Enzymological Characterization of ⁶⁴Cu-Labeled Neprilysin Substrates and Their Application for Modulating the Renal Clearance of Targeted Radiopharmaceuticals

Abstract

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced toxicity to healthy tissues, in particular to the kidneys as primary organs of elimination. The targeting of enzymes of the renal brush border membrane by cleavable linkers that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of 64 Culabeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure−activity relationships, and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr 3 ]octreotate. Radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small peptides entails their premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a kidneyspecific targeting of NEP, the additional targeting of albumin in the blood is highlighted.

Comparative Saturation Binding Analysis of 64Cu-Labeled Somatostatin Analogues Using Cell Homogenates and Intact Cells

“…While most of the serine and cysteine proteases might be inhibited by the used inhibitor cocktail during homogenization (see Materials and Methods), a residual proteolytic activity cannot be excluded. Therefore, for the two radioligands, [ 64 Cu]Cu-NODAGA-NES2-TATE and [ 64 Cu]Cu-NODAGA-NES5-TATE, the stability toward cell homogenates and intact cells under assay conditions were exemplarily examined (Figure S3 in the Supporting Informa- 17 Copyright 2023, American Chemical Society). Regression analysis was performed by using the model of "one site�total and nonspecific binding" as implemented in GraphPad Prism (Version 9.…”

“…The uptake fraction ("internalized" or not acid releasable radioligand) is expressed as percentage of specific total-bound radioligand (Figure S4 in the Supporting Information). d Data were previously published by us (adapted with permission from Brandt et al 16 Copyright 2022 American Chemical Society and Brandt et al 17 Copyright 2023, American Chemical Society). e Ratio of K d (cell homogenate)/K d (intact cells).…”

“…In Vitro SST 2 Binding Affinity Using Intact MPC Cells. 17 A number of 3 × 10 5 cells/cm 2 were seeded in collagen-coated 48-well microplates (CELLSTAR 48 Well Cell Culture Multiwell Plates, Polystyrene, Greiner bio-one, Item no. 677180) and grown for three days.…”

“…Based on the vector molecule [Tyr 3 ]­octreotate (TATE or TOCA) for SST 2 targeting, we recently reported on novel TATE derivatives modified with albumin binders ( [ 64 Cu]­Cu-NODAGA-cLAB-TATEs ), cleavage sequences for neprilysin, or both ( [ 64 Cu]­Cu-NODAGA-NES-TATEs ) to systematically explore the suitability of these structural modifications for modulating the pharmacokinetic properties of peptidic radioligands. As part of the in vitro radiopharmacological characterization, the SST 2 affinity of the [ 64 Cu]­Cu-NODAGA-cLAB-TATEs was characterized in saturation binding analyses using cell homogenates of mouse pheochromocytoma cells (MPC) exhibiting high levels of SST 2 .…”

Reduction of the Renal Radioactivity of 111In-DOTA-Labeled Antibody Fragments with a Linkage Cleaved by the Renal Brush Border Membrane Enzymes

“…Radiolabeled peptides also exhibit high and persistent radioactivity levels in the kidney, which causes dose-limiting nephrotoxicity . Since LMW Abs and peptides share similar metabolic pathways in the kidney, the renal brush border strategy was recently applied to peptides. ,, For the applications, further efforts may be needed to stabilize the cleavable linkage in the blood while liberating a urinary excretable radiometabolite at the renal brush border.…”