A DOTA-analog tetra-phosphinate chelator for preparation of tetrameric bioconjugates

Objectives Inspired by the favorable properties of TRAP chelators for ⁶⁸Ga, we developed and evaluated the analog DOTA-like chelator DOTPI for radiolanthanides and other radiometals. The DOTPI framework features four phosphinic acid moieties as primary coordination sites and four additional non-coordinating carboxylate groups, thus allowing for straightforward synthesis of homo- and heteromultimeric bioconjugates.

Methods DOTPI (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methyl(2-carboxyethyl)phosphinic acid]) was synthesized by Mannich reaction of 2-carboxyethylphospinic acid with 1,4,7,10-tetraazacyclododecane. Stabilities of complexes with selected di- and trivalent metal ions was determined by potentiometry. Radiolabeling was done using n.c.a. ¹⁷⁷Lu and ⁶⁴Cu at pH 6.5 and pH 5.6, respectively. Carboxylate functionalization with linkers & peptide receptor ligands was done by amide coupling using HATU. Integrin affinities of cyclo(RGDfK) conjugates were determined in competition assays on α_vβ₃-expressing M21 h-melanoma cells against ¹²⁵echistatin. Nude mice bearing M21/M21L xenografts were used for preliminary in vivo studies.

Results DOTPI can be synthesized easily with 20 % yield (TACN-based). Basicity (pK₁+pK₂ 20.5) is similar to that of other phosphinate DOTA analogs. Stable complexes (logK_ML >18) are formed with a wide range of metal ions. ¹⁷⁷Lu labeling is slightly more efficient compared to DOTA. ¹⁷⁷Lu- and ⁶⁴Cu-complexes were found stable upon challenge with 0.1 M EDTA. No protection of phosphinic acid moieties is necessary during carboxylate functionalization. A DOTPI-based cyclo(RGDfK) tetramer exhibited high α_vβ₃ integrin affinity (36 nM for Lu-DOTPI(RGD)₄, compared to 44 nM of Ga-TRAP(RGD)₃ and 336 nM of Ga-NODAGA-RGD) and favorable in vivo properties.

Conclusions DOTPI allows for facile synthesis of tetrameric bioconjugates and can be labeled with various radiometals, thus constituting a promising framework for further development of radiopharmaceuticals for imaging & therapy applications