Stability Estimation of Gallium Complexes of DOTA Derivatives for Radiotheranostic Applications

Abstract

1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid (DO3A) has been used to prepare ⁶⁸Ga-labeled probes for the diagnostic counterpart of radiotheranostic applications. While DO3A provides stable complexes with therapeutic radionuclides such as ⁹⁰Y, ¹⁷⁷Lu, and ²²⁵Ac, further improvement of the in vivo stability of the Ga-DO3A complex is required. Considering the high stability of an intact Ga-DOTA complex, the stability of Ga complexes of DOTA and DO3A derivatives, including benzyl-DOTA (Bn-DOTA), was evaluated to gain fundamental knowledge for developing the next-generation radiotheranostic probes using ⁶⁸Ga as a diagnostic counterpart. Following the complexation reaction to prepare ⁶⁷Ga-labeled DOTA and DO3A derivatives, the stability of the resulting ⁶⁷Ga-labeled compounds was evaluated in murine plasma and apo-transferrin challenge. [⁶⁷Ga]Ga-Bn-DOTA produced two isomers, and one of the isomers exhibited the highest stability among the tested complexes. The X-ray crystallography showed that the less stable isomer of Ga-Bn-DOTA suggested an N₃O₃ coordination geometry, while Ga-DOTA and Ga-Bn-DO3A show N₄O₂ coordination. To further evaluate the stability, a synthetic somatostatin analogue, [Tyr³]octreotide (TOC), was used as a model peptide, and p-COOH-Bn-DOTA and DO3A were conjugated with TOC to prepare DOTA-Bn-TOC and DOTATOC. [⁶⁷Ga]Ga-DOTA-Bn-TOC also yielded two isomers with varying stability, and one isomer exhibited significantly higher stability than [⁶⁷Ga]Ga-DOTATOC both in vitro and in vivo. These findings indicate that para-substituted Bn-DOTA would constitute a suitable chelating agent for developing next-generation radiotheranostic probes, although high-performance liquid chromatography purification is needed. Thus, further chemical modification on the Bn-DOTA molecule is also needed to avoid the formation of a Ga complex with the N₃O₃ configuration.

Figure 1

Chemical structures of model chelators used for the stability experiments; DO3AM^Bu (A), p-NO₂-Bn-DOTA (B), DOTAGAM^Bu (C), and p-NO₂-Bn-DO3A (D).

Figure 2

Chemical structures of octreotide analogues; DOTA-Bn-TOC (A) and DOTATOC (B).

Figure 3

HPLC chromatograms of p-NO₂-Bn-DOTA (yellow) and nonradioactive Ga-p-NO₂-Bn-DOTA (blue) and HPLC radiochromatograms of [⁶⁷Ga]Ga-p-NO₂-Bn-DOTA (red), isomer A (green), and isomer B (purple) on system A. [⁶⁷Ga]Ga-p-NO₂-Bn-DOTA exhibited two peaks at retention times of 17.6 min (isomer A) and 22.1 min (isomer B). Each isomer was separated by HPLC and reinjected to HPLC to exhibit a single peak.

Figure 4

X-ray crystal structures of isomer B (A) and Ga-p-NO₂-Bn-DO3A (B) and the chemical structure of p-SCN-Bn-oxo-DO3A (C). Hydrogen atoms and solvent molecules are omitted (A,B). Thermal ellipsoids are shown at a 50% probability (A,B).

Figure 5

HPLC radiochromatograms of [⁶⁷Ga]Ga-DOTA-Bn-TOC (red), ⁶⁷Ga-DOTATOC (blue), and the isolated isomer A′ (green) and isomer B′ (purple) on system B. [⁶⁷Ga]Ga-DOTA-Bn-TOC exhibited two peaks at retention times of 28.5 min (isomer A′) and 29.0 min (isomer B′). Each isomer was separated by HPLC and reinjected to HPLC to exhibit a single peak. In vitro stability assessment of ⁶⁷Ga-labeled TOC analogues.

Figure 6

Radioactivity in the blood, spleen, and bone after injection of ⁶⁷Ga-labeled TOC analogues in ICR mice at 1 h and 3 h postinjection. Significances were determined by Tukey’s test for the one-way ANOVA (*p < 0.05).