64Cu-Labeled Folate Radioconjugate for PET Imaging of Folate Receptor-Positive Tumors

Abstract

Purpose: Folate-receptor (FR)-targeted radioconjugates are promising for the imaging of a wide variety of tumor types [1]. So far, a number of folic acid conjugates have been developed for labeling with radiometals (e.g. 99mTc, 111In, 68Ga) for SPECT and PET imaging [2]. The aim of this study was to synthesize a folate conjugate with an albumin-binding entity, which is known to increase the conjugate's blood circulation time, and a NODAGA-chelator for 64Cu-labeling and [3]. 64Cu is a promising PET radionuclide for rf42 due to its excellent β+-energy (Eβ+=278 keV) and its reasonably long half-life (T1/2 = 12.7 h) [4]. The 64Cu-NODAGA-folate (64Curf42) was investigated in vitro and in vivo using mice bearing FR-positive tumor xenografts. Materials/methods: 64Cu was produced via the irradiation of, and subsequent chemical separation from, a Ni target at the research cyclotron at PSI. The synthesis of the NODAGAfolate conjugate (rf42) was carried out in eight steps and the resulting product was characterized by HPLC, MS and NMR. The radioconjugate was prepared by incubation of rf42 with 64CuCl2 in a mixture of HCl/Na-acetate (pH ∼5.5) at 45°C for 15 min. 64Cu-rf42 was investigated regarding its in vitro stability in PBS (pH 7.4) and in human plasma. FR-positive KB tumor cells were used for in vitro experiments. Biodistribution studies were carried out in KB tumor-bearing nude mice at 2 h, 4 h and 24 h post injection (p.i.) of 64Curf42. A small-animal scanner (Genisys8) was used for the PET/CT imaging studies Results: rf42 was synthesized with an overall yield of 5% and 95% purity. The radiochemical purity of 64Cu-rf42 was >99% at a specific activity of 10 MBq/nmol. 64Cu-rf42 was stable (>95%) over a period of 24 h in buffer and plasma. Cell internalization studies showed high uptake of 64Cu-rf42, while co-incubation of cells with excess folic acid to block FRs reduced the uptake to less than 1%. In vivo, high accumulation of radioactivity was found in KB tumors (13.36 ± 0.82% IA/g) 2 h after injection of 64Cu-rf42. As a consequence of the enhanced blood circulation time of 64Curf42, due to its albumin-binding properties, the tumor uptake increased over time (16.20 ± 3.58% IA/g, 24 h p.i.). Relevant uptake of 64Cu-rf42 was also found in the kidneys, which also express the FR. This resulted in a tumor-to-kidney ratio of 0.55 ± 0.11 at 24 h p.i. In non-targeted tissues undesired accumulation of radioactivity decreased over time. PET/CT imaging studies allowed excellent visualization of tumor xenografts up to 60 h p.i. of 64Cu-rf42. Conclusion: The novel folate-radioconjugate, 64Cu-rf42, was readily prepared and showed FR-specific accumulation in vitro and in vivo. The half-life of 64Cu is well suited to the enhanced circulation time of this novel albumin-binding conjugate. Based on the excellent PET images obtained from visualizing tumors in mice, 64Cu-rf42 may have the potential for future application in the clinics for diagnosis of FRpositive tumors and monitoring tumor response after therapy.