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

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.

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/natLu]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; logD_7.4: - 1.67 ± 0.08) and [^177/natLu]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; logD_7.4 =  - 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 [¹⁷⁷Lu]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 [¹⁷⁷Lu]Lu-DOTA-rhCCK-18.

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.