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

The folate receptor (FR) has emerged as an interesting diagnostic and therapeutic drug target with many potential applications in oncologic and inflammatory disorders. It was therefore the aim of this study to develop a folate-derived Ga-68-based positron emission tomography (PET) imaging tracer that is straightforward to radiolabel and could be broadly used in clinical studies. We validated its target binding affinity and specificity and compared it to [^99mTc]EC20, the folate single-photon emission computed tomography (SPECT) imaging tracer that has been most extensively studied clinically so far.

The new folic acid-derived PET imaging agent is linked via a polyethyleneglycol linker to the chelator 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA). This new compound, NOTA-folate, was labeled with gallium-68. We tested the probe's stability in human plasma and its selectivity in vitro, using the FR-positive KB cell line as well as the FR-negative A549 cell line. The pharmacokinetic profile of [⁶⁸Ga]NOTA-folate was evaluated in FR-positive KB mouse xenografts. Following intravenous injection of [⁶⁸Ga]NOTA-folate (383 ± 53 μCi), PET/computed tomography (CT) imaging studies as well as biodistribution studies were performed using KB tumor-bearing mice (n = 3). In vitro as well as in vivo studies were performed in parallel with the SPECT imaging tracer [^99mTc]EC20.

In comparison to [^99mTc]EC20 (radiochemical yield (RCY) = 82.0 ± 2.9 %, 91.8 ± 2.0 % purity), similar radiochemical yield (87.2 ± 6.9 %) and radiochemical purity (95.6 ± 1.8 %) could be achieved for [⁶⁸Ga]NOTA-folate. For both tracers, we observed high affinity for FR-positive cells in vitro and high plasma stability. In PET/CT and biodistribution studies, [⁶⁸Ga]NOTA-folate appeared to display slightly superior in vivo performance in comparison to [^99mTc]EC20. In detail, ⁶⁸Ga-NOTA-folate showed very good tumor uptake and retention (6.6 ± 1.1 %ID/g), relatively low kidney uptake (21.7 ± 1.1 %ID/g), and very low liver uptake (0.38 ± 0.08 %ID/g). In vivo blocking studies using a fivefold excess of EC20 reduced the tumor uptake to 2.5 ± 0.7 %ID/g, confirming receptor specific binding of [⁶⁸Ga]NOTA-folate in vivo.

We validated a new Ga-68 folate-based PET imaging agent with excellent pharmacokinetics and tumor uptake. Based on a head-to-head comparison between both tracers, [⁶⁸Ga]NOTA-folate is a suitable imaging probe for the delineation of FR-positive tumors and a promising candidate for clinical translation.