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

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 64Cu-labeled 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 [Tyr3]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 kidney-specific targeting of NEP, the additional targeting of albumin in the blood is highlighted

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

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 64Cu-labeled 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 [Tyr3]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 kidney-specific targeting of NEP, the additional targeting of albumin in the blood is highlighted

Cyclam Derivatives with a Bis(phosphinate) or a Phosphinato–Phosphonate Pendant Arm: Ligands for Fast and Efficient Copper(II) Complexation for Nuclear Medical Applications

Cyclam derivatives bearing one geminal bis­(phosphinic acid), −CH₂PO₂HCH₂PO₂H₂ (H₂<b>L</b>^<b>1</b>), or phosphinic–phosphonic acid, −CH₂PO₂HCH₂PO₃H₂ (H₃<b>L</b>^<b>2</b>), pendant arm were synthesized and studied as potential copper­(II) chelators for nuclear medical applications. The ligands showed good selectivity for copper­(II) over zinc­(II) and nickel­(II) ions (log <i>K</i>_CuL = 25.8 and 27.7 for H₂<b>L</b>^<b>1</b> and H₃<b>L</b>^<b>2</b>, respectively). Kinetic study revealed an unusual three-step complex formation mechanism. The initial equilibrium step leads to <i>out-of-cage</i> complexes with Cu²⁺ bound by the phosphorus-containing pendant arm. These species quickly rearrange to an <i>in-cage</i> complex with cyclam conformation <b>II</b>, which isomerizes to another <i>in-cage</i> complex with cyclam conformation <b>I</b>. The first <i>in-cage</i> complex is quantitatively formed in seconds (pH ≈5, 25 °C, Cu:L = 1:1, <i>c</i>_M ≈ 1 mM). At pH >12, <b>I</b> isomers undergo nitrogen atom inversion, leading to <b>III</b> isomers; the structure of the <b>III</b>-[Cu­(H<b>L</b>^<b>2</b>)] complex in the solid state was confirmed by X-ray diffraction analysis. In an alkaline solution, interconversion of the <b>I</b> and <b>III</b> isomers is mutual, leading to the same equilibrium isomeric mixture; such behavior has been observed here for the first time for copper­(II) complexes of cyclam derivatives. Quantum-chemical calculations showed small energetic differences between the isomeric complexes of H₃<b>L</b>^<b>2</b> compared with analogous data for isomeric complexes of cyclam derivatives with one or two methylphosphonic acid pendant arm(s). Acid-assisted dissociation proved the kinetic inertness of the complexes. Preliminary radiolabeling of H₂<b>L</b>^<b>1</b> and H₃<b>L</b>^<b>2</b> with ⁶⁴Cu was fast and efficient, even at room temperature, giving specific activities of around 70 GBq of ⁶⁴Cu per 1 μmol of the ligand (pH 6.2, 10 min, ca. 90 equiv of the ligand). These specific activities were much higher than those of H₃<b>nota</b> and H₄<b>dota</b> complexes prepared under identical conditions. The rare combination of simple ligand synthesis, very fast copper­(II) complex formation, high thermodynamic stability, kinetic inertness, efficient radiolabeling, and expected low bone tissue affinity makes such ligands suitably predisposed to serve as chelators of copper radioisotopes in nuclear medicine