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.

Highlight selection of radiochemistry and radiopharmacy developments by editorial board

“…A few research groups have reported analogues of SFB, with the aim for simplifying radiosynthesis protocols. Recently, a “fluorination on the Sep-Pak” method to prepare a prosthetic agent nicotinic acid N- hydroxysuccinimide ester (6-[ 18 F]SFPy) (Basuli et al, 2020 ) was published. The actual 18 F-fluorination takes place when passing the precursor compound through a solid-phase extraction cartridge pre-loaded with [ 18 F]fluoride.…”

Radiolabeled Peptides and Antibodies in Medicine

“… 92 Despite moderate results of 68 Ga-NOTA-NFB, other modifications to the T140 derivatives are under development. 16 , 93 …”

“…Unfortunately, the clearance pattern of 68 Ga-NOTA-NFB exhibited very high splenic and liver uptake, which challenged the application of 68 Ga-NOTA-NFB PET/CT in high contrast clinical imaging of CXCR4 expression . Despite moderate results of 68 Ga-NOTA-NFB, other modifications to the T140 derivatives are under development. , …”

“…This method was used in production 6- 18 F Fluoronicotinic acid N -hydroxysuccinimide ester (6-[ 18 F]SFPy) and then radiolabeling T140 peptide–CXCR4 PET imaging ligand. 16 Another possible method for binding the prosthetic group to a peptide is modification of the peptide to bear unnatural bioorthogonal functional groups like an azide and then by using “click” chemistry to achieve the radiolabeled biomolecule. 17 , 18 Tolmachev et al presented a large number of methods for binding prosthetic groups carrying various radionuclides to peptides in their review article.…”

“…Alcohols, amines, carboxylic acids, and thiols may be paired with the reactive site of a prosthetic group conjugated with a radionuclide to achieve the complete radiolabeled peptide. − A very effective method of 18 F radiolabeling peptides by using prosthetic groups is the automated “fluorination on the Sep-Pak” method. This method was used in production 6- 18 F Fluoronicotinic acid N -hydroxysuccinimide ester (6-[ 18 F]­SFPy) and then radiolabeling T140 peptide–CXCR4 PET imaging ligand . Another possible method for binding the prosthetic group to a peptide is modification of the peptide to bear unnatural bioorthogonal functional groups like an azide and then by using “click” chemistry to achieve the radiolabeled biomolecule. , Tolmachev et al presented a large number of methods for binding prosthetic groups carrying various radionuclides to peptides in their review article …”