Radiosynthesis and preliminary evaluations of 1-[18F]fluoroethyl-L- and -D-tryptophan for potential imaging of tryptophan metabolism

Objectives Tryptophan is an essential amino acid and over 95% of tryptophan is metabolized via kynurenine pathway, which is involved in the modulation of immune responses.1 Alpha-[11C]methyl-L-tryptophan (AMT) has been developed and used for clinical positron emission tomography (PET) imaging of tryptophan metabolism in tumors.2 However, the application of AMT was limited mainly due to the short half-life (20 min) of 11C. Therefore, it would be desirable to develop an AMT analog labeled with 18F (t1/2 = 110 min). Indeed, such efforts have been seen in the literature with chemical modifications made primarily to positions 2, 4, 5 and 6 of the indole ring, while biological data of these compounds are still lacking. Recently, the synthesis of 1-fluoroethyl-tryptophan (1-FETrp) was reported and importantly in vitro enzymatic assays demonstrated that the position 1 modified tryptophan analog remained to be a substrate of indoleamine-2,3-dioxygenase (IDO), the initial and rate-limiting enzyme in the kynurenine pathway.3 This makes it possible to use 1-[18F]fluoroethyl-tryptophan (1-[18F]-FETrp) for PET imaging of IDO expression, which is implicated in immunotherapy of cancer. Given the low radiochemical yield of the reported 1-[18F]-FETrp radiosynthesis (< 1%), in which the enantiopurity of 1-[18F]-FETrp was not mentioned,4 herein we describe an improved radiochemical method for the preparation of enantiopure L- and D-1-[18F]-FETrp for potential noninvasive imaging of IDO-mediated tryptophan metabolism.

Methods Enantiopure 1-[18F]-L-FETrp and 1-[18F]-D-FETrp were prepared by a nucleophilic reaction of N-boc-1-(2-tosylethyl)tryptophan tert-butyl ester with [18F]F- at 110 °C for 5 min followed by hydrolysis using 2 N HCl. The radiosynthesis was accomplished by a one-pot two-step procedure, which can be readily automated by a commercially available module. The separation of 1-[18F]-L-FETrp from 1-[18F]-D-FETrp was carried out by HPLC on an Astec CHIROBIOTIC T Chiral column (5 μm, 250 x 10 mm) using 90% ethanol in water as the mobile phase. In vitro cell uptake assays of 1-[18F]-L-FETrp and 1-[18F]-D-FETrp were performed with a breast cancer cell line MDA-MB-231. Small animal PET/CT imaging with 1-[18F]-L-FETrp and 1-[18F]-D-FETrp was carried out in a mouse model bearing MDA-MB-231 xenografts.

Results Radiotracers of 1-[18F]-L-FETrp and 1-[18F]-D-FETrp were obtained in decay-corrected radiochemical yields of 19 ± 7% and 9 ± 3%, respectively. The radiochemical purity of 1-[18F]-L-FETrp and 1-[18F]-D-FETrp was over 99%. As expected, the uptake of 1-[18F]-L-FETrp in MDA-MB-231 cells at 60 min (6.19 ± 0.92 %/mg) was about 47 times of that of 1-[18F]-D-FETrp (0.13 ± 0.07 %/mg). Further mechanistic assays indicated that system LAT (large amino acids transporter) and system ASC (alanine-, serine- and cysteine-preferring) are involved in the cell uptake of 1-[18F]-L-FETrp. Small animal PET/CT imaging studies with both radiotracers showed that the MDA-MB-231 xenografts can be visualized by 1-[18F]-L-FETrp but not 1-[18F]-D-FETrp. Quantitative analysis of the images revealed the expected preferential tumor uptake of 1-[18F]-L-FETrp (1-[18F]-L-FETrp: 4.2 ± 0.6 %ID/g; 1-[18F]-D-FETrp: 0.4 ± 0.1 %ID/g), which is consistent with the in vitro cell uptake results. In addition, a strikingly different distribution was observed between 1-[18F]-L-FETrp and 1-[18F]-D-FETrp in other tissues, especially pancreas and kidneys, which is probably associated with the differences in amino acid transporter expressions in these tissues.

Conclusions We have developed a practical radiochemical method to synthesize enantiopure 1-[18F]-L-FETrp and 1-[18F]-D-FETrp. While 1-[18F]-D-FETrp can be used to quantify the passive tissue distribution of tryptophan, L-[18F]-1-FETrp may find potential applications in IDO-mediated tryptophan metabolism. Further evaluations of both radiotracers are ongoing.