Radiosynthesis and preclinical development of [18F]labeled tryptophan for human indoleamine 2,3-dioxygenase PET imaging

Nowadays, Cancer is probably the global incidence disease which is the most widespread, the most varied, still constantly rising and that can affect all levels and social classes of the population. Its treatment and care of patients suffering from this disease made it undoubtedly a crucial medical and scientific challenge, but also and above all, a major political, economical and social challenge for the coming years. Since its early detection often leads to a positive prognostic for the future of the patient, numerous methods are currently being developed to make easier its screening and improve its treatment. Positron Emission Tomography (PET) is one of the best techniques used to diagnose and bring out the presence of tumor cells, but also to study enzymatic activities within them, allowing therefore the prescription of a more adequate treatment and better tailored to each individual patient. That’s in this spirit that a novel specific radiotracer of the human indoleamine 2,3-dioxygenase enzyme (hIDO) has been developed during this PhD thesis. This enzyme, highly expressed in numerous human tumors, is also currently subject to the development of various inhibitors. This report presents the development of N1-(2-[18F]fluoroethyl)-L-tryptophan (1-[18F]FE-L-Trp), a novel PET radiotracer derived from L-tryptophan and bearing a radioactive atom of fluorine-18 ([18F]F). This “story” starts with the synthesis of this compound and its selection as a specific substrate of hIDO among several potential candidates, and continues until its in vivo PET imaging on mice, while including also its enantiomerically pure and automated radiosynthesis, and numerous in vitro enzymatic and cellular assays, demonstrating the properties and the characteristics (specificity and selectivity) of this radiotracer with respect to hIDO. According to this study, in vitro cellular uptake have demonstrated that 1-[18F]FE-L-Trp is a radiotracer of choice to facilitate the development and the preclinical validation of novel potential inhibitors of hIDO (e.g. 1-methyl-L-tryptophan). Additionally, some in vivo PET imaging on mice bearing one tumor in each flank have shown that this radiotracer allows the in vivo detection of tumors expressing this enzyme. Nevertheless, in the conditions tested until now, only very small preferential uptake was observed with this radiotracer, in hIDO expressing tumors as compared to hTDO and control tumors. As a result, this radiotracer cannot be yet considered as a PET contrast agent in this animal model. Therefore, further additional investigation will be required to understand and characterize this animal model, to improve these in vivo results, and finally reach this objective. However, this PhD research constitutes the first step towards the preclinical development of radiopharmaceutical compounds allowing the molecular cancer imaging through the intermediary of hIDO enzyme

Mass spectrometry for detection of ruminant animal by-products in poultry feed

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Synthesis of N-fluoroalkyl-tryptophan and study of their biological activity as potential substrates for indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (rhIDO) is an enzyme mainly expressed in brain and tumor cells and catalyzing the oxidative cleavage of the indole ring of L-tryptophan through the kynurenine pathway. Furthermore this enzyme could be responsible for the eventual suppression of immune responses by blocking locally T-lymphocyte proliferation. The syntheses of 1-(2-fluoroethyl)-tryptophan (1-[19F]FETrp) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-tryptophan, two N-fluoroalkylated tryptophan derivatives, are described here. In vitro enzymatic assays with these two new potential substrates of rhIDO show that 1-[19F]FETrp is a good and specific substrate of hIDO

Fully automated radiosynthesis of N1-[18F]fluoroethyl-tryptophan and study of its biological activity as a new potential substrate for indoleamine 2,3-dioxygenase PET imaging

Introduction: Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of L-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumor tissues by blocking locally T-lymphocyte proliferation. Recently, 1-(2-[19F]fluoroethyl)-DL-tryptophan (1-[19F]FE-DL-Trp) was reported as a good and specific substrate of this enzyme. Herein, the radiosynthesis of its radioactive isotopomer (1-[18F]FE-DL-Trp, DL-[18F]5) is presented along with in vitro enzymatic and cellular uptake studies. Methods: The one-pot n.c.a. radiosynthesis of this novel potential PET imaging tracer, including HPLC purification and formulation, has been fully automated on a FASTlabTM synthesizer. Chiral separation of both isomers and their formulation were implemented on a second cassette. In vitro enzymatic and cellular uptake studies were then conducted with the D-, L- and DL-radiotracers. Results: The radiolabeling of the tosylate precursor was performed in DMF (in 5 min; RCY: 57% (d.c.), n=3). After hydrolysis, HPLC purification and formulation, DL-[18F]5 was obtained with a global radiochemical yield of 18±3% (not decay corrected, n=7, in 80 min) and a specific activity of 600±180 GBq/µmol (n=5). The subsequent separation of L- and D-enantiomers was performed by chiral HPLC and both were obtained after formulation with a RCY (d.c.) of 6.1% and 5.8%, respectively. In vitro enzymatic assays reveal that L-[18F]5 is a better substrate than D-[18F]5 for human IDO. In vitro cellular assays show an IDO-specific uptake of the racemate varying from 30% to 50% of that of L-[18F]5, and a negligible uptake of D-[18F]5. Conclusion: In vitro studies show that L-[18F]5 is a good and specific substrate of hIDO, while presenting a very low efflux. These results confirm that L-[18F]5 could be a very useful PET radiotracer for IDO expressing cells in cancer imaging