In vivo enzyme inhibition improves the targeting of [177Lu]DOTA-GRP(13-27) in GRPR-positive tumors in mice.

INTRODUCTION: Gastrin-releasing peptide receptors (GRPR) and GRP-derived analogs have attracted attention due to high receptor expression in frequently occurring human neoplasia. The authors recently synthesized a series of GRPR-affine peptide analogs based on the 27-mer GRP and derivatized with the DOTA chelator at the N-terminus for (111)In-labeling. In this study, the authors evaluated the most promising from these series, DOTA-GRP(13-27), after radiolabeling with (177)Lu for future therapeutic applications. In addition, to improve in vivo stability of the peptide against in vivo degradation by the protease neutral endopeptidase (NEP), the authors coinjected [(177)Lu]DOTA-GRP(13-27) with the potent NEP inhibitor phosphoramidon (PA). The authors also aimed at reducing renal uptake by coadministration of lysine. METHODS: In vivo stability studies were performed in Swiss albino mice. Biodistribution studies were conducted in NMRI nu/nu mice bearing prostate cancer (PC)-3 xenografts. Ex vivo autoradiography was performed using frozen sections from PC-3 xenografts and kidneys. RESULTS AND DISCUSSION: Coadministration of PA significantly increased the percentage of intact radiopeptide in the mouse circulation. From biodistribution and ex vivo autoradiography studies, coadministration of both lysine and PA with [(177)Lu]DOTA-GRP(13-27) appeared to induce a clear improvement of tumor uptake as well as lower levels of renal radioactivity, causing a promising ninefold increase in tumor/kidney ratios.

Tumor diagnosis with new 111In-radioligands based on truncated human gastrin releasing peptide sequences: synthesis and preclinical comparison.

Radiolabeled analogs of the frog tetradecapeptide bombesin (BBN) have been proposed for diagnosis and therapy of gastrin releasing peptide receptor (GRPR)-expressing tumors. Following a different and yet unexplored approach, we have developed four novel (111)In-labeled truncated analogs of the human 27-mer GRP after conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) at the N-terminus of GRP(13/14/17/18-27) fragments. Analog affinities for the human GRPR determined against [(125)I-Tyr(4)]BBN were at the nanomolar level and dependent on truncation site. The respective (111)In radioligands specifically internalized in GRPR-expressing PC-3 cells. The shorter chain [(111)In-DOTA]GRP(17/18-27) analogs showed higher metabolic stability in mice. Radioligands specifically localized in human PC-3 xenografts in SCID mice, with [(111)In-DOTA]GRP(17-27) exhibiting the most favorable pharmacokinetic profile. This study has demonstrated the efficacy of human GRP-based radiopeptides to target GRPR-positive lesions in vivo and has revealed the impact of GRP chain length on key biological parameters of resulting radiotracers.

99mTc radiotracers based on human GRP(18-27): synthesis and comparative evaluation.

UNLABELLED: Gastrin-releasing peptide receptors (GRPRs) expressed on human tumors can serve as molecular targets for radiolabeled peptide analogs based on the frog tetradecapeptide bombesin (BBN). We have recently expanded this approach toward human GRP(18-27) sequences and introduced (99m)Tc-demomedin C, our first radiotracer based on GRP(18-27), showing favorable biologic characteristics during preclinical evaluation in rodents. We now present a series of (99m)Tc-demomedin C analogs, generated by single-Gly(24) or double-Gly(24)/Met(27) substitutions in the peptide chain, and compare their performance in GRPR-positive in vitro and in vivo models. METHODS: The SARNC ([(N4)Gly(18)]GRP(18-27)) analogs (SARNC2 dAla(24), SARNC3 dAla(24)/Nle(27), SARNC4 dAla(24)/Leu(27), SARNC5 ßAla(24), and SARNC6 Sar(24)) were synthesized on the solid support and purified by high-performance liquid chromatography (HPLC). Competition binding experiments against [(125)I-Tyr(4)]BBN were conducted in GRPR-positive PC-3 cell membranes. Internalization of (99m)Tc radioligands was compared in PC-3 cells at 37°C. Metabolic stability was studied by HPLC analysis of blood samples collected 5 min after injection of radiopeptides in mice. Biodistribution was performed by injecting a (99m)Tc-SARNC bolus (185 kBq [5 µCi], 100 µL, 10 pmol of peptide ± 40 nmol of Tyr(4)-BBN: in vivo GRPR blockade) in severe combined immune deficient mice bearing PC-3 xenografts. RESULTS: SARNCs bound to GRPR with high affinity (range of 50% inhibitory concentration [IC50] values, 0.3 nM [SARNC5] to 9.3 nM [SARNC4]). (99m)Tc-SARNCs specifically internalized in PC-3 cells, with (99m)Tc-SARNC5 displaying the fastest internalization rate. (99m)Tc-SARNCs showed distinct degradation rates (17% [(99m)Tc-SARNC3] to >50% [(99m)Tc-SARNC4] remaining intact). All (99m)Tc-SARNCs efficiently and specifically localized in GRPR-positive PC-3 xenografts in mice (4.4 percentage injected dose per gram [%ID/g] [(99m)Tc-SARNC4] to 12.0 %ID/g [(99m)Tc-SARNC2] at 4 h after injection). (99m)Tc-SARNC6 displayed the highest tumor-to-nontumor ratios followed by (99m)Tc-SARNC2. CONCLUSION: This structure-activity relationship study has shown the impact of single-Gly(24) or double-Gly(24)/Met(27) substitutions in the (99m)Tc-SARNC1 motif on key biologic parameters, including GRPR affinity, internalization efficiency, and in vivo stability, which eventually determine the pharmacokinetic profile of resulting radiopeptides. By revealing improved analogs, this study has strengthened the applicability perspectives of radioligands based on human GRP sequences in the detection and therapy of GRPR-expressing tumors in humans.

Gastrin releasing peptide receptor-directed radioligands based on a bombesin antagonist: synthesis, (111)in-labeling, and preclinical profile.

Novel bombesin (BBN) antagonists were synthesized by coupling the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to H-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (JMV594) through linkers of increasing number of (ßAla)x residues (x = 1-3). Labeling with (111)In afforded the respective radiotracers in high purity and high specific activity. Bioconjugate affinity for the gastrin releasing peptide receptor (GRPR) as determined against [(125)I-Tyr(4)]BBN was high (IC50 values in the lower nanomolar range). Radioligands poorly internalized in PC-3 cells at 37 °C. Radiopeptides remained >60% intact 5 min after entering the bloodstream of healthy mice. After injection in SCID mice bearing human PC-3 xenografts all analogues showed high tumor uptake and rapid background clearance via the kidneys into urine. Interestingly, pancreatic uptake, albeit GRPR-specific, declined rapidly with time. (111)In-DOTA-(ßAla)2-JMV594 achieved the highest tumor values among the group (17.0 ± 2.8%ID/g vs. 8-10%ID/g, respectively, at 4 h pi) indicating that the (ßAla)2-linker favors in vivo interaction of radiopeptides with the GRPR.