Design, Synthesis and Biological Evaluation of a Novel [18F]AlF-H3RESCA-FAPI Radiotracer Targeting Fibroblast Activation Protein.

Design, Synthesis and Biological Evaluation of a Novel [18F]AlF-H3RESCA-FAPI Radiotracer Targeting Fibroblast Activation Protein. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design, Synthesis and Biological Evaluation of a Novel [18F]AlF-H3RESCA-FAPI Radiotracer Targeting Fibroblast Activation Protein. Qingyu Zhang, Zhoumi Hu, Haitao Zhao, Fuqiang Du, Chun Lv, Tukang Peng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5297123/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cancer-associated fibroblasts (CAFs), critical in tumor progression, overexpress fibroblast activation protein (FAP), presenting it as a promising target for tumor imaging and therapy. Our objective was to develop a novel radiotracer, [ 18 F]AlF-H 3 RESCA-FAPI, that achieves high labeling efficiency at room temperature for PET imaging of FAP-expressing tumors. Results The structure's feasibility was confirmed through molecular docking and ADMET prediction. H 3 RESCA-FAPI was synthesized and radiolabeled with [ 18 F]AlF 2+ . Optimal labeling conditions were identified as pH 5.0, a molar ratio of aluminum chloride to precursor of 0.58, and a precursor mass of 50 µg. The radiotracer demonstrated high binding affinity to FAP ( K D < 10.09 pM), favorable radiochemical yield (52.0 ± 3.0%), and radiochemical purity exceeding 95%. In vitro and in vivo studies revealed good stability and rapid clearance from non-target tissues. PET imaging in U87MG tumor-bearing mice showed substantial tumor uptake, which was specifically blocked by co-injection with unlabeled DOTA-FAPI-04, confirming tumor-specific uptake. Conclusions [ 18 F]AlF-H 3 RESCA-FAPI is a promising radiotracer for PET imaging of FAP-expressing tumors, exhibiting high tumor-specific uptake. With further structural modifications to enhance pharmacokinetic properties, it could become a potential candidate for clinical translation, providing a readily accessible new tool for future non-invasive tumor imaging research. H3RESCA Chelator Fibroblast Activation Protein Inhibitor Fluorine-18 Positron Emission Tomography Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Tumor microenvironment (TME), including fibroblasts, myeloid-derived suppressor cells (MDSCs), macrophages, lymphocytes, the extracellular matrix (ECM) and intertwined blood vessels constructed by endothelial cells and pericytes.(Xueman Chen and Song 2018 ) These stromal cells tightly interact with cancer cells, creating a supportive environment for tumor growth. Among these, cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression by contributing to processes such as tumorigenesis, neoangiogenesis, metastasis, immunosuppression, and drug resistance.(Lindner et al. 2019 ; Puré and Blomberg 2018 ) Fibroblast activation protein (FAP), a type II transmembrane serine protease belonging to the dipeptidyl peptidase 4 family with both dipeptidyl peptidase and endopeptidase activity,(Hamson et al. 2014 ) is overexpressed on CAFs in more than 90% of epithelial carcinomas.(Bu et al. 2019 ; Busek et al. 2018 ; Lamprecht et al. 2018 ; Šimková et al. 2020 ) Therefore, FAP is considered a promising target for both tumor imaging and therapy. In 2014, UAMC-1110 (Fig. 1 A) was identified as the most potent and selective small molecule inhibitor of FAP.(Jansen et al. 2013 ; Jansen et al. 2014 ) Since then, some classic UAMC-1110-based PET tracers have been developed,(Lindner et al. 2018 ; Loktev et al. 2019 ; Loktev et al. 2018 ) such as [ 68 Ga]Ga-DOTA-FAPI-04 (Fig. 1 B) and [ 18 F]AlF-NOTA-FAPI-42 (Fig. 1 C). Initially, [ 68 Ga]Ga-DOTA-FAPI-04 garnered the majority of attention within the FAPI-diagnostics field;(Kratochwil et al. 2019 ) however, current research is also focusing on 18 F-labeled FAPIs due to the excellent spatial resolution theoretically obtained by 18 F-PET imaging(N. Zhang et al. 2023 ) and the relatively long half-life of 18 F (t 1/2 = 109.8 min, compared with t 1/2 ( 68 Ga) = 67.7 min). Furthermore, the limited availability of 68 Ga, which must be produced in relatively small batches through 68 Ge/ 68 Ga generators, contributes to the growing interest in 18 F-labeled FAPIs. On the contrary, 18 F can be mass-produced in cyclotron or transported to PET imaging center which lacking radionuclide production capacity.(Cho et al. 1975 ; Hu et al. 2022 ) The labeling techniques for 18 F can be categorized into two groups: nucleophilic 18 F- derivatives generated by various substitution reactions,(Toms et al. 2020 ; Yu et al. 2023 ; N. Zhang et al. 2023 ) and the utilization of aluminum fluoride ([ 18 F]AlF 2+ ) for radiolabeling via coordination chemistry.(Zhou et al. 2024 ) The method of using [ 18 F]AlF labeling has been expanding, and a series of different chelator groups have achieved great success, such as NOTA, NODA, NODAGA, 2-AMPTA, DTPA, most of these require certain requirements for equipment.(Carroll et al. 2024 ) Recently, the H 3 RESCA has been widely used in 18 F labeling of antibodies because of its mild reaction conditions and straightforward labeling process,(Cleeren et al. 2018 ; Wegrzyniak et al. 2024 ) which has piqued our interest. Our research aims to utilize H 3 RESCA for the labeling of small molecules with 18 F, using FAPI as an example to broaden the potential applications of FAPI in nuclear medicine (Fig. 1 D). Methods and Materials General Except as indicated, compound 1 and Compound 2 were procured from TanzhenBio (Nanchang, China), and other chemicals and reagents were procured from Merck (Shanghai, China) and used without additional purification. Characterization of synthesized compounds was performed using LC-MS on an Infinity Lab mass spectrometer equipped with an Agilent 1260 series HPLC system and an Extend-C18 column (50 mm × 2.1 mm, 1.8 µm), monitores at a UV wavelength of 254 nm. Radioisotope 18 F − was produced in a medical cyclotron (HM-10, Sumitomo Heavy Industries Ltd., Tokyo, Japan) via the 18 O (p, n) 18 F reaction. Quality control of the radiotracers was conducted using HPLC with a C18 column (C18-TH, 5µm, 100Å, 150mm × 4.6mm, Morhchem Technologies Inc., United States) and a 1260 Quat pump VL, a 1260 DAD VL detector, a 1260 Vialsampler, and a γ-detector for radioactivity measurement (Eckert and Ziegler, United States). The mobile phase composition of water and acetonitrile was adjusted according to the specific compounds. Radioactivity measurements were taken with a CRC-55T activity meter (The China National Nuclear Corporation, Beijing, United States). Molecular Docking Three-dimensional structures of DOTA-FAPI-04, NOTA-FAPI-42 and H 3 RESCA-FAPI were constructed using Chem3D 20.0 (PerkinElmer). The resultant mol2 files were converted to .pdb format with PyMol 2.6.0. The target protein (PDB ID: 1Z68) underwent preparation for docking by adding hydrogen atoms with AutoDockTools 1.5.7 (The Scripps Research Institute). A docking grid, encompassing the catalytic site of FAPα, was defined. This grid was centered around the catalytic triad—Ser 624 , Asp 702 , and His 734 —using AutoGrid 4.0, which set the grid dimensions at 24 × 24 × 24 ų and a grid spacing of 1.000 Å, to ensure comprehensive coverage of the catalytic site. The coordinates for the grid box center were established at x = 25.071 Å, y = 10.676 Å, and z = 25.618 Å. Molecular docking, carried out with Autodock Vina 1.1.2, evaluated the influence uof various chelator groups on the binding affinity of FAPI-4 to FAP. Each compound underwent three independent docking simulations. The lowest-energy conformation from each set of runs, as identified by Autodock Vina, was selected for visualization with PyMol 2.6.0. ADMET Profiling Studies The ADMET properties of FAPI-based ligands were predicted using the ADMETlab 3.0 web tools, which facilitate a comprehensive ADMET analysis. Ligand structures, described by their Simplified Molecular Input Line Entry System (SMILES) representations, were input into the platform to generate 2D structural files. This enabled ADMETlab 3.0 to compute and evaluate essential ADMET parameters effectively. Notably, the SMILES strings for two specific ligands were generated with ChemDraw 20.0 and are detailed in Supplementary Texts S1 and S2 . Chemistry 2,2'-(((1R,2R)-2-((carboxymethyl)(4-(2-(4-(3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)benzyl)amino)cyclohexyl)azanediyl)diacetic acid (3). Compound 1 (50 mg, 103 µmol) was dissolved in DMSO (4 mL), followed by the addition of compound 2 (77 mg, 131 µmol) and triethylamine (Et 3 N, 100 µL, 719 µmol). The mixture was stirred at room temperature (20°C) for 4 hours and then concentrated under reduced pressure. Purification was achieved using semi-preparative HPLC with a COSMOSIL 5C18-MS-Ⅱ column (120 Å, 5 µm, 250 × 10.0 mm) and a gradient elution of acetonitrile/water (0.1% TFA) initiated at 16/84 (v/v) for 20 minutes at a flow rate of 2 mL/min. The solvent was removed under reduced pressure to yield compound 3 as a white powder (32.2 mg, 34.6%). LC-MS (ESI) analysis showed a molecular ion peak at m/z = 904.8 [M + H] + . Binding Affinity Measurement In order to determine the binding kinetics between ligands and human FAP protein (10464-H07H Sino Biological), the Biacore SPR interaction study was conducted. The binding affinity is expressed by the equilibrium dissociation constant ( K D ) (M), which is calculated from the values of the measured binding rate constant ( k a ) (M − 1 s − 1 ) and dissociation rate ( k d ) (s − 1 ). Radiochemistry and Quality Control 18 F − was loaded onto an activated QMA ion exchange column (Waters GmbH, Germany), which had been previously eluted with 1 mL of saline and air-dried. The column containing 18 F − was subsequently eluted with 1 mL of saline to obtain a Na 18 F solution. Reaction conditions were screened using five different pH buffers: a NaHP buffer at pH 6.0 (0.2 M) and a sodium acetate buffer ranging from pH 4 to 5.5 (0.2 M). For the reactions, 100 µL of Na 18 F solution (370 MBq) and 12 µL of AlCl 3 solution (1.28 mM, prepared in 0.2 M buffer solution) were added to a reaction vial. The mixture was incubated at room temperature for 5 minutes. Subsequently, 300 µL of buffer solution at varying pH values and 200 µL of H 3 RESCA-FAPI solution (0.1 mg/mL, prepared in deionized water) were added, and the mixture was allowed to react for 15 minutes at room temperature. The radiolabeling yields across different pH conditions were quantified by HPLC. Utilizing the pH buffer that yielded the highest radiolabeling efficiency, 10 µL of Na 18 F solution (37 MBq) and variable volumes of AlCl 3 solution (either 12 or 12.4 µL, 1.07 mM, prepared in the selected buffer) were added to a reaction vial and incubated at room temperature for 5 minutes. This was followed by the addition of 200 µL of H 3 RESCA-FAPI solution (0.1 mg/mL, prepared in the selected buffer) and 300 µL of the same buffer. The reaction proceeded for 15 minutes at room temperature. Radiolabeling efficiencies were then calculated by HPLC, analyzing different AlCl 3 to ligand molar ratios (0.58 or 0.60). In a subsequent experiment, the selected volume of AlCl 3 was added to a reaction flask along with 100 µL of Na 18 F solution (185 MBq) and incubated at room temperature for 5 minutes. Different volumes of H 3 RESCA-FAPI solution (200, 500, or 1000 µL, 0.1 mg/mL, prepared in the selected buffer) were then introduced, maintaining the same volume with the selected buffer. The reaction was sustained for 15 minutes at room temperature. Radiolabeling yields for varying ligand masses (20, 50, or 100 µg) were determined by HPLC. The purification protocol for the Sep-Pak Light C18 column involved first eluting the reaction solution with 5 mL of pure water through the activated column. The purified product was then collected into an aseptic vacuum bottle containing 1 mL of 50% ethanol solution, followed by the addition of 4 mL saline, rendering the preparation ready for use. For HPLC analysis, the mobile phase consisted of an aqueous solution containing 0.10% trifluoroacetic acid (Phase A) and acetonitrile (Phase B). An isocratic elution was employed, with a mobile phase consisting of 18% Phase B, maintained constant throughout the analysis. The flow rate was set at 1 mL/min. UV detection was performed at a wavelength of 254 nm. Partition Coefficient Radiotracers [ 68 Ga]Ga-DOTA-FAPI-04 and [ 18 F]AlF-H 3 RESCA-FAPI (0.37 MBq) was added to tubes containing n-octanol/PBS-mixture (1 mL, 1:1). The mixture was vortexed vigorously for 5 min at ambient temperature, and then centrifuged for five min at 8,000 rpm. In each phase, three samples (each 50 µL) was removed and measured in γ-counter (PerkinElmer). The partition coefficient was expressed as logD 7.4 = lg (CPM n−octanol /CPM PBS ) (n = 3). In Vitro and In Vivo Stability [ 18 F]AlF-H 3 RESCA-FAPI (10 MBq) in a mixture of ethanol and water was added to PBS (600 µL) or FBS (GE Healthcare, Chicago, IL, USA) (600 µL) and incubated at 37°C for 2 hours. And then, a sample of PBS or FBS (100 µL) was injected into the HPLC system for analysis. At 60 min after injection of [ 18 F]AlF-H 3 RESCA-FAPI (37 MBq/mouse), the mice were sacrificed. The urine was then collected and analysed with HPLC directly. Pharmacokinetics In Normal Mice The distribution of [ 18 F]AlF-H 3 RESCA-FAPI (0.93 MBq) in blood was assessed at 1, 2, 5, 10, 20, 30, 60, 90, 120 min using normal mice. The mice (n = 3 in each group) were adopted the blood at the selected time. All the collected blood were quickly removed and weighed, and the radio activity was counted using a γ-counter and the results were expressed as percentage of injected dose per gram of tissue (% ID/g). PET Imaging U87MG tumor-bearing mice (n = 3 per group) underwent PET imaging using an IRIS micro-PET/CT scanner (inviscan SAS, Strasbourg, France). Mice were anesthetized and positioned prone for the scans. Static PET images were acquired at 10, 30, 60, 90, and 120 min post-injection of [ 18 F]AlF-H 3 RESCA-FAPI (4 − 6 MBq/mouse). For the blocking study, [ 18 F]AlF-H 3 RESCA-FAPI was co-injecteded with unlabeled DOTA-FAPI-04 (0.5 mg/mouse), followed by a 1-hour static PET scan. The PET data were reconstructed using a three-dimensional ordered-subset expectation maximization (3D-OSEM) algorithm, which incorporates a Monte Carlo simulation for accurate modeling of the detector response. The reconstructed images were analyzed using Avatar 1.2 software (Pingseng China) for semi-quantitative assessment. Biodistribution Studies The biodistribution of [ 18 F]AlF-H 3 RESCA-FAPI (4.32–5.45 MBq) was evaluated in athymic nude mice bearing U87MG xenografts at 1 hour post-injection (p.i.). A subset of mice received a concurrent injection of the radiotracer and the competitor DOTA-FAPI-04 (0.5 mg per mouse) to assess blocking effects. At 60 min post-injection, the mice (n = 3 per group) were euthanized, and selected organs, tumors, and blood were harvested, weighed, and measured for radioactivity using a γ-counter. The results were expressed as % ID/g. Immunohistochemical Staining U87MG tumor tissues were harvested post-euthanasia and processed into paraffin-embedded sections for immunohistochemical analysis. Sections underwent deparaffinization in xylene and rehydration through graded ethanol. Antigen retrieval was performed, followed by overnight incubation with a rabbit polyclonal anti-FAP primary antibody (1:50, AF0739, Affinity) at 4℃. After rinsing, sections were incubated with a goat anti-rabbit secondary antibody (PV-9000, ZSGB-BIO) for 1 hour at room temperature. FAP expression was visualized using diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, cleared, and mounted. Positive staining was assessed and photographed under a microscope. Statistical Analysis Quantitative data are expressed as mean ± standard deviation (SD). The statistical significance between 2 independent groups was determined by the student t-test. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0.1 (Graph Software, Inc). Results Docking Simulations Molecular docking simulations were conducted to elucidate the interactions between FAPI-based ligands and the FAP enzyme, with the results depicted in Figure 2 demonstrating that the introduction of this innovative chelating agent group enhanced the interaction. Specifically, H 3 RESCA-FAPI exhibited a wider range of interactions with amino acid residues, including His 734 , Ser 624 , Arg 123 and Tyr 656 , Glu 203 , compared to DOTA-FAPI-04, which primarily interacted with Ser 624 , His 734 and Arg 123 , and NOTA-FAPI-42, which interacted with Glu 203 , Tyr 541 , Gln 547 and Arg 550 . These interactions were further characterized by the presence of potential hydrogen bonds, indicated by yellow dotted lines in the figure. Detailed scoring of these interactions is provided in Table 1. ADMET P rofiling S tudy In the preliminary stages of Computer-Aided Drug Design (CADD), the absorption, distribution, metabolism, excretion and toxicity (ADMET) profiles of chemical compounds are acknowledged as critical factors. The pharmacokinetic properties and drug-likeness metrics for these compounds are detailed in Tables S1 , S2 and S3 , respectively. Pharmacokinetic analysis indicates that H 3 RESCA-FAPI possesses a higher intestinal absorption (HIA) profile compared to DOTA-FAPI-04, which exhibits lower absorption rates. Both ligands demonstrated limited ability to cross the blood-brain barrier. The human colon epithelial cancer cell line, Caco-2, serves as a surrogate for studying drug intestinal absorption in humans. Permeability studies using the Caco-2 model revealed no significant difference between the two ligands in terms of membrane permeation. Additionally, drug-likeness was assessed based on the Lipinski Rule, Pfizer Rule, GSK Rule, and Golden Triangle Rule. Both compounds adhered to the Pfizer Rule; however, they did not comply with the Lipinski, GSK, and Golden Triangle criteria. For radioactive diagnostic agents, the administered doses are typically very low, thus mitigating concerns regarding the chemical toxicity of such drugs. In summary, the simulation experiments indicate that the two inhibitors are largely comparable, with the primary differences being their lipid solubility and intestinal uptake. The variance in lipid solubility may underlie the observed differences in intestinal absorption. Binding A ffinity Surface plasmon resonance (SPR) was utilized to evaluate the interaction between DOTA-FAPI-04 and H 3 RESCA-FAPI with recombinant human FAP protein (Figure 3). The equilibrium dissociation constant ( K D ) value of the 2 inhibitors binding to human FAP proteins exhibited a robust affinity, being in the picomole (pM) range. Specifically, The K D value of DOTA-FAPI-04 is less than 27.89 pM, while The K D value of H 3 RESCA-FAPI is less than 10.09 pM ( Figure 3A and B ). This shows that the affinity of H 3 RESCA-FAPI for FAP protein is slightly higher than that of DOTA-FAPI-04, which is consistent with the results of molecular docking simulation. Chemical and Radiochemical Syntheses H 3 RESCA-FAPI (compound 3 ) was synthesized ( Scheme 1 ), and its molecular weight was identified by LC-MS ( Figure S1 ). 18 F-labeled FAP tracer was generated via the formation of complexes of Al 18 F in a two-steps reaction. According to the lateral comparison, the reaction conditions with the highest labeling yield was as follows: buffer pH=5.0, the mole ratio of AlCl 3 to precursor was 0.58, and the weight of H 3 RESCA-FAPI was 50 μg ( Figure 4 and Table S4 ). The highly reproducible radiolabeling yield of 95% was obtained in the optimal conditions. Under the optimum labeling conditions, the total time needed for radiosynthesis was approximately 20 min, and the non-decay corrected radiochemistry yields (RCYs) of [ 18 F]AlF-H 3 RESCA-FAPI was 52.0 ± 3.0% (n = 6). The radiochemical purity (RCP) of [ 18 F]AlF-H 3 RESCA-FAPI was over 95% with molar activities of more than 14.5 GBq/μmol (n = 6) according to radioactivity measurements (Figure 5A, S2 and S3 ). The labeling yield is not only related to the three factors screened (pH of buffer, the ratio of AlCl 3 to precursor and the dosage of precursor), but is also influenced by other factors (room temperature and the concentration of reaction solution), resulting in significant variability in labeling yields under identical conditions. Octanol-Water Partition C oefficient and S tability A ssay The octanol-water partition coefficient, expressed as logD 7.4 , for the radiopharmaceuticals [ 68 Ga]Ga-DOTA-FAPI-04 and [ 18 F]AlF-H 3 RESCA-FAPI were calculated to be -3.53 ± 0.05 and -2.47 ± 0.16, respectively. This comparative analysis suggests that [ 18 F]AlF-H 3 RESCA-FAPI possesses a reduced hydrophilicity in comparison to [ 68 Ga]Ga-DOTA-FAPI-04. Given the constraints inherent in the ADMETLab 3.0 regarding the processing of SMILES files that include metal complexes, the distribution coefficients of the respective uncomplexed precursors served as a benchmark for assessing the hydrophilicity of these radiopharmaceutical agents (the logP of DOTA-FAPI-04 and H 3 RESCA-FAPI are -1.982 and -0.639, respectively). The observed hydrophilicity trends corroborate the simulation results obtained for the labeled precursors during ADMET prediction analyses. Furthermore, the stability assessment of [ 18 F]AlF-H 3 RESCA-FAPI in phosphate-buffered saline (PBS, pH=7.4), fetal bovine serum (FBS), and mouse urine—presented in Figure 5 , underscores the tracer's robust stability in both in vitro and in vivo conditions. Plasma Clearance The Plasma Clearance experiment involved the collection of blood drug concentration (%ID/g) at specific time points and fitting a curve to the data. The distribution-phase half-life (t 1/2α ) value of the [ 18 F]AlF-H 3 RESCA-FAPI was 0.76 min, and its clear-phase half-life (t 1/2β ) value was more than 60 min ( Figure 6 and Table S5 ). PET/CT Imaging Static PET imaging studies were performed in U87MG tumor-bearing nude mice to investigate the pharmacokinetics of [ 18 F]AlF-H 3 RESCA-FAPI. The coronal and axial images of [ 18 F]AlF-H 3 RESCA-FAPI at different scanning times are shown in Figure 7A . The tissue accumulation of tracer is described by standardized uptake value (SUV) scale. With regard to tumors, [ 18 F]AlF-H 3 RESCA-FAPI accumulated rapidly in U87MG tumor xenografts. A slow increase in tumor uptake was observed from 10 to 120 minute (SUV max , from 0.70 ± 0.02 to 0.72 ± 0.06). This observation indicates that [ 18 F]AlF-H 3 RESCA-FAPI exhibits a considerable retention time within the tumor. However, the liver and intestine exhibited significantly uptake values, possibly due to the certain lipid solubility of the tracer. Both ADMET prediction and LogD 7.4 measurement support this and predict increased intestinal absorption. In U87MG tumor model mice, from 10 to 120 minute, [ 18 F]AlF-H 3 RESCA-FAPI demonstrated rapid clearance kinetics in muscle (SUV max , from 0.029 ± 0.02 to 0.014 ± 0.02), intestine (SUV max , from 139.092 ± 43.447 to 53.872 ± 27.220) and liver (SUV max , from 2.251 ± 0.539 to 1.403 ± 0.370), while clearance from the kidney was comparatively slower. The relatively low uptake of [ 18 F]AlF-H 3 RESCA-FAPI in the kidney resulted in less noticeable numerical changes (SUV max , less than 0.10), whereas the significantly high uptake in the intestine (SUV max , more than 15.60) led to more pronounced numerical changes. It requires further validation to determine whether the observed uptake value in the intestine is attributable to direct absorption within the intestinal tissue or to metabolites generated by the radioactive tracer that subsequently enter the intestine through metabolic pathways. Like most 18 F-labeled tracers, [ 18 F]AlF-H 3 RESCA-FAPI shows a high bone uptake (because of its good stability, wiping is the bone uptake of tracer).(Xuedong Chen et al. 2024; Craig et al. 2023; Francis et al. 2024; Fu et al. 2023; Huang et al. 2023; Li et al. 2023; Liu et al. 2024; Poulie et al. 2023; Z. Wang et al. 2024; Yang et al. 2023; X. Zhang et al. 2023) The results of the blocking experiment, depicted in Figure 7B , demonstrate a successful reduction in tumor uptake following co-administration of unlabeled DOTA-FAPI-04 (SUV max , unblocking vs. blocking at 1h, 0.49 ± 0.18 vs. 0.04 ± 0.02, P = 0.0124). Moreover, significant reductions in uptake values were observed in the liver and bone. Biodistribution Further biodistribution experiments of tumor-bearing mice were performed to validate the findings from PET imaging. The results indicated a significant uptake of [ 18 F]AlF-H 3 RESCA-FAPI in the liver, where it is primarily metabolized. In comparison to the image results obtained from PET, the primary distinction lies in the reduced intestinal uptake ( Figure 8 and Table 2 ). This reduction can be attributed to the elimination of metabolites from the organs during the collection process, thereby demonstrating the rapid metabolism of [ 18 F]AlF-H 3 RESCA-FAPI in the liver and intestines. Apart from this difference, the imaging results are largely consistent with those of PET. The uptake of [ 18 F]AlF-H 3 RESCA-FAPI in tumor was 1.10 ± 0.12 %ID/g, which decreased to 0.05 ± 0.02 %ID/g after blocking, indicating a high level of specific uptake in tumor ( P = 0.004). Additionally, the tracer demonstrated significant tumor-muscle and tumor-blood ratios of 4.40 ± 0.71 and 4.56 ± 1.18, respectively ( Figure 8 and Table S6 ). The substantial bone uptake of most 18 F-labeled tracers remains evident, with the uptake quantified at 6.60 ± 1.16 %ID/g, which decreased to 0.49 ± 0.23 %ID/g after blocking ( P < 0.001). Notwithstanding this characteristic, [ 18 F]AlF-H 3 RESCA-FAPI demonstrates considerable promise as a PET tracer specifically targeting FAP. Immunohistochemical S taining The tumor of U87MG model mice was cut and soaked in formalin solution, and the staining pattern of FAP was obtained after immunohistochemical staining experiment, which confirmed that all animal experiments of this tracer were carried out in animal models with high FAP expression ( Figure 9 ). Discussion The present study introduces [ 18 F]AlF-H 3 RESCA-FAPI, a novel radiotracer for PET imaging of tumors expressing FAP. The development of this tracer is significant as FAP is overexpressed in numerous epithelial carcinomas, making it an attractive target for both diagnostic and therapeutic applications.(Lindner et al. 2019 ; Puré and Blomberg 2018 ) The results of our study provide a comprehensive evaluation of [ 18 F]AlF-H 3 RESCA-FAPI, from its synthesis and radiolabeling to its in vitro and in vivo performance. The molecular docking simulations and ADMET profiling study established the structural feasibility of H 3 RESCA-FAPI, predicting its interactions with FAP and its pharmacokinetic properties. The high binding affinity of [ 18 F]AlF-H 3 RESCA-FAPI to FAP, as determined by SPR, is consistent with the docking results and underscores the potential of this tracer for specific targeting of FAP-expressing tumors. The radiochemical synthesis of [ 18 F]AlF-H 3 RESCA-FAPI yielded a product with high RCP and RCYs, which is crucial for the practical application of the tracer in a clinical setting. The optimal conditions for radiolabeling, including pH, the molar ratio of aluminum chloride to precursor, and the mass of H 3 RESCA-FAPI, were identified, providing a reliable protocol for the production of the tracer. Considering that the pH value of colloidal Al(OH) 3 transformed from AlCl 3 is above 5.5,(X. Wang et al. 2020 ) and the labeling yield is unstable in the buffer with pH = 5.5, the buffer with pH = 5.0 is selected as the best condition. The capability of [ 18 F]AlF-H 3 RESCA-FAPI to achieve high radiolabeling yields at room temperature is a critical advantage. It simplifies the synthesis process, reducing the need for complex and costly equipment typically required for heating reactions. This not only lowers the barriers to production but also increases the feasibility of translating this tracer into a clinical setting. The high radiochemical yield of 52.0 ± 3.0% achieved under these mild conditions is particularly impressive and suggests that [ 18 F]AlF-H 3 RESCA-FAPI is a robust candidate for PET imaging. The octanol-water partition coefficient and stability assays demonstrated that [ 18 F]AlF-H 3 RESCA-FAPI has reduced hydrophilicity compared to [ 68 Ga]Ga-DOTA-FAPI-04, which may influence its biodistribution and pharmacokinetics. The plasma clearance data revealed a rapid distribution phase and a longer clearance phase, suggesting that the tracer is rapidly taken up by tissues and slowly cleared from the body. PET/CT imaging and biodistribution studies in U87MG tumor-bearing mice showed specific and significant uptake of [ 18 F]AlF-H 3 RESCA-FAPI in tumors, with high tumor-to-background ratios, indicating its potential for accurate tumor imaging. The high bone uptake observed is a common characteristic of 18 F-labeled tracers.(Xuedong Chen et al. 2024 ; Craig et al. 2023 ; Francis et al. 2024 ; Fu et al. 2023 ; Huang et al. 2023 ; Li et al. 2023 ; Liu et al. 2024 ; Poulie et al. 2023 ; Z. Wang et al. 2024 ; Yang et al. 2023 ; X. Zhang et al. 2023 ) The significant reduction in tumor uptake following co-administration of unlabeled DOTA-FAPI-04 further confirms the specificity of [ 18 F]AlF-H 3 RESCA-FAPI for FAP-expressing tumors. While the results are promising, there are limitations to consider. The study's findings are based on preclinical data, and further validation in clinical settings is necessary to confirm the tracer's performance in humans. Additionally, the high uptake in the liver and intestine, although possibly due to the tracer's lipid solubility, requires further investigation to understand its implications for diagnostic imaging. In comparison to existing FAPI-based tracers, [ 18 F]AlF-H 3 RESCA-FAPI demonstrates improved labeling techniques, which is a significant advancement. However, a direct comparison with other 18 F-labeled FAPI tracers in terms of tumor uptake and clearance rates is needed to fully assess its potential advantages. In comparison to existing FAPI-based tracers, [ 18 F]AlF-H 3 RESCA-FAPI demonstrates improved labeling-method, which is a significant advancement. However, a direct comparison with other 18 F-labeled FAPI tracers in terms of tumor uptake and clearance rates is needed to fully assess its potential advantages.The rapid metabolism of [ 18 F]AlF-H 3 RESCA-FAPI in the liver and intestines, as indicated by the biodistribution studies, suggests that the tracer may be suitable for imaging tumors with high FAP expression in these organs. In conclusion, [ 18 F]AlF-H 3 RESCA-FAPI shows considerable promise as a PET tracer for imaging FAP-expressing tumors. Its high specificity, favorable pharmacokinetics, and robust stability make it a potential candidate for clinical translation, offering a new tool for non-invasive tumor imaging in future studies. However, further research is needed to address the limitations identified and to fully realize the potential of this tracer in the clinical setting. Conclusions In this study, a novel radiopharmaceutical, [ 18 F]AlF-H 3 RESCA-FAPI, was synthesized utilizing the established FAPI scaffold. It was evaluated in vitro and in vivo, and showed considerable specific uptake of FAP-expressing tumors in mice. It exhibited a extremely mild labeling process and high availability. Therefore, this novel FAP-targeted radioactive tracer may be a promising tracer for non-invasive tumor imaging in subsequent clinical research, but its structure needs to be further modified to obtain better pharmacokinetic characteristics. Abbreviations HPLC High-performance liquid chromatography RCY Radiochemical yield RCP Radiochemical purity PET Positron emission tomography RESCA Restrained complexing agent NOTA Hexadentate ligand 1,4,7-triazacyclononane-1,4,7-triacetic acid DOTA 2,2’2”,2-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid QMA Quaternary methyl ammonium SUV Standardized uptake value [ 18 F] Fluorine-18 [ 68 Ga]Ga 68 Ga-gallium A m Molar activity Bq Becquerel FAP Fibroblast activation protein CAFs cancer-associated fibroblasts %ID/g percentage of injected dose per gram of tissue SPR Surface plasmon resonance Declarations Ethics approval and consent to participate The Institutional Committee for the Care and Use of Animals (Renji Hospital, Shanghai Jiao Tong University School of Medicine) approved all animal imaging studies. The current study did not include patient information. Consent for publication Informed consent was obtained from all participants included in the study. Competing interests C. Wang, Z. Hu, and J. Liu are co-inventors of pending patents describing the imaging technologies reported in the manuscript. Funding This work was funded by the National Key Research and Development Program of China (Grant No. 2021YFF0701900 and 2020YFA0909000), the Interdisciplinary Program of Shanghai Jiao Tong University (Grant No. ZH2018QNB20) and the construction project of Shanghai Key Laboratory of Molecular Imaging(18DZ2260400) (Grant No. KFKT-2024-27). Authors' contributions C. Wang, B. Zhang and J. Liu conceived and designed this research. Q. Zhang and Z. Hu were responsible for all the experiments, data collection and analysis and wrote the manuscript. H. Zhao, F. Du, and C. Lv were involved in the preparation of the radionuclide and radiopharmaceuticals and took part in most of the animal experiments. T. Peng and Y. Zhang were responsible for the small-animal image analysis. All the authors participated in the revision of the article. All authors read and approved the final manuscript. C. Wang obtained the funds supporting the work. Acknowledgements We gratefully appreciate all the chemists, nurses, and technicians from the Department of Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, for their contributions to the tracer administration and micro-PET/CT imaging. Availability of data and material Datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request. References Bu L, Baba H, Yoshida N, Miyake K, Yasuda T, et al. Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor microenvironment. Oncogene. 2019;38(25):4887–901. https://doi.org/10.1038/s41388-019-0765-y . Busek P, Mateu R, Zubal M, Kotackova L, Sedo A. Targeting fibroblast activation protein in cancer – Prospects and caveats. Front Biosci (Landmark Ed). 2018;23(10):1933–68. https://doi.org/10.2741/4682 . 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A radiohybrid theranostics ligand labeled with fluorine-18 and lutetium-177 for fibroblast activation protein-targeted imaging and radionuclide therapy. Eur J Nucl Med Mol Imaging. 2023;50(8):2331–41. https://doi.org/10.1007/s00259-023-06169-5 . Yu Z, Huang Y, Chen H, Jiang Z, Li C, et al. Design, Synthesis, and Evaluation of 18F-Labeled Tracers Targeting Fibroblast Activation Protein for Brain Imaging. ACS Pharmacol Transl Sci. 2023;6(11):1745–57. https://doi.org/10.1021/acsptsci.3c00187 . Zhang N, Pan F, Pan L, Diao W, Su F, et al. Synthesis, radiolabeling, and evaluation of a (4-quinolinoyl)glycyl-2-cyanopyrrolidine analogue for fibroblast activation protein (FAP) PET imaging. Front Bioeng Biotechnol. 2023;11:1167329. https://doi.org/10.3389/fbioe.2023.1167329 . Zhang X, Choi JY, Lee K-H, Choe YS. Synthesis and Evaluation of [18F]SiFA-Conjugated Ligands for Fibroblast Activation Protein Imaging. Mol Pharm. 2023;20(12):6441–50. https://doi.org/10.1021/acs.molpharmaceut.3c00824 . Zhou H, Zhong J, Peng S, Liu Y, Tang P, et al. Synthesis and preclinical evaluation of novel 18F-labeled fibroblast activation protein tracers for positron emission tomography imaging of cancer-associated fibroblasts. Eur J Med Chem. 2024;264:115993. https://doi.org/10.1016/j.ejmech.2023.115993 . Tables Table 1 and 2 are available in the Supplementary Files section. Scheme Scheme 1 is available in the Supplementary Files section. Supplementary Files Table12.docx Onlinefloatimage7.png Scheme 1. Synthesis of [ 18 F]AlF-H 3 RESCA-FAPI. RESCAFAPIsupportinginformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5297123","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373291485,"identity":"be115af6-87e6-432d-9a54-f9a5b1ac84b7","order_by":0,"name":"Qingyu Zhang","email":"","orcid":"","institution":"Shanghai Normal University","correspondingAuthor":false,"prefix":"","firstName":"Qingyu","middleName":"","lastName":"Zhang","suffix":""},{"id":373291486,"identity":"077ffc8d-c33d-47c6-b156-715fd15d0e95","order_by":1,"name":"Zhoumi Hu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhoumi","middleName":"","lastName":"Hu","suffix":""},{"id":373291487,"identity":"cb1e46df-f988-4f13-a391-fe9008531780","order_by":2,"name":"Haitao Zhao","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Zhao","suffix":""},{"id":373291488,"identity":"ac726649-72cd-4d21-9cbf-5019c7eea94d","order_by":3,"name":"Fuqiang Du","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fuqiang","middleName":"","lastName":"Du","suffix":""},{"id":373291489,"identity":"143c2ace-6ac5-44b5-a96f-ccbb7964e9a0","order_by":4,"name":"Chun Lv","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Lv","suffix":""},{"id":373291490,"identity":"bc8fdc27-01a3-4d85-8ecd-927aab2df85e","order_by":5,"name":"Tukang Peng","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tukang","middleName":"","lastName":"Peng","suffix":""},{"id":373291491,"identity":"6c47217a-5ba6-4416-97de-1dbe752dfd60","order_by":6,"name":"Yukai Zhang","email":"","orcid":"","institution":"Shanghai Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yukai","middleName":"","lastName":"Zhang","suffix":""},{"id":373291492,"identity":"5fb5a783-a662-436c-9326-0136529f5f4e","order_by":7,"name":"Bowu Zhang","email":"","orcid":"","institution":"Shanghai Normal University","correspondingAuthor":false,"prefix":"","firstName":"Bowu","middleName":"","lastName":"Zhang","suffix":""},{"id":373291493,"identity":"b7f56536-1016-4e3c-8772-70e9ba905b1b","order_by":8,"name":"Jianjun Liu","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Liu","suffix":""},{"id":373291494,"identity":"abcf6c7c-d513-4b6c-ad46-45178db4e5a4","order_by":9,"name":"Cheng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYLACxgYbOX72xsaHH0jQkmYs2XO42ViCBC2HEjfcSG8T4CFGtcHxs4df/NxxgHHmzIdtDBIMdnK6DYS0nMlLs+w9c4eZXzqx7UEBQ7Kx2QECWswO5JgZM7Y9Y5OcndhuIMFwIHEbQS3n34C0HOYxuHmwTYKHKC03cowfA7VIGNxgJFKL/Y03Zoy9bWkGkj2JwEA2IMIvkv05xh9+ttnU97Mff/jwQ4WdHEEtQMCGFIEGhJWDADPxyWQUjIJRMApGJgAA/E9IwffttakAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5312-4379","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-20 06:55:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5297123/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5297123/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70044128,"identity":"349288e6-3532-4440-984c-3fa8b96878aa","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27696,"visible":true,"origin":"","legend":"\u003cp\u003eThe structures of UAMC-1110 (A), [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04 (B), [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-FAPI-42 (C) and [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (D). The blue part is the linker, the red part is the chelator group, and the black part is FAPI pharmacophore.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/baf3af04eec5fedcd35014f3.png"},{"id":70044136,"identity":"2279e4f3-6e9a-49d2-ae6a-9effd1e77b8f","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":203716,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular modeling: (A) FAP ligand binding cavity of FAPIs and (B) Interaction diagram between amino acid residues of FAPIs and FAP in ligand binding pocket.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/3c2cb77bb44e642364ebe94d.png"},{"id":70044718,"identity":"16fd01eb-f76b-48c4-908a-c75e7c44db26","added_by":"auto","created_at":"2024-11-27 18:56:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24821,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DOTA-FAPI-04 and (B) H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI reactive curves with recombinant human FAP protein.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/0562c437cd2068a5d0299ba4.png"},{"id":70044137,"identity":"5e50ebfd-ac5c-4634-8e31-181e542e07fb","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41744,"visible":true,"origin":"","legend":"\u003cp\u003eLabeling yield of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI under different reaction conditions. Labeling yield under different pH values (4.0, 4.5, 5.0, 5.5, 6.0) of buffer system (A); The labeling yield was fixed (20 μg) with different AlCl\u003csub\u003e3\u003c/sub\u003e-ligand ratios (0.58, 0.60) (B); The ligand ratio of AlCl\u003csub\u003e3\u003c/sub\u003e is fixed (the molar ratio of substances is 0.58), and the labeling yield is different under different ligand mass (20 μg, 50 μg, 100 μg) (C).\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/f52c4d8dcfd9c62365314c68.png"},{"id":70044132,"identity":"20b83a22-60cc-4f49-89e1-3fd9191b39ea","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29972,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative HPLC graphs of quality control of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (A), \u003cem\u003ein vivo\u003c/em\u003e metabolism study of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI at 1 hour after injection (B) and its stability at 37°C for 2 hours in PBS (C) and FBS (D).\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/1348d8ed4d4dd493bb866706.png"},{"id":70044129,"identity":"116b89dc-f877-403f-9d9a-1f69011ab4f8","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":66552,"visible":true,"origin":"","legend":"\u003cp\u003eActivity curve of blood drug concentration (n = 3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/1e9ea6237431c9022cf407d2.png"},{"id":70044139,"identity":"af93e4d4-5862-4034-9f0d-1babfa29b660","added_by":"auto","created_at":"2024-11-27 18:48:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":106498,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative static PET images of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in U87MG xenograft models at different time points. (B) Representative static PET images of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in U87MG xenograft models with simultaneous injection of unlabeled DOTA-FAPI-04 as a competitor at 60 min. The yellow arrows point to the tumor.\u003c/p\u003e","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/46aa97c04f8386c0c515e3be.png"},{"id":70044810,"identity":"e0af5983-e033-4c38-9908-9a60b14ed4a9","added_by":"auto","created_at":"2024-11-27 19:04:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":18132,"visible":true,"origin":"","legend":"\u003cp\u003eThe ex vivo Biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in U87MG model mice 1 hour after injection (A) and the uptake ratio of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in tumor and blood, muscle, liver, kidney, intestine, bone (B) were co-administered with and without unlabeled DOTA-FAPI-04 as a blocking agent. All the data are expressed as mean ± SD values, n = 3.\u003c/p\u003e","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/668635fca7d1e4f76b126853.png"},{"id":70045200,"identity":"248f7dd8-08a5-47a1-8abf-6e7617b9b938","added_by":"auto","created_at":"2024-11-27 19:12:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":96296,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining of FAP expression in U87MG tumor tissues.\u003c/p\u003e","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/dd14edba13cec524e4421c23.png"},{"id":70045242,"identity":"063e2a2a-e20b-4f00-b3fa-6aeb6da19f6a","added_by":"auto","created_at":"2024-11-27 19:20:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1615897,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/71c21554-dbc1-40bb-bfe4-75c222cc022b.pdf"},{"id":70044127,"identity":"fa684fb1-9cdb-4139-9eb4-5fc6e3f3ed2e","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":42782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/fe05532ff78556d7385c9bf0.docx"},{"id":70044135,"identity":"881cbf3a-8c5b-42e6-97dd-8f2d2868d250","added_by":"auto","created_at":"2024-11-27 18:48:05","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eSynthesis of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/8e6e8e0984321ef5482fac39.png"},{"id":70044719,"identity":"8ad8b8cf-6f52-43ec-b117-b3b40bdbe1a1","added_by":"auto","created_at":"2024-11-27 18:56:05","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":647832,"visible":true,"origin":"","legend":"","description":"","filename":"RESCAFAPIsupportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5297123/v1/ddc308ee52380693338299d1.docx"}],"financialInterests":"","formattedTitle":"Design, Synthesis and Biological Evaluation of a Novel [18F]AlF-H3RESCA-FAPI Radiotracer Targeting Fibroblast Activation Protein.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTumor microenvironment (TME), including fibroblasts, myeloid-derived suppressor cells (MDSCs), macrophages, lymphocytes, the extracellular matrix (ECM) and intertwined blood vessels constructed by endothelial cells and pericytes.(Xueman Chen and Song \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) These stromal cells tightly interact with cancer cells, creating a supportive environment for tumor growth. Among these, cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression by contributing to processes such as tumorigenesis, neoangiogenesis, metastasis, immunosuppression, and drug resistance.(Lindner et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pur\u0026eacute; and Blomberg \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Fibroblast activation protein (FAP), a type II transmembrane serine protease belonging to the dipeptidyl peptidase 4 family with both dipeptidyl peptidase and endopeptidase activity,(Hamson et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) is overexpressed on CAFs in more than 90% of epithelial carcinomas.(Bu et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Busek et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lamprecht et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Šimkov\u0026aacute; et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Therefore, FAP is considered a promising target for both tumor imaging and therapy.\u003c/p\u003e \u003cp\u003eIn 2014, UAMC-1110 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was identified as the most potent and selective small molecule inhibitor of FAP.(Jansen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jansen et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Since then, some classic UAMC-1110-based PET tracers have been developed,(Lindner et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Loktev et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Loktev et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) such as [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and [\u003csup\u003e18\u003c/sup\u003eF]AlF-NOTA-FAPI-42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInitially, [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04 garnered the majority of attention within the FAPI-diagnostics field;(Kratochwil et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) however, current research is also focusing on \u003csup\u003e18\u003c/sup\u003eF-labeled FAPIs due to the excellent spatial resolution theoretically obtained by \u003csup\u003e18\u003c/sup\u003eF-PET imaging(N. Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the relatively long half-life of \u003csup\u003e18\u003c/sup\u003eF (t\u003csub\u003e1/2\u003c/sub\u003e = 109.8 min, compared with t\u003csub\u003e1/2\u003c/sub\u003e(\u003csup\u003e68\u003c/sup\u003eGa)\u0026thinsp;=\u0026thinsp;67.7 min). Furthermore, the limited availability of \u003csup\u003e68\u003c/sup\u003eGa, which must be produced in relatively small batches through \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa generators, contributes to the growing interest in \u003csup\u003e18\u003c/sup\u003eF-labeled FAPIs. On the contrary, \u003csup\u003e18\u003c/sup\u003eF can be mass-produced in cyclotron or transported to PET imaging center which lacking radionuclide production capacity.(Cho et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe labeling techniques for \u003csup\u003e18\u003c/sup\u003eF can be categorized into two groups: nucleophilic \u003csup\u003e18\u003c/sup\u003eF- derivatives generated by various substitution reactions,(Toms et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; N. Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the utilization of aluminum fluoride ([\u003csup\u003e18\u003c/sup\u003eF]AlF\u003csup\u003e2+\u003c/sup\u003e) for radiolabeling via coordination chemistry.(Zhou et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) The method of using [\u003csup\u003e18\u003c/sup\u003eF]AlF labeling has been expanding, and a series of different chelator groups have achieved great success, such as NOTA, NODA, NODAGA, 2-AMPTA, DTPA, most of these require certain requirements for equipment.(Carroll et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eRecently, the H\u003csub\u003e3\u003c/sub\u003eRESCA has been widely used in \u003csup\u003e18\u003c/sup\u003eF labeling of antibodies because of its mild reaction conditions and straightforward labeling process,(Cleeren et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wegrzyniak et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) which has piqued our interest. Our research aims to utilize H\u003csub\u003e3\u003c/sub\u003eRESCA for the labeling of small molecules with \u003csup\u003e18\u003c/sup\u003eF, using FAPI as an example to broaden the potential applications of FAPI in nuclear medicine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral\u003c/h2\u003e \u003cp\u003eExcept as indicated, compound \u003cb\u003e1 and\u003c/b\u003e Compound \u003cb\u003e2\u003c/b\u003e were procured from TanzhenBio (Nanchang, China), and other chemicals and reagents were procured from Merck (Shanghai, China) and used without additional purification. Characterization of synthesized compounds was performed using LC-MS on an Infinity Lab mass spectrometer equipped with an Agilent 1260 series HPLC system and an Extend-C18 column (50 mm \u0026times; 2.1 mm, 1.8 \u0026micro;m), monitores at a UV wavelength of 254 nm. Radioisotope \u003csup\u003e18\u003c/sup\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e was produced in a medical cyclotron (HM-10, Sumitomo Heavy Industries Ltd., Tokyo, Japan) via the \u003csup\u003e18\u003c/sup\u003eO (p, n) \u003csup\u003e18\u003c/sup\u003eF reaction. Quality control of the radiotracers was conducted using HPLC with a C18 column (C18-TH, 5\u0026micro;m, 100\u0026Aring;, 150mm \u0026times; 4.6mm, Morhchem Technologies Inc., United States) and a 1260 Quat pump VL, a 1260 DAD VL detector, a 1260 Vialsampler, and a γ-detector for radioactivity measurement (Eckert and Ziegler, United States). The mobile phase composition of water and acetonitrile was adjusted according to the specific compounds. Radioactivity measurements were taken with a CRC-55T activity meter (The China National Nuclear Corporation, Beijing, United States).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Docking\u003c/h3\u003e\n\u003cp\u003eThree-dimensional structures of DOTA-FAPI-04, NOTA-FAPI-42 and H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI were constructed using Chem3D 20.0 (PerkinElmer). The resultant mol2 files were converted to .pdb format with PyMol 2.6.0. The target protein (PDB ID: 1Z68) underwent preparation for docking by adding hydrogen atoms with AutoDockTools 1.5.7 (The Scripps Research Institute). A docking grid, encompassing the catalytic site of FAPα, was defined. This grid was centered around the catalytic triad\u0026mdash;Ser\u003csup\u003e624\u003c/sup\u003e, Asp\u003csup\u003e702\u003c/sup\u003e, and His\u003csup\u003e734\u003c/sup\u003e\u0026mdash;using AutoGrid 4.0, which set the grid dimensions at 24 \u0026times; 24 \u0026times; 24 \u0026Aring;\u0026sup3; and a grid spacing of 1.000 \u0026Aring;, to ensure comprehensive coverage of the catalytic site. The coordinates for the grid box center were established at x\u0026thinsp;=\u0026thinsp;25.071 \u0026Aring;, y\u0026thinsp;=\u0026thinsp;10.676 \u0026Aring;, and z\u0026thinsp;=\u0026thinsp;25.618 \u0026Aring;. Molecular docking, carried out with Autodock Vina 1.1.2, evaluated the influence uof various chelator groups on the binding affinity of FAPI-4 to FAP. Each compound underwent three independent docking simulations. The lowest-energy conformation from each set of runs, as identified by Autodock Vina, was selected for visualization with PyMol 2.6.0.\u003c/p\u003e\n\u003ch3\u003eADMET Profiling Studies\u003c/h3\u003e\n\u003cp\u003eThe ADMET properties of FAPI-based ligands were predicted using the ADMETlab 3.0 web tools, which facilitate a comprehensive ADMET analysis. Ligand structures, described by their Simplified Molecular Input Line Entry System (SMILES) representations, were input into the platform to generate 2D structural files. This enabled ADMETlab 3.0 to compute and evaluate essential ADMET parameters effectively. Notably, the SMILES strings for two specific ligands were generated with ChemDraw 20.0 and are detailed in Supplementary \u003cb\u003eTexts S1\u003c/b\u003e and \u003cb\u003eS2\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eChemistry\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003e2,2'-(((1R,2R)-2-((carboxymethyl)(4-(2-(4-(3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)benzyl)amino)cyclohexyl)azanediyl)diacetic acid\u003c/em\u003e \u003cb\u003e(3).\u003c/b\u003e Compound \u003cb\u003e1\u003c/b\u003e (50 mg, 103 \u0026micro;mol) was dissolved in DMSO (4 mL), followed by the addition of compound \u003cb\u003e2\u003c/b\u003e (77 mg, 131 \u0026micro;mol) and triethylamine (Et\u003csub\u003e3\u003c/sub\u003eN, 100 \u0026micro;L, 719 \u0026micro;mol). The mixture was stirred at room temperature (20\u0026deg;C) for 4 hours and then concentrated under reduced pressure. Purification was achieved using semi-preparative HPLC with a COSMOSIL 5C18-MS-Ⅱ column (120 \u0026Aring;, 5 \u0026micro;m, 250 \u0026times; 10.0 mm) and a gradient elution of acetonitrile/water (0.1% TFA) initiated at 16/84 (v/v) for 20 minutes at a flow rate of 2 mL/min. The solvent was removed under reduced pressure to yield compound \u003cb\u003e3\u003c/b\u003e as a white powder (32.2 mg, 34.6%). LC-MS (ESI) analysis showed a molecular ion peak at m/z\u0026thinsp;=\u0026thinsp;904.8 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eBinding Affinity Measurement\u003c/h3\u003e\n\u003cp\u003eIn order to determine the binding kinetics between ligands and human FAP protein (10464-H07H Sino Biological), the Biacore SPR interaction study was conducted. The binding affinity is expressed by the equilibrium dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) (M), which is calculated from the values of the measured binding rate constant (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e) (M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and dissociation rate (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) (s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRadiochemistry and Quality Control\u003c/h2\u003e \u003cp\u003e \u003csup\u003e18\u003c/sup\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e was loaded onto an activated QMA ion exchange column (Waters GmbH, Germany), which had been previously eluted with 1 mL of saline and air-dried. The column containing \u003csup\u003e18\u003c/sup\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e was subsequently eluted with 1 mL of saline to obtain a Na\u003csup\u003e18\u003c/sup\u003eF solution.\u003c/p\u003e \u003cp\u003eReaction conditions were screened using five different pH buffers: a NaHP buffer at pH 6.0 (0.2 M) and a sodium acetate buffer ranging from pH 4 to 5.5 (0.2 M). For the reactions, 100 \u0026micro;L of Na\u003csup\u003e18\u003c/sup\u003eF solution (370 MBq) and 12 \u0026micro;L of AlCl\u003csub\u003e3\u003c/sub\u003e solution (1.28 mM, prepared in 0.2 M buffer solution) were added to a reaction vial. The mixture was incubated at room temperature for 5 minutes. Subsequently, 300 \u0026micro;L of buffer solution at varying pH values and 200 \u0026micro;L of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI solution (0.1 mg/mL, prepared in deionized water) were added, and the mixture was allowed to react for 15 minutes at room temperature. The radiolabeling yields across different pH conditions were quantified by HPLC.\u003c/p\u003e \u003cp\u003eUtilizing the pH buffer that yielded the highest radiolabeling efficiency, 10 \u0026micro;L of Na\u003csup\u003e18\u003c/sup\u003eF solution (37 MBq) and variable volumes of AlCl\u003csub\u003e3\u003c/sub\u003e solution (either 12 or 12.4 \u0026micro;L, 1.07 mM, prepared in the selected buffer) were added to a reaction vial and incubated at room temperature for 5 minutes. This was followed by the addition of 200 \u0026micro;L of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI solution (0.1 mg/mL, prepared in the selected buffer) and 300 \u0026micro;L of the same buffer. The reaction proceeded for 15 minutes at room temperature. Radiolabeling efficiencies were then calculated by HPLC, analyzing different AlCl\u003csub\u003e3\u003c/sub\u003e to ligand molar ratios (0.58 or 0.60).\u003c/p\u003e \u003cp\u003eIn a subsequent experiment, the selected volume of AlCl\u003csub\u003e3\u003c/sub\u003e was added to a reaction flask along with 100 \u0026micro;L of Na\u003csup\u003e18\u003c/sup\u003eF solution (185 MBq) and incubated at room temperature for 5 minutes. Different volumes of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI solution (200, 500, or 1000 \u0026micro;L, 0.1 mg/mL, prepared in the selected buffer) were then introduced, maintaining the same volume with the selected buffer. The reaction was sustained for 15 minutes at room temperature. Radiolabeling yields for varying ligand masses (20, 50, or 100 \u0026micro;g) were determined by HPLC.\u003c/p\u003e \u003cp\u003eThe purification protocol for the Sep-Pak Light C18 column involved first eluting the reaction solution with 5 mL of pure water through the activated column. The purified product was then collected into an aseptic vacuum bottle containing 1 mL of 50% ethanol solution, followed by the addition of 4 mL saline, rendering the preparation ready for use. For HPLC analysis, the mobile phase consisted of an aqueous solution containing 0.10% trifluoroacetic acid (Phase A) and acetonitrile (Phase B). An isocratic elution was employed, with a mobile phase consisting of 18% Phase B, maintained constant throughout the analysis. The flow rate was set at 1 mL/min. UV detection was performed at a wavelength of 254 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePartition Coefficient\u003c/h3\u003e\n\u003cp\u003eRadiotracers [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04 and [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (0.37 MBq) was added to tubes containing n-octanol/PBS-mixture (1 mL, 1:1). The mixture was vortexed vigorously for 5 min at ambient temperature, and then centrifuged for five min at 8,000 rpm. In each phase, three samples (each 50 \u0026micro;L) was removed and measured in γ-counter (PerkinElmer). The partition coefficient was expressed as logD\u003csub\u003e7.4\u003c/sub\u003e = lg (CPM\u003csub\u003en\u0026minus;octanol\u003c/sub\u003e/CPM\u003csub\u003ePBS\u003c/sub\u003e) (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eStability\u003c/b\u003e\u003c/p\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (10 MBq) in a mixture of ethanol and water was added to PBS (600 \u0026micro;L) or FBS (GE Healthcare, Chicago, IL, USA) (600 \u0026micro;L) and incubated at 37\u0026deg;C for 2 hours. And then, a sample of PBS or FBS (100 \u0026micro;L) was injected into the HPLC system for analysis. At 60 min after injection of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (37 MBq/mouse), the mice were sacrificed. The urine was then collected and analysed with HPLC directly.\u003c/p\u003e\n\u003ch3\u003ePharmacokinetics In Normal Mice\u003c/h3\u003e\n\u003cp\u003eThe distribution of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (0.93 MBq) in blood was assessed at 1, 2, 5, 10, 20, 30, 60, 90, 120 min using normal mice. The mice (n\u0026thinsp;=\u0026thinsp;3 in each group) were adopted the blood at the selected time. All the collected blood were quickly removed and weighed, and the radio activity was counted using a γ-counter and the results were expressed as percentage of injected dose per gram of tissue (% ID/g).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePET Imaging\u003c/h2\u003e \u003cp\u003eU87MG tumor-bearing mice (n\u0026thinsp;=\u0026thinsp;3 per group) underwent PET imaging using an IRIS micro-PET/CT scanner (inviscan SAS, Strasbourg, France). Mice were anesthetized and positioned prone for the scans. Static PET images were acquired at 10, 30, 60, 90, and 120 min post-injection of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (4\u0026thinsp;\u0026minus;\u0026thinsp;6 MBq/mouse). For the blocking study, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI was co-injecteded with unlabeled DOTA-FAPI-04 (0.5 mg/mouse), followed by a 1-hour static PET scan. The PET data were reconstructed using a three-dimensional ordered-subset expectation maximization (3D-OSEM) algorithm, which incorporates a Monte Carlo simulation for accurate modeling of the detector response. The reconstructed images were analyzed using Avatar 1.2 software (Pingseng China) for semi-quantitative assessment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution Studies\u003c/h2\u003e \u003cp\u003eThe biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (4.32\u0026ndash;5.45 MBq) was evaluated in athymic nude mice bearing U87MG xenografts at 1 hour post-injection (p.i.). A subset of mice received a concurrent injection of the radiotracer and the competitor DOTA-FAPI-04 (0.5 mg per mouse) to assess blocking effects. At 60 min post-injection, the mice (n\u0026thinsp;=\u0026thinsp;3 per group) were euthanized, and selected organs, tumors, and blood were harvested, weighed, and measured for radioactivity using a γ-counter. The results were expressed as % ID/g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Staining\u003c/h2\u003e \u003cp\u003eU87MG tumor tissues were harvested post-euthanasia and processed into paraffin-embedded sections for immunohistochemical analysis. Sections underwent deparaffinization in xylene and rehydration through graded ethanol. Antigen retrieval was performed, followed by overnight incubation with a rabbit polyclonal anti-FAP primary antibody (1:50, AF0739, Affinity) at 4℃. After rinsing, sections were incubated with a goat anti-rabbit secondary antibody (PV-9000, ZSGB-BIO) for 1 hour at room temperature. FAP expression was visualized using diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, cleared, and mounted. Positive staining was assessed and photographed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eQuantitative data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The statistical significance between 2 independent groups was determined by the student t-test. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0.1 (Graph Software, Inc).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDocking\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSimulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking simulations were conducted to elucidate the interactions between FAPI-based ligands and the FAP enzyme, with the results depicted in \u003cstrong\u003eFigure 2\u0026nbsp;\u003c/strong\u003edemonstrating that the introduction of this innovative chelating agent group enhanced the interaction. Specifically, H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI exhibited a wider range of interactions with amino acid residues, including His\u003csup\u003e734\u003c/sup\u003e, Ser\u003csup\u003e624\u003c/sup\u003e, Arg\u003csup\u003e123\u003c/sup\u003e and Tyr\u003csup\u003e656\u003c/sup\u003e, Glu\u003csup\u003e203\u003c/sup\u003e, compared to DOTA-FAPI-04, which primarily interacted with Ser\u003csup\u003e624\u003c/sup\u003e, His\u003csup\u003e734\u003c/sup\u003e and Arg\u003csup\u003e123\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand NOTA-FAPI-42, which interacted with Glu\u003csup\u003e203\u003c/sup\u003e, Tyr\u003csup\u003e541\u003c/sup\u003e, Gln\u003csup\u003e547\u003c/sup\u003e and Arg\u003csup\u003e550\u003c/sup\u003e. These interactions were further characterized by the presence of potential hydrogen bonds, indicated by yellow dotted lines in the figure. Detailed scoring of these interactions is provided in \u003cstrong\u003eTable 1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADMET\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003erofiling\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003etudy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the preliminary stages of Computer-Aided Drug Design (CADD), the absorption, distribution, metabolism, excretion and toxicity (ADMET) profiles of chemical compounds are acknowledged as critical factors. The pharmacokinetic properties and drug-likeness metrics for these compounds are detailed in Tables\u003cstrong\u003e\u0026nbsp;S1\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eS2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;S3\u003c/strong\u003e, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePharmacokinetic analysis indicates that H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI possesses a higher intestinal absorption (HIA) profile compared to DOTA-FAPI-04, which exhibits lower absorption rates. Both ligands demonstrated limited ability to cross the blood-brain barrier. The human colon epithelial cancer cell line, Caco-2, serves as a surrogate for studying drug intestinal absorption in humans. Permeability studies using the Caco-2 model revealed no significant difference between the two ligands in terms of membrane permeation. Additionally, drug-likeness was assessed based on the Lipinski Rule, Pfizer Rule, GSK Rule, and Golden Triangle Rule. Both compounds adhered to the Pfizer Rule; however, they did not comply with the Lipinski, GSK, and Golden Triangle criteria. For radioactive diagnostic agents, the administered doses are typically very low, thus mitigating concerns regarding the chemical toxicity of such drugs. In summary, the simulation experiments indicate that the two inhibitors are largely comparable, with the primary differences being their lipid solubility and intestinal uptake. The variance in lipid solubility may underlie the observed differences in intestinal absorption.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBinding\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003effinity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurface plasmon resonance (SPR) was utilized to evaluate the interaction between DOTA-FAPI-04 and H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI with recombinant human FAP protein (Figure 3). The equilibrium dissociation constant (\u003cem\u003eK\u003csub\u003eD\u003c/sub\u003e\u003c/em\u003e) value of the 2 inhibitors binding to human FAP proteins exhibited a robust affinity, being in the picomole (pM) range. Specifically, The \u003cem\u003eK\u003csub\u003eD\u003c/sub\u003e\u003c/em\u003e value of DOTA-FAPI-04 is less than 27.89 pM, while The \u003cem\u003eK\u003csub\u003eD\u003c/sub\u003e\u003c/em\u003e value of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI is less than 10.09 pM (\u003cstrong\u003eFigure 3A\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e). This shows that the affinity of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI for FAP protein is slightly higher than that of DOTA-FAPI-04, which is consistent with the results of molecular docking simulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical and Radiochemical Syntheses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI (compound \u003cstrong\u003e3\u003c/strong\u003e) was synthesized (\u003cstrong\u003eScheme\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e), and its molecular weight was identified by LC-MS (\u003cstrong\u003eFigure S1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e18\u003c/sup\u003eF-labeled FAP tracer was generated via the formation of complexes of Al\u003csup\u003e18\u003c/sup\u003eF in a two-steps reaction. According to the lateral comparison, the reaction conditions with the highest labeling yield was as follows: buffer pH=5.0, the mole ratio of AlCl\u003csub\u003e3\u003c/sub\u003e to precursor was 0.58, and the weight of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI was 50 \u0026mu;g\u0026nbsp;(\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e and \u003cstrong\u003eTable S4\u003c/strong\u003e). The highly reproducible radiolabeling yield of 95% was obtained in the optimal conditions. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder the optimum labeling conditions, the total time needed for radiosynthesis was approximately 20 min, and the non-decay corrected radiochemistry yields (RCYs) of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI was 52.0 \u0026plusmn; 3.0% (n = 6). The radiochemical purity (RCP) of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI was over 95% with molar activities of more than 14.5 GBq/\u0026mu;mol (n = 6) according to radioactivity measurements\u0026nbsp;(Figure\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5A, S2\u003c/strong\u003e and\u0026nbsp;\u003cstrong\u003eS3\u003c/strong\u003e). The labeling yield is not only related to the three factors screened (pH of buffer, the ratio of AlCl\u003csub\u003e3\u003c/sub\u003e to precursor and the dosage of precursor), but is also influenced by other factors (room temperature and the concentration of reaction solution), resulting in significant variability in labeling yields under identical conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOctanol-Water\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Partition\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eoefficient and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003etability\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003essay\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;octanol-water partition coefficient, expressed as logD\u003csub\u003e7.4\u003c/sub\u003e, for the radiopharmaceuticals\u0026nbsp;[\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04\u0026nbsp;and [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI were calculated to be -3.53 \u0026plusmn; 0.05 and -2.47 \u0026plusmn; 0.16, respectively. This comparative analysis suggests that [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI possesses a reduced hydrophilicity in comparison to\u0026nbsp;[\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04. Given the constraints inherent in the ADMETLab 3.0 regarding the processing of SMILES files that include metal complexes, the distribution coefficients of the respective uncomplexed precursors served as a benchmark for assessing the hydrophilicity of these radiopharmaceutical agents\u0026nbsp;(the logP of DOTA-FAPI-04 and H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI are -1.982 and -0.639, respectively).\u0026nbsp;The observed hydrophilicity trends corroborate the simulation results obtained for the labeled precursors during ADMET prediction analyses. Furthermore, the stability assessment of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in phosphate-buffered saline (PBS, pH=7.4), fetal bovine serum (FBS), and mouse urine\u0026mdash;presented in \u003cstrong\u003eFigure 5\u003c/strong\u003e, underscores the tracer\u0026apos;s robust stability in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasma Clearance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Plasma Clearance experiment\u0026nbsp;involved the collection of\u0026nbsp;blood drug concentration (%ID/g) at\u0026nbsp;specific time points and\u0026nbsp;fitting a curve to the data. The distribution-phase half-life (t\u003csub\u003e1/2\u0026alpha;\u003c/sub\u003e) value of the [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI was 0.76 min, and its clear-phase half-life (t\u003csub\u003e1/2\u0026beta;\u003c/sub\u003e) value was more than 60 min (\u003cstrong\u003eFigure 6\u003c/strong\u003e and \u003cstrong\u003eTable S5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePET/CT Imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatic PET imaging studies were performed in U87MG tumor-bearing nude mice to investigate the pharmacokinetics of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI. The coronal and axial images of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI at different scanning times are shown in \u003cstrong\u003eFigure 7A\u003c/strong\u003e. The tissue accumulation of tracer is described by standardized uptake value (SUV) scale. With regard to tumors, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI accumulated rapidly in U87MG tumor xenografts. A slow increase in tumor uptake was observed from 10 to 120 minute (SUV\u003csub\u003emax\u003c/sub\u003e, from 0.70\u0026nbsp;\u0026plusmn;\u0026nbsp;0.02 to 0.72\u0026nbsp;\u0026plusmn;\u0026nbsp;0.06). This observation indicates that [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI exhibits a considerable retention time within the tumor.\u003c/p\u003e\n\u003cp\u003eHowever, the liver and intestine exhibited significantly uptake values, possibly due to the certain lipid solubility of the tracer. Both ADMET prediction and LogD\u003csub\u003e7.4\u003c/sub\u003e measurement support this and predict increased intestinal absorption. In U87MG tumor model mice, from 10 to 120 minute, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI demonstrated rapid clearance kinetics in muscle (SUV\u003csub\u003emax\u003c/sub\u003e, from 0.029\u0026nbsp;\u0026plusmn;\u0026nbsp;0.02 to 0.014\u0026nbsp;\u0026plusmn;\u0026nbsp;0.02), intestine (SUV\u003csub\u003emax\u003c/sub\u003e, from 139.092\u0026nbsp;\u0026plusmn;\u0026nbsp;43.447 to 53.872\u0026nbsp;\u0026plusmn;\u0026nbsp;27.220) and liver (SUV\u003csub\u003emax\u003c/sub\u003e, from 2.251\u0026nbsp;\u0026plusmn;\u0026nbsp;0.539 to 1.403\u0026nbsp;\u0026plusmn;\u0026nbsp;0.370), while clearance from the kidney was comparatively slower. The relatively low uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in the kidney resulted in less noticeable numerical changes (SUV\u003csub\u003emax\u003c/sub\u003e, less than 0.10), whereas the significantly high uptake in the intestine (SUV\u003csub\u003emax\u003c/sub\u003e, more than 15.60) led to more pronounced numerical changes. It requires further validation to determine whether the observed uptake value in the intestine is attributable to direct absorption within the intestinal tissue or to metabolites generated by the radioactive tracer that subsequently enter the intestine through metabolic pathways. Like most \u003csup\u003e18\u003c/sup\u003eF-labeled tracers, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI shows a high bone uptake (because of its good stability, wiping is the bone uptake of tracer).(Xuedong Chen et al. 2024; Craig et al. 2023; Francis et al. 2024; Fu et al. 2023; Huang et al. 2023; Li et al. 2023; Liu et al. 2024; Poulie et al. 2023; Z. Wang et al. 2024; Yang et al. 2023; X. Zhang et al. 2023)\u0026nbsp;The results of the blocking experiment, depicted in \u003cstrong\u003eFigure 7B\u003c/strong\u003e, demonstrate a successful reduction in tumor uptake following co-administration of unlabeled DOTA-FAPI-04 (SUV\u003csub\u003emax\u003c/sub\u003e, unblocking vs. blocking at 1h, 0.49 \u0026plusmn; 0.18 vs. 0.04 \u0026plusmn; 0.02, \u003cem\u003eP\u003c/em\u003e = 0.0124). Moreover, significant reductions in uptake values were observed in the liver and bone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiodistribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther biodistribution experiments of tumor-bearing mice were performed to\u0026nbsp;validate the findings from\u0026nbsp;PET imaging. The results\u0026nbsp;indicated a significant uptake of\u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI\u0026nbsp;in the liver,\u0026nbsp;where it is primarily metabolized.\u0026nbsp;In\u0026nbsp;comparison to the image results\u0026nbsp;obtained from\u0026nbsp;PET, the primary distinction lies in the reduced intestinal uptake (\u003cstrong\u003eFigure 8\u003c/strong\u003e and \u003cstrong\u003eTable 2\u003c/strong\u003e). This reduction can be attributed to the elimination of metabolites from the organs during the collection process, thereby demonstrating the rapid metabolism of\u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI\u0026nbsp;in the liver and intestines. Apart from this difference, the imaging results are largely consistent with those of PET.\u003c/p\u003e\n\u003cp\u003eThe uptake of\u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI\u0026nbsp;in tumor was\u0026nbsp;1.10 \u0026plusmn; 0.12\u0026nbsp;%ID/g, which decreased to 0.05 \u0026plusmn; 0.02 %ID/g after blocking, indicating a high level of specific uptake in tumor (\u003cem\u003eP\u003c/em\u003e = 0.004). Additionally, the tracer demonstrated significant tumor-muscle and tumor-blood ratios of\u0026nbsp;4.40 \u0026plusmn; 0.71\u0026nbsp;and\u0026nbsp;4.56 \u0026plusmn; 1.18, respectively (\u003cstrong\u003eFigure 8\u003c/strong\u003e and \u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS6\u003c/strong\u003e). The substantial bone uptake of most \u003csup\u003e18\u003c/sup\u003eF-labeled tracers remains evident, with the uptake quantified at\u0026nbsp;6.60 \u0026plusmn; 1.16\u0026nbsp;%ID/g, which decreased to 0.49 \u0026plusmn; 0.23 %ID/g after blocking (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). Notwithstanding this characteristic, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI demonstrates considerable promise as a PET tracer specifically targeting FAP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003etaining\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumor of U87MG model mice was cut and soaked in formalin solution, and the staining pattern of FAP was obtained after immunohistochemical staining experiment, which confirmed that all animal experiments of this tracer were carried out in animal models with high FAP expression (\u003cstrong\u003eFigure 9\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study introduces [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI, a novel radiotracer for PET imaging of tumors expressing FAP. The development of this tracer is significant as FAP is overexpressed in numerous epithelial carcinomas, making it an attractive target for both diagnostic and therapeutic applications.(Lindner et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pur\u0026eacute; and Blomberg \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) The results of our study provide a comprehensive evaluation of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI, from its synthesis and radiolabeling to its in vitro and in vivo performance.\u003c/p\u003e \u003cp\u003eThe molecular docking simulations and ADMET profiling study established the structural feasibility of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI, predicting its interactions with FAP and its pharmacokinetic properties. The high binding affinity of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI to FAP, as determined by SPR, is consistent with the docking results and underscores the potential of this tracer for specific targeting of FAP-expressing tumors.\u003c/p\u003e \u003cp\u003eThe radiochemical synthesis of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI yielded a product with high RCP and RCYs, which is crucial for the practical application of the tracer in a clinical setting. The optimal conditions for radiolabeling, including pH, the molar ratio of aluminum chloride to precursor, and the mass of H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI, were identified, providing a reliable protocol for the production of the tracer. Considering that the pH value of colloidal Al(OH)\u003csub\u003e3\u003c/sub\u003e transformed from AlCl\u003csub\u003e3\u003c/sub\u003e is above 5.5,(X. Wang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and the labeling yield is unstable in the buffer with pH\u0026thinsp;=\u0026thinsp;5.5, the buffer with pH\u0026thinsp;=\u0026thinsp;5.0 is selected as the best condition. The capability of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI to achieve high radiolabeling yields at room temperature is a critical advantage. It simplifies the synthesis process, reducing the need for complex and costly equipment typically required for heating reactions. This not only lowers the barriers to production but also increases the feasibility of translating this tracer into a clinical setting. The high radiochemical yield of 52.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0% achieved under these mild conditions is particularly impressive and suggests that [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI is a robust candidate for PET imaging.\u003c/p\u003e \u003cp\u003eThe octanol-water partition coefficient and stability assays demonstrated that [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI has reduced hydrophilicity compared to [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-FAPI-04, which may influence its biodistribution and pharmacokinetics. The plasma clearance data revealed a rapid distribution phase and a longer clearance phase, suggesting that the tracer is rapidly taken up by tissues and slowly cleared from the body.\u003c/p\u003e \u003cp\u003ePET/CT imaging and biodistribution studies in U87MG tumor-bearing mice showed specific and significant uptake of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in tumors, with high tumor-to-background ratios, indicating its potential for accurate tumor imaging. The high bone uptake observed is a common characteristic of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers.(Xuedong Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Craig et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Francis et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Poulie et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Z. Wang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; X. Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) The significant reduction in tumor uptake following co-administration of unlabeled DOTA-FAPI-04 further confirms the specificity of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI for FAP-expressing tumors.\u003c/p\u003e \u003cp\u003eWhile the results are promising, there are limitations to consider. The study's findings are based on preclinical data, and further validation in clinical settings is necessary to confirm the tracer's performance in humans. Additionally, the high uptake in the liver and intestine, although possibly due to the tracer's lipid solubility, requires further investigation to understand its implications for diagnostic imaging.\u003c/p\u003e \u003cp\u003eIn comparison to existing FAPI-based tracers, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI demonstrates improved labeling techniques, which is a significant advancement. However, a direct comparison with other \u003csup\u003e18\u003c/sup\u003eF-labeled FAPI tracers in terms of tumor uptake and clearance rates is needed to fully assess its potential advantages.\u003c/p\u003e \u003cp\u003eIn comparison to existing FAPI-based tracers, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI demonstrates improved labeling-method, which is a significant advancement. However, a direct comparison with other \u003csup\u003e18\u003c/sup\u003eF-labeled FAPI tracers in terms of tumor uptake and clearance rates is needed to fully assess its potential advantages.The rapid metabolism of [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI in the liver and intestines, as indicated by the biodistribution studies, suggests that the tracer may be suitable for imaging tumors with high FAP expression in these organs.\u003c/p\u003e \u003cp\u003eIn conclusion, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI shows considerable promise as a PET tracer for imaging FAP-expressing tumors. Its high specificity, favorable pharmacokinetics, and robust stability make it a potential candidate for clinical translation, offering a new tool for non-invasive tumor imaging in future studies. However, further research is needed to address the limitations identified and to fully realize the potential of this tracer in the clinical setting.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, a novel radiopharmaceutical, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI, was synthesized utilizing the established FAPI scaffold. It was evaluated in vitro and in vivo, and showed considerable specific uptake of FAP-expressing tumors in mice. It exhibited a extremely mild labeling process and high availability. Therefore, this novel FAP-targeted radioactive tracer may be a promising tracer for non-invasive tumor imaging in subsequent clinical research, but its structure needs to be further modified to obtain better pharmacokinetic characteristics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHPLC \u0026nbsp; \u0026nbsp; \u0026nbsp;High-performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eRCY \u0026nbsp; \u0026nbsp; \u0026nbsp; Radiochemical yield\u003c/p\u003e\n\u003cp\u003eRCP \u0026nbsp; \u0026nbsp; \u0026nbsp; Radiochemical purity\u003c/p\u003e\n\u003cp\u003ePET \u0026nbsp; \u0026nbsp; \u0026nbsp;Positron emission tomography\u003c/p\u003e\n\u003cp\u003eRESCA \u0026nbsp; Restrained complexing agent\u003c/p\u003e\n\u003cp\u003eNOTA \u0026nbsp; \u0026nbsp;Hexadentate ligand 1,4,7-triazacyclononane-1,4,7-triacetic acid\u003c/p\u003e\n\u003cp\u003eDOTA \u0026nbsp; \u0026nbsp;2,2\u0026rsquo;2\u0026rdquo;,2-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid\u003c/p\u003e\n\u003cp\u003eQMA \u0026nbsp; \u0026nbsp; Quaternary methyl ammonium\u003c/p\u003e\n\u003cp\u003eSUV \u0026nbsp; \u0026nbsp; \u0026nbsp; Standardized uptake value\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF] \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorine-18\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga \u0026nbsp;\u003csup\u003e68\u003c/sup\u003eGa-gallium\u003c/p\u003e\n\u003cp\u003eA\u003csub\u003em\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Molar activity\u003c/p\u003e\n\u003cp\u003eBq \u0026nbsp; \u0026nbsp; \u0026nbsp; Becquerel\u003c/p\u003e\n\u003cp\u003eFAP \u0026nbsp; \u0026nbsp; Fibroblast activation protein\u003c/p\u003e\n\u003cp\u003eCAFs \u0026nbsp; \u0026nbsp;cancer-associated fibroblasts\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e%ID/g \u0026nbsp;\u0026nbsp;percentage of injected dose per gram of tissue\u003c/p\u003e\n\u003cp\u003eSPR \u0026nbsp; \u0026nbsp; \u0026nbsp;Surface plasmon resonance\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The Institutional Committee for the Care and Use of Animals (Renji Hospital, Shanghai Jiao Tong University School of Medicine) approved all animal imaging studies. The current study did not include patient information.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eInformed consent was obtained from all participants included in the study.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eC. Wang, Z. Hu, and J. Liu are co-inventors of pending patents describing the imaging technologies reported in the manuscript.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the National Key Research and Development Program of China (Grant No. 2021YFF0701900 and 2020YFA0909000), the Interdisciplinary Program of Shanghai Jiao Tong University (Grant No. ZH2018QNB20) and the construction project of Shanghai Key Laboratory of Molecular Imaging(18DZ2260400) (Grant No. KFKT-2024-27).\u003c/p\u003e\u003ch2\u003eAuthors' contributions\u003c/h2\u003e \u003cp\u003eC. Wang, B. Zhang and J. Liu conceived and designed this research. Q. Zhang and Z. Hu were responsible for all the experiments, data collection and analysis and wrote the manuscript. H. Zhao, F. Du, and C. Lv were involved in the preparation of the radionuclide and radiopharmaceuticals and took part in most of the animal experiments. T. Peng and Y. Zhang were responsible for the small-animal image analysis. All the authors participated in the revision of the article. All authors read and approved the final manuscript. C. Wang obtained the funds supporting the work.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe gratefully appreciate all the chemists, nurses, and technicians from the Department of Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, for their contributions to the tracer administration and micro-PET/CT imaging.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eDatasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBu L, Baba H, Yoshida N, Miyake K, Yasuda T, et al. Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor microenvironment. 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Eur J Med Chem. 2024;264:115993. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejmech.2023.115993\u003c/span\u003e\u003cspan address=\"10.1016/j.ejmech.2023.115993\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"H3RESCA Chelator, Fibroblast Activation Protein Inhibitor, Fluorine-18, Positron Emission Tomography","lastPublishedDoi":"10.21203/rs.3.rs-5297123/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5297123/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCancer-associated fibroblasts (CAFs), critical in tumor progression, overexpress fibroblast activation protein (FAP), presenting it as a promising target for tumor imaging and therapy. Our objective was to develop a novel radiotracer, [\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI, that achieves high labeling efficiency at room temperature for PET imaging of FAP-expressing tumors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe structure's feasibility was confirmed through molecular docking and ADMET prediction. H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI was synthesized and radiolabeled with [\u003csup\u003e18\u003c/sup\u003eF]AlF\u003csup\u003e2+\u003c/sup\u003e. Optimal labeling conditions were identified as pH 5.0, a molar ratio of aluminum chloride to precursor of 0.58, and a precursor mass of 50 \u0026micro;g. The radiotracer demonstrated high binding affinity to FAP (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e \u0026lt; 10.09 pM), favorable radiochemical yield (52.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0%), and radiochemical purity exceeding 95%. In vitro and in vivo studies revealed good stability and rapid clearance from non-target tissues. PET imaging in U87MG tumor-bearing mice showed substantial tumor uptake, which was specifically blocked by co-injection with unlabeled DOTA-FAPI-04, confirming tumor-specific uptake.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]AlF-H\u003csub\u003e3\u003c/sub\u003eRESCA-FAPI is a promising radiotracer for PET imaging of FAP-expressing tumors, exhibiting high tumor-specific uptake. With further structural modifications to enhance pharmacokinetic properties, it could become a potential candidate for clinical translation, providing a readily accessible new tool for future non-invasive tumor imaging research.\u003c/p\u003e","manuscriptTitle":"Design, Synthesis and Biological Evaluation of a Novel [18F]AlF-H3RESCA-FAPI Radiotracer Targeting Fibroblast Activation Protein.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 18:48:00","doi":"10.21203/rs.3.rs-5297123/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"319d84be-c8eb-46d6-b654-7003e4abeb53","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-27T18:48:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-27 18:48:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5297123","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5297123","identity":"rs-5297123","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}