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

Abstract

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 (68)Ga production, the aim of our study was to develop a new (68)Ga-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 (68/67)Ga. 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 (68)Ga at room temperature within 10 min in labeling yields >95% and specific activity ~30 GBq/μmol. The K(d) values of (68/67)Ga-P3246 (5.61 ± 0.96 nM) and (68/67)Ga-P3238 (7.21 ± 2.46 nM) showed high affinity for the FR. (68/67)Ga-P3246 showed higher cell-associated uptake in vitro than (68/67)Ga-P3238 (approximately 72 and 60% at 4 h, respectively, P < 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 (68)Ga-P3246 and (68)Ga-P3238 (16.56 ± 3.67 and 10.95 ± 2.12% IA/g, respectively, P > 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. (68)Ga-P3246 and (68)Ga-P3238 showed very high tumor-to-background contrast in PET images; however, the tumor-to-kidney ratio remained low. The new radiotracers, especially (68)Ga-P3246, are promising as PET imaging probes for clinical application due to their facile preparation and improved in vivo profile...

Development of a New Folate-Derived Ga-68-Based PET Imaging Agent

“…Therefore, it is expected that [ 68 Ga]NOTA-folate and [ 99m Tc]EC20 show similar renal uptake. Both tumor-to-kidney (0.31 ± 0.11) and tumor-to-liver ratios (12.43 ± 0.73) seem comparable to other, previously published data for other folate-targeted radiopharmaceuticals [3, 14, 28–34]. For a direct comparison, however, the individual compounds would have to be tested side-by-side.…”

“…As a matter of fact, numerous of folate-conjugated PET agents have already been proven to visualize FR-positive tumors in vivo [3, 28–31, 33–38]. In the past, different approaches were utilized for the synthesis of folate-based PET radiotracers.…”

“…These therapies build on the high affinity of folic acid and its derivatives to folate receptors ( K D (folate) = 0.1 nM) [3, 4], paired with the receptor’s overexpression in a wide variety of human epithelial cancers [5–8]. In parallel to the development of FR-targeted therapeutics, radio and optical tracers have been developed, and folate receptor-targeted imaging has been used successfully in preclinical and clinical research [8–14].…”

Expression of folate receptors in nasopharyngeal and laryngeal carcinoma and folate receptor-mediated endocytosis by molecular targeted nanomedicine

“…Further, this molecular targeted effect could be blocked by preinjected free FA. [8][9][10] Some studies have linked a gene vector with FA and have made targeted gene vectors, such as FA-1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine-cholesterol and FA-poly(ethylene glycol)-polyethylenimine. 11,12 Compared with nontargeted carriers, these new vectors can improve the gene transfection efficiency of FR-positive HEK 293T cells, C6 cells, and KB cells in vitro, 11,12 and have had stronger antitumor effects in experiments in vivo.…”

Imaging the Folate Receptor on Cancer Cells with 99mTc-Etarfolatide: Properties, Clinical Use, and Future Potential of Folate Receptor Imaging

“…Both have shown good tumor visualization, with limited accumulation in nonmalignant tissues (24,25). 68 Ga-labeled radiofolates are also of interest because of their favorable biodistribution profile and 1-step radiolabeling (26,27). However, the short physical half-life of 68 Ga (68 min) limits the time window for imaging compared with 18 F (110 min) (21).…”