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

PurposeMelanocortin receptor 1 is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide.ProceduresThe 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.ResultsIn biodistribution studies, the 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 a decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracers for biodistribution. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake.ConclusionsWe 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.

68Ga-NODAGA-RGDyK for αvβ3 integrin PET imaging

Summary Aim: To visualize neovasculature and/or tumour integrin αvβ3 we selected the binding moiety Arg-Gly-Asp-D-Tyr-Lys (RGDyK) coupled to NODAGA for labeling with 68Ga. Methods: NODAGA-RGDyK (ABX) was labeled with the 68Ga eluate from the 68Ge generator IGG100 using the processor unit PharmTracer. Biodistribution was measured in female Hsd mice sacrificed 10, 30, 60 and 90 min after i. v. injection of 68Ga-NODAGA-RGDyK for OLINDA dosimetry extrapolated to humans. Tumour targeting was studied in SCID mice bearing A431 and other tumour transplants using microPET and biodistribution measurements. Results: Effective half-life of 68Ga-NODAGA-RGDyK was ∼25 min for total body and most organs except liver and spleen that showed stable activity retention. With a bladder voiding interval of 0.5 h the calculated effective dose (ED) was 0.012 and 0.016 mSv/MBq for males and females, respectively. Rapid uptake within 10 min was observed in A431 tumours with dynamic PET followed by a slow release. Biodistribution measurements showed a 68Ga-NODAGARGDyK uptake in A431 tumours of 3.4 ± 0.4 and 2.7 ±0.3%ID/g at 1 and 2 h, respectively. Similar uptakes were observed in a mouse and human breast and ovarian cancer xenografts. Co-injection of excess (5 mg/kg) unlabeled NODAGA- RGDyK with the radiotracer reduced tumour uptake at one hour to 0.23 ± 0.01%ID/g, but similarly decreased uptake in normal organs as well. When unlabeled peptide was injected 15 min after 68Ga- NODAGA- RGDyK, uptake diminished particularly in tumour and adrenals, suggestive of a different binding mode compared with other normal tissues. Conclusion: NODAGA- RGDyK was reliably labeled with 68Ga and revealed a predicted ED of 0.014 mSv/MBq. Tumour uptake was rapid and significant and was chased with unlabeled RGDyK in a similar manner as adrenal uptake.

Improved iodination of peptides for radioimmunoassay and membrane radioreceptor assay.

Generally applicable methods for iodinating and purifying small peptide radiolabels for radioimmunoassay and membrane radioreceptor are described in detail. Resulting improvements in radioreceptor assay and radioimmunoassay, as well as results of analyses of specific activity, separation from unlabeled peptide, and storage characteristics, are presented for luliberin, corticotropin, melanotropin, and calcitonin.