Automated radiosynthesis and evaluation of 18F-fluoropyridinated analogs of losartan and candesartan for imaging the angiotensin II Type 1 receptors by positron emission tomography

Numerous renal and cardiovascular diseases exhibit alterations in the Angiotensin type 1 receptor (AT1R) levels that are treated with candesartan or losartan. Our previous radiolabeled derivatives of these drugs displayed specific binding for renal AT1R in rats and pigs. [11C]Methyl-candesartan exhibited superior kinetics than [11C]methyl-losartan, but a hydrophobic radiometabolite interfered with the PET signal and AT1R quantification. High binding affinity, full antagonistic efficacy and minimal interference of labeled metabolites in rat kidneys were observed with [18F]fluoropyridine-losartan. However, the tracer’s low molar activity prevented visualization of low-density myocardial AT1R. The aim of this research was to develop automated radiosyntheses of 18F-fluoropyridinated analogs of candesartan and losartan in very high purities and molar activities and to evaluate [18F]fluoropyridine-candesartan efficacy as a selective AT1R PET tracer. [18F]Fluoropyridine-candesartan and [18F]fluoropyridine-losartan were synthesized via the copper(I)-catalyzed Huisgen cycloaddition between 2-[18F]fluoro-3-(pent-4-yn-1-yloxy)pyridine ([18F]FPyKYNE) and an azido-derivative of candesartan or losartan, respectively, followed by acid deprotection and C18-HPLC purification. [18F]FPyKYNE was obtained by radiofluorination of NO2PyKYNE and purified by silica-gel HPLC, preventing the formation of nitropyridinated by-products in the second step. The reaction parameters and purifications were optimized to provide tracers in 11% radiochemical yield with very high molar activities (>380 GBq/μmol), chemical and radiochemical purities (>97%). Full quality control testing of [18F]fluoropyridine-losartan was performed to validate its safe use in future clinical studies. The binding and pharmacokinetic properties of the novel [18F]fluoropyridine-candesartan were evaluated in vitro and ex/in vivo in male Sprague-Dawley rats. Competition binding assays in AT1R-expressing membranes revealed a high affinity of fluoropyridine-candesartan (Ki=5.9 nM), which is similar to fluoropyridine-losartan (Ki=5.6 nM) but lower than candesartan (Ki=0.4 nM). [18F]Fluoropyridine-candesartan bound strongly to plasma proteins (99.3%) as assessed by ultrafiltration. Specific binding was confirmed by ex vivo biodistribution 20 min after tracer injection, with a significant uptake reduction (-86%) in the AT1R-rich kidney cortex of pretreated (candesartan or losartan) rats and by in vitro autoradiography with losartan co-incubation. Radiolabeled metabolites in plasma and kidneys of control and pretreated (candesartan or losartan) animals were analyzed by column-switch HPLC. [18F]Fluoropyridine-candesartan was mainly metabolized to radiolabeled hydrophilic compounds, displaying minimal interference on renal AT1R binding with 82% of unchanged tracer in the kidneys at 20 min post-injection. Dynamic PET/CT images of [18F]fluoropyridine-candesartan, acquired for 60 min at baseline, revealed high accumulation and slow clearance from the kidney cortex and liver with high tissue-to-blood ratios. Binding specificity for AT1R was demonstrated with marked reductions in kidney-cortex (-84%) and liver (-93%) tissue-to-blood ratios at 20 min post-injection, when blocking with losartan. No change was observed in kidneys of rats pre-treated with the AT2R antagonist PD123,319, confirming binding selectivity for AT1 over AT2 receptors. The favorable binding properties and in vivo stability of [18F]fluoropyridine-candesartan support further studies to validate its potential as AT1R PET radioligand. The reproducible automated radiosyntheses developed in my research will allow innovative in-vivo PET imaging of AT1R-expression in several diseases, offering the possibility to follow disease progression in longitudinal studies and guide therapy.

Keyphrases: AT1R, [18F]FPyKYNE, IC50, TEP, THPTA, Automated radiosynthesis, Binding affinity, CuAAC click chemistry, Molar activity, PET, Radiolabeled metabolites