Synthesis and evaluation of O-(3-[18F]fluoropropyl)-L-tyrosine as an oncologic PET tracer

Nuclear Medicine and Biology 30 (2003) 733–739

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Synthesis and evaluation of O-(3-[18F]fluoropropyl)-L-tyrosine as an oncologic PET tracer Ganghua Tanga,*, Mingfang Wanga, Xiaolan Tangb, Lei Luoa, Manquan Gana a

Nan Fang PET Centre, Nan Fang Hospital, The First Military Medical University, Guangzhou 510515, China b Department of Applied Chemistry, South China Agricultural University, Guangzhou 510642 , China Received 25 April 2003; received in revised form 12 May 2003; accepted 15 May 2003

Abstract O-(3-[18F]fluoropropyl)-L-tyrosine (FPT), an analogue of O-(2-[18F]fluoroethyl)-L-tyrosine (FET) as an amino acid tracer for tumor imaging with positron emission tomography (PET), was synthesized and evaluated. FPT was prepared by [18F]fluoropropylation of L-tyrosine in a two-step procedure. Biodistribution of FPT was determined in normal mice. FPT, FET and [18F]fluorine-2-deoxy-D-glucose (FDG) uptake studies were performed in mice bearing S18 fibrosarcoma and S. aureus-inoculated mice. Also, carcinoma-bearing mice and S. aureus-inoculated mice were imaged using FPT PET imaging compared with FET and FDG PET imaging. Synthesis of FPT was accompished in about 60 min with an overall radiochemical yield of 25-30% (without decay correction) by manual operation. High uptake and long retention time of FPT and FET in kidney, liver, lung, blood, etc., and low uptake in brain were found. Furthermore, high FPT, FET and FDG uptake in tumor, and almost no FPT and FET uptake in inflammatory tissue, in contrast, high FDG uptake in inflammatory tissue, were observed. In conclusion, FPT is easy to prepare and superior to FDG in the differentiation of tumor and inflammation, and seems to be a potential amino acid tracer like FET for tumors imaging with PET. © 2003 Elsevier Inc. All rights reserved. Keywords: O-(3-[18F]fluoropropyl)-L-tyrosine; O-(2-[18F]fluoroethyl)-L-tyrosine; [18F]fluorine-2-deoxy-D-glucose; Experimental tumor; Inflammation

1. Introduction [18F]Fluorine-2-deoxy-D-glucose (FDG) as a parameter of glucose metabolism has been widely used in positron emission tomography (PET) for oncology, neurology and psychiatry, and treatment evaluation [1]. However, clinical FDG PET studies demonstrated several limitations such as differentiation of tumor from inflammation with difficulty because of high uptake of FDG in tumor and in nonmalignant, inflammatory tissue [2], motivating efforts to develop new oncologic PET tracers. Positron-labeled amino acids have proven to be useful for imaging tumors, especially for brain tumors, but also for peripheral tumors such as lung tumors, lymphoma, and breast cancer. Currently, [methyl-11C]-L-methionine is the commonly used amino acid tracer for PET imaging [3], but because of the short half-life of 11C (t1/2⫽20 min), the availability of this 11C-labeled agent is limited to PET * Corresponding author. Tel.: ⫹86-20-87795061; fax: ⫹86-2061642125. E-mail address: [email protected] (G. Tang). 0969-8051/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0969-8051(03)00099-4

centers with an in-house cyclotron, preventing the widespread use of this tracer. Inasmuch as 18F has the advantages of a longer half-life (t1/2⫽110 min), several 18F-labeled amino acids, such as 4-[18F]fluoro-L-phenylalanine [3], 2-[18F]fluoro-L-tyrosine [3] and [18F]fluoro-␣-methyl tyrosine (FMT) [3], are synthesized and evaluated. Unfortunately, the syntheses of these tracers are very difficult and only low yields are obtained [4]. Recently, a simple and efficient synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine (FET) with high radiochemical yield reported is sufficient for satellite distribution [4]. The physical properties of FET are suitable for the acquisition of whole-body PET scans and assist in the differentiation of infection and tumors with the advantage over FDG, and it seems promising for the use of clinical oncology [5,6,7,8]. The labeling of organic compounds by O-, N- or Sfluoroalkylation is an important method for the preparation of [18F]labeled compounds [9]. This approach has been adopted with some success to the synthesis of new receptor radioligands, examples being N-2-[18F]fluoroethyl-2-␤-carbomethoxy-3-␤-(4-iodophenyl)nortropane (18F-FE-CIT) 18 and N-2-[ F]fluoropropyl-2-␤-carbomethoxy-3-␤-(4-iodo-

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phenyl)nortropane (18F-FP-CIT), which are effective radioligands for imaging dopamine transporter with PET [10,11]. However, 18F-FP-CIT demonstrates higher dopamine transporter (DAT) affinity than 18F-FE-CIT for the DAT and is superior to 18F-FE-CIT for the DAT imaging with PET [10], which has revived the interest in developing an O-fluoroalkyl analogue of L-tyrosine, O-(3-[18F]fluoropropyl)-L-tyrosine (FPT) [12] for tumor imaging with PET. In this article, we report the easy-to-automate synthesis and biodistribution of FPT, a FET analogue. The uptake and retention of this agent is studied in mice implanted with S18 fibrosarcoma cell and in activated inflammatory cells using an experimental acute abscess model, and compared with FDG and FET. Furthermore, carcinoma-bearing mice and S. aureus-inoculated mice are studied using FPT PET imaging compared with FDG and FET PET imaging.

Fig. 1. Synthesis of FPT by direct [18F]alkylation of L-tyrosine.

The pH was adjusted to about 7.5 and the resulting precipitate was filtered off, washed with water and dried. The crude product was recrystallized from 60% acetic acid to give 0.260 g (1.1 mmol, 31%) of cold standard. 1H NMR (CDCl3) ␦: 6.7-7.2 (m, 4H), 4.7 (t, 2H), 4.3 (t, 2H), 3.3 (m, 1H), 2.6 (m, 2H), 1.8 (m, 2H) . MS (EI) m/z: 241 (M⫹).

2. Materials and methods 2.1. Materials 1H-NMR spectra were recorded on DPX 300 model spectrometer (IBM-Bruker, Germany) in DCCl3 as an internal reference. Electron-Ionization mass spectra were recorded on LC/MS/MS from USA. HPLC was carried out on a modular HPLC system, consisting of two LC-10ATvp pump (Shimadzu Corporation, Japan) and a variable wavelength SPD-10ATvp UV detector (Shimadzu Corporation, Japan), a LB 508 Radioflow Detector with two channels analyzer (EG&G, Germany) and a computer (Japan). The UV signal was monitored with a UV Lambda Max detector at 254 nm. The C18 reverse-phase analytical columns was purchased from Shimadzu Corporation of Japan (4.6 mm ⫻ 150 mm). The system permitted the simultaneous detection of UV absorbance and radioactivity. Analytical TLC plates used were active silica gel on glass or plastic plates. Spots were detected by ultraviolet (UV) illumination or TLC plates were cut into many equal sections, who were analyzed for radioactivity with ␥ counter. Aminopolyether Kryptofix 222 (K222) was from Merck. C18, QMA and Silica Sep-Pak cartridges were from Waters Corp.. Unless otherwise noted, all other materials were obtained from commercial suppliers in China. 2.2. Synthesis of O-(3-[19F]fluoropropyl)-l-tyrosine (19FPT) 19

FPT was prepared according to the method described by Solar et al [13]. A solution of 0.646 g (3.5 mmol) L-tyrosine in 2.800 g (7.0 mmol) 10% aqueous sodium hydroxide was added to 14 mL of dimethyl sulfoxide and heated in a oil bath to 80°C. To this was added, with stirring, 0.500 g (3.5 mmol) 1-fluoro-3-bromopropane. Heating and stirring were continued for 2 hours and the reaction mixture was then poured into 18 g of crushed ice.

2.3. Radiosynthesis of O-(3-[18F]fluoropropyl)-l-tyrosine (FPT) No-carrier-added (NCA) aqueous [18F]fluoride was prepared by the 18O(p, n) [18F] reaction on a small volume of enriched water H218O (1.5 mL, 85%) in a silver target. A typical production run (16.5 MeV and 25 ␮ bombardment for 30 min) yield 400 to 500 mCi of [18F]fluoride at the PETtrace cyclotron (GE Co.), which was delivered to the radiochemical laboratory. The radiochemical synthesis of FPT was modified from the original method [4,8] by a two-step reaction sequence as follows (see Fig. 1). The generated [18F]fluoride target water passed through a QMA Sep-Pak cartridge, where [18F]fluoride was trapped, and [18O]water was collected for recycling. The aqueous mobile phase containing 1 mL of a solution of Kryptofix 222 (11 mg, 29.4 ␮mol) and anhydrous potassium carbonate (2 mg, 14.5 ␮mol) in water (0.04 mL) and acetonitrile (0.96 mL) was eluted through the QMA Sep-Pak cartridge, in which the trapped [18F]fluoride was eluted into the reaction vessel. The solvent was evaporated under a stream of nitrogen at 90°C. Azeotropic drying was repeated twice with 0.5 mL portions of acetonitrile. 1,3Ditosyloxypropane (1) (7.5 mg, 20 ␮mol) dissolved in dry acetonitrile (1 mL) was added to the dried [K/K222]⫹ 18Fcomplex, and the vessel was heated for 15 min at 90°C. Afterwards, the reaction mixture was cooled to room temperature, diluted with anhydrous ether (4 mL) and passed through a silica Sep-Pak cartridge (Waters) [14] into the maxi-vial. The Sep-Pak was eluted with additional anhydrous ether (4 mL), which was added to the maxi-vial. The resulting ethereal solution was evaporated to dryness by nitrogen flow and heating and 1-[18F]fluoro-3-tosyloxypropane (2) was yielded. L-Tyrosine (4.6 mg, 25 ␮mol), 20 ␮L (2 mg, 50 ␮mol) of 100 g/L sodium hydroxide solution and 0.5 mL of dimethylsulfoxide (DMSO) [8] were added the dry residue. The vial was closed and heated at 90°C for 15

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min and the reaction mixture was cooled to room temperature. After the mixture was passed through silica Sep-Pak cartridge and C-18 Sep-Pak cartridge, which were eluted sequentially with anhydrous ether (10 mL) and water (1 mL) to remove the unreacted L-tyrosine, 1-18F-3-tosyloxypropane and DMSO, FPT was eluted from the serial silica Sep-Pak cartridge and C-18 Sep-Pak cartridge with 4 mL 0.15 mol/L phosphate-buffered saline (PBS, 0.74). Dilution with 2 mL isotonic saline solution and 0.22 ␮m sterile filtration into a sterile vial led to the final formulation. Quality control was performed by HPLC (RP-18, 150 ⫻ 4 mm, 10/87.5/2.5 ethanol/water/acetic acid (volume-to-volume-to-volume ratio), 2.5 g/L ammonium acetate, pH 3.0, 1 mL/min, the retention time 11.2 min), and thin-layer chromatography (SiO2; 95/5, acetonitrile/water (volume-to-volume ratio), Rf⫽0.5). In addition, K222 was not visualized (developed in an iodine chamber) on the silica gel 60 coated plate with 0.1% (volume-to-volume ratio) triethylamine in methanol as an eluant. FET was synthesized as published previously by Tang et al. [8]. FDG was synthesized by direct nucleophilic exchange on a quaternary 4-aminopyridinium resin [15]. 2.4. Measurement of partition coefficient The partition coefficient of FPT was measured using 2.0 mL 1-octanol as the organic phase and 2.0 mL 0.1 mol/L phosphate buffer (pH 7.0) as the aqueous phase. Ten microliters of the radioactive sample in PBS buffer were added and mixed twice for 2 min at room temperature. The radioactivity of 200 ␮L of each phase was measured after centrifugation [4]. 2.5. Tumor-bearing model and inflammation model The study was performed according to the guidelines and recommendations of the committee on animal research at Nan Fang Hospital, First Military Medical University. The protocol was fully approved by the local institutional review committee on animal care. Fifteen mice weighing 18-25 g were used to prepare tumor-bearing model. All the animals were kept in cages with standardized conditions of light, asepsis and free access to water and food. Mice were inoculated with S18 fibrosarcoma cell in the left-sided, a unilateral axillary region. Experiments were performed 7-14 d after inoculation of the tumor cells. At this time, the tumors weighed 80-200 mg and a tumor diameter of more than 0.5-1.0 cm was observed. Fifteen mice weighing 18-25 g were used to prepare inflammation (abscess) model. All the animals were kept in cages with standardized conditions. Right-sided, a unilateral deep thigh muscle abscess was induced by intramuscular inoculation of 0.2 mL of bacterial suspension (S. aureus, 1.2 ⫻ 108 CFU/0.2 mL) [6]. Only those animals who developed a palpable fluctuating mass (abscess) in the right thigh

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muscle within 72 hours after bacterial inoculation were used to experimental studies. No systemic infection occurred. 2.6. In vivo biodistribution studies The animals were anesthetized with pentobarbital (75 mg/kg) before injection of radiotracer and remained anesthetized through the study. Mice were injected with 0.741.48 MBq (20-40 ␮ Ci) of FPT, FET and FDG in 100-200 ␮ L of PBS buffer through the tail vein, respectively. A prescribed duration of time was allowed before procurement of organs and tissues. Arterial blood was obtained through cardiac puncture, and the tissue samples of interest, including blood, brain, heart, lung, liver, kidney, spleen, small intestine, muscle, bone, and skin in normal mice, blood, normal muscle and tumor tissue in tumor-bearing mice, and blood, muscle and inflammatory tissue in inflammation model mice were rapidly dissected, weighed and 18F radioactivity was counted with an autogamma counter. All measurements were background subtracted and decay corrected to the time of killing, then averaged together [15]. Data were expressed as the differential uptake ratio (DUR) [16] as follows. DUR ⫽

Tissue counts (cpm)/Tissue weight (g) Injected dose (cpm)/Animal body weight (g)

DUR for tumor and inflammatory tissue can be also calculated from the above formula. 2.7. PET studies PET was performed using an Advanced scanner (General Electric Medical System, Milwaukee, WI) operated in 2-dimensional model. The scanner consists of 18 rings of bismuth germanate detectors yielding 35 transverse slices spaced by 4.25 mm. The imaging field of view is 55 cm in diameter and 15.2 cm in axial length. All measurements were corrected for scatter, random counts, and dead time. Attenuation correction was performed with 2 rotating rod sources containing 68Ge/68Ge (total, 350 million counts). Imaging reconstruction was performed using an iterative procedure with Ordered Subset Expectation Maximum algorithm and a cutoff frequency of 4.6 mm to a 128 ⫻ 128 imaging matrix, which resulted in image pixels of 2.34 ⫻ 2.34 mm. Before FPT, FET and FDG studies, the experimental animals had to fast at least 4 h. The animals including tumor-bearing mice and inflammation model mice were anesthetized with pentobarbital (75 mg/kg) before injection of radiotracer and remained anesthetized through the study. Mice were injected with 10 MBq of FPT, or FET or FDG in 100-200 ␮L of PBS buffer through the tail vein, respectively. Emission and transmission data with one bed position (4 minutes for transmission, 4 minutes of emission per position) were acquired 1 hour after administration of FPT,

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Table 1 Differential uptake ratio (DUR) of FPT in normal mice

Blood Brain Heart Lung Liver Kidney Spleen Small intestine Muscle Bone Skin

10 min

30 min

60 min

120 min

180 min

0.90 ⫾ 0.26 0.25 ⫾ 0.10 0.89 ⫾ 0.33 0.96 ⫾ 0.27 1.14 ⫾ 0.31 1.24 ⫾ 0.40 0.81 ⫾ 0.29 0.91 ⫾ 0.40 0.84 ⫾ 0.30 0.63 ⫾ 0.14 0.87 ⫾ 0.20

0.64 ⫾ 0.10 0.23 ⫾ 0.05 0.58 ⫾ 0.07 0.62 ⫾ 0.10 0.71 ⫾ 0.14 0.74 ⫾ 0.09 0.65 ⫾ 0.20 0.46 ⫾ 0.08 0.47 ⫾ 0.08 0.35 ⫾ 0.05 0.49 ⫾ 0.17

0.52 ⫾ 0.08 0.27 ⫾ 0.04 0.48 ⫾ 0.07 0.54 ⫾ 0.07 0.64 ⫾ 0.12 0.78 ⫾ 0.31 0.48 ⫾ 0.05 0.44 ⫾ 0.08 0.46 ⫾ 0.07 0.39 ⫾ 0.07 0.49 ⫾ 0.33

0.38 ⫾ 0.05 0.23 ⫾ 0.05 0.31 ⫾ 0.03 0.35 ⫾ 0.04 0.35 ⫾ 0.03 0.42 ⫾ 0.06 0.43 ⫾ 0.13 0.31 ⫾ 0.04 0.33 ⫾ 0.06 0.29 ⫾ 0.03 0.32 ⫾ 0.11

0.32 ⫾ 0.03 0.17 ⫾ 0.03 0.26 ⫾ 0.02 0.30 ⫾ 0.04 0.31 ⫾ 0.04 0.35 ⫾ 0.05 0.28 ⫾ 0.03 0.26 ⫾ 0.04 0.31 ⫾ 0.07 0.28 ⫾ 0.05 0.23 ⫾ 0.03

Note.-Numbers are the mean differential uptake ratio (DUR) (five injections per time period)(⫾ standard deviation).

or FET or FDG. The coronal, transaxial, and sagittal views of a PET image of tumor-bearing mice and inflammation model mice were obtained after image reconstruction.

The same protocol above was also used for the synthesis of FET with high chemical and radiochemical purity, but with low radiochemical yield of 20-25% [8].

2.8. Statistical analysis

3.2. Partition coefficient

The SPSS 10.0 soft and t-test was used to analyze the data. A two-tailed P value of less than 0.05 was considered statistically significant.

The lipophilicity of FPT was determined to be lgP ⫽ ⫺1.42, a value slightly higher than that of FET (lgP ⫽ ⫺1.51). The similar P value suggested FPT was similar hydrophilic to FET.

3. Results

3.3. Tracer uptake in mice

3.1. Radiosynthesis

The biodisribution data of FPT in normal mice are summarized in Table 1. The highest uptakes of 18F-activity in most tissues except brain was found to exceed 0.60 (DUR) at 10 min postinjection. The DUR in the blood decreased from 0.90 ⫾ 0.26 at 10 min postinjection to 0.32 ⫾ 0.03 at 180 min postinjection. No elevated organ uptake of FPT in most organs sampled was found at 60 min postinjection. Furthermore, the uptake of FPT in the bone did not exceed 0.40 over the whole time of observation except 10 min postinjection. The uptake of FPT into the brain was slow and slightly increased at 60 min postinjection. These results were similar to those of FET reported by Heiss et al. [2], Wester et al. [4] and Tang et al. [8]. The DUR and uptake ratios of the inoculated tumor side to the contralateral muscle or blood for FPT, FDG, and FET at 30, 60, and 90 min postinjection are shown in Table 2. There was significant difference in FPT or FDG or FET uptake between the inoculated side and the contralateral muscle or blood. Except for the tumor/blood FPT ratio less than 1.5 at 30 min postinjection, tumor to muscle and tumor to blood ratios reached the high values and exceeded 1.5 over the whole time of experiment. Tumor to muscle and tumor to blood ratios for FPT, FDG and FET reached the maximum value exceeding 2.0 at 90 min postinjection, but these values were much higher for FDG than for FPT or FET.

18

F-fluorination of 1,3-ditosyloxypropane and purification by a silica Sep-Pak cartridge led to a yield of approximately 40-50% with no decay correction. After separation, 1-[18F]fluoro-3-tosyloxypropane with the retention time of 2.8 min, only little 1,3-ditosyloxypropane (less than 100 ␮g/mL) and no K222 were detected by HPLC or TLC. The radiochemical purity of 1-[18F]fluoro-3-tosyloxypropane was about 100% and 18F was not dectected. Subsequently, fluoropropylation with unprotected L-tyrosine and purification steps on Sep-Pak cartridges were performed to give FPT injection in 60% yield. Unreacted 1-18F-3-tosyloxypropane, unreacted L-tyrosine, and DMSO were removed through silica Sep-Pak cartridge eluted with anhydrous ether, C-18 Sep-Pak cartridge eluted with 1 mL water, and Sep-Pak cartridges eluted with ether and water, respectively. After separation, FPT with the retention time of 11.2 min and radiochemical purity of above 95%, 1-[18F]fluoro3-tosyloxypropane with radiochemical purity of below 5%, only little L-tyrosine (less than 50 ␮g/mL), and no DMSO in the formulated FPT-solution were detected by HPLC. The synthesis was completed by manual operation in 60 min with an overall radiochemical yield of 25-30% without decay correction and with an radiochemical purity of more than 95%.

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Table 2 The Differential uptake ratio (DUR) and uptake ratio of the tumor or infected tissue to the contralateral side muscle or blood for FPT, FDG and FET Tissue or Ratio

FPT

FDG

FET

30 min

60 min

90 min

30 min

60 min

90 min

30 min

60 min

90 min

T TM TB I IM IB T/TM T/TB I/IM I/IB

0.98 ⫾ 0.54 0.52 ⫾ 0.21 0.73 ⫾ 0.15 0.58 ⫾ 0.23 0.63 ⫾ 0.15 0.66 ⫾ 0.36 1.86 ⫾ 0.53 1.33 ⫾ 0.67 0.92 ⫾ 0.10 0.88 ⫾ 0.24

0.99 ⫾ 0.31 0.50 ⫾ 0.15 0.54 ⫾ 0.14 0.53 ⫾ 0.37 0.54 ⫾ 0.15 0.59 ⫾ 0.28 1.97 ⫾ 0.67 1.83 ⫾ 0.63 0.98 ⫾ 0.12 0.90 ⫾ 0.21

0.98 ⫾ 0.19 0.34 ⫾ 0.10 0.49 ⫾ 0.15 0.41 ⫾ 0.12 0.43 ⫾ 0.15 0.40 ⫾ 0.24 2.85 ⫾ 0.65 2.01 ⫾ 0.43 0.95 ⫾ 0.03 1.02 ⫾ 0.18

0.90 ⫾ 0.21 0.56 ⫾ 0.26 0.49 ⫾ 0.12 0.74 ⫾ 0.16 0.48 ⫾ 0.26 0.33 ⫾ 0.12 1.62 ⫾ 0.58 1.84 ⫾ 0.83 1.53 ⫾ 0.11 2.25 ⫾ 0.43

0.99 ⫾ 0.17 0.46 ⫾ 0.18 0.42 ⫾ 0.15 1.04 ⫾ 0.46 0.37 ⫾ 0.14 0.33 ⫾ 0.17 2.16 ⫾ 1.22 2.38 ⫾ 1.03 2.83 ⫾ 1.30 3.17 ⫾ 0.28

0.97 ⫾ 0.20 0.36 ⫾ 0.30 0.18 ⫾ 0.15 1.02 ⫾ 1.40 0.25 ⫾ 0.07 0.13 ⫾ 0.06 2.67 ⫾ 1.92 5.41 ⫾ 2.02 4.08 ⫾ 1.84 7.64 ⫾ 5.88

0.96 ⫾ 0.30 0.49 ⫾ 0.12 0.59 ⫾ 0.17 0.69 ⫾ 0.26 0.68 ⫾ 0.26 0.69 ⫾ 0.28 1.94 ⫾ 0.26 1.60 ⫾ 0.15 1.01 ⫾ 0.33 0.99 ⫾ 0.32

1.05 ⫾ 0.23 0.48 ⫾ 0.13 0.54 ⫾ 0.17 0.53 ⫾ 0.25 0.51 ⫾ 0.17 0.50 ⫾ 0.21 2.15 ⫾ 0.63 1.96 ⫾ 0.70 1.02 ⫾ 0.18 1.07 ⫾ 0.18

1.01 ⫾ 0.24 0.43 ⫾ 0.34 0.46 ⫾ 0.23 0.39 ⫾ 0.15 0.39 ⫾ 0.14 0.42 ⫾ 0.17 2.35 ⫾ 0.78 2.19 ⫾ 0.44 0.99 ⫾ 0.01 0.93 ⫾ 0.07

Note.-Numbers are the mean differential uptake ratio (DUR) (five injections per time period)(⫾ standard deviation), or the mean model side/normal muscle or model side/blood uptake ratio (N/NT) (five injections per time period)(⫾ standard deviation). T, Tumor tissue; TM, Muscle of tumor-bearing mice; TB, Blood of tumor-bearing mice; I, Infected tissue; IM, Muscle of infected mice; IB, Blood of infected mice; T/TM, T/TB, I/IM, and I/IB are uptake ratio (N/NT).

Table 2 also shows the DUR, uptake ratios and changes of the infected side to the noninfected contralateral muscle or blood over time postinjection of FPT, FDG and FET. In the saline control group, there was no significant difference in FDG uptake or FPT uptake or FET uptake between the saline inoculated side and the contralateral muscle (data not shown). Similarly, there was no significant difference in FPT or FET uptake between the inoculated side and the contralateral muscle or blood, and inflammatory side to muscle or blood ratio for FPT uptake was less than or approximate to 1.0 (background level) at 30, 60, and 90 min postinjection, so was for FET uptake. However, these values (I/IM and I/IB ratio) were slightly higher for FET than for FPT except I/IB ratio at 90 min postinjection. On the other hand, FDG uptake of the operated side in the S. aureusinoculated group was significantly increased compared with uptake in the saline control group (data not shown) and the contralateral muscle. Inflammatory side to the controalateral muscle ratio and inflammatory side to blood ratio for FDG uptake with more than 1.5 at 30 min, more than 2.5 at 60 min, and more than 4.0 at 90 min postinjection respectively, were much higher than those for FPT or FET.

4. Discussion The tumor uptake is thought to reflect increased amino acid metabolism of cancer cells, including increased active transport and protein synthesis [3,17]. For example, several 18 F-labeled amino acids such as 4-[18F]fluoro-L-phenylalanine and 2-[18F]fluoro-L-tyrosine show protein incorporation [3], [18F]fluoro-␣-methyl tyrosine (FMT) and FET, an O-fluoroethyl analogue of L-tyrosine, are not metabolized and not incorporated into proteins but are actively transported into tumor cells [3,4] and thus show active transport,

3.4. PET imaging The coronal views of PET images of tumor-bearing mice and inflammation model mice obtained 1 hour after administration of FPT, FDG and FET are shown in Fig. 2. High FDG, FPT and FET uptakes in tumor were found in the S18 fibrosarcoma - inoculated group. Similarly, high FDG uptake in inflammatory tissue was observed in the S. aureusinoculated group. On the contrary, hardly any FPT and FET uptakes in inflammatory tissue were observed in the S. aureus- inoculated group, however, inflammatory tissue with slightly lower FPT uptake than FET uptake was found.

Fig. 2. PET Images in tumor-bearing mice (A, B, C) and inflammatory mice (D, E, F) obtained with FPT, FET and FDG PET imaging 1 hour after injection. The tumor is located in the left axillary area (A, B, C) and inflammation is located in the right thigh muscle region (D, E, F). High FPT, FET and FDG uptakes of tumor (A, B, C) were observed, similarly, high FDG uptake of inflammatory tissue (F) was also observed. However, almost no FPT and FET uptake of inflammatory tissue (D and E) was observed.

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and [methyl-11C]-L-methionine mainly exhibits active transport with minor protein incorporation [17]. On the other hand, radiolabelled amino acids such as FMT and FET are not significantly incorporated into inflammatory cells [3,6]. Therefore, positron-labeled amino acids have clinical potential in the differentiation of tumor from inflammation. FET as a positron-emitting radiopharmaceutical for the differentiation of tumor from inflammation was confirmed by animal experiments [5,6]. FPT, an analogue to FET, could exhibit similar biologic activity to FET. Although FPT could be prepared by a similar synthetic method to FET, it was advantageous to synthesize FPT by a simple method without HPLC system purification. This work demonstrated that FPT could be prepared routinely for clinical use by a simple two-step procedure. The Sep-Pak cartridges were used to purify and separate the radiolabeled intermediate product and the last product instead of HPLC purification [4], which resulted in simplification of the operation steps and shortening of the whole synthesis time. This improved separation could be also used for the synthesis of FET with a slightly low radiochemical yield. Also, of great important in the synthesis of FPT was the selective Oalkylation of L-tyrosine [13]. Dimethyl sulfoxide (DMSO) was a particularly effective reagent in promoting base-catalyzed condensation, a lot of alkyl ethers of L-tyrosine were synthesized from O-alkylation of L-tyrosine in this solvent without protecting the amino group. In this reaction, the major by-product was the ether ester resulting from dialkylation, however, this by-product could be avoided by carrying out the reaction with a slight excess of the amino acid or eliminated by saponification with dilute base before isolation of the product. The highest yield of FPT was obtained from L-tyrosine (1 equivalent) pre-incubated with 10% sodium hydroxide (2 equivalents) and about 3-10 volume of DMSO at 70-90° before adding 1-[18F]fluoro-3-tosyloxypropane to avoid hydrolization of activated precursor. In order to avoid undesired excess of base, it would be of advantage to use only e.g. 1.8 equivalents of sodium hydroxide or to use the di-potassium salt of L-tyrosine without further addition of any base. Recently, a simple one-pot synthesis of FET with high radiochemical yield of about 60% has been reported which allows large scale production for routine clinical purposes [18,19]. However, this new synthesis approach exhibiting some limitations, such as the long total synthesis time (80 min), O-(2-tosyloxyethyl)-Ntrityl-L-tyrosine tert.butylester as precursor purchased commercially with difficulty, and purification of the final product by a troublesome HPLC system which made a fully automated synthesis of FET or FPT difficult, was not a best practical alternative to produce FPT or FET. Hence, the two-step reaction sequence of FPT synthesis provided a practical and easily automated synthetic procedure, which made it possible to synthesize FPT or FET employed by an automated FDG-synthesis unit with only minor modifications. The biodistribution of FPT in normal mice and tumor-

bearing mice demonstrated that high uptake and long retention time of FPT in kidney, liver, lung, blood, etc. and low uptake in brain were found, and accumulation of FPT in the tumor with a continuous increase up to 60 min postinjection and tumor-to-blood ratio more than 2.0 at 90 min postinjection were also observed. Decreasing accumulation of radioactivity with time was observed in organs and tissues. The tissue-to-blood ratios were very low at each time investigated. Most tissues and organs took up FPT rapidly from the blood, and subsequent clearance from the blood was somewhat slow. Thus, the fast tumor uptake kinetics of FPT, and its low accumulation in brain and nontumor tissue showed that FPT was a potential amino acid PET tracer for cerebral and peripheral tumors. Furthermore, the time course of FPT in the blood was found to be as expected for an unnatural amino acid, and the similar results of FPT uptakes to FET data [2,4,8] indicated that FPT was not incorporated into proteins but was actively transported into tumor cells. All these studies show that FPT resembles FET in the uptake kinetics and transport mechanism, and seems to be a promising tracer for tumor imaging with PET like FET. Several investigators showed high accumulation of FDG in bacterial inflammation models by inoculating S. aureus [6] or Escherichia coli (E. coli) [20] and turpentine-induced inflammatory tissue [16]. However, since many infections are caused by bacteria or fungi and abscess formation is more common in infections with pyogenic organisms, a bacterial infection model, even if it produces somewhat more heterogeneous results, seems to be more physiologically relevant than nonbacterial models, and may be more applicable to clinical conditions [16,20]. We tried to use a bacterial inflammation model by inoculating S. aureus. High accumulations of FDG in the S. aureus-induced inflammatory tissue and in the S18 fibrosarcoma tumors were found. Inflammatory side to background ratios were much higher than tumor to background ratios for FDG. Low FPT and FET uptake in inflammatory tissue, and inflammatory side to background ratios close to 1.0 were observed, though high accumulation of FPT and FET in the S18 fibrosarcoma tumors was found. Additionally, it deserved to be mentioned that inflammatory side to background ratios (I/IM and I/IB) with slightly higher for FET uptake than for FPT uptake and the similar results in mice with PET imaging were also observed. All these results indicated that FPT could assist in the differentiation of tumor from inflammation like FET or with a slight advantage over FET, whereas FDG couldn’t, but which remained to be further confirmed.

5. Conclusion FPT is easy to prepare and easy to automate synthesis, and our initial study results show that FPT is superior to FDG and similar to FET or a little advantage over FET in the differentiation of tumor and inflammation, and seems to

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be a highly sensitive and specific amino acid tracer for differentiation of tumor from inflammation. Therefore, FPT PET can be used as a functional imaging method like FET PET but has to be further confirmed in clinical studies. Acknowledgments The authors thank Xiaojun Guo, Jinmei Zhong, Professor Zuhan Huang, and Hubing Wu for their assistance in this work. This work was supported by the Director’s Foundation of Nan Fang Hospital, First Military Medical University. References [1] Tang GH. Positron emission tomography imaging and drug development (in Chinese). Acta Pharm Sin 2001;36:470 – 4. [2] Heiss P, Mayer S, Herz M, Wester HJ, Schwaiger M, SenekowitschSchmidtke R. Investigation of transport mechanism and uptake kinetics of O-(2-[18F]fluoroethyl)-L-tyrosine in vitro and in vivo. J Nucl Med 1999;40:1367–73. [3] Laverman P, Boerman OC, Corstens FHM, Oyen WJG. Fluorinated amino acids for tumour imaging with positron emission tomography. Eur J Nucl Med 2002;29:681–90. [4] Wester HJ, Herz M, Weber W, Heiss P, Senekowitsch-Schmidtke R. Synthesis and radiopharmacology of O-(2-[18F]fluoroethyl)-L-tyrosine for tomor imaging. J Nucl Med 1999;40:205–12. [5] Rau RC, Weber WA, Wester HJ, Herz M, Becker I, Kruger A, Schwaiger M, Senekowitsch-Schmidtke R. O-(2-[18F]fluoroethyl)-Ltyrosine (FET): a tracer for differentiation of tumour from inflammation in murine lymph nodes. Eur J Nucl Med 2002;29:1039 – 46. [6] Kaim AH, Weber B, Kurrer MQ, Westera G, Schweitzer A, Gottschalk J, von Schulthess GK, Buck A. 18F-FDG and 18F-FET uptake in experimental soft tissue infection. Eur J Nucl Med 2002; 29:648 –54. [7] Weber WA, Wester HJ, Grosu AL, Herz M, Dzewas B. O-(2-[18F]fluoroethyl)-L-tyrosine and L-[methyl-11C]methionine uptake in brain tumours: initial results of a comparative study. Eur J Nucl Med 2000;27:542–9.

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