Synthesis and Evaluation of Novel 68Ga-Labeled [D-Phe6,Leu13ψThz14]bombesin(6-14) Analogs for Cancer Imaging with Positron Emission Tomography

Gastrin-releasing peptide receptor (GRPR) is overexpressed in various cancers and is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake and/or metabolic instability observed for most reported GRPR-targeted radioligands might limit their clinical applications. Our group recently reported a GRPR-targeted antagonist tracer, [68Ga]Ga-TacsBOMB2 ([68Ga]Ga-DOTA-Pip-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13ψThz14-NH2), which showed a minimal pancreas uptake in a preclinical mouse model. In this study, we synthesized four derivatives with unnatural amino acid substitutions (Tle10-derived Ga-LW01158, NMe-His12-derived Ga-LW01160, α-Me-Trp8- and Tle10-derived Ga-LW01186, and Tle10- and N-Me-Gly11-derived Ga-LW02002) and evaluated their potential for detecting GRPR-expressing tumors with positron emission tomography (PET). The binding affinities (Ki(GRPR)) of Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 were 5.11 ± 0.47, 187 ± 17.8, 6.94 ± 0.95, and 11.0 ± 0.39 nM, respectively. [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 enabled clear visualization of subcutaneously implanted human prostate cancer PC-3 tumor xenografts in mice in PET images. Ex vivo biodistribution studies showed that [68Ga]Ga-LW01158 had the highest tumor uptake (11.2 ± 0.65 %ID/g) and good tumor-to-background uptake ratios at 1 h post-injection. Comparable in vivo stabilities were observed for [68Ga]Ga-LW01158, [68Ga]Ga-LW01186, and [68Ga]Ga-LW02002 (76.5–80.7% remaining intact in mouse plasma at 15 min post-injection). In summary, the Tle10 substitution, either alone or combined with α-Me-Trp8 or NMe-Gly11 substitution, in Ga-TacsBOMB2 generates derivatives that retained good GRPR binding affinity and in vivo stability. With good tumor uptake and tumor-to-background imaging contrast, [68Ga]Ga-LW01158 is promising for detecting GRPR-expressing lesions with PET.


Introduction
As a member of the transmembrane G protein-coupled receptors, gastrin-releasing peptide receptor (GRPR) is expressed in the pancreas, gastrointestinal tract, and central nervous system, and it regulates a series of physiological functions such as hormone secretion, smooth muscle contraction, and synaptic plasticity [1][2][3].Moreover, GRPR is overexpressed in a variety of malignancies, including breast, prostate, lung, and colon cancers, and the activation of GRPR leads to the proliferation of cancer cells [4][5][6][7].Thus, GRPR is considered a promising target for the design of targeted radiopharmaceuticals for the diagnosis and radioligand therapy of GRPR-expressing cancers.Bombesin (BBN), isolated from the skin of the European frog, Bombina bombina, is a natural exogenous ligand with a good binding affinity toward GRPR.The heptapeptide sequence at the C-terminus (bombesin (8)(9)(10)(11)(12)(13)(14)) is the minimal sequence needed for binding to GRPR with a high affinity.Thus, this peptide sequence has been used for the design of GRPR-targeted radiopharmaceuticals for cancer diagnosis and radioligand therapy [8][9][10][11][12][13][14][15].Although several GRPR-targeted radiotracers have been evaluated in the clinic, the extraordinarily high pancreas uptake might limit the detection of pancreatic cancer and the metastatic lesions of other cancers in and/or adjacent to the pancreas.In addition, to avoid damage to the pancreas, the maximum tolerated dose might have to be lowered, and this could potentially lead to a suboptimal treatment efficacy for radiotherapeutic application [9,13,14,16].

Syntheses of GRPR-Targeted Ligands
The yields for the synthesis of LW01158, LW01160, LW01186, and LW02002 ranged from 8 to 32%, and the yields for the synthesis of their nonradioactive Ga-complexed standards ranged from 76 to 81% (Tables S1 and S2).The identities of all precursors and nonradioactive Ga-complexed standards were confirmed by MS analyses (Tables S1 and  S2 and Figures S1-S8). 68Ga-labeled LW01158, LW01186, and LW02002 were purified by Similar to most of the reported GRPR-targeted ligands, Ga-TacsBOMB2 could potentially be enzymatically degraded in vivo, especially by the neutral endopeptidase 24.11 (NEP, EC 3.4.24.11, neprilysin) [20,21].The amide bonds between Gln 7 -Trp 8 , Trp 8 -Ala 9 , Ala 9 -Val 10 , and His 12 -Leu 13 have been identified as the cleavage sites of clinically validated GRPR-targeted radioligands derived from RM2 and AMBA [10,22].In this study, we hypothesized that (1) the amide bonds between Gln 7 -Trp 8 , Trp 8 -Ala 9 , Ala 9 -Val 10 , and His 12 -Leu 13 in Ga-TacsBOMB2 (Figure 1A) are also potential cleavage sites of peptidases, and (2) replacing the amino acids adjacent to the potential cleavage sites in Ga-TacsBOMB2 with a closely related unnatural amino acid could improve its vivo stability and potentially retain a high GRPR binding affinity and low pancreas uptake characteristics.
Hence, in this study, we synthesized Ga-labeled LW01158, LW01160, LW01186, and LW02002 (Figure 1B-E) by replacing the natural amino acids adjacent to the cleavage sites with a closely related unnatural amino acid.We determined their antagonist/agonist characteristics with an in vitro fluorescence-based calcium release assay.The potential of these ligands for detecting GRPR-expressing cancer was evaluated by an in vitro competition binding assay, PET imaging, and ex vivo biodistribution studies in PC-3 tumor-bearing mice.The biodistribution data of these novel tracers were compared with previously reported data on [ 68 Ga]Ga-RM2 (Figure 1F) obtained using the same preclinical tumor model [19].

PET Imaging and Ex Vivo Biodistribution
The PC-3 tumor xenografts were clearly visualized in PET images acquired at 1 h postinjection using [ 68 Ga]Ga-LW01158, [ 68 Ga]Ga-LW01186, and [ 68 Ga]Ga-LW02002 (Figure 4).All three tracers were primarily excreted via the renal pathway.[ 68 Ga]Ga-LW01158 had the best tumor-to-background contrast among all three tracers.While [ 68 Ga]Ga-LW01186 showed significant pancreas and liver uptake, the uptake in these two organs was much lower for [ 68 Ga]Ga-LW01158 and [ 68 Ga]Ga-LW02002.Co-injection with 100 µg of nonradioactive standard decreased the uptake of [ 68 Ga]Ga-LW01158 in the PC-3 tumor xenograft to a value that was close to the background level.
We first determined the binding affinities of Ga-LW01158 and Ga-LW01160 using an in vitro competition binding assay (Figure 2).The K i value of Ga-LW01158 was 5.11 ± 0.47 nM, which was better than that of Ga-TacsBOMB2 (7.08 ± 0.65 nM) [19].This observation is consistent with our previous finding showing that Tle 10 substitution on the GRPR agonist Ga-TacBOMB2 improves binding affinity [23].However, Ga-LW01160 showed very poor binding toward GRPR (K i = 187 ± 17.8 nM), while the previously reported NMe-His 12 substitution significantly improved the binding affinity of Ga-TacBOMB2 from 7.62 ± 0.19 nM to 2.98 ± 0.69 nM [23,24].These data demonstrate that Tle 10 substitution is tolerable in both GRPR agonists and antagonists, while NMe-His 12 substitution can only be applied to GRPR agonists without significantly reducing the binding affinity.One possible explanation for this observation is that GRPR agonists and antagonists might bind to the receptors in different configurations so that modifications to some specific amino acids are tolerable only by either antagonists or agonists.
Next, we introduced an additional αMe-Trp 8 substitution to LW01158 to obtain LW01186 (Figure 1D).αMe-Trp 8 substitution has been successfully used by the Wester group for the design of the potent and in vivo-stable GRPR-targeted antagonist AMTG, derived from RM2 [25].NMe-Gly 11 substitution has also been reported for the design of GRPR-targeted ligands used to improve in vivo stability [26,27].Previously, we developed a GRPR antagonist, Ga-TacsBOMB5, by introducing the NMe-Gly 11 substitution to Ga-TacsBOMB2 [19].Though [ 68 Ga]Ga-TacsBOMB5 was not metabolically more stable than [ 68 Ga]Ga-TacsBOMB2, it had a better PC-3 tumor uptake and tumor-to-background imaging contrast than [ 68 Ga]Ga-TacsBOMB2 at 1 h post-injection [19].Therefore, in this study, we also combined NMe-Gly 11 and Tle 10 substitutions to generate Ga-LW02002 (Figure 1E).As expected, good GRPR binding affinities for both Ga-LW01186 and Ga-LW02002 were observed (K i = 6.94 ± 0.95 and 11.0 ± 0.39 nM, respectively) (Figure 2).These data also support that the configurations of GRPR binding with agonists and antagonists might be different.While αMe-Trp 8 and NMe-Gly 11 substitutions are tolerable in antagonists, we previously showed that αMe-Trp 8 and NMe-Gly 11 substitutions significantly reduce the binding affinity of GRPR agonists.
The GRPR antagonist characteristics of the three potent Ga-TacsBOMB2 derivatives were determined using in vitro intracellular calcium release assays (Figure 3).In comparison with the positive control (ATP) and agonist control (bombesin), Ga-LW01158, Ga-LW01186, and Ga-LW02002 induced significantly lower intracellular Ca 2+ efflux.This indicates that Tle 10 substitution in Ga-TacsBOMB2, either alone or in combination with αMe-Trp 8 or NMe-Gly 11 substitution, retains antagonist characteristics.
Compared with the previously reported biodistribution data of [ 68 Ga]Ga-RM2 (Table S4) [19], all three [ 68 Ga]Ga-TacsBOMB2 derivatives showed significantly lower uptake in the pancreas.This is consistent with our previous finding showing that [D-Phe 6 ,Leu 13 ψThz 14 ]Bombesin(6-14) is a promising pharmacophore for the design of GRPRtargeted radiopharmaceuticals with a minimal pancreas uptake.One possible explanation is that these three [ 68 Ga]Ga-TacsBOMB2 derivatives are more selective for binding to the human GRPR expressed in PC-3 tumors in comparison with the mouse GRPR expressed in mouse pancreas.The low pancreas uptake of these three [ 68 Ga]Ga-TacsBOMB2 derivatives also demonstrates that αMe-Trp 8 , NMe-Gly 11 , and Tle 10 substitutions do not significantly increase the pancreas uptake of the resulting GRPR-targeted tracers.With a significantly lower uptake in the pancreas and a comparable tumor uptake compared with the clinically validated [ 68 Ga]Ga-RM2, [ 68 Ga]Ga-LW01158 is a promising radiopharmaceutical for detecting GRPR-expressing lesions with PET, especially for lesions in or adjacent to the pancreas.Similarly, LW01158 might be promising for labeling with 177 Lu for radioligand therapy to minimize toxicity to the pancreas.
A blocking study (Figure 7 and Table S4) was conducted to tease out the specificity of our top candidate, [ 68 Ga]Ga-LW01158.The uptake in GRPR-expressing PC-3 tumor xenografts was reduced by >80% with the co-injection of 100 µg of nonradioactive standard, confirming the tumor uptake of [ 68 Ga]Ga-LW01158 is specific.Moreover, significant reductions were also observed in the pancreas (12.0 ± 1.41 to 0.66 ± 0.28%ID/g; p < 0.001), stomach (1.30 ± 0.41 to 0.42 ± 0.15 %ID/g, p < 0.01), and small intestine (2.46 ± 0.30 to 1.17 ± 0.39%ID/g, p < 0.01).This is in agreement with the physiological expression pattern of GRPR in normal tissue/organs [1].In addition, a significantly increased uptake was observed in kidneys (2.98 ± 0.34 to 21.1 ± 11.0%ID/g; p < 0.01).This is most likely due to the competitive binding of the nonradioactive standard to the GRPR in PC-3 tumors, increasing the amount of free [ 68 Ga]Ga-LW01158 to be metabolized and excreted via the renal pathway.Furthermore, GRPR-targeted ligands are mainly metabolized by NEP, which is highly expressed in kidneys [20,21].Co-injection with a significant amount of nonradioactive standard could saturate the metabolism of [ 68 Ga]Ga-LW01158 caused by NEP in kidneys, leading to higher kidney absorption and the retention of [ 68 Ga]Ga-LW01158.
In vivo stability studies revealed that all three [ 68 Ga]Ga-TacsBOMB2 derivatives were relatively stable in vivo with 76.5 to 80.7% of the tracer remaining intact in mouse plasma at 15 min post-injection.These values were comparable to that of the previously reported [ 68 Ga]Ga-TacsBOMB2 (83.3 ± 1.45%) [19].This suggests that, among the potential cleavage sites on the [ 68 Ga]Ga-TacsBOMB2 pharmacophore for peptidases, the amide bond between His 12 -Leu 13 is the major one.Since the amide bond between His 12 -Leu 13 was already stabilized by the introduction of a reduced peptide bond (Leu 13 ψThz 14 ), no further improvements in in vivo stability were observed with the additional Tle 10 substitution, either alone or in combination with αMe-Trp 8 or NMe-Gly 11 substitution.

General Methods
Fmoc-LeuψThz-OH hydrochloride was synthesized following our previously published procedures [19].All other chemicals and solvents were purchased from commercial sources and used without further purification.GRPR-targeted peptides were synthesized on solid phase using an AAPPTec (Louisville, KY, USA) Endeavor 90 peptide synthesizer.Purification and quality control of DOTA-conjugated peptides and their nat Ga/ 68 Gacomplexed analogs were conducted on 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 operation of Agilent HPLC systems was controlled using the Agilent ChemStation software (Version A.01.05 (1.3.19.115)).A semi-preparative column (Luna C18; 5 µm; 250 × 10 mm) and an analytical column (Luna C18; 5 µm; 250 × 4.6 mm) purchased from Phenomenex (Torrance, CA, USA) were used for purification and quality control, respectively.The HPLC eluates were collected and lyophilized with a Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze-drier.MS analyses of DOTA-conjugated peptides and their nat Gacomplexed analogs were performed with a Waters (Milford, MA, USA) Acquity QDa mass spectrometer equipped with a 2489 UV/Vis detector and an e2695 Separations module.C18 Sep-Pak cartridges (1 cm 3 , 50 mg) were purchased from Waters. 68Ga was eluted from an ITM Medical Isotopes GmbH (Munich, Germany) generator and purified according to previously published procedures using a DGA resin column from Eichrom Technologies LLC (Lisle, IL, USA) [28].The radioactivity of 68 Ga-labeled peptides was measured using a Capintec (Ramsey, NJ, USA) CRC ® -25R/W dose calibrator.The radioactivity measurements for samples collected from biodistribution studies, binding assays, in vivo stability tests, and LogD 7.4 assays were counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.

Synthesis of Nonradioactive Ga-Complexed Standards
The nonradioactive Ga-complexed standards were synthesized by incubating the DOTA-conjugated precursor (1 eq.) and GaCl 3 (1.0M; 5 eq.) in NaOAc buffer (0.1 M; 500 µL; pH 4.5) at 80 • C for 15 min.The reaction mixture was then purified with HPLC (semipreparative column).The HPLC eluates containing the desired peptide were collected and lyophilized.The HPLC conditions, retention times, isolated yields, and MS confirmations of the nonradioactive Ga-complexed standards are provided in Table S2 and Figures S5-S8.

Synthesis of 68 Ga-Labeled Tracers
The radiolabeling experiments were performed following previously published procedures [28][29][30].Purified 68 GaCl 3 in 0.5 mL of water was added to a vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and 10 µL of precursor solution (1 mM).The radiolabeling reaction was conducted by 100 • C microwave heating for 1 min (Monowave 200, Anton Paar, Graz, Austria) followed by HPLC purification using the semi-preparative column.The eluate fraction containing the radiolabeled product was collected, diluted with water (50 mL), and passed through a C18 Sep-Pak cartridge that was pre-washed with ethanol (1 mL) and water (2 mL).The 68 Ga-labeled product was eluted off the cartridge with ethanol (0.4 mL) containing 1% ascorbic acid and diluted with PBS containing 1% ascorbic acid for imaging and biodistribution studies.Quality control was performed with HPLC on the analytical column.The HPLC conditions and retention times for purification and quality control are provided in Table S3.

LogD 7.4 Measurement
The LogD 7.4 values of [ 68 Ga]Ga-LW01158, [ 68 Ga]Ga-LW01186, and [ 68 Ga]Ga-LW02002 were measured using the shake flask method following previously published procedures [28].Briefly, an aliquot of 68 Ga-labeled peptide was added to a 15 mL falcon tube containing a mixture of n-octanol (3 mL) and DPBS (3 mL; 0.1 M; pH 7.4).The mixture was vortexed for 1 min followed by centrifugation at 3000 rpm for 15 min.Samples of the n-octanol (1 mL) and buffer (1 mL) layers were collected and measured in a gamma counter.LogD 7.4 was calculated with the following equation: LogD 7.4 = log 10 [(counts in the n-octanol phase)/(counts in the buffer phase)].

Cell Culture
Known to overexpress GRPR, the PC-3 cell line, a human prostate cancer cell line, has been widely used for the in vitro and in vivo evaluation of GRPR-targeted ligands for decades [4,8].Thus, our group chose the PC-3 cell line for this study.The PC-3 cells obtained from ATCC (via Cedarlane, Burlington, Canada) were cultured in RPMI 1640 medium (Life Technologies Corporations, Carlsbad, CA, USA) supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 .The cells were confirmed to be pathogen-free via the IMPACT Rodent Pathogen Test (IDEXX BioAnalytics, Columbia, MO, USA).Cells grown to 80-90% confluence were washed with sterile DPBS (pH 7.4) and collected after 1 min trypsinization at 37 • C. The cell concentration was measured in duplicate using a Moxi mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA).

In Vitro Competition Binding Assay
Inhibition constants (K i ) of GRPR-targeted ligands were measured by in vitro competition binding assay using PC-3 cells and [ 125 I-Tyr 4 ]Bombesin as the radioligand.PC-3 cells were seeded in 24-well poly-D-lysine plates at 2 × 10 5 cells/well 48 h prior to the assay.The growth medium was replaced with 400 µL of reaction medium (RPMI 1640 containing 2 mg/mL of BSA and 20 mM of HEPES).After 1 h incubation at 37 • C. Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 in 50 µL of reaction medium with decreasing concentrations (10 µM to 1 pM) and 50 µL of 0.01 nM [ 125 I-Tyr 4 ]Bombesin were added to the wells followed by incubation with moderate agitation for 1 h at 37 • C. Cells were gently washed with ice-cold PBS twice, harvested via trypsinization, and counted for radioactivity on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.Data were analyzed using nonlinear regression (one binding site model for competition assay) with the GraphPad (San Diego, CA, USA) Prism 8 software (Version 8.4.3).

Ex Vivo Biodistribution, PET/CT Imaging, and In Vivo Stability Studies
PET/CT imaging, biodistribution, and in vivo stability studies were conducted using male NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG) mice following previously published procedures [28,[31][32][33].The experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia.The mice were anesthetized through inhalation of 2.5% isoflurane in 2 mL/min oxygen and implanted subcutaneously with 5 × 10 6 PC-3 cells (100 µL; 1:1 PBS:Matrigel) behind the left shoulder.Mice were used for PET/CT imaging and biodistribution studies when the tumor grew to 5-8 mm in diameter over around 4 weeks.PET/CT imaging experiments were performed on a Siemens (Knoxville, TN, USA) Inveon micro-PET/CT scanner.The tumor-bearing mice were injected with 3-5 MBq of 68 Ga-labeled tracer through a lateral caudal tail vein under anesthesia, followed by recovery and roaming freely in their cages during the uptake period.At 50 min post-injection, a 10 min CT scan was conducted first for localization and attenuation correction after segmentation to reconstruct the PET images, followed by a 10 min static PET imaging acquisition.
For biodistribution studies, the mice were injected with the radiotracer (2-4 MBq) via the tail vein as described above.For blocking, the mice were co-injected with [ 68 Ga]Ga-LW01158 and 100 µg of its nonradioactive standard.At 1 h post-injection, the mice were anesthetized via isoflurane inhalation and euthanized via CO 2 inhalation.Blood was collected through cardiac puncture, and organs/tissues of interest were collected, weighed, and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.
For in vivo stability studies, 5-13 MBq of [ 68 Ga]Ga-LW01158, [ 68 Ga]Ga-LW01186, or [ 68 Ga]Ga -LW02002 was injected via a lateral caudal tail vein into healthy male NRG mice (n = 3).At 15 min post-injection, the urine and blood samples were collected after the mice were anesthetized and euthanized.The plasma was extracted from whole blood by adding CH 3 CN (500 µL), 1 min of vortex, centrifugation, and the separation of supernatant.The plasma and urine samples were analyzed via radio-HPLC by using the conditions for the quality control of these 68 Ga-labeled radioligands (Table S3).

Statistical Analysis
Statistical analyses were performed with Student's t-test using the Microsoft (Redmond, WA, USA) Excel software (Version 16.84 (24041420)).A comparison of biodistribution data between two tracers was conducted using an unpaired two-tailed test.The unpaired one-tailed test was used to compare the biodistribution data of [ 68 Ga]Ga-LW01158 with/without co-injection of nonradioactive Ga-LW01158.A statistically significant difference was considered when the adjusted p-value was <0.05.

Conclusions
The Tle 10 substitution, either alone or in combination with αMe-Trp 8 or NMe-Gly 11 , in the GRPR binding sequence of Ga-TacsBOMB2 generates derivatives that retained good GRPR binding affinity, antagonist characteristics, and good in vivo stability.However, the substitution of His 12 with NMe-His leads to a significant decrease in GRPR binding affinity.In comparison with the clinically validated [ 68 Ga]Ga-RM2, [ 68 Ga]Ga-LW01158 has comparable tumor uptake but much less pancreas uptake.Therefore, [ 68 Ga]Ga-LW01158 is promising for clinical development for detecting GRPR-expressing lesions with PET, particularly for lesions in or adjacent to the pancreas.With a superior tumor-to-pancreas uptake ratio, [ 68 Ga]Ga-LW02002 might be more promising for detecting cancer lesions adjacent to and in the pancreas.

Patents
The compounds disclosed in this report are covered by a recent patent application (PCT/CA2023/050401; filing date: 23 March 2023).Lei Wang, Zhengxing Zhang, Chengcheng Zhang, François Bénard, and Kuo-Shyan Lin are listed as inventors in this filed patent application.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ph17050621/s1:Table S1: MS characterizations, yields, and HPLC purification conditions of LW01158, LW01160, LW01186, and LW02002.Table S2: MS characterizations, yields, and HPLC purification conditions of Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002.Table S3: HPLC conditions for the purification and quality control of 68 Ga-labeled LW01158, LW01186, and LW02002.Table S4: Biodistribution and uptake ratios of 68 Ga-labeled GRPRtargeted tracers in PC-3 tumor-bearing mice.Figure S1: The MS spectrum of LW01158. Figure S2: The MS spectrum of LW01160. Figure S3: The MS spectrum of LW01186. Figure S4: The MS spectrum of LW02002. Figure S5: The MS spectrum of Ga-LW01158. Figure S6: The MS spectrum of Ga-LW01160. Figure S7: The MS spectrum of Ga-LW01186. Figure S8: The MS spectrum of Ga-LW02002. Figure S9: Representative radio-HPLC chromatograms from analysis of an intact fraction of [ 68 Ga]Ga-LW01158 in mouse plasma and urine samples.Figure S10: Representative radio-HPLC chromatograms from analysis of an intact fraction of [ 68 Ga]Ga-LW01186 in mouse plasma and urine samples.Figure S11: Representative radio-HPLC chromatograms from analysis of an intact fraction of [ 68 Ga]Ga-LW02002 in mouse plasma and urine samples.