Fluorine-18-labeled PET Radiotracers for Imaging Tryptophan Uptake and metabolism: a Systematic Review

Abstract

Due to its metabolism via the serotonin and kynurenine pathways, tryptophan plays a key role in multiple disease processes including cancer. Imaging tryptophan uptake and metabolism in vivo can be achieved with tryptophan-derivative positron emission tomography (PET) radiotracers. While human studies with such tracers have been confined to C-11 labeled compounds, pre-clinical development of F-18 labeled tryptophan-based radiotracers has surged in recent years. We performed a systematic review of studies reporting on such F-18 labeled tryptophan tracers to summarize and compare their biological characteristics and their potential for tumor imaging, with a particular focus on key enzymes of the kynurenine pathway (indoleamine 2,3-dioxygenase [IDO] and tryptophan 2,3-dioxygenase [TDO]), which play an important role in tumoral immune resistance. From a PubMed search, English language articles including data on the preparation, radiochemical and/or biological characteristics of F-18 labeled tryptophan derivative radiotracers were reviewed. A total of 19 original papers included data on 15 unique radiotracers, the majority of which were synthesized with an adequate radiochemical yield. Automated synthesis was reported for 1-(2-[¹⁸F]fluoroethyl)-L-tryptophan, the most extensively evaluated tracer thus far. Biodistribution studies showed high uptake in the pancreas, while the L-type amino acid transporter was the dominant transport mechanism for most of the reviewed tracers. Tracers tested for tumor uptake showed accumulation in tumor cell lines in vitro and in xenografts in vivo, often with favorable tumor-to-background uptake ratios in comparison to clinically used F-18 labeled radiotracers. Five tracers showed promise for imaging IDO activity, including 1-(2-[¹⁸F]fluoroethyl)-L-tryptophan and a F-18 labeled analog of alpha-[¹¹C]methyl-L-tryptophan tested clinically in previous studies. Two radiotracers were metabolized by TDO but showed defluorination in vivo. In summary, most F-18 labeled tryptophan derivative PET tracers share common transport mechanisms and biodistribution characteristics. Several reported tracers could be candidates for further testing and validation toward PET imaging applications in a variety of human diseases.

Introduction

Tryptophan is an essential amino acid involved in protein synthesis and in the formation of niacin and the neurotransmitter serotonin [1]. Serotonin and its receptors play key roles in neuronal functions and brain plasticity in the central nervous system [2–3]. Emerging data also indicate the role of gut microbiota in the regulation of tryptophan metabolism with implications on brain function and behavior [4]. Tryptophan can also be metabolized via the kynurenine pathway and produce metabolites that control classic neurotransmitter systems [5]. In addition, tryptophan, kynurenine, and their metabolites play key roles in the pathomechanism of a wide variety of human diseases such as cancer, neurodegenerative diseases, depression, and diabetes [6–11]. In oncology, the interest in the dysregulated kynurenine pathway has been sparked by a 2003 report demonstrating that upregulation of indoleamine 2,3-dioxygenase (IDO), the initial and rate-limiting enzyme of the pathway, is associated with tumoral immune resistance, which could be reversed by inhibition of IDO [12]. Subsequent studies confirmed and expanded these findings and demonstrated the upregulation of additional key enzymes (such as IDO2 and tryptophan 2,3-dioxygenase [TDO]) in malignant tumors [11, 13–14]. These data facilitated the development and testing of IDO/TDO enzyme blockers, acting as potent immune checkpoint inhibitors, with the aim of breaking tumoral immune resistance and enhance the efficacy of tumoral immunotherapy [15–17]. It has also become apparent that preclinical and clinical studies with such inhibitors could greatly benefit from molecular imaging methods that could interrogate the activity of these enzymes and their response to pharmacologic targeting.

The value of radiolabeled amino acids, especially tryptophan derivatives, as potential imaging probes to visualize organs and tumors, has been long recognized. In a 1959 study of C-14 labeled amino acids, tryptophan showed the highest uptake in multiple organs in rats, especially in the liver, pancreas, and kidney; and tryptophan uptake was also high in Walker 256 rat breast cancer tumors [18]. Carbon-11 labeled DL-tryptophan was used in one of the earliest published human positron emission tomography (PET) studies to visualize the pancreas and pancreatic tumors [19]. While F-18 labeled tryptophan derivatives have been developed in the early 1970s [20], the clinical oncology applications of F-18-labeled amino acid PET radiotracers emerged only more than two decades later, when promising results of brain tumor imaging were reported with O-(2-[¹⁸F]fluoroethyl)-L-tyrosine ([¹⁸F]FET) and [¹⁸F]fluoro-deoxy-phenylalanine ([¹⁸F]FDOPA) [21–23]. In the same period, C-11-labeled tryptophan-based PET tracers also gained human applications. [¹¹C]5-hydroxytrypophan has been useful in detecting neuroendocrine tumors [24], while alpha-[¹¹C]methyl-L-tryptophan (AMT), a tracer originally developed for evaluating brain serotonin synthesis rates [25], have found more widespread utility in neuropsychiatric disorders, epilepsy, and brain tumor imaging [26–30]. While AMT shares transport mechanisms with other commonly used amino acid radiotracers (such as [¹⁸F]FET, [¹⁸F]FDOPA, and C-11 labeled methionine) [31], its imaging ability extends beyond amino acid transport, as it can map the activity of both the serotonin and kynurenine pathways [32]. This latter ability makes AMT a particularly attractive PET tracer for preclinical and clinical studies involving inhibitors of the kynurenine pathway. As a proof of concept, we recently demonstrated that AMT uptake on PET can respond to IDO blockade in patients treated for recurrent glioblastoma [33]. Despite these promising data, AMT could not gain widespread clinical application due to its short half-life and difficult radiosynthesis.

After decades of limited progress, a surge of novel F-18 labeled tryptophan-derived PET radiotracers have been developed in the past few years. Most studies were motivated by developing a superior molecular imaging probe for tumor detection based on increased tryptophan accumulation, while a handful of them also tested the ability of these compounds to image the activity of the immunosuppressive kynurenine pathway.

In this review, we summarize detailed characteristics of these probes, including their radiosynthesis, biodistribution, tumor uptake mechanisms and, where available, their potential ability to image tumoral activation of key enzymes of the kynurenine pathway. The collected data provide a direct, comprehensive review and comparison of these radioligands, help understand their advantages and limitations, and identify the ones that are most likely to make an impact in future clinical PET applications.

Systematic Literature Review

PubMed search strategy and selection process

The initial search was performed in PubMed (last updated on 4/26/2019), where the key words “tryptophan” AND “PET” OR “positron emission tomography” were used to identify all articles that might include the description and use of a tryptophan derivative PET tracer. All abstracts identified by the initial search were reviewed, and all English language full papers including data on the preparation and/or radiochemical or biological characteristics of a F-18 labeled tryptophan derivative PET tracer were retrieved. In addition, reference lists of these selected articles were searched for potential additional relevant publications. Review papers were combed through to identify additional original relevant papers that may have been missed in our initial PubMed search.

Data collection from the identified studies

The following key data were collected from each of the identified studies (where available): (i) molecular structure of the tryptophan-derivative compound; (ii) radiosynthesis data (including radiochemical yield and purity); (iii) tracer transport mechanisms between blood and tissue; (iv) biological organ distribution; (v) tumoral uptake and tumor types tested; (vi) comparison of uptake characteristics with clinically used F-18 labeled radiotracers (such as 2-deoxy-2-[¹⁸F]-fluoro-D-glucose [FDG], [¹⁸F]FET, and [¹⁸F]FDOPA); (vii) stability with particular focus on in vivo defluorination; and (viii) whether the compound was found to be a substrate of enzymes of the serotonin pathway or IDO and/or TDO enzymatic activity.

Findings

The initial PubMed search yielded 309 articles. After the detailed review and selection process outlined above, a total of 19 original full-length articles (Table 1) were identified to be included in this review. One paper was published before 2010 [20], 8 papers between 2010-2015 [34–41], and 10 additional papers between 2016-2019 [42–51]. These articles included data on a total of 15 different F-18 labeled tryptophan derivative PET tracers.

Table 1.

List of studies with F-18 labeled tryptophan derivative PET tracers and their basic radiochemical characteristics. In the first 7 studies, F-18 was directly attached to the indole ring, while the rest of the compounds are grouped according to the F-18 labeled chain, such as ethyl, ethoxy-, propyl-, and prop(yl)oxy-groups. Numbers in brackets indicate references of publications where the molecules were included.

	Author	Journal	Year	Tracer name	Radiochemical yield/synthesis time	Radiochemical purity (methods)
[18F]FTrp	Atkins, et al. [20]	J Nucl Med	1972	5/6-[18F] fluoro-L-tryptophan	9-10%/255 min	>99% (TLC)
	Weiss, et al. [41]	Bioorg Med Chem	2015	4-[18F] fluoro-L-tryptophan	13%/100 min	N/A
	Schäfer, et al. [44]	Eur JOC	2016	6-[18F] fluoro-L-tryptophan	15.8±4%/110 min	>99% (HPLC)
	Tang, et al. [46]	Nucl Med Biol	2017	5-[18F] fluoro-L-tryptophan 5-[18F]fluoro-D-tryptophan	1.5±0.6%/193±46 min	>99% (HPLC)
	Giglio, et al. [47]	Theranostics	2017	4-[18F]fluorotryptophan 5-[18F]fluorotryptophan 6-[18F]fluorotryptophan 7-[18F]fluorotryptophan	5.4% (no DC); 7.9% (DC) 8% (no DC); 12.0% (DC) 2.8% (no DC); 4.2% (DC) 6.4% (no DC); 8.8% (DC)	N/A
	Zlatopolskiy, et al. [50]	J Med Chem	2018	4-[18F]fluorotryptophan 5-[18F]fluorotryptophan 6-[18F]fluorotryptophan 7-[18F]fluorotryptophan	40± 12%/100-110 min 53±5%/100-110 min 30±3%/100-110 min 41±3%/100-110 min	>98% (HPLC)
[18F]F-AMT	Giglio, et al. [47]	Theranostics	2017	5-[18F] fluoro-α-methyl-tryptophan	10.9-14.9% (DC)	>97% (HPLC)
[18F]FEHTrp	Li, et al. [34]	Appl Radiat Isot	2010	5-(2-[18F]fluoroethoxy)-L-tryptophan	12-16% (no DC)/ 65 min	>98% (HPLC/TLC)
	Krämer, et al. [35]	J Nucl Med	2012	5-(2-[18F]fluoroethoxy)-L-tryptophan	23% (DC); 15% (no DC)	>95% (HPLC)
	He, et al. [38]	Nucl Med Biol	2013	5-(2-[18F]fluoroethoxy)-L-tryptophan	N/A	N/A
	Chiotellis, et al. [39]	Mol Pharm	2014	4-(2-[18F]fluoroethoxy)-DL-tryptophan 6-(2-[18F]fluoroethoxy)-DL-tryptophan 7-(2-[18F] fluoroethoxy)-DL-tryptophan	18% (DC)/90 min 13% (DC)/90 min 8% (DC)/75 min	≥95% (HPLC)
	Abbas, et al. [45]	F1000Res	2016	5-(2-[18F]fluoroethoxy)-L-tryptophan	50% (DC)	>98% (HPLC)
[18F]FPTrp	Chiotellis, et al. [37]	Eur J Med Chem	2013	2-(3-[18F]fluoropropyl)-DL-tryptophan 5-(3-[18F]fluoropropyl)-DL-tryptophan	34% (DC)/90 min 29% (DC)/90 min	>99% (HPLC)
	Chiotellis, et al. [42]	J Med Chem	2016	5-hydroxy-2-(3-[18F]fluoropropyl)-DL-tryptophan	39% (DC)/70 min	>95% (HPLC)
[18F]FPOTrp	He, et al. [38]	Nucl Med Biol	2013	5-(3-[18F]fluoropropyloxy)-L-tryptophan	21.1±4.4% (no DC)/60 min	>99% (HPLC)
	Shih, et al. [40]	Biomed Res Int	2014	5-(3-[18F]fluoropropoxy)-tryptophan	37.7% (DC)/90 min	>96% (HPLC/TLC)
[18F]FETrp	Sun, et al. [36]	Appl Radiat Isot	2012	1-(2-[18F]fluoroethyl)-L-tryptophan	0.9±0.2% (no DC)/65 min	95-97% (HPLC)
	Henrottin, et al. [43]	Nucl Med Biol	2016	1-(2-[18F]fluoroethyl)-DL-tryptophan 1-(2-[18F]fluoroethyl)-L-tryptophan 1-(2-[18F]fluoroethyl)-D-tryptophan	18±3% (no DC); 30±4% (DC)/80 min 6.1% (DC) 5.8% (DC)	>98% (HPLC)
	Chiotellis, et al. [42]	J Med Chem	2016	5-hydroxy-2-(2-[18F]fluoroethyl)-DL-tryptophan	15% (DC)/70 min	>95% (HPLC)
	Xin, et al. [48]	Mol Imaging Biol	2017	1-(2-[18F]fluoroethyl)-L-tryptophan 1-(2-[18F]fluoroethyl)-D-tryptophan	19±7% (DC)/90 min 9±3% (DC)/90 min	>99 % (HPLC)
	Michelhaugh, et al. [49]	J Nucl Med	2017	1-(2-[18F]fluoroethyl)-L-tryptophan	25% (DC)/50 min	>98% (HPLC)
	Xin, et al. [51]	Mol Imaging Biol	2019	1-(2-[18F]fluoroethyl)-L-tryptophan	N/A	N/A

Abbreviations: DC: decay correction; TLC: thin-layer chromatography; HPLC: high-performance liquid chromatography; N/A: not available.

Strategies of F-18 labeling of tryptophan

Fluorine-18 labeling of tryptophan has been performed by replacing a hydrogen atom with F-18 or by attaching fluorinated carbon chains to various positions of the indole ring. Fluorinated ring positions included both the benzene and the pyrrole rings. Among the 15 radiofluorinated tryptophan derivatives reported, the benzene ring was F-18 labeled in 11 compounds (Fig. 1), among which the most common target was the 5-position in 5 tracers. Direct benzene ring fluorination has been applied for 5 radiotracers: 4-, 5-, 6-, 7-[¹⁸F]fluorotryptophan (4/5/6/7-[¹⁸F]FTrp) [20, 41, 44, 46–47, 50] and 5-[¹⁸F]fluoro-α-methyl-tryptophan (5-[¹⁸F]F-AMT) [47]. A fluoroethoxy group was attached to benzene ring positions in 4 compounds: 4-, 5-, 6-, 7-(2-[¹⁸F]fluoroethoxy)-tryptophan (4/5/6/7-[¹⁸F]FEHTrp) [34–35, 38–39, 45]. In addition, fluoropropyl or fluoropropoxy groups in the 5-position of the benzene ring were applied in three additional studies [37–38, 40].

Figure 1.

Tryptophan derivative radiotracers F-18-labeled on the benzene ring. For each molecule, the F-18 containing group is in a red frame, while the non-labeled methyl group (where present) is framed in black. Numbers in brackets indicate references of publications where the molecules were included.

Four tracers had the pyrrole ring labeled with F-18 (Fig. 2): the 1-position was fluorinated with ethyl in 1-(2-[¹⁸F]fluoroethyl)-tryptophan (1-[¹⁸F]FETrp), which has been the most widely reported among all the 15 tracers [36, 43, 48–49, 51], and an automated radiosynthesis method has been also developed for this tracer [43, 48]. The 2-position was labeled in 5-hydroxy-2-(2-[¹⁸F]fluoroethyl)-DL-tryptophan (5-OH-2-[¹⁸F]FETrp) [42], and also, utilizing a fluorinated propyl group, in 2-(3-[¹⁸F]fluoropropyl)-DL-tryptophan (2-[¹⁸F]FPTrp) and 5-OH-2-(3-[¹⁸F]fluoropropyl)-DL-tryptophan (5-OH-2-[¹⁸F]FPTrp) [37, 42].

Figure 2.

Tryptophan derivative radiotracers F-18-labeled on the pyrrole ring. For each molecule, the F-18 containing group is in a red frame, while the non-labeled hydroxyl group (where present) is framed in black. Numbers in brackets indicate references of publications where the molecules were included.

Radiochemical characteristics of the F-18 labeled tryptophan derivatives

Radiochemical yield (RCY) of a radioactive tracer refers to the yield of a radiochemical separation, expressed as a fraction of the activity originally present, while radiochemical purity indicates the fraction of the stated isotope present in the stated chemical form. The first studies creating radiolabeled tryptophan tracers (4/5/6-[¹⁸F]FTrps) provided 9–13% RCY [20, 41], while a more recent study could reach higher RCY using copper-mediated radiofluorination for 6-[¹⁸F]fluoro-L-tryptophan (15.8±4%) with an excellent radiochemical purity (>99%) detected by high-performance liquid chromatography (HPLC) [44]. In the subsequent study of the same group, high RCYs were obtained for 4/5/6/7-[¹⁸F]FTrp by a modified alcohol-enhanced copper-mediated radiofluorination (40±12%, 53±5%, 30±3%, and 41±3%, respectively) with a high (>98%) radiochemical purity for all tracers within 100–110 min [50] (Table 1).

Among tracers labeled on the 5-position of the benzene ring, RCY varied between 1.5±0.6% and 53±5% with decay correction (DC) [34–35, 37–38, 40, 45–47, 50]. The highest RCYs were achieved for 5-[¹⁸F]FTrp and 5-[¹⁸F]FEHTrp (53% and 50%, respectively) [45, 50] (Table 1). The radiochemical purity was above 95% in almost all C5-labeled radiofluorinated tryptophan derivative studies detected by HPLC or thin-layer chromatography [34–35, 37–38, 40, 45, 47, 50] (Table 1). Radiochemical purity of 85–90% was detected for L-5-[¹⁸F]FTrp which also had the lowest RCY (1.5±0.6%) after 193 min using the original copper-mediated radiofluorination procedure [46]. In the study comparing different labeling positions of [¹⁸F]fluoroethoxy-DL-tryptophan, all tracers reached 95% radiochemical purity with relatively low or moderate RCYs (18%, 13%, and 8% for 4/6/7-[¹⁸F]FEHTrp, respectively) [39].

In 1-[¹⁸F]fluoroethyl-L-tryptophan studies, radiosynthesis reached only poor or moderate radiochemical yield (0.9% without DC to 25% with DC) but good radiochemical purity (95–99%) [36, 43, 48–49] (Table 1). Chiotellis, et al. also synthesized two hydroxy-fluorotryptophan derivatives, 5-OH-2-[¹⁸F]FETrp and 5-OH-2-[¹⁸F]FPTrp, with reasonable RCYs (15% and 39% with DC, respectively) and with high radiochemical purity (>95%) [42].

Tracer transport mechanisms

Cells in various tissue types can possess a number of different transport systems located in their plasma membranes [52]. Native tryptophan, similar to other branched and aromatic amino acids, enters the cells via the Na⁺-independent system L [53]. Four subtypes of system L have been isolated: L-type amino acid transporter 1 (LAT1), LAT2, LAT3, and LAT4. LAT1 is widely expressed in primary human cancers and cancer cell lines and plays an essential role in the survival and growth of tumors [54–56]. LAT2 is predominantly expressed in other cell types such as epithelial cells of the kidneys and intestines, and it carries small neutral amino acids [57], whereas LAT3 and LAT4 subtypes have a narrower substrate selectivity (preferring phenylalanine) [58].

Similar to native tryptophan, the majority of the F-18 labeled tryptophan tracers have been found to enter the cells via the LAT system (Table 2, Fig. 3). In vitro cell uptake studies verified the LAT system as the transport mechanism for 2-[¹⁸F]FPTrp and 5-[¹⁸F]FPTrp from the extracellular space but did not specify the particular subtype [37]. In case of L-1-[¹⁸F]FETrp, the main transport was facilitated by the LAT system, but system ASC (preferentially transporting alanine, serine, and cysteine) was also involved in the tumor cell uptake of the tracer; while the D-isomer of the same tracer showed negligible uptake in MDA-MB-231 breast cancer cells, where the mechanism of LAT system is significant [48].

Table 2.

Transport, stability, and in vivo defluorination (based on bone uptake) of different F-18 labeled tryptophan derivative tracers.

Author	Year	Tracer name	Transport	Stability	In vivo defluorination
Li, et al.	2010	5-(2-[18F]fluoroethoxy)-L-tryptophan	N/A	stable in saline >6 h p.i. at room temperature	N/A
Krämer, et al.	2012	5-(2-[18F]fluoroethoxy)-L-tryptophan	LAT1/2	stable in vivo at 60 min p.i.	negligible
Chiotellis, et al.	2014	6-(2-[18F]fluoroethoxy)-DL-tryptophan	LAT1	only intact compound was detected in plasma at 60 min p.i.	minor
Abbas, et al.	2016	5-(2-[18F]fluoroethoxy)-L-tryptophan	LAT1 suggested	N/A	N/A
Chiotellis et al.	2013	2-(3-[18F]fluoropropyl)-DL-tryptophan 5-(3-[18F]fluoropropyl)-DL-tryptophan	LAT	only intact compound was detected in plasma at 60 min p.i.	minor
					negligible
Chiotellis, et al.	2016	5-hydroxy-2-(3-[18F]fluoropropyl)-DL-tryptophan 5-hydroxy-2-(2-[18F]fluoroethyl)-DL-tryptophan	LAT1/2 (system b0,+, ATB)	only intact compound was detected in plasma at 60 min p.i.	minor
He, et al.	2013	5-(3-[18F]fluoropropyloxy)-L-tryptophan	system b0,+, LAT2, (ASC)	good stability in vitro	negligible
Shih, et al.	2014	5-(3-[18F]fluoropropoxy)-tryptophan	LAT1 suggested	N/A	N/A
Tang, et al.	2017	5-[18F]fluoro-L-tryptophan 5-[18F]fluoro-D-tryptophan	N/A	good stability at 4 h in vitro	marked
					negligible
Zlatopolskiy, et al.	2018	4-18F]fluorotryptophan 5-[18F]fluorotryptophan 6-18F]fluorotryptophan 7-18F]fluorotryptophan	LAT1	stable in human serum at 37 °C stable in presence rat liver microsomes	rapid
				stable in human serum at 37 °C slow defluorination with rat liver microsomes	negligible
Henrottin, et al.	2016	1-(2-[18F]fluoroethyl)-L-tryptophan	N/A	good stability at 4 h in vitro	N/A
Xin, et al.	2017	1-(2-[18F]fluoroethyl)-L-tryptophan	LAT, ASC	good stability in vitro in vivo stability 97% in plasma at 2 h p.i.	negligible
Michelhaugh, et al.	2017	1-(2-[18F]fluoroethyl)-L-tryptophan	N/A	N/A	negligible

Abbreviations: N/A: not available; LAT: L-type amino acid transport system; ASC: alanine-, serine-, cysteine-preferring transport system; p.i.: post injection.

Figure 3.

Summary of F-18 labeled tryptophan-derivative radiotracers that have been tested for transport via the L-type amino acid transporter 1 (LAT1) and/or metabolism via key enzymes of the kynurenine and serotonin pathways. Tracers transported via LAT1 or metabolized via specific enzymes are in blue, while others that have been tested but showed no enzyme activity are in red color. Abbreviations: KMO: kynurenine 3-monooxygenase; IDO1: indoleamine 2,3-dioxygenase 1; TDO: tryptophan dioxygenase; TPH: tryptophan hydroxylase; AADC: amino acid decarboxylase; 5-[¹⁸F]F-AMT: 5-[¹⁸F]fluoro-α-methyl-tryptophan; 4-,5-,6-, 7-[¹⁸F]FTrp: 4-,5-,6-,7-[¹⁸F]fluorotryptophan; 1-[¹⁸F]FETrp: 1-(2-[¹⁸F]fluoroethyl)-tryptophan; 4-,5-,6-,7-[¹⁸F]FEHTrp: 4-,5-,6-,7-(2-[¹⁸F]fluoroethoxy)-tryptophan; (5-OH)-2-[¹⁸F]FPTrp: (5-hydroxy)-2-(3-[¹⁸F]fluoropropyl)-tryptophan; 5-OH-2-[¹⁸F]FETrp: 5-hydroxy-2-(2-[¹⁸F]fluoroethyl)-tryptophan.

LAT1 was reported to play the main role in the transport mechanism of 5/6-[¹⁸F]FEHTrp, [¹⁸F]fluoropropoxy-tryptophan ([¹⁸F]FPOTrp), and 4/5/6/7-[¹⁸F]fluorotryptophan [35, 39–40, 50] (Table 2). Abbas and coworkers also suggested the role of LAT1 in transport of 5-[¹⁸F]FEHTrp, as they found reduced tracer uptake and LAT1 expression in the pancreas of Akita mice compared to wild-type mice [45]. The main role of LAT1/2 and possibly system B^0,+ and/or ASC was hypothesized in the uptake of 5-OH-2-[¹⁸F]FPTrp and 5-OH-2-[¹⁸F]FETrp tracers based on in vitro cell uptake studies [42]. In the uptake studies performed for [¹⁸F]FPOTrp in hepatocellular cancer cells, the amino acid transport system B^0,+ appeared to play the main role, but the LAT system also took part in the transport mechanism of the tracer [38]. In the same study, [¹⁸F]FPOTrp uptake was also blocked by serine, suggesting that [¹⁸F]FPOTrp transport was shared by serine, while system ASC might contribute to its uptake. Serine is a substrate for both system LAT2 and B^0,+, but not LAT1, which suggested that [¹⁸F]FPOTrp is mainly transported by the amino acid transport systems B^0,+ and LAT2, but the system ASC has also a minor effect on tracer cell uptake [38].

In vitro and in vivo tracer stability

Stability of the various tracers have been investigated in both in vitro (using buffer, blood serum, or diverse cell lines at different temperatures), as well as in vivo models. Good in vitro stability was reported with 5-[¹⁸F]FEHTrp, [¹⁸F]FPOTrp, 5-[¹⁸F]FTrp, and 1-[¹⁸F]FETrp [34, 38, 43, 46, 48] (Table 2). Although 4-, 5-, and 6-[¹⁸F]FTrps were stable in vitro with a decomposition less than 4% in human blood serum after 1 hour at 37 °C and remained stable in the presence of rat liver microsomes for at least 1 hour, they suffered from rapid in vivo defluorination [50]. In contrast, 7-[¹⁸F]FTrp demonstrated a slow defluorination (<6% after 1 hour) using rat liver microsomes and good in vivo stability in healthy rats as reflected by low skull uptake [50].

Ex vivo metabolite studies of the tracers detected only intact compounds in the plasma, tumor, and urine for 2-[¹⁸F]FPTrp, 5-[¹⁸F]FPTrp, 6-[¹⁸F]FEHTrp, 5-OH-2-[¹⁸F]FPTrp, 5-OH-2-[¹⁸F]FETrp at 60 min post injection in mice bearing different tumor xenografts (lung, prostate carcinoma, and glioma), thus making the possible involvement of a biotransformation step in tumor accumulation unlikely [37, 39, 42] (Table 2).

In vivo defluorination of F-18 labeled PET tracers is mediated by hepatic cytochrome P450 enzymes affecting mostly aliphatic fluorides, and the detached F-18 can be visualized by PET as an increased uptake in bones, which can also contribute to the tracer’s toxicity. Defluorination of 5-[¹⁸F]FEHTrp, 1-[¹⁸F]FETrp, and 5-[¹⁸F]FPTrp was negligible in PET studies suggesting a good in vivo stability of all three tracers [35, 37, 49]. Negligible bone uptake was also reported using [¹⁸F]FPOTrp and 7-[¹⁸F]FTrp [38, 50], while a faint bone accumulation was observed for 6-[¹⁸F]FEHTrp and 2-[¹⁸F]FPTrp [37, 39], as well as for 5-OH-2-[¹⁸F]FPTrp and 5-OH-2-[¹⁸F]FETrp, where the uptake was higher than that of [¹⁸F]FET indicating a minor defluorination of the F-18 labeled tryptophan tracers [42]. High bone uptake was reported for the L isomer of 5-[¹⁸F]FTrp but not for the D isomer [46].

Biodistribution

Most of the F-18 labeled tryptophan tracers, including 5-[¹⁸F]FEHTrp, [¹⁸F]FPOTrp, 2-[¹⁸F]FPTrp, 5-OH-2-[¹⁸F]FPTrp, 5-OH-2-[¹⁸F]FETrp, 1-[¹⁸F]FETrp, and 5-[¹⁸F]F-AMT, showed high uptake in kidney suggesting urinary clearance [34–35, 37–38, 40, 42, 47–48, 51] (Table 3). Elevated accumulation in liver, blood, intestines, and heart was reported for 5-[¹⁸F]FEHTrp in one study [34], although subsequent studies with the same tracer showed lower uptake in liver [35, 45]. Similar inconsistent results were reported for 1-[¹⁸F]FETrp, where high uptake was described in the kidney and liver by Xin, et al. [48], while our group reported relatively low uptake in both organs [49]. Besides the elevated kidney uptake, high tracer accumulation in intestines was observed for 2-[¹⁸F]FPTrp and [¹⁸F]FPOTrp [37–38]. Among those reports where pancreas was included in biodistribution studies, pancreas had the highest uptake in most of the cases [35, 38, 45, 51] (Table 3). One exception was 5-[¹⁸F]FTrp that showed low uptake in most tissues except for urinary bladder and gut [46] (Table 3).

Table 3.

Biodistribution of F-18 labeled tryptophan derivative tracers in mice. The highest uptakes are indicated in bold in each row.

Tracer	Uptake distribution
	Kidneys	Liver	Pancreas	Brain	Bone	Other
5-[18F]FEHTrp
Li, et al. 2010a (normal mice)	high (4.67±0.59)	high (5.92±0.69)	N/A	high (3.06±0.47)	high (2.59±1.19)	high in intestines, heart
Li, et al. 2010a (tumor-bearing mice)	high (4.47±0.71)	high (5.69±0.89)	N/A	high (2.97±0.76)	high (2.14±1.36)	high in intestines, heart
Krämer, et al. 2012b	high (2.05±0.59)	low (0.81±0.10)	high (8.50±1.64)	low (0.84±0.32)	no uptake	N/A
Abbas, et al. 2016c (wild-type mice)	low (7.64±1.6)	high (17.89±4.2)	high (35.47±6.7)	low (2.76±0.7)	N/A	high in heart, low in intestines
Abbas, et al. 2016c (Akita mice)	low (11.36±5.4)	low (4.32±1.9)	high (43.81±24.7)	low (3.50±1.6)	N/A	low in heart, intestines
4/6/7-[18F]FEHTrp
Chiotellis, et al. 2014#	N/A	N/A	N/A	N/A	faint for 6-[18]FEHTrp	high in intestines
2-[18F]FPTrp
Chiotellis, et al. 2013#	high	N/A	N/A	N/A	faint uptake	high in intestines
5-[18F]FPTrp
Chiotellis, et al. 2013#	low	N/A	N/A	N/A	no uptake	high in intestines
5-OH-2-[18F]FPTrp
Chiotellis, et al. 2016d	high (7.2±1.4)	N/A	N/A	low (0.96±0.09)	high 1.4±0.13	N/A
5-OH-2-[18F]FETrp
Chiotellis, et al. 2016d	high (12.1±2.2)	N/A	N/A	low (0.94±0.07)	high 2.0±0.5	N/A
[18F]FPOTrp
He, et al. 2013e	high (3.17±1.45)	high (3.91±0.87)	high (4.66±1.33)	low (1.16±0.38)	low (1.33±0.22)	high in intestines
Shih, et al. 2014#	high	N/A	N/A	low	N/A	high in heart
5[18F]FTrp
Tang, et al. 2017f#	low	low	low	low	high for L isomer	high in intestines
4/5/6/7-[18F]FTrp
Zlatopolskiy, et al. 2018g	N/A	N/A	N/A	low (76.5±9.8)	low for 7-[18F]FTrp, high in 4/5/6-[18F]FTrp	N/A
5[18F]F-AMT
Giglio, et al. 2017#	high at 30 min p.i.	N/A	N/A	N/A	N/A	N/A
1-[18F]FETrp
Xin, et al. 2017h#	high	high	high	low	no uptake	high in heart
Michelhaugh, et al. 2017i#	low	low	high	low	no uptake	N/A
Xin, etal. 2019j#	high (3.48±0.45)	high (2.41±0.07)	high (16.7±0.80)	low (1.19±0.01)	low (1.03±0.33)	low in lungs

^a:in vivo %ID/g at 15 min p.i. (highest values);

^b:ex vivo SUV at 70 min p.i.;

^c:ex vivo %ID/g at 90 min p.i.;

^d:organ/muscle SUV ratios at 60-75 minp.i.;

^e:in vivo %ID/g at 30 min p.i. (highest values in normal mice);

^f:based on %ID/g at 45 min p.i. data;

^g:in vivo %ID at 120 min p.i.;

^h:based on %ID/g at 120 min p.i. data;

^i:based on SUVs/TAC btw 30-60 min p.i. data

^j:ex vivo %ID/g at 240 min p.i.

^#:uptake based on the text or figures/graphs presented in the paper (no exact values were shown in the article).

Abbreviations: [¹⁸F]FEHTrp: (2-[¹⁸F]fluoroethoxy)-tryptophan; [¹⁸F]FPTrp: (3-[¹⁸F]fhioropropyl)-tryptophan; [¹⁸F]FETrp: (2-[¹⁸F]fluoroethyl)-tryptophan; [¹⁸F]FPOTrp: (3-[¹⁸F]fluoropropyloxy)-tryptophan; [¹⁸F]FTrp: [¹⁸F]fluorotryptophan; [¹⁸F]F-AMT: [¹⁸F]fluoro-α-methyl-tryptophan; N/A: not available; %ID/g: percent injected dose/gram; p.i.: post injection; SUV: standard uptake value; TAC: time activity curve.

Low radiotracer uptake in normal brain is essential to create a low-uptake background for brain tumor imaging. In one of the first F-18 labeled tryptophan tracer studies, 5-[¹⁸F]FEHTrp uptake was elevated in the brain (3.06±0.47 and 2.97±0.76 percent injected dose/gram [%ID/g], in normal and tumor-bearing mice, respectively) [34], however, subsequent studies found relatively low 5-[¹⁸F]FEHTrp uptake in brain [35, 45] (Table 3). Low brain uptake was also observed for [¹⁸F]FPOTrp, 5-OH-2-[¹⁸F]FPTrp, 5-OH-2-[¹⁸F]FETrp, 1-[¹⁸F]FETrp, and 5-[¹⁸F]FTrp [38, 40, 42, 46, 48–49, 51]. Moreover, 7-[¹⁸F]FTrp showed overall low cortical uptake associated with increased accumulation in pineal gland and hypothalamus [50].

Uptake in tumor xenografts

The initial PET studies with 5-[¹⁸F]FEHTrp showed high tumoral uptake in different tumor xenografts, including fibrosarcoma, small cell lung cancer (SCLC), prostate, breast cancer, and hepatocellular carcinoma [34–35, 38], and tracer uptake was able to differentiate tumors from inflammation [34, 38] (Table 4). 4-, 6-, 7-[¹⁸F]FEHTrp showed increased SUVs in NCI-H69 (SCLC) xenografts with 6-[¹⁸F]FEHTrp showing the highest tumor-to-muscle SUV ratio (2.6±0.2) among them [39] and also higher than 5-[¹⁸F]FEHTrp in the same lung cancer model [35]. Compared to inflammation, the uptake ratios in fibrosarcoma and hepatocellular carcinoma xenografts were also higher with [¹⁸F]FPOTrp [38] (Table 4).

Table 4.

Tracer uptake of F-18 labeled tryptophan derivative tracers in various tumor models.

Tracer	Tumor model (all in mice subcutaneous xenografts unless otherwise indicated)	Tracer uptake in xenografts
		Absolute values	Tumor/background ratios
5-[18F]FEHTrp
Li, et al. 2010a	S180 (fibrosarcoma)	5.19±0.79	tumor/muscle ratio: 2.79 tumor/blood ratio: 2.87
	Intramuscular inflammation	1.82±0.28	inflammation/muscle ratio: 0.94 inflammation/blood ratio: 0.91
Krämer, et al. 2012b#	NCI-H69 (SCLC)	~1.6	tumor/reference ratio: ~1.9
	PC-3 (prostate)	~1.5	tumor/reference ratio: ~1.8
	MDA-MB-231 (breast)	~1.3	tumor/reference ratio: ~1.3
He, et al. 2013c#	S180, Hepa1-6 cell	N/A	tumor/muscle ratio: 2.51±0.14 tumor/inflammation ratio: 1.53±0.09
	Subcutaneous inflammation	N/A	inflammation/muscle ratio: ~2.0 inflammation/blood ratio: ~1.0
4/6/7-[18F]FEHTrp
Chiotellis, et al. 2014d#	NCI-H69	~0.7 (4-[18F]FEHTrp); ~0.9 (6-[18F]FEHTrp); ~1.1 (7-[18F]FEHTrp)	tumor/muscle: 2.6±0.2*
2-[18F]FPTrp
Chiotellis, et al. 2013e	NCI-H69	N/A	tumor/reference region ratio: 2.38±0.02
	PC-3	N/A	tumor/reference region ratio: 2.4
5-[18F]FPTrp
Chiotellis, et al. 2013e	NCI-H69	N/A	tumor/reference region ratio: 1.8
	PC-3	N/A	tumor/reference region ratio: 1.8
5-OH-2-[18F]FPTrp
Chiotellis, et al. 2016d#	NCI-H69	~0.6	tumor/muscle ratios: 4.3±0.9
	PC-3	~0.5	tumor/muscle ratios: 4.2±0.6
	C6 (glioma)	~0.4	tumor/muscle ratios: 2.7±0.3
5-OH-2-[18F]FETrp
Chiotellis, et al. 2016d#	NCI-H69	~0.6	tumor/muscle ratios: 3.9±0.2
	PC-3	~0.5	tumor/muscle ratios: 4.2±0.4
	C6	~0.4	tumor/muscle ratios: 3.2±1.1
[18F]FPOTrp
He, et al. 2013c#	S180, Hepal-6 (hepatocellular carcinoma)	N/A	tumor/muscle ratio: 3.1±0.11 tumor/inflammation ratio: 2.53±0.06
	Subcutaneous inflammation	N/A	inflammation/muscle ratio: −1.3 inflammation/blood ratio: −0.5
Shih, et al. 2014#	NCI-H187Lu (SCLC)	high uptake at 45 min p. i.
	PC-3	low uptake (<2 %ID/g) at 22 min p. i.
5[18F]FTrp
Tang, et al. 2017#	17095A, 17082A (NSCLC)	high uptake (~4 %ID/g) at 20 min p. i.
	CT26 (colon)	high uptake (5-8 %ID/g) at 20 min p.i.
4/5/6/7[18F]FTrp
Zlatopolskiy, et al. 2018#	MCF-7 (ER negative breast), PC-3, NCI-H69 xenograft in chicken embryos	high uptake in all tumor xenograft at 30 min p.i., but no values shown
5[18F]F-AMT
Giglio, et al. 2017#	B16F10 cell (skin melanoma)	high uptake in tumor xenograft, but no values shown
1-[18F]FETrp
Xin, et al. 2017f	MDA-MB-231	4.6±0.4 (L-isomer) 1.0±0.2 (D-isomer)	N/A
Michelhaugh, et al. 2017g	glioblastoma	1.17±0.28	N/A
	breast cancer metastasis	0.70±0.10	N/A
	NSCLC metastasis	0.51±0.05	N/A
Xin, et al. 2019f	PC-3	7.5±0.6 6.4±1.0h	tumor/muscle ratio: 4.3 tumor/muscle ratio: 4.9h
	H2009 and H460 (NSCLC)	5.3±0.8 (H2009); 9.0±1.4 (H460)	N/A
	orthotopic A549 (lung)	4.5±0.5	tumor/normal lung ratio: 2.6
	intracranial 73 C (glioma)	4.1±0.7	tumor/brain ratio: 2.9

^a:%ID/g uptake at 60 min p.i.;

^b:SUV at 30-45 min p.i.;

^c:%ID/g ratio at 30-60 min p.i.;

^d:SUV and SUV ratio at 60-75 min p.i.;

^e:xenograft to reference SUV ratio at 60 min p.i;

^f:%ID/g at 120 min p.i.;

^g:patient-derived xenografts, SUV at 30-60 min p.i.;

^h:%ID/g at 240 min p.i.

^#:uptake based on the text or figures/graphs presented in the paper (values after ~ sign are estimates);

^*:all tracers accumulated in the tumor xenograft, values for 6-[¹⁸F]FEHTrp are shown – uptake of other two tracers ranged from 1.9 to 2.1.

Abbreviations: [¹⁸F]FEHTrp: (2-[¹⁸F]fluoroethoxy)-tryptophan; [¹⁸F]FPTrp: (3-[¹⁸F]fhioropropyl)-tryptophan; [¹⁸F]FETrp: (2-[¹⁸F]fluoroethyl)-tryptophan; [¹⁸F]FPOTrp: (3-[¹⁸F]fluoropropyloxy)-tryptophan; [¹⁸F]FTrp: [¹⁸F]fluorotryptophan; [¹⁸F]F-AMT: [¹⁸F]fluoro-α-methyl-tryptophan; SCLC: small cell lung cancer; NSCLC: non-small cell lung cancer; ER: estrogen receptor; N/A: not available; %ID/g: percent injected dose/gram; p.i.: post injection; SUV: standard uptake value.

Among fluorotryptophans, 5-[¹⁸F]FTrp showed low uptake (approximately 4%ID/g) both in high and low IDO-expressing non-small cell lung cancer (NSCLC) xenografts, while its uptake was slightly higher in CT26 (colon cancer) xenografts (up to 8%ID/g) [46], while 7-[¹⁸F]FTrp could clearly delineate all the three types of tumor tissue (breast, prostate, and lung) in a chicken chorioallantoic membrane model [50] (Table 4).

1-[¹⁸F]FETrp, especially the L isomer, also showed increased uptake in several subcutaneous tumor xenografts, including MDA-MB-231 (breast), PC-3 (prostate), H460, H2009 (lung), and patient-derived glioblastoma, metastatic breast cancer and NSCLC [48–49, 51], as well as orthotopic A549 lung cancers and intracranial 73C gliomas [51] (Table 4); while the D isomer of this tracer did not visualize the tumor tissue [48]. In a direct comparison, our group noted that this radiotracer showed higher tumor SUVs compared with AMT [49]. A recent study reported that 5-[¹⁸F]F-AMT accumulated in skin melanoma xenograft-bearing mice [47] (Table 4).

Comparison of tumoral uptake of tryptophan-derived vs. clinically used F-18 labeled tracers

In vitro cell uptake studies showed similar higher uptake of 4,- 5-, 6-, 7-[¹⁸F]fluoro-L-tryptophans than [¹⁸F]FET in medulloblastoma, prostate, and interferon-γ (IFN-γ)-treated breast cancer cells [50], while these tracers showed similar levels of uptake in glioblastoma and untreated breast cancer cells.

In animal PET studies involving tumor xenografts, 5-[¹⁸F]FEHTrp was superior to FDG in differentiation of tumor from inflammation in fibrosarcoma [34], and the same tracer demonstrated high uptake in SCLC and prostate carcinoma xenografts, comparable to [¹⁸F]FDOPA uptake, in a subsequent study [35], 4-, 6-, 7-[¹⁸F]FEHTrps showed lower SUVs in subcutaneous SCLC tumor xenografts compared with [¹⁸F]FET, however, they also presented lower background uptake than [¹⁸F]FET, thus their SUV tumor/background ratios were higher [39]. In the same study, 6-[¹⁸F]FEHTrp was found to be superior to [¹⁸F]FET in delineation of SCLC tumors [39], [¹⁸F]FPOTrp was also better for differentiating tumor from inflammation than FDG and even 5-[¹⁸F]FEHTrp [38]. Interestingly, a subsequent study with [¹⁸F]FPOTrp (derived from a different precursor of the automated synthesis) found lower uptake in prostate carcinoma xenograft but higher uptake in SCLC xenograft compared to FDG (Table 4) [40]. This difference was suggested to be due to variations of LAT1 activity in different animal models.

Tumor/background SUV ratios of 2-[¹⁸F]FPTrp and [¹⁸F]FET were comparable in lung and prostate cancer models, while showing higher uptake compared with 5-[¹⁸F]FPTrp [37], 5-OH-2-[¹⁸F]FPTrp and 5-OH-2-[¹⁸F]FETrp showed higher tumor/muscle SUV ratios compared with [¹⁸F]FET in SCLC, prostate, and glioma xenografts in head-to-head comparisons of the scans performed with different tracers in the same animals [42].

Tracer metabolism via enzymes of the serotonin pathway

As mentioned earlier, tryptophan can be metabolized by two major pathways, the serotonin and the kynurenine pathways. The serotonin pathway, which converts tryptophan to 5-hydroxytryptamine (i.e., serotonin), has two key enzymes, tryptophan hydroxylase (TPH) and amino acid decarboxylase (AADC) [59]. TPH-1 seems to be a regulator of immunity not only by moderating serotonin levels, but more likely by exhausting tryptophan as it was shown in a bladder cell carcinoma mice model, where TPH-1^−/− mice had reduced tumor growth kinetics and ~50% completely reject the tumor [60].

Tracer metabolism by TPH has been evaluated for 5-[¹⁸F]F-AMT, a fluorinated analog of C-11 labeled AMT that had been tested extensively in human PET studies and showed metabolic activity via both TPH and, to a certain degree, IDO [26–28, 31–33, 61–62]. Compared with tryptophan and AMT, 5-[¹⁸F]F-AMT was reported be a worse substrate for TPH [47], thus suggesting that this radiotracer is more specific for imaging activity of the kynurenine pathway than AMT.

Several studies evaluated metabolism of radiofluorinated tryptophan tracers by AADC, and none of the evaluated tracers were decarboxylated by this enzyme [35, 37, 39, 42] (Fig. 3). He, et al. also reported that [¹⁸F]FPOTrp was not incorporated into proteins after cellular uptake [38]. Subsequent studies focused on the kynurenine pathway, especially whether the F-18-labeled tryptophan tracers were substrates for IDO1 or TDO [42–43, 46–48, 50–51], as discussed in the next section.

Tracer metabolism via IDO1 and TDO

The importance of the kynurenine pathway was originally ascribed to its role in the biogenesis of nicotinamide adenine dinucleotide. However, tumoral immune tolerance made this pathway an attractive target for developing inhibitors to its first and rate-limiting enzyme, IDO1 and 2 [12–13]. Moreover, TDO1 and 2 can also convert tryptophan to kynurenine, and TDO2 appears to play a key role in immune resistance of gliomas and meningiomas [14, 61]. IDO expressing tumors showed escape from immune surveillance by inhibiting effector T cells due to tryptophan depletion and accumulation of kynurenine metabolites, which latter mechanism could be the most important among them [12–13, 63]. Experiments with recombinant human IDO and TDO (rhIDO and rhTDO) in E. coli cultures showed that rhIDO tolerated only substitutions at the 5-position of the indole ring, while rhTDO could only oxidize 5-fluoro-L-tryptophan and 5-methyl-L-tryptophan [64].

Several recent F-18 labeled tryptophan tracer studies focused on the kynurenine pathway, especially on their metabolism via IDO or TDO, to evaluate their potential for imaging IDO activity in tumor tissue [42–43, 46–48, 50–51] (Table 5; Fig. 3). No metabolites of 5-[¹⁸F]FEHTrp were detected in breast cancer cells with high IDO activity suggesting that this tracer is not a substrate for IDO [35]. Furthermore, none of the tracers 6-[¹⁸F]FEHTrp, 2-[¹⁸F]FPTrp, 5-OH-2-[¹⁸F]FPTrp, and 5-OH-2-[¹⁸F]FETrp were substrate for IDO evaluated by in vitro enzymatic assays [42] (Table 5).

Table 5.

Data on metabolism of F-18 labeled tryptophan derivative tracers by IDO and TDO enzymes.

Author	Year	Tracer name	Study type	Result
Chiotellis, et al.	2016	6-(2-[18F]fluoroethoxy)-DL-tryptophan 2-(3-[18F]fluoropropyl)-DL-tryptophan 5-hydroxy-2-(3-[18F]fluoropropyl)-DL-tryptophan 5-hydroxy-2-(2-[18F]fluoroethyl)-DL-tryptophan	in vitro enzyme assays with rhIDO	not substrate for IDO (no oxygenation was found)
Tang, et al.	2017	5-[18F] fluoro-L-tryptophan 5-[18F]fluoro-D-tryptophan	in vitro enzyme assays with hIDO1 and hTDO2 in vitro cell uptake studies of cells with diverse IDO1 and TDO2 expression	substrate for IDO1 and TDO2 not substrate for IDO1 and TDO2
Zlatopolskiy, et al.	2018	4-[18F]fluorotryptophan 5-[18F]fluorotryptophan 6-[18F]fluorotryptophan 7-[18F]fluorotryptophan	in vitro cell uptake studies using IFN-γ treated and non-treated cells in vivo dynamic uptake studies	substrate for IDO substrate for IDO substrate for IDO not substrate for TDO and IDO, inhibitor of both enzymes
Giglio, et al.	2017	5-[18F] fluoro-α-methyl-tryptophan	in vitro enzyme assays in vitro uptake study using IFN-γ treated cells	substrate for IDO1
Henrottin, et al.	2016	1-(2-[18F]fluoroethyl)-L-tryptophan	in vitro enzyme assays with rhIDO in vitro cell uptake studies of cells with diverse IDO expression	good and specific substrate for IDO
Xin, et al.	2017	1-(2-[18F]fluoroethyl)-L-tryptophan	in vitro cell uptake studies using IFN-γ treated and non-treated cells	substrate for IDO
Xin, et al.	2019	1-(2-[18F] fluoroethyl)-L-tryptophan	radio-HPLC of tumor extracts	30% of the tracer was converted to a radioactive kynurenine metabolite

Abbreviations: rhIDO: recombinant human indoleamine 2,3-dioxygenase; hTDO: human tryptophan 2,3-dioxygenase; IFNγ: interferon-γ; HPLC: high-performance liquid chromatography.

In vitro enzymatic assays with rhIDO and rhTDO confirmed that L-5-[¹⁸F]FTrp was substrate for both enzymes, whereas D isomer was not [46]. Further in vitro cell uptake experiments using colon carcinoma cell lines revealed an elevated cell uptake of L-5-[¹⁸F]FTrp upon induction of IDO1 or TDO2 enzymes compared with baseline; however, the uptake was observed only in the presence of low L-tryptophan levels in media [46]. In the same study, in PET imaging experiments of NSCLC tumor bearing mice, the time-activity profiles of both isomers were indistinguishable between low vs. high IDO-expressing tumors suggesting that IDO1 activity does not play a significant role in observed radioactivity accumulation in the tumor tissue. Moreover, the time-activity profiles of the tracer uptake were not different between mice bearing colon cancer xenografts with and without induced IDO expression. Although L-5-[¹⁸F]FTrp was a substrate for both IDO1 and TDO2 in vitro and accumulated in tumor cells in an enzyme-dependent manner at low tryptophan concentrations, the authors concluded that in vivo tumor uptake of the tracer could not be attributed to IDO1 or TDO2 enzyme activity presumably due to competition with endogenous tryptophan and rapid tracer metabolism [46].

In a subsequent study with 4-, 5-, 6-, 7-[¹⁸F]FTrp, in vitro cell uptake experiments performed with different tumor cell lines, including low IDO-expressing estrogen receptor positive breast cancer cells, showed no significant difference in accumulation of [¹⁸F]FTrps vs. [¹⁸F]FET in untreated cancer cells [50]. IDO expression was stimulated by IFN-γ treatment in estrogen receptor positive breast cancer cells, where the uptake of 6-[¹⁸F]FTrp and 7-[¹⁸F]FTrp was markedly increased during the incubation time after IFN-γ stimulation (2-fold and 7-fold after 2 hours, respectively), while [¹⁸F]FET uptake (driven mostly by amino acid transport) showed only a slight alteration after treatment [50]. Moreover, in vivo dynamic PET analysis suggested that 4-, 5-, and 6-[¹⁸F]FTrp were metabolized by IDO with V_max/K_m values of 14%, 37%, and 47%, respectively; even though 7-[¹⁸F]FTrp was not a substrate of IDO and TDO, it was an inhibitor of both enzymes [50].

In in vitro enzymatic assays with rhIDO and rhTDO, the non-radioactive 1-FETrp showed a variable consumption depending on the rhIDO concentration and incubation time [65], 100% consumption was measured at 5μM after 4 hours, 27% at 0.5 μM after 1 hour, and 7% at 0.1 μM rhIDO concentration (where L-tryptophan still showed 94% consumption); however, 1-FETrp showed only 2% conversion with 10 μM rhTDO concentration. These findings suggest that this tracer is a good and specific substrate for rhIDO but not for rhTDO, which could facilitate a more specific detection of rhIDO expressing cells in cancer imaging [65]. A subsequent study of the same group extended this observation to 1-[¹⁸F]FETrp where high IDO-specific uptake was associated with very low efflux in in vitro enzymatic cellular assays for the L-isomer [43]. This radiotracer has been suggested as a promising tool for imaging of IDO-expressing tumor cells by other groups too [48–49, 51], 1-[¹⁸F]FETrp uptake in IFN-γ-stimulated MDA-MB-231 cells was similar to non-treated (low IDO-expressing) cells after 10 min incubation period but significantly increased compared with native MDA-MB-231 cells at a longer incubation time (60 min) [48]. This increased uptake could be inhibited by the IDO inhibitor NLG919. These findings support that cell uptake of L-1-[¹⁸F]FETrp was driven by IDO activity [48]. The subsequent study of the same group found that about 30% of the L-1-[¹⁸F]FETrp was converted into a highly polar radioactive metabolite, probably a kynurenine analogue, in highly IDO-expressing PC-3 prostate tumor extracts, and also showed higher tracer uptake in tumors with known high IDO expression [51]. Our group also demonstrated the increased accumulation of L-1-[¹⁸F]FETrp in different IDO/TDO-expressing patient derived tumor xenografts [49]; notably these xenografts showed stronger expression of IDO2 and TDO2 than IDO1 in a previous study [66].

In a recent study, 5-[¹⁸F]F-AMT, an analog of the clinically tested C-11 labeled AMT, was also evaluated as a potential substrate for IDO [47]. The uptake of this tracer in IFN-γ-treated melanoma cells was significantly higher than in control cells after a 1-hour incubation; moreover, incubation of treated cells with NLG919 has led to a decrease in 5-[¹⁸F]F-AMT accumulation comparable with control cells indicating that cell uptake of 5-[¹⁸F]F-AMT is associated with IDO1 activity levels.

Discussion

This review of the published data demonstrates that most F-18 labeled tryptophan derivative radiotracers can be produced with a sufficient radiochemical yield and high purity. Although detailed, quantitative organ biodistribution data have been limited to selected studies, high tracer uptake in the pancreas appears to be a common feature across several tracers. This indicates that these compounds may not be practical for pancreas tumor imaging due to high background activity in normal pancreas tissue. Still, almost all tryptophan-derivative radiotracers showed moderate to high accumulation in a variety of tumor xenografts, with only a few exceptions, such as [¹⁸F]FPOTrp in PC-3 (prostate), and the D-isomer of 1-[¹⁸F]FETrp in MDA-MB-231 (breast cancer) xenografts. Comparisons with clinically used F-18 labeled PET tracers showed favorable uptake characteristics of multiple tryptophan derivative radiotracers in multiple tumor models. The L-type amino acid transporter (mainly LAT1) was found to be the main transporter system in the majority of studies where transport mechanisms were assessed. This confirms the notion that tryptophan-based radiotracers can be useful to evaluate the activity of LAT1, which is often upregulated in cancer tissue and also highly expressed in the blood-brain barrier. Several tracers, such as 6-[¹⁸F]FEHTrp, 5-OH-2-[¹⁸F]FPTrp, and 5-OH-2-[¹⁸F]FETrp, have been tested and showed no substrate activity to AADC or IDO, suggesting that their tumoral accumulation is driven by transporter activity. Thus, these tracers may be the best to assess LAT1 transporter upregulation quantitatively in tumors. While these initial data are promising, the potential superiority of F-18 labeled tryptophan derivatives to image various tumors in vivo needs further, comparative studies involving other, current F-18 labeled amino acid PET radiotracers that have already been in clinical use.

The most unique aspect of tryptophan-derive radiotracers is their potential ability to provide information about tumoral activity of IDO1 and TDO, the key enzymes of the immunosuppressive kynurenine pathway. Upregulation of these enzymes is associated with tumoral immune resistance and shorter survival in several different cancer types [11–14], In vitro cell uptake and enzymatic studies reported data indicating that five of the tracers are substrates of IDO: 4-, 5-, 6-[¹⁸F]fluoro-L-tryptophan, 1-[¹⁸F]FETrp, and 5-[¹⁸F]F-AMT. 5-[¹⁸F]F-AMT is a F-18 labeled analog of AMT, a C-11 labeled tryptophan-derivative that has shown promise in multiple neuro-oncology applications [27–28, 31, 33]. Thus, 5-[¹⁸F]F-AMT, along with 1-[¹⁸F]FETrp whose automated radiosynthesis has been reported [43, 48], deserve further studies for their ability to quantify IDO activity in vivo. 5-[¹⁸F]F-AMT has been also tested and showed less substrate activity with TPH as compared to C-11 labeled AMT, thus demonstrating its potential as an IDO-specific PET tracer. This can be useful in cases when both the serotonin and kynurenine pathways are activated as seen for example in certain types of breast cancers [67]. The questionable in vivo stability for 5-[¹⁸F]F-AMT remains a concern, because the only report with this compound did not have data on in vivo defluorination and did not report in vivo uptake data beyond 30 min after tracer injection [47]. Rapid in vivo defluorination, likely due to peripheral (such as liver) TDO activity, indeed has been reported for 5- and 6--[¹⁸F]fluoro-L-tryptophan [46, 50], which would be tracers of interest for imaging TDO activity [68], a substrate-selective enzyme of the kynurenine pathway [50]. Although TDO imaging could be of great value in brain tumors [11, 14], the reported rapid in vivo defluorination of these TDO-selective compounds, render them less likely candidates for human PET imaging.

Based on the preclinical data summarized in this review, a number of F-18 labeled tryptophan-derivative PET tracers may find clinical applications covering a wide range of human diseases. 1-[¹⁸F]FETrp and 5-[¹⁸F]F-AMT have the potential to replicate the clinical findings reported with C-11-labeled AMT, such as visualizing certain epileptic foci and brain lesions (such as cortical developmental malformations and low-grade tumors) for epilepsy surgery [26, 55] and detecting epilepsy-associated neuroinflammatory abnormalities [69]. These two compounds could also be candidates for developing PET probes to evaluate various cancers in the conjunction of emerging immunotherapies that incorporate IDO enzyme inhibitors. Since fluorination on the 5-position of the benzene ring makes the tracer resistant to metabolism via TPH, 5-[¹⁸F]F-AMT also has the advantage over C-11 labeled AMT to be a more IDO-specific PET probe. However, in vivo stability of this radiotracer remains a concern and requires further studies before considering it for human applications. Several other fluorinated tryptophan tracers, which are not substrates of IDO (e.g., 6-[¹⁸F]FEHTrp, 5-OH-2-[¹⁸F]FPTrp, and 5-OH-2-[¹⁸F]FETrp), may be specific imaging probes to assess LAT1 transporter activity in human cancer. Imaging tumoral amino acid transport is a main interest in response assessment in clinical neuro-oncology [70]. Considering that some other fluorinated amino acid PET tracers with an ability of imaging LAT1 transport activity are already in clinical use, rigorous head-to-head comparisons are needed to determine if tryptophan-based compounds have any advantage in this respect.

This review also identified a few limitations and challenges of tryptophan-based PET tracers toward their potential clinical applications. As mentioned above, one limitation is the lack of an ideal candidate for imaging specific TDO enzyme activity. While TDO may be an important treatment target to break cancer immune suppression, especially for gliomas [14], as well as in a wider spectrum of brain disorders (from schizophrenia to Alzheimer’s disease) [71], currently there is no specific TDO inhibitor in clinical trials. However, such compounds along with combined IDO1/TDO dual inhibitors are under intense development [72–73], and, if successful, in vivo imaging of TDO activity may become a highly relevant clinical application in the foreseeable future. Another limitation is the paucity of data on the ability of the new fluorinated tryptophan-based tracers to characterize the activity of TPH and the serotonin pathway. This application also has a broad clinical interest in neuro-psychiatric conditions, which motivated the original development of AMT [25]. The importance of this application has been further amplified by the recent FDA approval of telotristat ethyl, a selective TPH inhibitor, for the treatment of neuroendocrine tumor-associated diarrhea [74]. Therefore, future development and validation of tryptophan-derivative PET tracers should include testing of the new compounds for their ability to quantify TPH activity.

Conclusions

In summary, radiosynthesis, including automated synthesis, of F-18 labeled tryptophan-derivative PET radiotracers is feasible, and several of these compounds showed promise and deserve further assessment to evaluate LAT1 transporter activity and/or IDO1 activity in tumor tissue in vivo. Development of radiotracers with sufficient in vivo stability for TDO imaging requires further studies. Fluorine-18 labeled tryptophan-derivative PET tracers may have a wide range of clinical applications including imaging and monitoring the activity of the immunosuppressive kynurenine pathway in cancers and inflammatory conditions, detecting upregulated amino acid transport, and imaging epileptic foci.