Reduction of the Renal Radioactivity of 111In-DOTA-Labeled Antibody Fragments with a Linkage Cleaved by the Renal Brush Border Membrane Enzymes.

The interposition of a cleavable linkage by enzymes on the renal brush border membrane constitutes a promising approach for reducing the renal radioactivity levels of radiolabeled low-molecular-weight antibody fragments and constructs (LMW Abs). Herein, we applied the molecular design to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based reagents for radiotheranostic applications with trivalent radiometals. DOTA or a derivative thereof was conjugated to a Fab through an FGK linkage ([111In]In-DO3AiBu-Bn-FGK-Fab or [111In]In-DOTA-Bn-FGK-Fab). When injected into mice, both generated radiometabolites, [111In]In-DO3AiBu-Bn-F and [111In]In-DOTA-Bn-F, by the angiotensin-converting enzyme at similar rates. Both exhibited significantly lower renal radioactivity levels than a 111In-labeled Fab prepared by the conventional procedure ([111In]In-DOTA-Bn-SCN-Fab). The different elimination rates of each radiometabolite from the kidney significantly affected the renal radioactivity levels. [111In]In-DO3AiBu-Bn-FGK-Fab preferentially reduced the renal localization without impairing tumor accumulation. These findings would pave the way for developing a DOTA-based radiotheranostic platform for LMW Abs bearing cleavable linkers for renal brush border enzymes.

Stability Estimation of Gallium Complexes of DOTA Derivatives for Radiotheranostic Applications.

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.

Synthesis and evaluation of a para-carboxylated benzyl-DOTA for labeling peptides and polypeptides.

INTRODUCTION: Radiolabeled peptides and low-molecular-weight (LMW) polypeptides show high and persistent radioactivity levels in the kidney. To develop a DOTA-based bifunctional chelating agent that provides a radiometabolite with a rapid elimination rate from the kidney, a para-carboxyl Bn-DOTA (p-COOH-Bn-DOTA) was designed, synthesized, and evaluated. METHODS: A precursor compound, p-COOH-Bn-DOTA(tBu)4, was synthesized in 9 steps using N-Boc-p-iodo-L-phenylalanine as the starting material. A synthetic somatostatin analog (TOC) was used as a representative peptide metabolized in the renal lysosomes. p-COOH-Bn-DOTA-conjugated TOC (DOTA-Bn-TOC) was synthesized by the conventional solid-phase peptide synthesis using p-COOH-Bn-DOTA(tBu)4. DOTA-tris(tBu ester) was also conjugated with TOC to prepare DOTATOC. 111In-labeling of the peptides was conducted under similar conditions. The radiochemical conversions, stability against apo-transferrin (apoTf), and in vivo behaviors were compared. RESULTS: [111In]In-DOTA-Bn-TOC was obtained with higher radiochemical conversions than [111In]In-DOTATOC. Both 111In-labeled TOC derivatives remained stable against apoTf. In biodistribution studies, [111In]In-DOTA-Bn-TOC exhibited higher initial uptake in the kidney, followed by a faster elimination rate of radioactivity into the urine than [111In]In-DOTATOC. The metabolic studies showed that the shorter residence time of the radiometabolite from [111In]In-DOTA-Bn-TOC was responsible for the renal radioactivity decline. CONCLUSION: p-COOH-Bn-DOTA provides stable 111In-labeled peptides in high yields at low peptide concentrations. p-COOH-Bn-DOTA also provides a radiometabolite with a short residence time in the kidney. Such characteristics would render p-COOH-Bn-DOTA useful to the future application to radiolabeled LMW polypeptides with low renal radioactivity levels.