Increasing molar activity by HPLC purification improves ⁶⁸Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model

by Jan Lennart von Hacht, Sarah Erdmann, Lars Niederstadt, Sonal Prasad, Asja Wagener, Samantha Exner, Nicola Beindorff, Winfried Brenner, Carsten Grötzinger

Purpose

Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. ⁶⁸Gallium (⁶⁸Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the ⁶⁸Ga-DOTA peptide purified from the excess of unlabeled peptide.

Procedures

The MC1R ligand DOTA-NAPamide was labeled with ⁶⁸Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and ⁶⁸Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors.

Results

In biodistribution studies, non-purified ⁶⁸Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified ⁶⁸Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake.

Conclusions

We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as ⁶⁸Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.