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 FASTlab™ 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 an 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

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry–based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development. Graphical abstract: [Figure not available: see fulltext.