Establishment of two complementary in vitro assays for radiocopper complexes achieving reliable and comparable evaluation of in vivo stability

Abstract

The development of novel radiopharmaceuticals for imaging and therapy requires rapid and reproducible in vitro assays to estimate their in vivo stability and dissociation behaviour. In general, these assays should allow an assessment of dissociation of the radiometal from the radiopharmaceuticals. In the past, a series of chemical challenges has been widely used to estimate complex stability under non-physiological and non-radiotracer conditions providing limited information on the potential in vivo stability. In contrast, we herein present two independent in vitro methods to measure the stability of radiocopper complexes under physiologically relevant conditions. To quantify and compare the dissociation behaviour of six wellestablished 64 Cu chelates (TETA, DOTA, NOTA, Cyclam, diamSar and EDTA), we combine a protein challenge experiment considering the stability of the chelates in the presence of human superoxide dismutase with a serum assay measuring the stability of the radiometal complexes against human serum. Unlike HPLC-and TLC-based analytical techniques, we describe the stability assessments by standard gel electrophoretic procedures, which allow a timesaving workflow as well as simultaneous processing and comparative analysis of a variety of copper-containing chelates and conjugates thereof. [ 64 Cu]Cu-diamSar is the most kinetically stable ligand, whereas the acyclic chelate [ 64 Cu]Cu-EDTA underwent an almost complete complex dissociation. Furthermore, kinetic stability studies in human serum carried out for [ 64 Cu]Cu-diamSar revealed no substantial time-dependent influence under commonly used labelling conditions. Both described assays, the protein challenge experiment as well as the serum stability assay, are not restricted to radiocopper, but may be adopted for other radiometal containing chelates.

Polyglycerol‐Based Copper Chelators for the Transport and Release of Copper Ions in Biological Environments

“…Additionally, it is important to understand the stability of the CSNP in the presence of blood serum. The stability of 64 Cu( ii ) labeled CSNPs was studied in blood serum by gel electrophoresis . However, due to the polydispersity of the CSNPs, a high background was seen that made it impossible to quantify the results.…”

“…The stability of 64 Cu( II ) labeled CSNPs was studied in blood serum by gel electrophoresis. [ 53 ] However, due to the polydispersity of the CSNPs, a high background was seen that made it impossible to quantify the results. Moreover, the similar behavior of CSNPs to serum proteins in radio-TLC and radio-SEC made it diffi cult to quantify their stability in whole serum conditions.…”

Synthesis and Structural Analysis of 1,4,7,10‐Tetraazacyclododecane‐1,4,7,10‐­tetraazidoethylacetic Acid (DOTAZA) Complexes

“…Although 64 Cu (the use of 64 Cu-DOTA in vivo is currently discussed controversially) [15] and 68 Ga can be used with DOTA for PET, [16,17] DOTA is the current "gold standard" for 111 In in SPECT imaging and for 90 Y in radiotherapy. [1] An important factor in these applications is the targeting of the complexes to tissue or cell types, which might be realized by the conjugation of targeting vectors to the DOTA scaffold.…”

Versatile Bispidine‐Based Bifunctional Chelators for 64CuII‐Labelling of Biomolecules

“…[13] Cetuximab containsi nt otal 86 lysine residues, eleven in each light and 32 in each heavy chain. [14] Similart oaprevious study, [15] the mAb was modified with 5 via random isothiocyanate-mediated coupling. After purification,t he degree of conjugation was determined by MALDI-TOF MS analysis (Supporting Information, FigureS8).…”