Automated Radiosynthesis of 1-(2-[¹⁸F]Fluoroethyl)-L-Tryptophan ([¹⁸F]FETrp) for PET Imaging of Cancer in Humans

Abstract

The radiotracer 1-(2-[¹⁸F]fluoroethyl)-L-tryptophan (L-[¹⁸F]FETrp or [¹⁸F]FETrp) is a substrate of indoleamine 2,3-dioxygenase (IDO), the initial and key enzyme of the kynurenine pathway associated with tumoral immune resistance. In preclinical PET studies, [¹⁸F]FETrp is highly accumulated in a wide range of primary and metastatic cancers, such as lung cancer, prostate cancer, and gliomas. However, the clinical translation of this radiotracer into the first-in-human trial has not been reported, partially due to its racemization during radiofluorination which renders the purification of the final product challenging. However, efficient purification is essential for human studies in order to assure radiochemical and enantiomeric purity. In this work, we report a fully automated radiosynthesis of [¹⁸F]FETrp on a Synthra RNPlus research module, including a one-pot two steps radiosynthesis, dual independent chiral and reverse-phase semi-preparative HPLC purifications, and solid-phase extraction (SPE) assisted formulation. The presented approach has led to its Investigational New Drug (IND) application and approval that allows the testing of this tracer in humans.

1. Introduction

Tryptophan is metabolized via the kynurenine pathway mediated by indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which are prominent targets for cancer imaging and therapy.^1–4 Positron emission tomography (PET) is a powerful and highly sensitive technique for non-invasive imaging of metabolic processes. In the past, the radiotracer α-[¹¹C]methyl-L-tryptophan ([¹¹C]AMT) has been investigated to assess neuropsychiatric disorders, epilepsy, primary and metastatic brain tumors,^5–9 but is only available in limited sites due to the short half-life of carbon-11 (t1/2 = 20.3 min) and the challenging radiochemistry. To facilitate the clinical translation of tryptophan PET imaging, several fluorine −18 labeled tryptophan analogs haven been developed as potential radiotracers for tryptophan transport and metabolism imaging in a wide range of diseases, especially in cancer.^10–11 With a desired fluorine-18 half-life of 109.8 min, 1-(2-[¹⁸F]fluoroethyl)-L-tryptophan (L-[¹⁸F]FETrp or [¹⁸F]FETrp) is one of the most promising radiolabeled tryptophan analogs for PET imaging of IDO activity in various primary and metastatic cancers.^10,12–13 In preclinical studies, L-[¹⁸F]FETrp showed favorable uptake in IDO-overexpressed tumors and up to 10-fold higher uptake than that of D-[¹⁸F]FETrp in breast cancer xenografts mice model.¹⁴

The radiosynthesis of [¹⁸F]FETrp is well documented.^13–16 However, the clinical translation of [¹⁸F]FETrp has not been reported, partially due to its racemization during radiofluorination,¹³ and thereby complicating purification procedures to reach the required radiochemical and enantiomeric purities for human studies. The radiosynthesis of [¹⁸F]FETrp is a typical one-pot two steps process that includes the radiofluorination of the precursor with azeotropically dried [¹⁸F]F⁻/K_2.2.2/K₂CO₃, followed by 2N HCl deprotection. L-/D-[¹⁸F]FETrp racemization (L-/D-[¹⁸F]FETrp = ~2:1) was observed at the radiofluorination step under heating and basic conditions,¹⁴ requiring dual chiral and reverse-phase HPLC separations.^15–16

We have performed a pilot preclinical PET study of [¹⁸F]FETrp in different patient-derived xenograft tumor mouse models, including glioblastoma and metastatic brain tumors;¹² where tracer uptake and kinetics was favorable in comparison to [¹¹C]AMT. In that preclinical study, [¹⁸F]FETrp radiosynthesis was performed on an in-house built module with low yield. To facilitate the clinical translation, we have recently developed an automated radiosynthesis of [¹⁸F]FETrp on a Synthra RNPlus research module, which was designed to allow advanced synthesis of research radiotracers. In this work, we report a fully automated production of [¹⁸F]FETrp (Fig.1), including a one-pot two steps radiosynthesis, dual semi-preparative HPLC purifications and assisted solid-phase extraction (SPE) formulation. The goal of this approach was to develop a standardized radiosynthesis procedure that allows human studies using this tracer under an Exploratory Investigational New Drug (eIND) application approved by the Food and Drug Administration (FDA).

Figure 1.

Scheme and flowchart for [¹⁸F]FETrp radiosynthesis.

2. Materials and methods

2.1. General

Unless otherwise stated, reagents, solvents, and chemicals were purchased from commercially available vendors and used without further purification. The tert-Butyl N^α-(tert-butoxycarbonyl)-1-(2-(tosyloxy)ethyl)-L-tryptophanate (>99% purity; >99% enantiomeric excess (ee)) precursor, 1-(2-Fluorethyl)-L-tryptophan hydrochloride (L-[¹⁹F]FETrp; >98% purity; >99% ee) and 1-(2-Fluorethyl)-D-tryptophan hydrochloride (D-[¹⁹F]FETrp; >99% purity; >95% ee) reference standard compounds were purchased from Affinity Research Chemical Inc (Wilmington, DE, USA). Acetonitrile (MeCN; anhydrous 99.8%), potassium carbonate (K₂CO₃; 99.995% trace metals basis), potassium phosphate monobasic (KH₂PO₄; ≥99%), trifluoroacetic acid (TFA; >99.0%), and 0.22 μm Millex-GS syringe filter unit (33 mm) were purchased from Millipore Sigma (St. Louis, MO, USA). Acetonitrile (MeCN; HPLC grade), hydrochloric acid (2.0 N solution), acetic acid (ACS grade) and ammonium hydroxide (28.0–30.0%) were purchased from Fisher Scientific (Hampton, NH, USA). Kryptofix 2.2.2 (K_2.2.2; chemical grade) and [¹⁸O]H₂O (≥98%) were purchased from ABX (Radeberg, Germany). QMA carbonate plus light cartridge (46 mg sorbent per cartridge; 40 μm) and Oasis MCX plus short cartridge (225 mg sorbent per cartridge; 60 μm) were purchased from Waters (Milford, MA, USA). Absolute ethanol (EtOH; USP grade) was purchased from Greenfield Global USA Inc (Shelbyville KY, USA). Sterile water for injection, USP and 0.9% sodium chloride (NaCl) for injection, USP were purchased from Hospira (Lake Forest, IL, USA). Fresh deionized water (18.2 MΩ∙cm at 25 °C) was generated from Milli-Q Direct water purification system (Millipore Sigma, Billerica, MA, USA), and used for the preparation of all the standard and eluent solutions. The K_2.2.2/ K₂CO₃ stock solution for [¹⁸F]fluoride elution was prepared with K_2.2.2 (240 mg) and K₂CO₃ (40 mg) in acetonitrile (19.4 mL) and deionized water (0.6 mL), and passed through a Millex-LG PTFE filter (Millipore Sigma, St. Louis, MO, USA). Prior to use, QMA cartridge was conditioned with 5 mL deionized water, and MCX cartridge was conditioned with 5 mL ethanol and 5 mL deionized water, respectively. Radioactivity was determined with a Capintec^® CRC-712M dose calibrator (Capintec, Inc., Florham Park, NJ, USA).

2.2. Chromatographic method

Chiral and reverse-phase semi-preparative high-performance liquid chromatography (HPLC) purifications were carried out on a RNplus Research module (Synthra, Hamburg, Germany), which included a built-in semi-preparative HPLC system with ultraviolet (UV) and radioactivity detectors. The chiral semi-preparative HPLC was conducted on an Astec CHIROBIOTIC T column (5 μm, 250 × 10 mm; Millipore Sigma, St. Louis, MO, USA) with 90% EtOH mobile phase at a flow rate of 4 mL/min. The retention times of L- and D-[¹⁸F]FETrp were about 12 and 18 min, respectively. The reverse-phase semi-preparative HPLC was conducted on a Reeperbahn C18 column (5 μm, 250 × 10 mm; Synthra, Hamburg, Germany) with MeCN/0.1% AcOH (v/v = 20/80) mobile phase at a flow rate of 4 mL/min. The retention times of [¹⁸F]FETrp were about 15 min. Chiral and reverse-phase analytical analyses were performed on an Acquity ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA ) with ultraviolet (UV) and Ortec-556 radioactivity detectors (Oak Ridge, TN, USA). The enantiomeric purity analysis was conducted on an ASTEC CHIROBIOTIC T column (5 μm, 100 × 2.1 mm; Millipore Sigma, St. Louis, MO, USA) with 90% EtOH mobile phase at a flow rate of 0.3 mL/min. The chemical and radiochemical purities analyses were conducted on an analytical ACQUITY BEH C18 column (1.7 μm, 2.1 × 100 mm; Waters, Milford, MA, USA) at the UV wavelength of 254 nm. The sample injection volume was 10 μL, and the mobile phase was a mixture of 10 mM KH₂PO₄ in water and acetonitrile (v/v = 85/15) with a flow rate of 0.3 mL/min.

2.3. RNplus Research Module configuration

Synthra RNplus research module configuration and setup for [¹⁸F]FETrp production were detailed in Figure 2. and Table 1. In the module configuration, vials A1–7, B1–2, B5, C1-C3 and reaction vessels 1 and 2 were used for [¹⁸F] FETrp radiosynthesis, chiral HPLC (column #1), and reverse-phase HPLC (column #2) separation, and formulation.

Figure 2.

Diagram of Synthra RNplus Research module for [¹⁸F]FETrp production.

Table 1.

Synthra RNplus module setup for [¹⁸F]FETrp production.

Location	Items
A1	1 mL of K2.2.2/ K2CO3 solution
A2	0.5 mL of 2 N HCl
A3	4 mg of precursor in 1 mL of MeCN
A4	0.4 mL of 20% AcOH
A5	3 mL of water
A6	5 mL of water
A7	2 mL of 5% NH4OH in ethanol
B1	0.4 mL of 20% AcOH
B2	2 mL of water
B5	2 mL of 5% NH4OH in ethanol
C1	10 mL of sterile water for injection, USP
C2	2 mL of 5% NH4OH in ethanol
C3	10 mL of 0.9% NaCl, USP and 90 μL of AcOH
D1	Reverse-phase HPLC eluent: MeCN/0.1% AcOH (v/v = 20/80)
D2	Chiral-phase HPLC eluent: 90% EtOH in water
HPLC column #1	C18 Synthra Reeperbahn semi-prep HPLC column (250 × 10 mm)
HPLC column #2	Chiral Astec Chirobiotic T semi-prep HPLC column (250 × 10 mm)
E1	QMA cartridge (46 mg)
E2	MCX Plus cartridge
E3	MCX Plus cartridge
E4	MCX Plus cartridge
F1	20 mL of water and 0.2 mL of 2 N HCl
F2	20 mL of water and 0.2 mL of 2 N HCl
G1	Millex-GS filter
G2	30 mL product vial with preloaded 10 mL of 0.9% NaCl, USP

2.4. Radiosynthesis, purification, and formulation of [¹⁸F]FETrp

The radiosynthesis of [¹⁸F]FETrp, including two HPLC separations and SPE formulation, is fully automated on the Synthra RNPlus research module. Briefly, with a standard Kryptofix_2.2.2/K₂CO₃ elution from a QMA cartridge and azeotropic drying procedures, [¹⁸F]fluoride was reacted with the precursor (4 mg; 7 μmol) in anhydrous acetonitrile at 110 °C for 5 min. After 2N HCl deprotection, the afforded crude D/L-[¹⁸F]FETrp mixture was diluted with water (V5-V6) and trapped on an Oasis MCX Plus cartridge (V15-V44-V16-V7), washed with water (V6-V15-V44-V16-V7), eluted with 5% NH₄OH/EtOH solution (V7-V16-V44-V15), neutralized with 20% AcOH (V4-V5-V6), and then loaded to a chiral semi-preparative HPLC column (V15-V44-loading syringe-HPLC loop). The enantiomerically pure L-form [¹⁸F]FETrp fraction was collected and extracted with a second MCX cartridge (V28-V39-V40), and eluted with 5% NH₄OH/EtOH solution (V11-V39-V40-V42), neutralized with 20% AcOH (V8-V9-V10-V49). After removal of ethanol under vacuum and helium blowing (V23-V20-V22), the crude product was diluted with 2 mL water (V9-V10-V49) and then purified with a C18 reverse-phase semi-preparative HPLC (V24-V57-V25-loading syringe-HPLC loop) to remove the chemical impurities. The collected [¹⁸F]FETrp fraction was diluted with 20 mL water, trapped on the third MCX cartridge (V33-V35-V36), washed with 10 mL sterile water (V29-V30-V31-V35-V36), and eluted with 2 mL of 5% NH₄OH/EtOH solution (V30-V31-V35-V36). The final product was formulated with 20 mL of 0.9% sodium chloride USP and 90 μL of acetic acid (V31-V35-V36), and then passed through a sterile Millex-GS syringe filter to a sterile vial (V34-V37).

2.5. Quality control

A sample of ~0.5 mL was taken from the finished final product for quality control (QC) tests following United States Pharmacopeia (USP) and GMP guidelines. The detailed QC procedure was described in supplemental data.

3. Results

3.1. Radiosynthesis

Three consecutive batches for the validation of [¹⁸F]FETrp production were completed successfully in a total synthesis time of 90 min from end of bombardment (EOB). [¹⁸F]FETrp product was obtained in 5.4 ± 0.3 GBq at end of synthesis (EOS), with a typical irradiation of 60 min at 60 μA. The radiochemical yields were 6.0 ± 0.3% (decay corrected to EOB). The volumes of final formulated [¹⁸F]FETrp product were 22.3 ± 0.3 mL.

3.2. Quality control

The QC results exhibited that the [¹⁸F]FETrp product met all the release criteria for human use, as shown in Table 2. Three batch products were clear, colorless solutions, and free from particulate matter. The pH and half-life values were within the ranges of 4.0 – 7.0 and 105 – 115 min. From analytical HPLC results (Fig. 5 and S1–3), the radiochemical purities of [¹⁸F]FETrp were 96–97% with chemical impurities of 0.7 ± 0.3 ug/mL, and enantiomeric purities of L-isomer were >99% ee. The specific activities were 412 ± 25 GBq/μmol at EOS. The retention time of FETrp standard on analytical revers-phase HPLC was about 2.6 min. The retention times of L- and D-FETrp references on analytical chiral HPLC were about 3.7 and 7.2 min, respectively. The radionuclidic purities were determined by a Multi-Channel Analyzer (MCA). The residual solvents in product were determined by GC to be 5.2 – 5.8% ethanol. The integrity of the final filter was demonstrated by a bubble-point filter test with holding ≥ 345 KPa pressure. The formulated products were sterile and nonpyrogenic from the sterility and endotoxin results. Stability at 4 hours after EOS was evaluated by performing the repeated assessment of appearance, radiochemical identity/purity, chemical purity, pH, and bacterial endotoxin, showing no significant changes at 4 hours post EOS (Table S2).

Table 2.

Summary of QC results from three [¹⁸F]FETrp validation runs.

QC Test	Acceptance Criteria	Result
		Run 1	Run 2	Run 3
Appearance	Clean, colorless and no particles	pass	pass	pass
Concentration (mCi/mL)	≥ 2 mCi/mL @ EOS	6.9	6.2	6.4
Filter integrity	Bubble point: ≥ 345 KPa (50 psi)	pass	pass	pass
Radionuclidic identity	Half-life (min): 105 – 115	109.9	109.9	110.1
Radionuclidic purity	≥99.5% observed gamma emission should correspond to 0.511 and 1.022 MeV	pass	pass	pass
pH	pH value: 4.0 – 7.0	5.0	5.0	5.0
Radiochemical purity	[18F]FETrp peak: ≥ 90%	97.3%	96.5%	96.6%
Radiochemical identity	RSD of [18F]FETrp Rf values: ≤ 10%	2.8%	1.3%	2.9%
Chemical purity:	FETrp mass: ≤ 2 μg/mL	0.15	0.17	0.18
	Total impurities: ≤ 4 μg/mL	0.34	1.04	0.86
Enantiomeric purity	L- [18F]FETrp ≥ 90%	>99%	>99%	>99%
Chemical purity: residual solvent	Ethanol ≤ 10 % (w/v)	5.2%	5.4%	5.8%
	MeCN ≤ 0.04 % (w/v)	0.00%	0.00%	0.00%
Chemical purity: K2.2.2	Intensity of product is less than K2.2.2 STD	pass	pass	pass
Pyrogen test	LAL Endotoxins test: <175 EU/vial	pass	pass	pass
Sterility test	No growth after 2 weeks incubation.	pass	pass	pass

Figure 5.

Representative analytical reverse-phase C18 HPLC chromatograms of [¹⁸F]FETrp. (Radioactivity and UV channels).

4. Discussion

With a desired fluorine-18 half-life of 109.8 min, [¹⁸F]FETrp is one of the most promising radiolabeled tryptophan analogs for PET imaging of IDO-mediated kynurenine pathway in various primary and metastatic tumors. In preclinical studies, L-[¹⁸F]FETrp showed favorable tumoral uptake in IDO overexpressed glioblastomas,¹² and up to 10-fold higher uptake than that of D-[¹⁸F]FETrp in breast cancer cell lines and xenografts mice mode.¹⁴ During a typical one-pot two steps radiosynthesis, L-/D-[¹⁸F]FETrp racemization (L-/D-[¹⁸F]FETrp = ~2:1) was observed at the radiofluorination step under heating and basic conditions,¹⁴ which was confirmed using enantiomerically pure L-form precursor (>99% ee) for the radiosynthesis in our approach.

To achieve enantiomerically pure L-[¹⁸F]FETrp for in-human administration, chiral HPLC separation was first performed. Following the method of Xin et al.,¹⁴ L-[¹⁸F]FETrp was smoothly separated from D-[¹⁸F]FETrp (figure 3), using an Astec CHIROBIOTIC T column semi-preparative HPLC column and 90% ethanol as the mobile phase. Astec CHIROBIOTIC T column has macrocyclic glycopeptide-based HPLC phases, and its chiral separation relies on a combination of factors, such as morphology, molecular composition, and multiple covalent linkages to the silica surface, with mixed types of interactions of H-bond, ionic, dispersive, π-π, dipole stacking, steric, and inclusion mechanisms. Although the L-[¹⁸F]FETrp peak was successfully separated from D-[¹⁸F]FETrp during chiral HPLC separation, the chemical impurities were also collected in the broad L-[¹⁸F]FETrp peak fraction (Figure 3). Optimization of mobile phase for better chemical purity on chiral HPLC separation was unsuccessful with adjustment of pH, ethanol or acetonitrile percentage, and acid or base additives. Although the product from chiral HPLC may be acceptable for preclinical imaging, further purification of [¹⁸F]FETrp is required for human use. Therefore, another reverse-phase semi-preparative HPLC was performed on the crude [¹⁸F]FETrp product collected from chiral HPLC. The Synthra RNPlus research module is designed for multiple-step synthesis of research radiotracers and has the functionality of allowing dual independent HPLC purifications (Figure 2) for [¹⁸F]FETrp synthesis and purification. As a result, both chemical and enantiomeric pure [¹⁸F]FETrp was obtained after the reverse-phase C18 HPLC semi-preparative separation with the removal of the chemical impurities (Figure 4). In the final product, the unintegrated UV peaks from 0.5 to 1.1 min were from the ammonium hydroxide and acetic acid formulation (Figure 5 and S5). An unknown impurity with retention time of 1.14 min was also found during QC analysis. It is likely to be the hydrolyzed precursor, but LCMS analysis failed to find the desired information to identify it (data not shown). Considering the low concentration of this impurity (0.34 – 1.04 μg/mL), the release criteria for the total impurities was set at 4 μg/mL.

Figure 3.

Representative chiral semi-preparative HPLC chromatograms for L-[¹⁸F]FETrp purification (Radioactivity and UV channels).

Figure 4.

Representative reverse-phase semi-preparative HPLC chromatograms for [¹⁸F]FETrp purification (Radioactivity and UV channels).

The Oasis MCX Sep-Pak cartridge was found suitable for [¹⁸F]FETrp extraction during the radiosynthesis to avoid time-consuming evaporation steps. Crude [¹⁸F]FETrp solution is readily trapped on an Oasis MCX Sep-Pak cartridge and can be eluted with 1 – 2 mL of ammonium hydroxide in ethanol solution (v/v = 5:95). Therefore, three solid phase MCX extractions procedures were used to assist the purification and formulation during [¹⁸F]FETrp production (Figure 2). The first Oasis MCX Sep-Pak cartridge extraction was performed prior to chiral HPLC separation, and the second one was used to enrich [¹⁸F]FETrp from ~20 mL of 90% ethanol for reverse-phase HPLC loading. And finally, the third Oasis MCX Sep-Pak cartridge was applied to extract [¹⁸F]FETrp product from acetonitrile before the formulation step. As a result, the enantiomeric and chemical pure [¹⁸F]FETrp product was obtained in a total synthesis time of 90 min, which is slightly shorter than the reported method.¹⁶

5. Conclusions

A fully automated radiosynthesis of [¹⁸F]FETrp was achieved with a reasonable radiochemical yield and desired radiochemical/chemical and enantiomeric purities. Based on these results, an eIND from FDA has been obtained, which will allow for the first-in-human use of [¹⁸F]FETrp for PET imaging of brain tumors and various other cancer types.