Abstract
Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid)], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), based on the building block DOTPI(azide)4. A detailed investigation of kinetic properties of Cu(II)-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II)-, Zn(II), and Cu(II)-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx)4 (a model for DOTPI conjugates) as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA) were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of CaII-, ZnII, and CuII-complexes of DOTPI (logK(CaL) = 8.65, logK(ZnL = 15.40, logK(CuL) = 20.30) and DOTPI(Chx)4 (logK(CaL) = 8.99, logK(ZnL) = 15.13, logK(CuL) = 20.42) were found. Transchelation of Cu(II)-complexes occurs via proton-assisted dissociation, whereafter released Cu(II) is scavenged by EDTA. The corresponding dissociation rates [kd = 25 × 10−7 and 5 × 10−7 s−1 for Cu(DOTPI) and Cu(DOTPI(Chx)4), respectively, at pH 4 and 298 K] indicate that conjugation increases the kinetic inertness by a factor of 5. However, demetallation is completed within 4.5 and 7.2 h at pH 2 and 25°C, respectively, indicating that Cu(II) removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess H4EDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA) targeting motif Lys-urea-Glu (KuE) were synthesized via CuAAC as well as dibenzo-azacyclooctine (DBCO) based, strain-promoted click chemistry (SPAAC), which were labeled with Lu-177 and subsequently evaluated in vitro and in SCID mice bearing subcutaneous LNCaP tumor (PSMA+ human prostate carcinoma) xenografts. High affinities (3.4 and 1.4 nM, respectively) and persistent tumor uptakes (approx. 3.5% 24 h after injection) confirm suitability of DOTPI-based tetramers for application in targeted radionuclide therapy.
Highlights
Endoradiotherapy ( termed molecular radiotherapy, radioligand therapy or, if addressing peptide receptors, peptide receptor radionuclide therapy (PRRT) or, if involving alphaemitting nuclides, targeted alpha therapy) (Oyen et al, 2007) refers to the internal application of radionuclides for therapeutic purposes, above all, for curing cancer
We investigated the metal coordination properties of DOTPI and a model conjugate thereof, DOTPI(Chx)4 (Figure 1), with a particular emphasis on CuII complexes, in order to facilitate optimization of respective demetallation protocols and to support application of CuAAC for elaboration of DOTPI-based multimers
The animal experiments were conducted in accordance with the German Animal Welfare Act (Tierschutzgesetz), and an ethics approval was obtained from the responsible authority (Regierung von Oberbayern)
Summary
Endoradiotherapy ( termed molecular radiotherapy, radioligand therapy or, if addressing peptide receptors, peptide receptor radionuclide therapy (PRRT) or, if involving alphaemitting nuclides, targeted alpha therapy) (Oyen et al, 2007) refers to the internal application of radionuclides for therapeutic purposes, above all, for curing cancer. In a tandem application of both types of agents, referred to as “theranostics,” one targeting mechanism is exploited for delivery of different sorts of radionuclides to tissues, either for diagnostic purposes, that is, with the intention to localize lesions by means of external detection of emitted gamma photons, or to achieve a therapeutic effect via local absorption of particle (i.e., alpha- or beta) radiation. The corresponding radiopharmaceuticals are frequently based on peptides, peptidomimetics, enzyme inhibitors, or similar molecules capable of recognizing a specific cell surface receptor, membrane-bound enzyme, ion channel, or comparable target. Recognizing malignant cells by overexpression of above-mentioned surface markers, the radiolabeled bioconjugates deliver these beta- or alpha-emitting isotopes in the lesion, resulting in a local irradiation which kills malignant cells
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