Article pubs.acs.org/molecularpharmaceutics
64
Cu- and 68Ga-Based PET Imaging of Folate Receptor-Positive Tumors: Development and Evaluation of an Albumin-Binding NODAGA−Folate
Renáta Farkas,† Klaudia Siwowska,† Simon M. Ametamey,‡ Roger Schibli,†,‡ Nicholas P. van der Meulen,†,§ and Cristina Müller*,†,‡ †
Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, Villigen-PSI, Switzerland Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland § Laboratory of Radiochemistry, Paul Scherrer Institut, Villigen-PSI, Switzerland ‡
S Supporting Information *
ABSTRACT: A number of folate-based radioconjugates have been synthesized and evaluated for nuclear imaging purposes of folate receptor (FR)-positive tumors and potential therapeutic application. A common shortcoming of radiofolates is, however, a significant accumulation of radioactivity in the kidneys. This situation has been faced by modifying the folate conjugate with an albumin-binding entity to increase the circulation time of the radiofolate, which led to significantly improved tumor-to-kidney ratios. The aim of this study was to develop an albuminbinding folate conjugate with a NODAGA-chelator (rf42) for labeling with 64Cu and 68Ga, allowing application for PET imaging. The folate conjugate rf42 was synthesized in 8 steps, with an overall yield of 5%. Radiolabeling with 64Cu and 68Ga was carried out at room temperature within 10 min resulting in 64Cu-rf42 and 68Ga-rf42 with >95% radiochemical purity. 64Cu-rf42 and 68Ga-rf42 were stable (>95% intact) in phosphate-buffered saline over more than 4 half-lives of the corresponding radionuclide. In vitro, the plasma protein-bound fraction of 64Cu-rf42 and 68 Ga-rf42 was determined to be >96%. Cell experiments proved FR-specific uptake of both radiofolates, as it was reduced to 95%. Traces of unreacted 64Cu2+ and 68Ga3+ appeared with a retention time of 2.0 min. Experiments to investigate the highest achievable specific activity revealed 20 MBq/nmol for 64Cu-rf42 and 68Ga-rf42, respectively. Both radiofolates, 64Cu-rf42 and 68Ga-rf42, were stable (>95%) over a period of 4 half-lives of the corresponding radionuclide when they were incubated in PBS pH 7.4. TLC analysis of the samples incubated in PBS showed the same results as obtained when using HPLC. All results were in agreement with those obtained with the albumin-binding DOTA−folate conjugates, 64Cu-cm10 and 68Ga-cm10, respectively (Supporting Information). Distribution Coefficients (LogD Values). Distribution coefficients (logD values) were determined to obtain information about the hydrophilicity of the radiolabeled folate conjugate rf42. The 64Cu-rf42 experiments revealed a logD value of −4.04 ± 0.48, and for 68Ga-rf42 a logD value of −3.85 ± 0.09 was obtained. There was no significant difference (p > 0.05) between these values. Moreover, the logD values obtained for 64Cu-rf42 and 68Ga-rf42 were in good agreement with the logD values obtained for 64Cu-cm10 and 68Ga-cm10 (p > 0.05) (Supporting Information). Protein Binding. The filtered fraction of plasma samples incubated with the two radiofolates contained minor amounts of radioactivity, indicating that the majority of both radiofolates was bound to plasma proteins. The determined bound fraction of 64Cu-rf42 (96.3 ± 1.3%, n = 3) did not differ significantly from the value obtained for 68Ga-rf42 (98.0 ± 0.2%, n = 4, p > 0.5). After filtration of the folate radioconjugates incubated in PBS pH 7.4, almost the entire quantity of loaded radioactivity (>95%) was detected in the filtrate. This control experiment confirmed that the retention in plasma samples was due to the binding of the radiofolates to plasma proteins. The results obtained in these experiments showed slightly increased values for the plasma protein-bound fraction of 64Cu-rf42 as compared to 64Cu-cm10 and 68Ga-cm10. The protein-bound fraction of 68 Ga-rf42 was significantly greater than the protein-bound fraction of 64Cu-cm10 (p < 0.05) and 68Ga-cm10 (p < 0.05) (Table S1). Cell Internalization Experiments. FR-specific uptake of 64 Cu-rf42 and 68Ga-rf42 was demonstrated in vitro using KB tumor cells. The experiment showed high cell uptake of both radiofolates whereof the internalized fractions accounted for about 55% and 30% of the totally bound 64Cu-rf42 and 68Garf42, respectively. The addition of excess folic acid resulted in a significant reduction of the radiofolate uptake (
