In Vivo Imaging of Folate Receptor Positive Tumor Xenografts Using Novel ⁶⁸Ga-NODAGA-Folate Conjugates

The overexpression of the folate receptor (FR) in a variety of malignant tumors, along with its limited expression in healthy tissues, makes it an attractive tumor-specific molecular target. Noninvasive imaging of FR using radiolabeled folate derivatives is therefore highly desirable. Given the advantages of positron emission tomography (PET) and the convenience of ⁶⁸Ga production, the aim of our study was to develop a new ⁶⁸Ga-folate-based radiotracer for clinical application. The chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) was conjugated to folic acid and to 5,8-dideazafolic acid using 1,2-diaminoethane as a spacer, resulting in two novel conjugates, namely, P3246 and P3238, respectively. Both conjugates were labeled with ^68/67Ga. In vitro internalization, efflux, and saturation binding studies were performed using the FR-positive KB cell line. Biodistribution and small-animal PET imaging studies were performed in nude mice bearing subcutaneous KB xenografts. Both conjugates were labeled with ⁶⁸Ga at room temperature within 10 min in labeling yields >95% and specific activity ∼30 GBq/μmol. The <i>K</i>_d values of ^68/67Ga-P3246 (5.61 ± 0.96 nM) and ^68/67Ga-P3238 (7.21 ± 2.46 nM) showed high affinity for the FR. ^68/67Ga-P3246 showed higher cell-associated uptake in vitro than ^68/67Ga-P3238 (approximately 72 and 60% at 4 h, respectively, <i>P</i> < 0.01), while both radiotracers exhibited similar cellular retention up to 4 h (approximately 76 and 71%, respectively). Their biodistribution profile is characterized by high tumor uptake, fast blood clearance, low hepatobiliary excretion, and almost negligible background. Tumor uptake was already high at 1 h for both ⁶⁸Ga-P3246 and ⁶⁸Ga-P3238 (16.56 ± 3.67 and 10.95 ± 2.12% IA/g, respectively, <i>P</i> > 0.05) and remained at about the same level up to 4 h. Radioactivity also accumulated in the FR-positive organs, such as kidneys (91.52 ± 21.05 and 62.26 ± 14.32% IA/g, respectively, 1 h pi) and salivary glands (9.05 ± 2.03 and 10.39 ± 1.19% IA/g, respectively, 1 h pi). The specificity of the radiotracers for the FR was confirmed by blocking experiments where tumor uptake was reduced by more than 85%, while the uptake in the kidneys and the salivary glands was reduced by more than 90%. Reduction of the kidney uptake was achieved by administration of the antifolate pemetrexed 1 h prior to the injection of the radiotracers, which resulted in an improvement of tumor-to-kidney ratios by more than a factor of 3. In line with the biodistribution results, small-animal PET images showed high uptake in the kidneys, clear visualization of the tumor, accumulation of radioactivity in the salivary glands, and no uptake in the gastrointestinal tract. ⁶⁸Ga-P3246 and ⁶⁸Ga-P3238 showed very high tumor-to-background contrast in PET images; however, the tumor-to-kidney ratio remained low. The new radiotracers, especially ⁶⁸Ga-P3246, are promising as PET imaging probes for clinical application due to their facile preparation and improved in vivo profile as compared to the other folate-based PET radiotracers