/g, 1 h p.i.) in mice. Since Met is prone to oxidation to generate methionine sulfoxide (Met(O) or M(O)) [16]
The DOTA-conjugated peptides and peptidomimetics including DOTA-AmBz-MVK(Ac)-OH, DOTA-AmBz-MVK(HTK01166)-OH, DOTA-AmBz-Met-OH, and DOTA-AmBz-M(O)VK(HTK01166)-OH ( Figure 2) were assembled on solid phase (Schemes 2 and 3). After cleavage/deprotection with TFA and HPLC purification, these DOTA-conjugated peptides and peptidomimetics were obtained in 1-35% isolated yields. Their nonradioactive Ga-complexed standards were obtained by incubating the DOTA-conjugated peptides and peptidomimetics with excess GaCl3 in acetate buffer (0.1 M, pH 4.2) at 80 °C. After HPLC purification, the nonradioactive Ga-complexed standards were obtained in 16-78% isolated yields. Detailed HPLC conditions and retention times for the purification of the DOTA-conjugated peptides and peptidomimetics and their nonradioactive Ga-complexed standards are provided in Supplemental Tables S1 and S2 (see the Supplemental Materials). The identities of the DOTA-conjugated peptides and peptidomimetics and their nonradioactive Ga-complexed standards were confirmed by MS analysis. 68 Ga labeling of the DOTA-conjugated peptides and peptidomimetics was conducted in HEPES buffer (2 M, pH 5.0) with microwave heating for 1 min. After HPLC purification, [ 68 Ga]Ga-DOTA-
The DOTA-conjugated peptides and peptidomimetics including DOTA-AmBz-MVK(Ac)-OH, DOTA-AmBz-MVK(HTK01166)-OH, DOTA-AmBz-Met-OH, and DOTA-AmBz-M(O)VK(HTK01166)-OH ( Figure 2) were assembled on solid phase (Schemes 2 and 3). After cleavage/deprotection with TFA and HPLC purification, these DOTA-conjugated peptides and peptidomimetics were obtained in 1-35% isolated yields. Their nonradioactive Ga-complexed standards were obtained by incubating the DOTA-conjugated peptides and peptidomimetics with excess GaCl 3 in acetate buffer (0.1 M, pH 4.2) at 80 • C. After HPLC purification, the nonradioactive Ga-complexed standards were obtained in 16-78% isolated yields. Detailed HPLC conditions and retention times for the purification of the DOTA-conjugated peptides and peptidomimetics and their nonradioactive Ga-complexed standards are provided in Supplemental Tables S1 and S2 (see the Supplemental Materials). The identities of the DOTA-conjugated peptides and peptidomimetics and their nonradioactive Ga-complexed standards were confirmed by MS analysis.

In Vitro Enzyme Assays
In vitro enzyme assays revealed very efficient cleavage (>95%) of the 68 Ga-labeled DOTA-

PET/CT Imaging and Ex Vivo Biodistribution Studies
The pharmacokinetics of [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was first evaluated in mice via PET/CT imaging studies. As shown in Figure 6, radioactivity from the injected [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was excreted rapidly from blood pool and all background organs/tissues predominately via the renal pathway. At 1 h p.i., only kidneys and urinary bladder were clearly visualized in PET images, with low radioactivity (<2.5 %ID/g) retained in kidneys.

PET/CT Imaging and Ex Vivo Biodistribution Studies
The pharmacokinetics of [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was first evaluated in mice via PET/CT imaging studies. As shown in Figure 6, radioactivity from the injected [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was excreted rapidly from blood pool and all background organs/tissues predominately via the renal pathway. At 1 h p.i., only kidneys and urinary bladder were clearly visualized in PET images, with low radioactivity (<2.5 %ID/g) retained in kidneys.  Table 1. The ex vivo biodistribution data acquired at 1 h p.i. were consistent with the observations from their PET images ( Figure 7A and Figure 8A, and Table  1) with low background in blood pool and non-target tissues/organs, good uptake in LNCaP tumor xenografts, and high uptake in kidneys.          Table 1. The ex vivo biodistribution data acquired at 1 h p.i. were consistent with the observations from their PET images ( Figures 7A and 8A, and Table 1) with low background in blood pool and non-target tissues/organs, good uptake in LNCaP tumor xenografts, and high uptake in kidneys. [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH also showed 30% reduction in average kidney uptake (147 vs. 104 %ID/g), but the difference is not statistically significant (p = 0.055). Table 1. Biodistribution data and uptake ratios of 68 Ga-labeled PSMA-targeted tracers in LNCaP tumor-bearing mice acquired at 1 h p.i. (*** p < 0.001).

Quantification of Radio-Metabolites in Blood and Urine
Radio-metabolites of [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH and [ 68 Ga]Ga-DOTA-AmBz-MVK (HTK01166)-OH in mouse blood and urine samples were analyzed by HPLC. The blood samples were collected at 5 min p.i. due to the fast excretion nature of the small radio-metabolites, whereas the urine samples were collected at 15 min p.i. to allow sufficient time for the accumulation of enough radio-metabolites for analysis.
As shown in Figure Figure 10A). However, a major unidentified radio-metabolite, accounting for 56% of the recovered radioactivity, was also observed. Analysis of the urine samples revealed that only 20% of the recovered radioactivity was presented as the intact tracer and the remaining 80% was the expected fragment [ 68 Ga]Ga-DOTA-AmBz-Met-OH ( Figure 10B).
Analysis of blood samples from mice injected with [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH showed only 9% remaining intact and 35% of the tracer was metabolized to the expected fragment [ 68 Ga]Ga-DOTA-AmBz-Met-OH ( Figure 10A). However, a major unidentified radiometabolite, accounting for 56% of the recovered radioactivity, was also observed. Analysis of the urine samples revealed that only 20% of the recovered radioactivity was presented as the intact tracer and the remaining 80% was the expected fragment [ 68 Ga]Ga-DOTA-AmBz-Met-OH ( Figure 10B).

Discussion
The potential of NEP to recognize and cleave specific sequences has been exploited by various groups in the recent years to reduce kidney uptake of radiopharmaceuticals [1,13,18]. This is attributable to the abundant expression of NEP in the brush border membrane lining primarily of the proximal convoluted tubules of the juxtamedullary nephrons [11]. The mechanism underlying

Discussion
The potential of NEP to recognize and cleave specific sequences has been exploited by various groups in the recent years to reduce kidney uptake of radiopharmaceuticals [1,13,18]. This is attributable to the abundant expression of NEP in the brush border membrane lining primarily of the proximal convoluted tubules of the juxtamedullary nephrons [11]. The mechanism underlying degradation of certain specific linker sequences has been well elucidated [1]. The type of amino acid in the radio-metabolite(s) as well as the radiometal chelate used plays a critical role in deciding the kidney residence time of the generated radio-metabolite(s).
NEP is a type-II integral membrane glycoprotein with metalloendopeptidase activity, and also presents an even better carboxydipeptidase activity when the two situations are possible [19]. To apply this strategy to reduce the kidney uptake of radiolabeled peptides and peptidomimetics, we modified the original design as shown in Figure 2. We followed the same design with the targeting vector conjugated to the cleavable linker at the Lys side chain. Such design preserves the free carboxylic group of Lys and enhances the cleavage of the MVK linker by NEP via its carboxydipeptidase activity.
We first synthesized the model tracer [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH with an acetyl group coupled to the ε-amino group of Lys to provide the amide linkage which would be present when a targeting vector is coupled to this linker. Enzyme assays using the brush border membrane vesicles (BBMVs) extracted from mouse kidneys confirmed that [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH can be efficiently cleaved (>95%) by renal brush border enzymes (Figure 3 Figure 9A). This indicates that [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was cleared rapidly from the blood pool either as its intact form or as radio-metabolite(s). Besides kidneys, NEP is also present in some tissues although in a much lower expression level. Therefore, the cleavage of However, due to a high expression level of PSMA in kidneys, high and sustained renal uptake of PSMA-targeting radioligands are constantly observed, leading to suboptimal detection sensitivity for lesions adjacent to kidneys, and concerns for renal toxicity when radiotherapeutic agents are used. The idea of incorporating the MVK cleavable linker to a radiolabeled DOTA-conjugated PSMA-targeting radioligand is to have the radioligand cleaved by the renal brush border enzymes when it is excreted through kidneys. Since the expected radiometal-complexed DOTA-AmBz-Met-OH is not retained in kidneys, the overall renal uptake of the PSMA-targeting radioligand containing the MVK cleavable linker will be reduced.
Enzyme assays revealed that unlike the instability of [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH against renal brush border enzymes, [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01116)-OH was relatively stable with 80% remaining intact and 16% converted to the expected radio-metabolite [ 68 Ga]Ga-DOTA-AmBz-Met-OH under the same assay conditions (Figure 4). The enhanced stability could be due to the steric hindrance introduced by replacing the acetyl group with the much bulkier HTK01166 motif.  (Figures 7 and 8). The observed PET images were consistent with the ex vivo biodistribution data showing comparable uptake for all collected organs/tissues ( Table 1). The brain uptake (≤0.03 %ID/g) of both tracers was negligible indicating that they cannot freely cross the blood-brain barrier. Their fast blood clearance (~0.55 %ID/g at 1 h p.i.) suggests that the [ 68 Ga]Ga-DOTA complex was stable as free 68 Ga would be captured by transferrins leading to prolonged retention in blood pool [20]. Minimal uptake (≤0.60 %ID/g) of both tracers in liver and intestines indicates that they were excreted mainly by the renal pathway. Higher uptake was observed in PSMA-expressing LNCaP tumors (~4 %ID/g), kidneys (90-104 %ID/g) and spleen (8.9-11.5 %ID/g) suggesting that the uptake of both tracers in these tissues was PSMA-mediated [15]. This was further confirmed by PET imaging studies as co-injection of the PSMA inhibitor, 2-PMPA (0.2 mg), reduced the tumor uptake of both tracers to the background level (Figures 7 and 8).
The average kidney uptake of [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was lower than that of the previously reported [ 68 Ga]Ga-HTK01166 (104 vs. 147 %ID/g), suggesting the insertion of the cleavable MVK linker might be a useful strategy to reduce kidney uptake of radiopharmaceuticals. However, although statistically not significant (p = 0.15), a slightly higher kidney retention was observed for mice injected with  Figure 7B).
The retained kidney uptake was unlikely to have resulted from the intact [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH, as the mouse was co-injected with excess 2-PMPA to block PSMA. The retained uptake was unlikely to have resulted from the expected radio-metabolite [ 68 Ga]Ga-DOTA-AmBz-Met-OH either, as we have shown that no significant kidney retention was observed in the mouse injected with [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH ( Figure 6). The only possibility for the kidney retention would be unidentified radio-metabolite(s), which cannot bind PSMA but can be retained in kidneys. Therefore, the in vivo stability study was further conducted for [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH to discern the cause of its higher kidney retention in mice. Figure 10A, unlike the good stability observed in enzyme assays, [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was rapidly metabolized in vivo with only 9% of the tracer remaining intact in the blood at 5 min p.i. However, the expected radio-metabolite [ 68 Ga]Ga-DOTA-AmBz-Met-OH was accounted for only 35% of the recovered radioactivity, while 56% consists of an unidentified radio-metabolite.

As shown in
Interestingly, in urine samples, only [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH (20%) and [ 68 Ga]Ga-DOTA-AmBz-Met-OH (80%) were detected, and there was no presence of the unidentified radio-metabolite ( Figure 10B). The absence of the unidentified radio-metabolite in urine samples suggests that it might be retained in kidneys. This would explain the higher kidney uptake of [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH than [ 68 Ga]Ga-DOTA-AmBz-M(O)VK(HTK01166)-OH (Table 1), as well as its higher kidney retention when co-injected with 2-PMPA (Figures 7 and 8). This would also explain the insufficient reduction (~30%) in kidney uptake when compared [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH and [ 68 Ga]Ga-HTK01166 [15], which is far less than the~80% and~67% reduction in kidney uptake reported by Uehara et al. [12] and Zhang et al. [13], respectively, using the radiolabeled NOTA-MVK(Targeting vector)-OH design. The insufficient reduction reported here using the DOTA-AmBz-MVK(Targeting vector)-OH design is likely caused by the unidentified radio-metabolite that could be trapped in kidneys. Therefore, further optimization of the design of DOTA-conjugated cleavable linkers should avoid the generation of radio-metabolites that could be trapped in kidneys and cause high and sustained kidney uptake.
The identity of the unidentified fragment remains unknown ( Figure 10). This is because the core structure of HTK01166 contains no amide bonds formed by two natural amino acids. Moreover, the data from our [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH, [ 68 Ga]Ga-DOTA-AmBz-M(O)VK(HTK01166)-OH and the previously reported [ 68 Ga]Ga-NOTA-MVK-conjugated antibody fragment [12] and Exendin 4 [13], did not suggest the cleavage of the Val-Lys amide bond. Therefore, we did not expect cleavage of [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH at locations other than the Met-Val amide bond, and more investigations are needed to verify the identity of this unknown radio-metabolite.
To conclude, we showed that replacing NOTA and the aminobenzyl group in the reported NOTA-MVK linker with DOTA and AmBz, respectively, generated the model compound [ 68 Ga]Ga-DOTA-AmBz-MVK(Ac)-OH, which was still recognized and specifically cleaved at the Met-Val amide bond by NEP. Coupling a bulkier PSMA-targeting vector to the side chain of Lys in DOTA-AmBz-MVK enhanced its stability against NEP, but possibly also rendered its vulnerability against other enzyme(s) as evident by the formation of an unidentified radio-metabolite that could be retained in kidneys. Nevertheless, the renal uptake of the resulting [ 68 Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was still lower than that of [ 68 Ga]Ga-HTK01166. These data demonstrated that MVK could be a promising cleavable linker for use to reduce renal uptake of radiolabeled DOTA-conjugated tumor-targeting peptides and peptidomimetics. This strategy can be used to enhance detection sensitivity of the imaging agents for lesions adjacent to kidneys, and improve the tumor-to-kidney absorbed dose ratio for the radiotherapeutic agents.

General Methods
Fmoc-l-Lys(pentynoyl)-OH (1) was synthesized according to the literature procedures [17]. Brush border membrane vesicles (BBMVs) were extracted from mouse kidneys following literature procedures [21]. All other chemicals were procured from commercial sources and used without further purification. All peptides and peptidomimetics were synthesized either on an AAPPTec (Louisville, KY, USA) Endeavor 90 peptide synthesizer or a CEM (Matthews, NC, USA) Liberty Blue™ automated microwave peptide synthesizer. Purification and quality control of radiolabeling precursor, nonradioactive Ga-complexed standards and 68 Ga-labeled peptides and peptidomimetics were performed on Agilent (Santa Clara, CA, USA) HPLC systems equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 220 nm), and a Bioscan (Washington, DC, USA) NaI scintillation detector. The operation of Agilent HPLC systems was controlled using the Agilent ChemStation software. HPLC columns used were a semipreparative column (Luna C18, 5 µm particle size, 100 Å pore size, 250 × 10 mm) and an analytical column (Luna C18, 5 µm particle size, 100 Å pore size, 250 × 4.6 mm) from Phenomenex (Torrance, CA, USA). The HPLC solvents were A: H 2 O containing 0.1% TFA; B: CH 3 CN containing 0.1% TFA. The collected HPLC eluates containing the desired peptides were lyophilized using a Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze drier. 1 H-NMR spectrum was acquired using an AVANCE Bruker 400 MHz NMR spectrometer equipped with BBI probe with Z gradients. Mass analyses were performed using an AB SCIEX (Framingham, MA, USA) 4000 QTRAP mass spectrometer system with an ESI ion source. C18 Sep-Pak cartridges (1 cm 3 , 50 mg) were obtained from Waters (Milford, MA, USA). 68 Ga was eluted from an iThemba Laboratories (Somerset West, South Africa) generator and purified according to the previously published procedures using a DGA resin column from Eichrom Technologies LLC (Lisle, IL, USA) [22,23]. Radioactivity of 68 Ga-labeled peptides and peptidomimetics was measured using a Capintec (Ramsey, NJ, USA) CRC-25R/W dose calibrator. PET/CT imaging was performed using a Siemens Inveon (Knoxville, TN, USA) micro PET/CT scanner. The radioactivity of mouse tissues collected from biodistribution studies was counted using a PerkinElmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.

Synthesis of 68 Ga-Labeled Peptides and Peptidomimetics
Purification of 68 Ga eluated from 68 Ge/ 68 Ga generator and labeling experiments were performed following our previously published procedures [22,23]. Purified 68 Ga in 0.5 mL of water was added into a 4-mL glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and 25 µg of the precursor. The radiolabeling reaction was carried out under microwave heating for 1 min. The reaction mixtures were purified using the semipreparative HPLC column and eluted with 84/16 A/B for The eluate fraction containing the radiolabeled product was collected, diluted with water (50 mL), and passed through a C18 Sep-Pak cartridge that was prewashed with ethanol (10 mL) and water (10 mL). After washing the C18 Sep-Pak cartridge with water (10 mL), the 68 Ga-labeled product was eluted off the cartridge with ethanol (0.4 mL) and diluted with saline for all the in vitro enzyme assays, PET imaging, ex vivo biodistribution and in vivo stability studies.

In Vitro Enzyme Assay
The enzymatic recognition of synthesized peptides and peptidomimetics was determined by incubating the 68 Ga-labeled peptides and peptidomimetics with the extracted BBMVs at 37 • C for 1 h. For the assay, aliquots (25 µL) of the enzyme solution (1.52 mg/mL) and enzyme buffer (250 mM NaCl, 57.5 mM Tris-base; adjusted to a pH 7.5) were mixed in a 96-well clear bottom plate and incubated at 37 • C for 10 min. The mixtures also contained 100 ppm ascorbic acid to prevent oxidation of the Met residue in the tested peptides and peptidomimetics during the assay. The radiolabeled peptide (50 µL, 3.7 MBq) was then added to the test well. The control well contained phosphoramidon, a potent NEP inhibitor, at a final concentration of 1 mmol/L in addition to the contents of the test well. After 1 h incubation, all reactions were quenched using equal volume of CH 3 CN and centrifuged at 13,000 rpm for 10 min. The resulting supernatant was collected and analyzed using the analytical HPLC column to identify and quantify radio-metabolite(s). The assay was performed in duplicates.

PET/CT Imaging and Ex Vivo Biodistribution in Tumor-Bearing Mice
All imaging and biodistribution studies were performed using male NOD-scid IL2Rg null (NSG) mice and conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. For tumor inoculations, mice were anesthetized by inhalation with 2% isoflurane in oxygen and implanted subcutaneously with 5 × 10 6 LNCaP cells below the left shoulder. Imaging and biodistribution studies were performed only after tumors grew to 5−8 mm in diameter over a period of 5−7 weeks.
For PET/CT imaging studies,~3−6 MBq of the 68 Ga-labeled tracer was injected through the tail vein. For the blocking study, 2-PMPA (0.2 mg) was co-injected with the tracer. Mice were allowed to recover and roam freely in the cages after injecting the tracer. At 45 min p.i., mice were sedated again and positioned on the scanner. First, a 10 min CT scan was conducted for localization and attenuation correction for reconstruction of PET images, before a 10 min PET image was acquired. Heating pads were used during the entire procedure to keep the mice warm.
For ex vivo biodistribution studies, mice were injected with~1.5−3 MBq of the 68 Ga-labeled tracer. At 1 h p.i., mice were euthanized, blood was drawn from heart, and organs/tissues of interest were collected, rinsed with PBS, blotted dry, weighed, and counted using an automated gamma counter. The uptake in each organ/tissue was normalized to the injected dose and expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Quantification of Radio-Metabolites in Blood and Urine
Male NSG mice were injected with 3-17 MBq of the 68 Ga-labeled peptide. For blood profiling, mice were anesthetized with 2% isoflurane in O 2 and euthanized by CO 2 inhalation at 5 min p.i. Blood draw was then performed by cardiac puncture and blood was collected in an eppendorf tube with equal volume of CH 3 CN. Each tube was then centrifuged at RT for 10-15 min and the resulting supernatant was collected and analyzed using the analytical HPLC column to identify and quantify radio-metabolite(s) in blood. For the purpose of urine profiling, urine was collected after euthanizing the mice at 15 min p.i. The urine samples were also collected and analyzed using the analytical HPLC column to identify and quantify radio-metabolite(s) in urine.  [15]. The difference was considered statistically significant when the p value was <0.05.