[18F]Fluoronicotinic-Acid-Conjugated Folate as a Novel Candidate Positron Emission Tomography Tracer for Inflammation

Abstract

Folate receptors are clinically relevant targets, as evidenced by therapeutic agents, including mirvetuximab soravtansine-gynx, an antibody-drug conjugate recently approved for cancer treatment. In this study, we report the development of a novel positron emission tomography (PET) imaging agent, [¹⁸F]-fluoronicotinic-acid-conjugated folate ([¹⁸F]-FNA-folate), for the evaluation of folate receptor expression. The [¹⁸F]-FNA-folate was synthesized via the N-acylation of an amino-functionalized folate derivative with [¹⁸F]-FNA 4-nitrophenyl ester under mild reaction conditions. The resulting radiotracer exhibited excellent in vitro and in vivo stability, rapid blood clearance, and minimal bone uptake in mice and rats. In vitro tissue binding studies using heart sections from an experimental rat model of myocardial infarction demonstrated focal, intense, and heterogeneous uptake of [¹⁸F]-FNA-folate, and the binding specificity to macrophage folate receptors was confirmed. The straightforward radiosynthesis, excellent in vivo stability, and target-specific binding support further development of [¹⁸F]-FNA-folate as a promising PET imaging agent for inflammatory diseases.

1

Chemical scheme for the preparation of [¹⁸F]­FNA–folate. The prosthetic compound [¹⁸F]­FNA 4-nitrophenyl ester was prepared by on-resin ¹⁸F-fluorination of compound 1 and conjugation with folate precursor 2 in the presence of triethylamine as a base.

2

HPLC analysis of the radiochemical purity and chemical identity of [¹⁸F]­FNA–folate. Radiochemical purity (A) at the end of synthesis and (B) 6 h after synthesis. (C) Chemical identity of [¹⁸F]­FNA–folate was confirmed by HPLC comparison with FNA–folate as the standard under the same analytical conditions. In panel C, the HPLC trace of FNA–folate was shown in red and [¹⁸F]­FNA–folate was shown in black.

3

Coronal PET/CT images (maximum intensity projection) of a rat injected with [¹⁸F]­FNA–folate, showing weighted mean standardized uptake values (SUV_mean) at 1–10, 10 – 30, and 30–60 min post-injection, respectively.

4

Time–activity curves (TACs) of selected organs and tissues in healthy rats injected with [¹⁸F]­FNA–folate (n = 4). Heart radioactivity was quantified from the whole heart volume.

5

Coronal PET/CT images (maximum intensity projection) of a mouse injected with [¹⁸F]­FNA–folate, showing weighted mean standardized uptake values (SUV_mean) at 1–10, 10 – 30, and 30–60 min post-injection, respectively.

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Time–activity curves (TACs) of selected organs and tissues in healthy mice injected with [¹⁸F]­FNA–folate (n = 8). Heart radioactivity was quantified from the entire heart volume.

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Ex vivo tissue biodistribution of [¹⁸F]­FNA–folate in mice at 60 min post-injection (n = 4). Stomach and intestines were measured after removal of contents.

8

In vivo stability of [¹⁸F]­FNA–folate analyzed by HPLC. (A) Mouse plasma sample collected 60 min post-injection. (B) [¹⁸F]­FNA–folate tracer standard.

9

Histological, immunohistochemical, and autoradiographic analysis of rat heart tissue cryosections. (A) H&E staining revealed myocardial infarction-induced inflammatory lesions. (B) Immunohistochemistry showed activated macrophages within the infarcted (inflamed) regions. (C) Autoradiography demonstrated focal and intense binding of [¹⁸F]­FNA–folate in adjacent tissue sections, consistent with macrophage-rich inflammatory sites. (D) In vitro blocking experiments were performed on adjacent tissue sections in the presence of folate glucosamine. (E) Intensity of [¹⁸F]­FNA–folate in vitro total and blocked binding in myocardial infarction (MI)-induced inflamed tissue was significantly higher than in remote tissues.