Development and evaluation of radiotracers for tumor imaging via positron emission tomography
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Abstract
Positron emission tomography (PET) enables the non-invasive imaging of metabolical processes in the human body. Thus, the use of radiolabeled cancer-specific targeting vectors can facilitate early diagnosis of tumors. A robust and reliable attachment of the radionuclide to the bioactive compound without major influences on its pharmacokinetic behavior is mandatory for routine PET imaging. In the herein presented work two different 18F-prosthetic groups for the radiolabeling of biomolecule-azides via click chemistry were evaluated.
First, a strained prosthetic group based on (aza)dibenzocyclooctyne (DBCO) was developed to enable copper-free strain-promoted alkyne-azide cycloadditions (SPAAC) with biomolecule-azides. The radiolabeling conditions were evaluated and optimized providing [18F]fluoro-DBCO in good radiochemical yields and within a suitable synthesis time. Click reactions with various azides (cRGD, ɲMSH peptides, maltohexaose, PDGF-ɴ peptide) showed very high yields under mild conditions, which proved the general feasibility of this novel prosthetic group for the labeling of sensitive biomolecules with fluorine-18.
The oxidized form of folic acid (5,6,7,8-tetrahydrofolate, THF) plays an important role as one-carbon donor during DNA synthesis. The reduced folate carrier (RFC) provides sufficient supply of THF for normal cell proliferation. Tumors additionally (over)express the folate receptor (FR) due to their increased proliferation rate. This makes the FR an ideal oncological target for diagnosis and therapy. Most of the 18F-labeled folate derivatives until now suffer from either insufficient radiolabeling or unfavorable in vivo behavior. Especially the need of cytotoxic copper during click radiolabeling might hinder the application of [18F]fluoro-click-folates for human use. On one hand the novel [18F]fluoroDBCO was used for the copper-free radiofluorination of a folate-azide. On the other hand, the recently reported [18F]fluoro-alakyne was applied to the same folate-azide to improve its pharmacokinetic behavior. Both [18F]fluoro-folates were available in good radiochemical yields and showed high affinities to the FR. Unfortunately, the DBCO-moiety increased the overall lipophilicity of this novel folate-based radiotracer. Thus, high unspecific binding was observed in cell studies and the tumor was not visible in µPET imaging studies due to high abdominal background. In contrast, the [18f]fluoro-alafolate showed a much more favorable lipophilitity reducing the unspecific binding in vitro and provided moderate tumor accumulation combined with a suitable tumor-to-background contrast.
In a second part of this work, [18F]fluoro-alakyne was evaluated as a tumor imaging candidate. Tumors in general show a high demand on amino acids due to an increased protein synthesis rate. Most of the amino acid based radiotracers suffer from unwanted accumulation in the abdominal region, limiting the application of these radiotracers for the detection of brain tumors. [18F]fluoro-alakyne showed low accumulation in non-small cell lung cancer xenografts but no unspecific binding in other organs combined with an exclusively renal excretion pathway. Therefore, despite the low tumor uptake a good tumor visibility was achieved. These findings enable a 2in1 approach where [18F]fluoro-alakyne cannot only be used as a polar 18F-prosthetic group but also as a highly potential tumor imaging radiotracer. This approach broadens the application range of [18F]fluoro-alakyne for various different tumor types.
In the last part of this work, a microbody with specificity to the fibroblast activation protein (FAP) was derivatized with a DOTA chelator and labeled with gallium-68. In vitro cell studies with [68Ga]Ga-DOTAMC-FA-012 showed high specific accumulation in huFAP-positive cells compared to huFAP-negative cells. In addition, the multivalency approach was evaluated with a microbody neutrAvidin-biotin tetramer, showing an enhanced uptake compared to the monomer and increased specificity. This proved the high potential of this novel microbody for the imaging of epithelial cancers (breast, lung or colorectal).