Radiopharmaceutical for imaging


Folate is a type of B vitamin that acts as a coenzyme for the synthesis and repair of DNA and is thus essential for the development and proliferation of cells. Folate is water soluble and so cannot be stored in fat cells. A constant intake of folate in the diet is thus required to ensure that these vital processes are not interrupted1.

Ingested folate entering the blood stream gains access to cells via the anionic reduced folate carrier and the proton-coupled folate transporter. In addition, oxidized folate is carried into some cells by folate-receptor-mediated endocytosis.

The folate receptor is present at limited sites in the body, including kidney, choroid plexus, lung, salivary glands and placenta. It has a high affinity for folic acid.

Using folate in oncological imaging

The folate receptor is over-expressed on a variety of highly proliferating cancer cells, especially ovarian and endometrial cancers. In contrast, there is limited expression of the folate receptor in healthy cells. Together, this makes the folate receptor a prime target for the detection and evaluation of tumours in vivo. Indeed, expression of the folate receptor has been shown to correlate with tumour differentiation in a number of cancers and serves as a reliable prognostic indicator1.

Whole-body radionuclide imaging using radiolabelled folate provides a valuable tool for determining folate receptor expression, and consequently tumour status. Folate has several characteristics that make it a favourable candidate for an imaging ligand. It has a low molecular weight, is non-immunogenic and can withstand the harsh chemical conditions required for radiolabelling. Furthermore, such labelling does not interfere with its affinity for the folate receptor2.

The combination of properties of folate making it an ideal target for cancer imaging and therapy have resulted in a range of radiolabelled folates being developed2.

Folate-based radiopharmaceuticals

The majority of effective radiofolates produced feature radionuclides suitable for use in single photon emission computed tomography (SPECT). These include 111In-diethylenetriamine pentaacetic acid-folates, 99mTc-folates and 67Ga-folates, for which preclinical in vivo tumour targeting investigations have been promising3. In contrast, there are few radiofolates suitable for clinical positron emission tomography (PET) imaging.



The development of folate-based PET radiopharmaceuticals has been heavily centred on a variety of 18F-folate derivatives. These can be prepared using a range of strategies involving either direct labelling or conjugation with 18F-labeled prosthetic groups. With either production route, the challenge is to maintain the desired pharmacokinetics (reduced abdominal background) whilst achieving good radiochemistry.

An 18F-folate produced by amide coupling of the prosthetic group 4-[18 F]fluorobenzylamine and native folic acid achieved good visualization of tumours expressing the folate receptor but, despite time-consuming synthesis, the radiochemical yield was low4. Yields were increased by using the copper-catalyzed azide-alkyne cycloaddition (click) reaction, but the in vivo behaviour was poor due to poor signal-to-noise ratio and a very high abdominal background5. Several alternatives were developed in attempts to improve the properties of the folate tracer, but none achieve the ideal ligand for PET imaging6. Good radiochemical yield and high tissue uptake was achieved by attaching 18F-FDG to a folate derivative using the click reaction6, but preparation times were long and the click reaction had detrimental effect on the pharmacokinetics. Most recently a novel clickable 18F prosthetic group has been developed based on alanine (18F-alakyne)7.

In addition, due to the cytotoxicity of the copper catalyst needed in the click reaction, 18F-dibenzocyclooctyne (18F-DBCO) has been used as a prosthetic group that can be coupled to folate using a copper-free click reaction (strain-promoted azide alkyne cycloaddition, SPAAC)8.

Evaluating folates for clinical PET imaging

A recent study compared the copper-free 18F-DBCO folate derivative with 18F-alakyne folate produced using the traditional copper click reaction9. This is the first time a copper-free clicked folate derivative has been tested in a PET imaging study. The two folate radioligands were assessed for in vitro characteristics and in vivo performance in a KB-xenograft-bearing mouse model. A Bruker AC-300-spectrometer was used to obtain 1H and 19F nuclear magnetic resonance (NMR) spectra, and 13C NMR was performed using a Bruker Advance II-400-spectrometer. PET/MRI-studies were performed on a nanoScan PET/MRI. The relative lipophilicities of the folate derivatives were determined by HPLC.



The radiochemical yield for 18F-alakyne folate was 22%. Mice inoculated with human KB cancer cells were intravenously injected with 18F-DBCO folate and 18F-alakyne folate. Both folate derivatives demonstrated stability in human serum albumin and high affinity for the folate receptor. However, 18F-DBCO folate was more lipophilic than 18F-alakyne folate, which resulted in a greater degree of non-specific binding. The in vivo performance was consequently higher for 18F-alakyne folate, giving much clearer visualization of folate receptor-positive tumours in PET scans7. There was lower tumour-to-liver contrast than expected due to accumulation of 18F-alakyne folate in the liver and gal bladder.

In view of high radiochemical yield, radiochemical purity and favourable pharmacokinetics, 18F-alakyne folate is expected to be a promising candidate for folate receptor PET imaging. However, there remains the need to fine-tune the metabolism profile of 18F-alakyne folate.


1. Teng L, et al. Clinical translation of folate receptor-targeted therapeutics. Expert Opin Drug Deliv. 2012;9(8):901‑908.

2. Muller C, Schibli R. Folic acid conjugates for nuclear imaging of folate receptor-positive cancer. J Nucl Med 2011;52:1-4.

3. Kim MH, et al. Synthesis and Evaluation of 99mTc-Labeled Folate-Tripeptide Conjugate as a Folate Receptor-Targeted Imaging Agent in a Tumor-Bearing Mouse Model. Nucl Med Mol Imaging. 2015;49(3):200-207.

4. Bettio A, et al. Synthesis and preclinical evaluation of a folic acid derivative labeled with 18F for PET imaging of folate receptor-positive tumors. J Nucl Med. 2006;47(7):1153‑1160.

5. Ross TL, et al. Fluorine-18 click radiosynthesis and preclinical evaluation of a new 18F-labeled folic acid derivative. Bioconjug. Chem. 2008;19:2462-2470.

6. Schieferstein H, et al. 18 F-click labeling and preclinical evaluation of a new 18 F-folate for PET imaging. EJNMMI Res. 2013;3:68.

7. Schieferstein H, Ross TL. A polar 18F-labeled amino acid derivative for click labeling of biomolecules. Eur. J. Org. Chem. 2014, 17, 3546-3550.

8. Kettenbach K, et al. A 18F-labeled dibenzocyclooctyne (DBCO) derivative for copper-free click labeling of biomolecules. Med. Chem. Commun. 2016;7:654-657.

9. Kettenbach K, et al. Comparison Study of Two Differently Clicked 18F-Folates-Lipophilicity Plays a Key Role. Pharmaceuticals 2018;11:30.