Automated Synthesis of Fluorine-18 Labeled CXCR4 Ligand via the Conjugation with Nicotinic Acid N-Hydroxysuccinimide Ester (6-[18F]SFPy)

Abstract

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[¹⁸F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[¹⁸F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[¹⁸F]SFPy) have been prepared using 'fluorination on the Sep-Pak' method. Conjugation of 6-[¹⁸F]SFPy or 6-[¹⁸F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[¹⁸F]FPy-T140. The overall radiochemical yields were 6-17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[¹⁸F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [¹⁸F]SFPy has been prepared using 'fluorination on the Sep-Pak' method that allows rapid automated synthesis of 6-[¹⁸F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

Keywords: CXCR4; SFPy; T140; fluorination on the Sep-Pak; fluorine-18.

Figure 1

Structures of C-X-C motif chemokine receptor 4 (CXCR4) ligands.

Figure 2

HPLC analysis (method B) of 6-[¹⁸F]SFPy (A) Crude reaction mixture; (B) Crude reaction mixture co-injected with the non-radioactive standard; (C) Pure; (D) Pure co-injected with the non-radioactive standard; (E) After 4h of labeling at room temperature co-injected with the non-radioactive standard, 6-fluoronicotinic acid. Solid line, in-line radio detector; dotted line, UV detector at 254 nm. Retention time (min): 6-[¹⁸F]SFPy, 4.0; compound 4, 4.1; 6-[¹⁸F]fluoronicotinic acid, 2.8.

Figure 2

HPLC analysis (method B) of 6-[¹⁸F]SFPy (A) Crude reaction mixture; (B) Crude reaction mixture co-injected with the non-radioactive standard; (C) Pure; (D) Pure co-injected with the non-radioactive standard; (E) After 4h of labeling at room temperature co-injected with the non-radioactive standard, 6-fluoronicotinic acid. Solid line, in-line radio detector; dotted line, UV detector at 254 nm. Retention time (min): 6-[¹⁸F]SFPy, 4.0; compound 4, 4.1; 6-[¹⁸F]fluoronicotinic acid, 2.8.

Figure 2

HPLC analysis (method B) of 6-[¹⁸F]SFPy (A) Crude reaction mixture; (B) Crude reaction mixture co-injected with the non-radioactive standard; (C) Pure; (D) Pure co-injected with the non-radioactive standard; (E) After 4h of labeling at room temperature co-injected with the non-radioactive standard, 6-fluoronicotinic acid. Solid line, in-line radio detector; dotted line, UV detector at 254 nm. Retention time (min): 6-[¹⁸F]SFPy, 4.0; compound 4, 4.1; 6-[¹⁸F]fluoronicotinic acid, 2.8.

Figure 3

Schematic diagram of the automated synthesis of 6-[¹⁸F]FPy-CONH-T140 on GE FX-N Pro Module.

Figure 4

HPLC analysis (method B) of (A) 6-[¹⁸F]FPy-T140; (B) 6-[¹⁸F]FPy-T140 co-injected with the non-radioactive standard. Solid line, in-line radio detector; dotted line, UV detector at 254 nm. Retention time (min): 6-[¹⁸F]FPy-T140, 7.3.

Figure 4

HPLC analysis (method B) of (A) 6-[¹⁸F]FPy-T140; (B) 6-[¹⁸F]FPy-T140 co-injected with the non-radioactive standard. Solid line, in-line radio detector; dotted line, UV detector at 254 nm. Retention time (min): 6-[¹⁸F]FPy-T140, 7.3.

Figure 5

Representative plot from an in vitro 6-[¹⁸F]FPy-T140 saturation binding assay using HeLa cells with each point representing the average of duplicates. B_t = Btotal; Bns = Bnon-specific (determined in the presence of 10⁻⁶ M unlabeled T-140 peptide); Bsp = Bspecific (Bt-Bns).

Scheme 1

Preparation of [¹⁸F]SFPy by Sep-Pak fluorination method and conjugation with T140.