Preparation and evaluation of L- and D-5-[18F]fluorotryptophan as PET imaging probes for indoleamine and tryptophan 2,3-dioxygenases

Abstract Indoleamine and tryptophan 2,3-dioxygenases (IDO1 and TDO2) are pyrrolases catalyzing the oxidative cleavage of the 2,3-double bond of L-tryptophan in kynurenine pathway. In the tumor microenvironment, their increased activity prevents normal immune function, i.e. tumor cell recognition and elimination by cytotoxic T-cells. Consequently, inhibition of the kynurenine pathway may enhance the activity of cancer immunotherapeutics by reversing immune dysfunction. We sought to investigate the properties of radiolabeled 5-[ 18 F]fluorotryptophan with respect to its ability for measuring IDO1 and TDO2 activity by positron emission tomography (PET). Results L-5-[ 18 F]fluorotryptophan and D-5-[ 18 F]fluorotryptophan were synthesized by Cu(I) catalyzed [ 18 F]fluorodeboronylation of Boc/tBu protected precursors in moderate yields (1.5±0.6%) sufficient for pre-clinical studies. The specific activity of the product was 407–740GBq/μmol, radiochemical purity >99% and enantiomeric excess 90–99%. Enzymatic assay confirmed that L-5-fluorotryptophan is an IDO1 and TDO2 substrate whereas the D-isomer is not. In-vitro cell uptake experiments using CT26 cells with doxycycline-induced overexpression of human-IDO1 and human-TDO2 revealed an elevated cell uptake of L-5-[ 18 F]fluorotryptophan upon induction of IDO1 or TDO2 enzymes compared to baseline; however, the uptake was observed only in the presence of low L-tryptophan levels in media. PET imaging experiments performed using tumor bearing mouse models expressing IDO1 at various levels (CT26, CT26-hIDO1, 17082A, 17095A) showed tumor uptake of the tracer elevated up to 8%ID/g; however, the observed tumor uptake could not be attributed to IDO1 activity in the tumor tissue. The metabolism of L- and D- isomers was markedly different in vivo, the D-isomer was excreted by a combination of hepatobiliary and renal routes, the L-isomer underwent extensive metabolism to [ 18 F]fluoride. Conclusion The observed in vivo tumor uptake of the tracer could not be attributed to IDO1 or TDO2 enzyme activity in the tumor, presumably due to competition with endogenous tryptophan as well as rapid tracer metabolism.