Fluorine-18 Click Radiosynthesis and Preclinical Evaluation of a New ¹⁸F-Labeled Folic Acid Derivative

Abstract

The folate receptor (FR) is highly expressed on most epithelial cancer cells, while normal cells show only restricted expression of FR. As a result, the FR is an ideal target for receptor-based molecular imaging and therapy of cancer and has become a promising target in oncology. To date, several folate-based chemotherapeutics and imaging probes such as radiopharmaceuticals for single photon emission computed tomography (SPECT) have been developed. However, an (18)F-labeled folic acid derivative suitable for positron emission tomography (PET) imaging that can be routinely applied is still lacking. In this study, a new fluorinated and radiofluorinated folic acid derivative, (18/19)F-click folate, was synthesized using click chemistry. In a convenient and very efficient two-step radiosynthesis, the isolated (18)F-click folate was obtained in good radiochemical yields of 25-35% with a specific activity of 160+/-70 GBq/micromol after <or=90 min synthesis time. The new compound was pharmacologically evaluated in vitro and in vivo. The affinity of the non-radioactive (19)F-click folate to the FR was determined in displacement studies with FR expressing KB tumor cells using (3)H-folic acid. In these in vitro binding studies, a nanomolar affinity with a K(i) of 9.76+/-3.13 nM was found for (19)F-click folate. The (18)F-labeled click folate derivative was then applied for in vivo PET studies and ex vivo biodistribution experiments using nude mice bearing KB tumor xenografts. The post mortem dissection experiments showed a high specific uptake of (18)F-click folate derivative in FR-expressing tissues. Uptake in KB tumor xenografts and kidneys (FR-positive tissue) amounted to 3.13%ID/g (94% specific blockade) and 16.53%ID/g (75% specific blockade), respectively. PET imaging using (18)F-click folate permitted a visualization of KB tumors, and blockade studies confirmed the specific accumulation of the radiotracer in vivo. However, strong hepatobiliary excretion of the new tracer led to elevated accumulation of radioactivity in the abdominal region. In conclusion, the click chemistry approach is convenient to accomplish and provided high radiochemical yields of (18)F-click folate. The new tracer showed good in vitro but limited in vivo properties. Ultimately, the (18)F-click folate emphasizes the potential of (18)F-labeled folates for receptor-based tumor PET imaging.

A New 18F-Labeled Folic Acid Derivative with Improved Properties for the PET Imaging of Folate Receptor–Positive Tumors

“…In vitro displacement experiments using KB tumor cells and 3 H-folic acid as radioligand revealed a K i value of 1.8 nM, the highest in vitro binding affinity obtained so far in our group for fluorinated folates (19,20). In vivo PET studies in nude mice found highly specific uptake of 29-18 F-fluorofolic acid in the tumor as well as in the folate receptor-positive kidneys as demonstrated by blockade experiments (Fig.…”

“…In the same assay, folic acid exhibited an inhibitory concentration of 50% of 1.1 6 0.4 nM (K i 5 0.6 6 0.2 nM) (20). The displacement curves of 29-fluorofolic acid and folic acid are depicted in Figure 2.…”

“…1) was determined on a reversed-phase HPLC system. The same HPLC system was already used in previous studies for the determination of the relative lipophilicity of native folic acid (k9 5 0.30), a/g-FBA folate (both regioisomeres: k9 5 0.67), and Fclick folate (k9 5 2.28) (20). For 29-fluorofolic acid, a k9 value of 0.53 was obtained.…”

“…More recently, we reported another 18 F-labeled folate conjugate designated 18 F-click folate (Supplemental Fig. 1) (20). An efficient 2-step procedure provided the 18 F-click folate in high radiochemical yields of 25%-35% in less than 90 min.…”

Click Reaction: An Applicable Radiolabeling Method for Molecular Imaging

“…Thus, 18 F-CuAAC is a very attractive strategy for the design and synthesis of radiotracers for molecular imaging purposes. Ross and coworkers used 6-[ 18 F]fluoro-1-hexyne to generate, by click reaction, a folic acid derivative as a promising target molecule in oncology [4]. 18 F-click folate was synthesized by a convenient and efficient two-step radiosynthesis.…”