18 F-click labeling and preclinical evaluation of a new 18 F-folate for PET imaging

Abstract

Background: The folate receptor (FR) is a well-established target for tumor imaging and therapy. To date, only a few 18 F-folate conjugates via 18 F-prosthetic group labeling for positron emission tomography (PET) imaging have been developed. To some extent, they all lack the optimal balance between efficient radiochemistry and favorable in vivo characteristics.

Methods: A new clickable olate precursor was synthesized by regioselective coupling of folic acid to 11-azido-3,6,9-trioxaundecan-1-amine at the γ-position of the glutamic acid residue. The non-radioactive reference compound was synthesized via copper-catalyzed azide-alkyne cycloaddition of 3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)prop-1-yne and γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide. The radiosynthesis was accomplished in two steps: at first a 18 F-fluorination of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate, followed by a 18 F-click reaction with the γ-azido folate. The in vitro, ex vivo, and in vivo behaviors of the new 18 F-folate were investigated using FR-positive human KB cells in displacement assays and microPET studies using KB tumor-bearing mice.

Results: The new 18 F-folate with oligoethylene spacers showed reduced lipophilicity in respect to the previously developed 18 F-click folate with alkyl spacers and excellent affinity (Ki = 1.6 nM) to the FR. Combining the highly efficient 18 F-click chemistry and a polar oligoethylene-based 18 F-prosthetic group facilitated these results. The overall radiochemical yield of the isolated and formulated product averages 8.7%. In vivo PET imaging in KB tumor-bearing mice showed a tumor uptake of 3.4% ID/g tissue, which could be reduced by FR blockade with native folic acid. Although the new 18 F-oligoethyleneglycole (OEG)-folate showed reduced hepatobiliary excretion over time, a distinct unspecific abdominal background was still observed.

Conclusions: A new 18 F-folate was developed, being available in very high radiochemical yields via a fast and convenient two-step radiosynthesis. The new 18 F-OEG-folate showed good in vivo behavior and lines up with several recently evaluated 18 F-labeled folates.

Scheme 1

Synthesis of the azido-folate (5) and the reference compound (6). (a) COMU, TMP, MeCN, rt. (b) DCM/TFA (1:1), rt. (c) COMU, TMP, DMF, rt. (d) 1 M NaOH (aq.), rt. (e) NaH, DMF, 0°C to rt. (f) DCM, DAST, 0°C to rt. (g) Cu(I)I, sodium ascorbate, MeCN, 0.05 M phosphate buffer, DIPEA/2,6-lutidine (1:1), microwave.

Scheme 2

Synthesis of the prosthetic group precursor (10), followed by radiosynthesis of the 18F-labeled prosthetic group [18F]11 and 18F-labeled radiofolate [18F]12. Synthesis of the prosthetic group precursor (10), followed by radiosynthesis of the ¹⁸ F-labeled prosthetic group [¹⁸ F]11 and ¹⁸ F-labeled radiofolate [¹⁸ F]12. (a) DCM, TEA, Tos-Cl, rt. (b) TBA-OH, acetonitrile, 110°C. (c) Cu(II)acetate, sodium ascorbate, 110°C, acetonitrile/water/0.05 M phosphate buffer.

Figure 1

Displacement assay. Displacement assay of native folic acid and ¹⁸ F-OEG-folate 6 using [³H]folic acid and human KB cells in cell suspension. Experiments were conducted in triplicate (n = 3).

Figure 2

Results of ex vivo biodistribution studies under control and blockade conditions using [¹⁸ F]12 in mice. The human KB tumor-bearing nude mice were euthanized 60 min post-injection. Data are expressed as percentage injected dose per gram tissue (%ID/g). In the blockade group (n = 3), 100 μg of native folic acid was injected into each animal 5 min prior to the radiotracer administration, whereas the control group (n = 3) received a corresponding volume of PBS.

Figure 3

PET images of a KB tumor-bearing mouse. Scanned 60 to 90 min after injection of [¹⁸ F]-OEG-folate 12 (approximately 13 MBq). (A) Maximal intensity projection. (B) Two representative coronal slices. Tu, tumor; Ki, kidneys; Bl, urinary bladder; Int, intestines/feces; SUV, standardized uptake value.