Benefits of NOPO as chelator in gallium-68 peptides, exemplified by preclinical characterization of ⁶⁸Ga-NOPO-c(RGDfK)

The α_vβ₃-integrin addressing cyclic pentapeptide cyclo(RGDfK) was conjugated to NOPO, 1,4,7-triazacyclononane-1,4- bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl) phosphinic acid], a bifunctional chelator with exceptional gallium-68 labeling properties. NOPO-c(RGDfK) and its Ga(III) and Cu(II) complexes showed high affinity to α_vβ₃ integrin (IC₅₀ = 0.94 ± 0.06, 1.02 ± 0.09, and 0.51 ± 0.06 nM, respectively). ⁶⁸Ga labeling of NOPO-c(RGDfK) in an automated GMP-compliant procedure was performed with near-quantitative radiochemical yield, using precursor amounts as low as 0.5 nmol (approximately 0.6 μg). ⁶⁸Ga-NOPO-c(RGDfK) was obtained with high purity (>99% by radio-HPLC/TLC) and, optionally, could be produced with specific activities up to 6 TBq/μmol. M21/M21L (human melanoma with high/low α _vβ₃ integrin expression) xenografted athymic CD-1 nude mice were used for biodistribution, in vivo stability studies, and PET imaging. ⁶⁸Ga-NOPO-c(RGDfK) showed rapid and specific uptake in M21 tumor xenografts (2.02 ± 0.34% ID/g at 60 min p.i.) and was found stable in vivo. Its high hydrophilicity is reflected by an octanol-water distribution coefficient (log D = -4.6) which is more than 1 order of magnitude lower compared to respective NOTA or DOTA analogues. As expected, ⁶⁸Ga- NOPO-c(RGDfK) thus showed fast renal clearance from nontargeted tissues. We conclude that NOPO might generally prove a useful means to improve renal clearance of corresponding radiopharmaceuticals by increasing the polarity of its bioconjugates. Favorable labeling properties render NOPO conjugates highly recommendable for reliable routine production of ⁶⁸Ga- radiopharmaceuticals in a clinical setting.