Abstract
Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H–NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.
Highlights
Introduction distributed under the terms andFibroblast activation protein (FAP) is a type II serine protease that cleaves peptides after proline residues with endopeptidase and dipeptidyl-peptidase activities
Considering that the 6-hidrazinylnicotinic acid (HYNIC) contains two nitrogen atoms of the hydrazine group that could function as additional anchoring sites in the active site of fibroblast activation protein (FAP), as well as HYNIC’s ability to function as a 99m Tc chelator, the objective of this research was to design, synthesize and preclinically evaluate, a new FAP inhibitor radioligand based on the 99m Tc-HYNIC-D-alanine-boroPro (99m Tc-iFAP) structure
The design of the iFAP molecule [((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin2-yl) boronic acid] was carried out by molecular docking, which was compared in affinity and inhibition constant (Ki) with two of the most representative structures of the boroPro family reported in the literature: N-acyl-Gly-boroPro [4] and N-(pyridine-4-carbonyl)-D
Summary
Fibroblast activation protein (FAP) is a type II serine protease that cleaves peptides after proline residues with endopeptidase and dipeptidyl-peptidase activities. FAP is primarily expressed on activated stromal fibroblasts present in most human epithelial tumors but not in normal fibroblasts [1]. Specific FAP inhibitors were initially developed as potential anticancer drugs [2,3,4]. Jansen et al reported the synthesis of 39 new FAP inhibitors to assess the 4-quinolinoyl-Gly-cyanopyrrolidine activity [5]; they found that FAP forms covalent bonds with the nitrile group [6]. The authors reported that derivatives containing N-pyridines produce FAP inhibitors recognized by other cleavage enzymes, such as prolyl-oligopeptidase (PREP) and dipeptidyl-peptidases (DPP), while the molecules containing the quinolinoyl fragment showed a higher affinity for FAP
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