Reduced 18F-Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on F-18-labeled 6S- and 6R-5-MTHF radiotracers for imaging folate receptor (FR)-alpha-positive tissues. Therefore, the goal of this study was to synthesize four F-18-labeled 5-MTHF derivatives conjugated at either the alpha- or gamma-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6S-alpha, 6S-gamma, 6R-alpha, and 6R-gamma-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the alpha- and gamma-conjugated F-18-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1-7% d.c.) and high molar activities (139-245 GBq/mu mol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and L-cysteine. In vitro, all four 5-MTHF conjugates showed similar binding affinities to FR-alpha (IC50 = 17.7-24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-a. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high in vivo stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6S- and 6R-5-MTHF conjugates exhibited a different accumulation pattern in various organs induding the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the alpha- and gamma-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8-11% IA/g). Based on these results, we conclude that F-18-labeled 5-MTHF conjugates are a promising new dass of radiotracers for targeting FR-positive tumor tissues.