S-¹¹C-Methyl-L-Cysteine: A New Amino Acid PET Tracer for Cancer Imaging

Chemicals

All reagents used in the synthesis were commercial products applied without further purification unless otherwise indicated. Acetonitrile, dimethylsulfoxide, N,N-dimethylformamide, tetrahydrofuran, lithium aluminum hydride (1.0 M in tetrahydrofuran), hydroiodic acid, and L-cysteine were purchased from Sigma-Aldrich.

Cell Culture

A hepatocellular carcinoma cell line (Hepa1–6) was obtained from the Laboratory Animal Center of Sun Yat-Sen University, Guangzhou, China. The cells were cultivated in RPMI 1640 medium with a physiologic glucose concentration (1.0 g/L) containing 5% fetal calf serum at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The medium was routinely renewed 3 times a week. Two days before the in vitro experiments, the cells were trypsinized and 2 × 10⁵ cells per well were seeded into 24-well plates.

Animals and Tumors

Female and male C57BL/6J mice (6 wk old) were obtained from the Laboratory Animal Center of Sun Yat-Sen University. Mice and nude mice were housed 5 animals per cage under standard laboratory conditions at 25°C and 50% humidity. They were allowed free access to food and water. We prepared 3 groups of mice, namely normal mice (n = 30), tumor nude mice (n = 20), and inflammation mice (n = 20). Tumor nude mice were inoculated subcutaneously in the femoral region with 1.0 × 10⁷ Hepa1–6 cells. Inflammation mice were inoculated subcutaneously with 0.2 mL of turpentine oil (Wako Chemical) ( 12). These turpentine-induced masses showed acute inflammation histologically as described in an earlier report ( 13). All animals were kept in cages under standardized conditions. At the time of the experiments, the mice were 9–10 wk old and weighed 18–25 g. The mass of the tumors and inflammatory tissue grew to 10–25 mm during the experiments. The study was performed according to the guidelines and recommendations of the Committee on Animal and Human Research at the First Affiliated Hospital, Sun Yat-Sen University. The protocol was fully approved by the local institutional review committee on animal care.

Radiosynthesis Procedures

¹¹CO₂ was produced by ¹⁴N(p,α)¹¹C nuclear reactions using a Cyclone 10/5 cyclotron (IBA) and was delivered to the radiochemical laboratory. ¹¹CO₂ was trapped in a loop ring cooled at –160°C with liquid nitrogen. After a flush with helium, the ring was warmed and ¹¹CO₂ released into the reactor module under a helium flow (20 mL/min), through a dehydrating agent, P₂O₅. ¹¹CH₃I was produced through reduction of ¹¹CO₂ with LiAlH₄, hydrolysis of the intermediately formed organometallic complex, and subsequent iodination of ¹¹C-methanol with hydrogen iodide. ¹¹C-MCYS were synthesized according to the solid-phase ¹¹C-methylation of the precursor L-cysteine loaded into a C18 column with ¹¹CH₃I ( Fig. 1). Briefly, ¹¹CH₃I produced as described above was delivered under a helium flow (20 mL/min) to a Sep-Pak Plus C18 cartridge (Waters) previously loaded with a solution of L-cysteine (∼2 to 3 mg) dissolved in NaOH 0.5 mol/L in 50:50 ethanol:water (v/v, 0.210 mL). ¹¹C-MCYS was eluted with NaH₂PO₄ 0.05 M buffer (5 mL, pH ∼3 to 4) and collected in a vented sterile vial. The solution was finally sterilized by a 0.22-mm filter to give the final formulation. The fully automated synthesis of ¹¹C-MCYS was performed on a simple, remotely controlled commercial ¹¹C-methylation module (PET-CS-I-IT-I ¹¹C-methylation synthesizer; PET (Beijing) Technology Co., Ltd.) as shown in Supplemental Figure 1 (supplemental materials are available online only at http://jnm.snmjournals.org).

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FIGURE 1.

Radiosynthesis of ¹¹C-MCYS by on-column ¹¹C-methylation of L-cysteine.

¹¹C-MET and ¹⁸F-FDG were synthesized as published previously by Tang et al. ( 14, 15).

Quality Control Procedures

Radiochemical purity and chemical purity of ¹¹C-MCYS were measured by analytic high-performance liquid chromatography with an Eclipse XDB-C18 analytic column (4.6 × 150 mm, 5 μm; Agilent) using 55:45 CH₃OH:3 mM NaH₂PO₄ (v/v) as the mobile phase at a flow rate of 1 mL/min. The elution was monitored by an ultraviolet detector (254 nm) and radiodetector. The pH was measured by a standard pH meter. Absence of pyrogen in the injectable solutions was checked by limulus amebocyte lysate testing using a gel clotting assay, and samples were tested for sterility.

Transport Assays

Transport assays with Hep1–6 cells were performed at 2 d after seeding during the exponential growth phase. The method of measuring transport was similar to a method reported previously ( 2, 16, 17). The competitive inhibition experiments to characterize the transport system were performed in the presence of the specific competitive inhibitors: for system L, 2′-aminobicyclo( 2, 2, 1)-heptane-2′-carboxylic acid (BCH); for system A, N-methylamino-isobutyric acid (MeAIB); and for system ASC, MeAIB plus serine. The concentration of the inhibitors used was 15 mmol/L. Parallel experiments with increasing concentrations of MCYS were performed to investigate the capacity of the transport system. All experiments were performed in the presence and absence of Na⁺. In Na⁺-free experiments, sodium salts were replaced by choline chloride. Each experiment was done in triplicate and averaged and was repeated 5 times on different days. After preincubation of the cells in 200 μL of the medium for 30 min, 200 μL of ¹¹C-MCYS (1.85 MBq/mL) and 200 μL of one of the inhibitors or MCYS (50, 100, 200, 300, or 350 μmol/L) were added and samples were incubated at 37°C for 4 min. After tracer uptake had been stopped with 1 mL of ice-cold 1× phosphate-buffered saline, the cells were washed 3 times with phosphate-buffered saline at 4°C and dissolved in 1.5 mL of 0.1 M NaOH plus 2% Triton X (Sigma Chemical Co.) and the activity measured by a γ-counter.

In Vivo Biodistribution Studies

The animals were anesthetized with pentobarbital (75 mg/kg) before injection of radiotracer and remained anesthetized through the study. They were injected with 0.74–1.48 MBq (20–40 μCi) of radiotracer in 100–200 μL of saline through the tail vein (¹¹C-MCYS and ¹¹C-MET in phosphate-buffered saline and ¹⁸F-FDG in normal saline). A prescribed duration was allowed before procurement of organs and tissues. Blood was obtained through the eyeball, tissue samples of interest (blood, brain, heart, lung, liver, kidney, and small intestine in normal mice) were rapidly dissected and weighed, and ¹¹C radioactivity was counted with an automatic γ-counter. All measurements were background-subtracted and decay-corrected to the time of killing and then averaged ( 2). Data were expressed as a percentage of the injected dose per gram of tissue (%ID/g).

Incorporation of ¹¹C-MCYS into Proteins

To determine the extent of protein incorporation of ¹¹C-MCYS, protein-bound activity in the brain, pancreas, tumor, and blood samples was determined at 30 min after injection. For this purpose, tumor, pancreatic, and cerebral tissue (∼50 to 100 mg in wet weight) was homogenized by the addition of 1 mL of distilled water and by ultrasonication at 0°C. Blood samples were immediately centrifuged at 4,000g, and 250 μL of 50% trichloroacetic acid were added to 750 μL of plasma and stirred in a vortex mixer for 1 min. After centrifugation, the supernatants were removed from the pellets. The radioactivity in the acid solution and acid-precipitable fraction was measured with a γ-counter. Sampling of the ¹¹C-activity was performed using the supernatant solution after spiking with a small amount of MCYS by high-performance liquid chromatography (55:45 CH₃OH:3 mM NaH₂PO₄ (v/v)), and the elution profile was detected with an ultraviolet detector at 254 nm. Incorporation of ¹¹C-MCYS into tumor, pancreatic, and cerebral tissue was measured as described. Radioactivities contained in the acid solution (supernatant solution) and acid-precipitable fraction (protein pellet) were also determined.

PET Studies

PET was performed using a Gemini GXL scanner (Philips). Before undergoing ¹¹C-MCYS, ¹¹C-MET, and ¹⁸F-FDG PET studies, the animals were kept fasting for at least 4 h. The tumor-bearing mice and inflammation model mice were anesthetized with pentobarbital (75 mg/kg) before injection of the radiotracer and remained anesthetized throughout the study. ¹¹C-MCYS, ¹¹C-MET, or ¹⁸F-FDG (10 MBq) was injected in 100–200 μL of phosphate-buffered saline or normal saline through the tail vein. Then, image acquisition was performed at 60 min after intravenous injection in 3-dimensional mode, with emission scans of 4 min per bed position. Imaging started with a low-dose CT scan (30 mAs), immediately followed by a PET scan. The CT scan was used for attenuation correction and localization of the lesion site. In addition, a 45-y-old patient with a history of attempted resection of grade IV glioma 1 y previously was also examined with ¹⁸F-FDG and ¹¹C-MCYS PET. The coronal, transaxial, and sagittal PET views of tumor-bearing mice, inflammation model mice, and the patient were obtained after image reconstruction with a slice thickness of 2.0 mm. A region of interest was placed on each tumor, each inflammatory lesion, and a region of femoral muscle. The human study was approved by the Institutional Review Board of the First Affiliated Hospital, Sun Yat-Sen University. Informed consent was obtained from the patient before injection.

Statistical Analysis

Statistical analysis was performed with SPSS software, version 15.0 (SPSS Inc.), for Windows (Microsoft). All data were expressed as mean ± SD. Comparisons between conditions were performed using the unpaired, 2-tailed Student t test. A P value of less than 0.05 was considered to indicate statistical significance.