Positron Emission Tomography Imaging of the Gastrin-Releasing Peptide Receptor with a Novel Bombesin Analogue

Abstract

The gastrin-releasing peptide receptor (GRPR), a G protein-coupled receptor, is overexpressed in solid malignancies and particularly in prostate cancer. We synthesized a novel bombesin derivative, [68Ga]Ga-ProBOMB1, evaluated its pharmacokinetics and potential to image GRPR expression with positron emission tomography (PET), and compared it with [68Ga]Ga-NeoBOMB1. ProBOMB1 (DOTA-pABzA-DIG-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ(CH2N)-Pro-NH2) was synthesized by solid-phase peptide synthesis. The polyaminocarboxylate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminal and separated from the GRPR-targeting sequence by a p-aminomethylaniline-diglycolic acid (pABzA-DIG) linker. The binding affinity to GRPR was determined using a cell-based competition assay, whereas the agonist/antagonist property was determined with a calcium efflux assay. ProBOMB1 was radiolabeled with 68GaCl3. PET imaging and biodistribution studies were performed in male immunocompromised mice bearing PC-3 prostate cancer xenografts. Blocking experiments were performed with coinjection of [d-Phe6,Leu-NHEt13,des-Met14]bombesin(6-14). Dosimetry calculations were performed with OLINDA software. ProBOMB1 and the nonradioactive Ga-ProBOMB were obtained in 1.1 and 67% yield, respectively. The Ki value of Ga-ProBOMB1 for GRPR was 3.97 ± 0.76 nM. Ga-ProBOMB1 behaved as an antagonist for GRPR. [68Ga]Ga-ProBOMB1 was obtained in 48.2 ± 10.9% decay-corrected radiochemical yield with 121 ± 46.9 GBq/μmol molar activity and >95% radiochemical purity. Imaging/biodistribution studies showed that the excretion of [68Ga]Ga-ProBOMB1 was primarily through the renal pathway. At 1 h postinjection (p.i.), PC-3 tumor xenografts were clearly delineated in PET images with excellent contrast. The tumor uptake for [68Ga]Ga-ProBOMB1 was 8.17 ± 2.57 percent injected dose per gram (% ID/g) and 9.83 ± 1.48% ID/g for [68Ga]Ga-NeoBOMB1, based on biodistribution studies at 1 h p.i. This corresponded to tumor-to-blood and tumor-to-muscle uptake ratios of 20.6 ± 6.79 and 106 ± 57.7 for [68Ga]Ga-ProBOMB1 and 8.38 ± 0.78 and 39.0 ± 12.6 for [68Ga]Ga-NeoBOMB1, respectively. Blockade with [d-Phe6,Leu-NHEt13,des-Met14]bombesin(6-14) significantly reduced the average uptake of [68Ga]Ga-ProBOMB1 in tumors by 62%. The total absorbed dose was lower for [68Ga]Ga-ProBOMB1 in all organs except for bladder compared with [68Ga]Ga-NeoBOMB1. Our data suggest that [68Ga]Ga-ProBOMB1 is an excellent radiotracer for imaging GRPR expression with PET. [68Ga]Ga-ProBOMB1 achieved a similar uptake as [68Ga]Ga-NeoBOMB1 in tumors, with enhanced contrast and lower whole-body absorbed dose.

Comparison of biological properties of [177Lu]Lu‐ProBOMB1 and [177Lu]Lu‐NeoBOMB1 for GRPR targeting

“…was added to 25 μg of ProBOMB1 in 0.5 mL of sodium acetate buffer (0.1M; pH 4.5) and incubated at 95 C for 15 minutes. 15 To obtain the nonradioactive standard, NeoBOMB1 (3.0 mg, 1.9 μmol) and LuCl 3 (0.2M, 48 μL, 9.6 μmol) in 450 μL of sodium acetate buffer (0.1 M, pH 4.2) and 60 μL of MeCN were incubated at 90 C for 15 minutes and purified by HPLC using the preparative column (29.6% acetonitrile and 0.1% TFA in water; flow rate: 30 mL/min). Determination of molar activity was conducted using the analytical column (24% acetonitrile and 0.1% TFA in water; flow rate: 2.0 mL/min, retention time: 7.6 minutes).…”

“…ProBOMB1 ( Figure 1) was synthesized according to published procedures. 15 To obtain the nonradioactive standard, ProBOMB1 (1.46 mg, 88.7 μmol) and LuCl 3 (0.2M, 22.2 μL, 4.43 mmol) in 500 μL of sodium acetate buffer (0.1M, pH 4.5) were incubated at 95 C for 30 minutes and purified by HPLC using the semipreparative column (23% acetonitrile and 0.1% TFA in water; flow rate: 4.5 mL/min). was added to 25 μg of ProBOMB1 in 0.5 mL of sodium acetate buffer (0.1M; pH 4.5) and incubated at 95 C for 15 minutes.…”

“…[1][2][3][4][5] BBN, a peptide isolated from the frog, Bombina bombina, has a mammalian ortholog named the gastrin-releasing peptide (GRP) and both bind GRPR. 7,[9][10][11][12] 13,14 In a previous article, we modified a sequence initially published by the Schally group, RC-3950-II, to adapt it for theranostic application yielding DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH 2 (ProBOMB1), which we characterized after labeling with nat/68 Ga. 8,15 The radiotracer, [ 68 Ga]Ga-ProBOMB1, had excellent characteristics for imaging GRPR. In nuclear medicine, the use of peptides as delivery vectors is an effective strategy for targeting GPCRs (ie, somatostatin, CXC chemokine receptor 4, cholecystokinin-2, and substance P).…”

“…,15 Labeled with 68 Ga, the radiotracer had lower background activity, a faster clearance, and low pancreas uptake compared with [ 68 Ga]Ga-Neo-BOMB1. We substituted 68 Ga with 177 Lu to leverage the excellent contrast to obtain a high therapeutic index candidate for peptide receptor radioligand therapy.While there are numerous examples in the literature of such substitutions having limited effects on biodistribution (ie, some somatostatin receptor ligands, α v β 3 binding peptides, and GRPR ligands), this was not the case with ProBOMB1.…”

“…7,[9][10][11][12] Examples of agents tested in the clinic include [ 68 Ga]Ga-NeoBOMB1, [ 68 Ga]Ga-RM2, [ 68 Ga]Ga-SB3, [ 64 Cu]Cu-CB-TE2A-AR06, [ 68 Ga]Ga-NOTA-Aca-BBN, and [ 99m Tc]Tc-demobesin-4. 13,14 In a previous article, we modified a sequence initially published by the Schally group, RC-3950-II, to adapt it for theranostic application yielding DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH 2 (ProBOMB1), which we characterized after labeling with nat/68 Ga. 8,15 The radiotracer, [ 68 Ga]Ga-ProBOMB1, had excellent characteristics for imaging GRPR. In particular, contrast between tumor tissue and healthy tissue was better than that of other leading compounds that have been trialed in clinical settings.…”