Significant reduction of activity retention in the kidneys via optimized linker sequences in radiohybrid-based minigastrin analogs.

BACKGROUND: We recently introduced radiohybrid (rh)-based minigastrin analogs e.g., DOTA-rhCCK-18 (DOTA-D-Dap(p-SiFA)-(D-γ-Glu)8 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ), that revealed substantially increased activity retention in the tumor. However, one major drawback of these first generation rh-based cholecystokinin-2 receptor (CCK-2R) ligands is their elevated activity levels in the kidneys, especially at later time points (24 h p.i.). Therefore, this study aimed to reduce kidney retention with regard to a therapeutic use via substitution of negatively charged D-glutamic acid moieties by hydrophilic uncharged polyethylene glycol (PEG) linkers of various length ((PEG)4 to (PEG)11 ). Furthermore, the influence of differently charged silicon-based fluoride acceptor (SiFA)-moieties (p-SiFA: neutral, SiFA-ipa: negatively charged, and SiFAlin: positively charged) on in vitro properties of minigastrin analogs was evaluated. Out of all compounds evaluated in vitro, the two most promising minigastrin analogs were further investigated in vivo.

RESULTS: CCK-2R affinity of most compounds evaluated was found to be in a range of 8-20 nM (by means of apparent IC50 ), while ligands containing a SiFA-ipa moiety displayed elevated IC50 values. Lipophilicity was noticeably lower for compounds containing a D-γ-glutamate (D-γ-Glu) moiety next to the D-Dap(SiFA) unit as compared to their counterparts lacking the additional negative charge. Within this study, combining the most favorable CCK-2R affinity and lipophilicity, [177/nat Lu]Lu-DOTA-rhCCK-70 (DOTA-D-Dap(p-SiFA)-D-γ-Glu-(PEG)7 -D-γ-Glu-(PEG)3 -Trp-(N-Me)Nle-Asp-1-Nal-NH2 ; IC50 : 12.6 ± 2.0 nM; logD7.4 : - 1.67 ± 0.08) and [177/nat Lu]Lu-DOTA-rhCCK-91 (DOTA-D-Dap(SiFAlin)-D-γ-Glu-(PEG)4 -D-γ-Glu-(PEG)3 -Trp-(N-Me)Nle-Asp-1-Nal-NH2 ; IC50 : 8.6 ± 0.7 nM; logD7.4  =  - 1.66 ± 0.07) were further evaluated in vivo. Biodistribution data of both compounds revealed significantly reduced (p < 0.0001) activity accumulation in the kidneys compared to [177 Lu]Lu-DOTA-rhCCK-18 at 24 h p.i., leading to enhanced tumor-to-kidney ratios despite lower tumor uptake. However, overall tumor-to-background ratios of the novel compounds were lower than those of [177 Lu]Lu-DOTA-rhCCK-18.

CONCLUSION: We could show that the reduction of negative charges within the linker section of radiohybrid-based minigastrin analogs led to decreased activity levels in the kidneys at 24 h p.i., while maintaining a good tumor uptake. Thus, favorable tumor-to-kidney ratios were accomplished in vivo. However, further optimization has to be done in order to improve tumor retention and general biodistribution profile.