A convenient and efficient total solid-phase synthesis of DOTA-functionalized tumor-targeting peptides for PET imaging of cancer

An efficient and cost-effective synthesis of the metal chelating agents that couple to tumor-targeting peptides is required to enhance the process of preclinical research toward the clinical translation of molecular imaging agents. DOTA is one of the most widely used macrocyclic ligands for the development of new metal-based imaging and therapeutic agents owing to its ability to form stable and inert complexes under physiological conditions. Although solid-phase synthesis compatible DOTA-tris-(t-Bu ester) is a commercial product, it is expensive and contain chemical impurities. There is a need to explore new and cost-effective methods for the preparation of metal chelating agents, i.e., DOTA, directly on solid support to facilitate rapid, cost-effective, and high purity preparation of DOTA-linked peptides for imaging and therapy. In the present study, we describe a facile synthetic strategy of DOTA preparation and its linkage to peptides directly on solid-phase support.

Bombesin (BN) peptides were functionalized with DOTA chelator prepared from cyclen precursor on solid-phase and from commercial DOTA-tris and radiolabeled with ⁶⁸Ga. In vitro BN/GRP receptor binding affinities of the corresponding radiolabeled peptides were determined by saturation binding assays on human breast MDA-MB-231, MCF7, T47D, and PC3 prostate cancer cells. Pharmacokinetics were studied in Balb/c mice and in vivo tumor targeting in MDA-MB-231 tumor-bearing nude mice.

DOTA was prepared successfully from cyclen on solid-phase support, linked specifically to BN peptides and resultant DOTA-coupled peptides were radiolabeled efficiently with ⁶⁸Ga. The binding affinities of all the six BN peptides were comparable and in the low nanomolar range. All ⁶⁸Ga-labeled peptides showed high metabolic stability in plasma. These radiopeptides exhibited rapid pharmacokinetics in Balb/c mice with excretion mainly through the urinary system. In nude mice, MDA-MB-231 tumor uptake profiles were slightly different; the BN peptide with Ahx spacer and linked to DOTA through cyclen exhibited higher tumor uptake (2.32% ID/g at 1 h post-injection) than other radiolabeled BN peptides investigated in this study. The same leading BN peptide also displayed favorable pharmacokinetic profile in Balb/c mice. The PET images clearly visualized the MDA-MB-231 tumor.

DOTA prepared from cyclen on solid-phase support showed comparable potency and efficiency to DOTA-tris in both in vitro and in vivo evaluation. The synthetic methodology described here allows versatile, site-specific introduction of DOTA into peptides to facilitate the development of DOTA-linked molecular imaging and therapy agents for clinical translation.