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Clinical Investigations |
1 Department of Radiology, International Medical Center of Japan, Tokyo, Japan
2 Department of Urology, International Medical Center of Japan, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: 18F • choline • PET • prostate cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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We previously developed 11C-choline as a PET tracer for cancer detection and have succeeded in visualizing brain tumor (5), lung cancer (6), esophageal cancer (7), colon cancer (8), bladder cancer (8), prostate cancer (9), and many other cancers (8). Motivated by this success, we attempted to develop an 18F-labeled choline analog as a PET tracer, with an idea that 18F labeling would be superior to 11C labeling because of the longer half-life and the shorter positron range of 18F. We thought that 18F-fluoroethylcholine (FECh) would be appropriate for this purpose. The following evidence supports our idea: Deves and Krupka (10) studied the binding affinity of the choline transport system for synthetic choline analogs, using red blood cells, and found that 2 methyl groups were essential, but the third methyl group was replaceable with a longer alkyl group. Clary et al. (11) studied the substrate specificity of choline kinase for synthetic choline analogs, using yeast choline kinase, and found that the 2 methyl groups and the hydroxylethyl side chain were essential, but the third methyl group was replaceable with a longer alkyl group. We had already synthesized 18F-FECh and studied its biodistribution in normal and tumor-bearing rabbits; our results are reported in a preliminary form (12). In this article, we report the details of the synthesis, biochemistry, and clinical application of this compound.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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2-Fluoroethyl Tosylate (Fluoroethanol Method).
At 0°C while stirring in an argon atmosphere, 2-fluoroethanol (1.92 g, 30 mmol) and tosyl chloride (6.87 g, 36 mmol) were dissolved in dichloromethane (30 mL), and pyridine (10 mL) and 4-dimethylaminopyridine (100 mg) were added. After remaining at room temperature for 3 d, the mixture was diluted with dichloromethane (100 mL) and then washed with 5% HCl (3 times) and brine. It was dried over MgSO4 and evaporated in vacuo to give an oil. Thin-layer chromatography (TLC) (silica gel; n-hexane:ethyl acetate, 3:1) of the oil showed 3 reaction products: vinyl tosylate (Rf, 0.57), 2-fluoroethyl tosylate (Rf, 0.36), and 1,2-bis(tosyloxy)ethane (Rf, 0.21). The product was purified by column chromatography (silica gel; n-hexane:ethyl acetate, 5:1) to give 2-fluoroethyl tosylate as a colorless oil (4.4 g, 67%). 1H NMR (CDCl3): 
2.46 (s, 3H), 4.28 (dt, J = 27.3, 4.0 Hz, 2H), 4.56 (dt, J = 47.0, 4.0 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 7.9 Hz, 2H). MS: m/z 218, 172, 155, and 91. High-resolution MS: calculated for C9H11FSO3, 218.041; observed, 218.042. The product was analyzed by high-performance liquid chromatography (HPLC): Column, ODS-silica gel (ODS-A; YMC), 250 x 10 mm; solvent, 50 mmol/L phosphoric acid + 1 mmol/L 2-naphthalenesulfonic acid; flow rate, 5 mL/min; detector, refractometer. The retention time of the product was 3.9 min.
FECh Tosylate (Fluoroethanol Method).
2-Fluoroethyl tosylate (996 mg, 4.56 mmol) was dissolved in N,N-dimethylethanolamine (407 mg, 4.56 mmol) and, under argon, heated at 100°C for 10 min. The resultant syrup was dissolved in methyl acetate:methanol (10:1), and the solvent was removed by evaporation to give the product as colorless rhomboid crystals. After recrystallization from the same solvent, mp 63.9°C–64.5°C (1.43 g, 100%). 1H NMR (CD3OD): 2.37 (s, 3H), 3.23 (s, 6H), 3.54–3.61 (m, 2H), 3.76–4.04 (m, 4H), 4.90 (bd, J = 48.0 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H). IR (KBr): 3,375, 2,976, 1,478, 1,213, 1,195, 1,124, 1,035, 1,012, and 684 cm-1.
FECh Chloride (Fluoroethanol Method).
FECh tosylate (50 mg, 0.16 mmol) was dissolved in methanol (20 mL) and passed through an anion-exchange resin, Amberlite IRA-900 (Cl-1) (Sigma, St. Louis, MO) (3 g). Removal of methanol gave FECh chloride as colorless needle crystals (28 mg, 100%). 1H NMR (CD3OD): 3.27 (s, 6H), 3.58–3.65 (m, 2H), 3.82–4.07 (m, 4H), 4.95 (bd, J = 50.0 Hz, 2H). IR (KBr): 3,383, 3,020, 1,475, 1,082, 957, 931, and 689 cm-1.
FECh Hydroxide (Fluoroethanol Method).
FECh tosylate (40 mg, 0.13 mmol) was dissolved in water (30 mL) and passed through an anion-exchange resin, Amberlite IRA-900 (OH-1) (2 g). Removal of water gave FECh hydroxide as a colorless oil (18 mg, 92%). 1H NMR (CD3OD): 3.26 (s, 6H), 3.55–3.69 (m, 2H), 3.78–4.07 (m, 4H), 4.98 (bd, J = 50.0 Hz, 2H). IR (KBr): 3,444, 1,474, 1,387, 1,350, 1,084, 1,052, and 957 cm-1. On the ODS-silica gel HPLC performed under the same condition as above, the retention time of FECh hydroxide was 4.6 min.
2-18F-Fluoroethyl Tosylate (TBA Method).
No-carrier-added 18F-fluoride (approximately 370 MBq), collected from an anion-exchange cartridge by elution with 2 mL 40 mmol/L TBA bicarbonate in acetonitrile:water (4:1), was dried by evaporation at 100°C and dried again with 2 mL dry acetonitrile. After addition of 1,2-bis(tosyloxy)ethane (20 mg) in dry acetonitrile (1 mL), the mixture was heated at 80°C for 20 min. After the solvent was evaporated at 80°C under reduced pressure, the dry residue was analyzed on TLC and HPLC. The Rf on TLC was identical with that of 2-fluoroethyl tosylate from the fluoroethanol method. On HPLC, the retention time of the product was identical with that of 2-fluoroethyl tosylate. A small amount of radioactivity remained in the column head.
18F-FECh Hydroxide (TBA Method).
No-carrier-added 2-18F-fluoroethyl tosylate prepared as above was dried and then dissolved in N,N-dimethylethanolamine (0.3 mL). The mixture was heated at 100°C for 5 min. After evaporation of N,N-dimethylethanolamine at 100°C under high vacuum, the dry residue was analyzed by HPLC. On the ODS-silica gel HPLC, a single radioactive peak, corresponding to 18F-FECh hydroxide, was found at 4.6 min. There was no radioactive peak of 2-18F-fluoroethyl tosylate. The radiochemical yield of 18F-FECh hydroxide compared with 18F-fluoride was 46.3% (decay corrected). The rest of the radioactivity was found in the reaction vessel and the column head. On this HPLC, a sharp mass peak of TBA (detected by a refractometer) appeared far behind the radioactive peak of 18F-FECh hydroxide. There was no other mass peak that eluted closely to 18F-FECh hydroxide.
Automated Synthesis of No-Carrier-Added 18F-FECh Chloride
Design of Automated Apparatus.
We constructed an automated apparatus for the synthesis of 18F-FECh. The function of this apparatus was as follows: (a) transfer of proton-irradiated 18O-water into a transit vessel and separation of 18F-fluoride ions using an ion-exchange cartridge, (b) transfer of 18F-fluoride ions into a reaction vessel, (c) 2-step chemical reactions (2-18F-fluoroethyl tosylate synthesis followed by 18F-FECh synthesis) in the reaction vessel, (d) passage of the product through anion-exchange cartridges to remove anionic by-products, (e) transfer of the effluent from cartridges to an HPLC apparatus, (f) HPLC, (g) passage of the effluent through anion-exchange cartridges to remove anionic molecular species in the HPLC solvent (phosphoric acid and 2-naphthalenesulfonic acid), (h) passage of the effluent from the cartridges through a cation-exchange cartridge to trap 18F-FECh, (i) washing of the cartridge with water, and (j) elution of 18F-FECh from the cartridge with saline. A close-up illustration of the reaction vessel is shown in Figure 2A, where the top of the vessel is sealed by a rubber septum, and 3 side arms are fixed on the side of the vessel. All chemical reactions were performed in this reaction vessel. Delivery of reagents to the reaction vessel was achieved by moving the upper and lower turntables; the upper table was equipped with needle-and-syringe units, and the lower table was equipped with reagent reservoirs. After one of the reagents was sucked from a reservoir into the corresponding needle-and-syringe unit, the upper and lower turntables moved vertically and rotationally, and the reagent was injected then into the reaction vessel from the needle pierced through the rubber septum. The purpose of the side arms was (a) to receive 18F-fluoride ions into the vessel; (b) to create negative, neutral, and positive pressures within the vessel; and (c) to send helium gas to the bottom of the vessel or transport the product of synthesis to the next HPLC unit. The reaction vessel could be heated by blowing hot air toward the bottom or toward the top and bottom simultaneously. The reaction vessel could be cooled by blowing chilled air. The temperature, pressure, and radioactivity of the reaction vessel were displayed continuously on a computer screen. A charged-coupled device camera and a monitor screen were used for the display of an actual view of the reaction vessel.
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Property of Product.
The final product from this apparatus was essentially sterile and pyrogen free because it was obtained by the elution of the product from the sterile cation-exchange cartridge with sterile saline, after the cartridge was thoroughly washed with sterile distilled water. (The cation-exchange cartridge was sterilized previously by ethylene oxide.)
The OnGuard A effluent (not the final product that contained saline) was analyzed in ion chromatography using a column of IonPac CS14 (Dionex, Sunnyvale, CA) and the elution solution of 2 mmol/L H2SO4, 2 mmol/L methanesulfonic acid, and 2.5% acetonitrile at the flow rate of 1 mL/min. The substances detected in this analysis were N,N-dimethylethanolamine at the retention time of 8.1 min and FECh at the retention time of 13.7 min. The chemical quantities of N,N-dimethylethanolamine and FECh chloride in the final product were calculated from the ion chromatography data, thus obtained, using standards of N,N-dimethylethanolamine and FECh. The FECh standard was prepared by carrier-added 18F-FECh synthesis from 10 µmol TBA 18F-fluoride, and the chemical quantity of FECh was determined by the isotopic dilution method. The chemical quantity of 2-naphthalenesulfonic acid in the final product (the eluant from the cation-exchange cartridge with saline) was measured by anion-exchange resin HPLC with ultraviolet detection at 226 nm (column, MCI Gel CQA 35S [Mitsubishi Chemicals, Tokyo, Japan], 7.5-mm inner diameter [ID] x 75 mm; elution, 0.2N NaOH + 0.5 mol/L NaCl at 2 mL/min). The retention time of 2-naphthalenesulfonic acid was 18 min.
Toxicity of Product.
The toxicity of 18F-FECh chloride was examined in mice. No-carrier-added 18F-FECh chloride (synthesized in the automated apparatus, 1 mL per batch) was cooled until the radioactivity disappeared and injected intravenously into 10 mice (body weight, 25 g; injection volume, 0.2 mL). The general condition of the mice was observed for 2 wk.
In addition, the median lethal dose (LD50) of FECh chloride was determined in mice. FECh chloride (synthesized by the fluoroethanol method) dissolved in saline at various concentrations was injected intravenously into 20 mice (body weight, 25 g; injection volume, 0.2 mL). The general condition of the mice was observed for 2 wk.
Biochemical Study
Treatment of 18F-FECh with Choline Kinase and ATP and with Choline Oxidase.
Yeast choline kinase, dithiothreitol, adenosine triphosphate (ATP), and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma-Aldrich Japan. Bacterial choline oxidase (from Alcaligenes species) and other reagents were purchased from Wako. MCI Gel CQK 31S and MCI Gel CQA 35S columns were purchased from Mitsubishi Chemicals.
The treatment of 18F-FECh with yeast choline kinase was performed as follows. No-carrier-added 18F-FECh chloride solution (0.2 mL) was incubated with 2.5 U choline kinase, 15 µmol dithiothreitol, 15 µmol ATP, and 15 µmol MgCl2 and dissolved in 1.5 mL 57 mmol/L Tris-HCl, pH 8.5. The reaction was performed for 30 min at 37°C, then stopped by addition of 2 mL cold methanol, and followed by addition of 4 mL chloroform. The mixture was centrifuged to give an upper methanol-water layer, lower chloroform layer, and the proteins in the interface. The upper methanol-water layer was separated, evaporated to dryness, and then dissolved in HPLC solvents. This sample was analyzed by HPLC using 2 kinds of ion-exchange columns (injection volume, 10 µL each): (a) MCI Gel CQK 31S column (containing carboxylmethyl group for cation exchange), 7.5-mm ID x 75 mm, eluted with 20 mmol/L sodium phosphate buffer, pH 6.5, at 1 mL/min; and (b) MCI Gel CQA 35S column (containing quaternary ammonium group for anion exchange), 7.5-mm ID x 75 mm, eluted with 10 mmol/L Tris-HCl buffer, pH 8.0, at 1 mL/min. One-milliliter fractions were collected, and the radioactivity of each fraction was measured in a well counter.
The treatment of 18F-FECh with bacterial choline oxidase was performed as follows. No-carrier-added 18F-FECh chloride solution (0.2 mL) was incubated with 2.5 U choline oxidase, 15 µmol dithiothreitol, and 15 µmol MgCl2 in 1.5 mL 57 mmol/L Tris-HCl, pH 8.5. The reaction was performed for 30 min at 37°C and stopped by addition of 2 mL cold methanol and 4 mL chloroform. The subsequent treatment was the same as the above.
Treatment of 18F-FECh with Choline Kinase and 
-32P-ATP.
-32P-ATP (product of New England Nuclear, Boston, MA) was purchased from Daiichi Pure Chemicals (Tokyo, Japan). A double-labeling experiment with 18F-FECh and -32P-ATP was performed as follows. No-carrier-added 18F-FECh chloride solution (0.2 mL, 370 MBq) containing approximately 0.005 µmol FECh was added to the reaction solution containing 2.5 U yeast choline kinase, 10 µmol dithiothreitol, 0.01 µmol -32P-ATP (instead of 15 µmol nonradioactive ATP), 0.01 µmol MgCl2, and 0.8 mL 62.5 mmol/L Tris-HCl, pH 8.5. The mixture was incubated at 37°C for 60 min. After the reaction was stopped by cooling in ice, 1 µmol nonradioactive phosphoric acid was added to the mixture to lower the specific activity of inorganic 32P-phosphate that might be formed from -32P-ATP during the reaction. After addition of water, methanol, and chloroform, the methanol-water layer was separated, evaporated to dryness, and dissolved in 0.2 mL of the HPLC solvent. After injection of 10 µL of the sample to the HPLC column (MCI Gel CQA 35S), it was eluted with 10 mmol/L Tris-HCl buffer, pH 8.0, at a flow rate of 1 mL/min. One-milliliter fractions were collected, and the radioactivity of 18F in each fraction was measured in a well counter; then, after the decay of 18F, the radioactivity of 32P was measured in a liquid scintillation counter. Inorganic phosphate and ATP were not eluted from the column into these fractions and only eluted by washing the column with 0.2N NaOH.
Metabolism of 18F-FECh in Tumor Cells.
Ehrlich ascites tumor cells were obtained from Human Science Research Resource Bank (Osaka, Japan). Authentic phospholipid samples (phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine) and silica gel 60 plates were obtained from Sigma-Aldrich Japan. Hanks’ balanced salt solution was obtained from Wako. An imaging plate-scanner-printer system (BAS-1800II) was obtained from Fuji Film (Tokyo, Japan).
The metabolism of 18F-FECh in tumor cells was measured as follows. Ehrlich ascites tumor cells (approximately105 cells) were implanted intraperitoneally in Institute for Cancer Research (ICR) mice (Japan Clea, Tokyo, Japan), and the proliferated tumor cells were collected 2–3 wk later. The tumor cells were washed twice with 0.6% glucose-fortified Hanks’ balanced salt solution, pH 7.4, and suspended in the same solution to give a cell density of approximately 5 x 106 cells/mL; the volume density of the cells was measured by hematocrit. No-carrier-added 18F-FECh chloride solution (40 µL, 74 MBq) was added to the tumor-cell suspension (200 µL), and the mixture was incubated then at 37°C for 30 min. After the reaction, the cells were cooled in ice and washed 3 times by addition of 5 mL unlabeled medium and centrifugation. The precipitated cells were mixed with 1.5 mL cold water, 2 mL methanol, and 4 mL chloroform, successively. After centrifugation, the upper methanol-water layer, lower chloroform layer, and precipitate were separated. The radioactivity of each fraction was measured in a well counter. The methanol-water layer was analyzed further by HPLC: After the sample was evaporated to dryness, it was dissolved in a small volume of HPLC solvents and passed through HPLC MCI Gel CQK 31S and MCI Gel CQA 35S columns (injection volume, 10 µL) to obtain 1-mL fractions. The radioactivity of each fraction was measured in a well counter. In addition to this experiment, the chloroform layer was analyzed, separately, in the following manner: After the chloroform layer was washed with methanol-water and then concentrated, TLC was performed on the silica gel 60 thin-layer plates using the chloroform, methanol, and 28% ammonia (65:35:5) and benzene, pyridine, and formic acid (50:40:10) solvents. The distribution of radioactivity on the plates was measured using an imaging plate-scanner-printer system. As standards of TLC, 3 representative choline-containing phospholipids (phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine) were used, and the location of them was detected after staining with sulfuric acid.
Time Course of Uptake and Metabolism of 18F-FECh in Tumor Cells.
The time course of 18F-FECh uptake and metabolism in tumor cells was measured as follows. Ehrlich ascites tumor cells in 50 µL 0.6% glucose-fortified Hanks’ solution (approximately 2 x 106 cells/mL) were mixed with 10 µL no-carrier-added 18F-FECh chloride solution, and the mixture was incubated for 0, 5, 10, 20, 30, 40, 50, and 60 min at 37°C. At the determined time of incubation, each sample was diluted with 5 mL ice-cold unlabeled medium and left in ice until the end of the incubation schedule. After washing the cells 3 times by centrifugation with glucose-fortified Hanks’ solution, the precipitated cells were treated successively with 1.5 mL cold water, 2 mL methanol, and 4 mL chloroform. After centrifugation, the upper methanol-water layer, lower chloroform layer, and precipitate were separated. The methanol-water layer was diluted then with 2 mL water and filtered through an anion-exchange resin cartridge (OnGuard A, OH-1). By this procedure, 18F-labeled anionic substances (i.e., phosphoryl-18F-FECh) were trapped in this anion-exchange resin cartridge and unreacted 18F-FECh passed through it. Subsequently, the radioactivity of each fraction was measured in a well counter. The total radioactivity in the cells and the chemical constitution of the total radioactivity is presented as the cell-to-medium ratio = (radioactivity concentration in cells)/(radioactivity concentration in medium). The (volume of cells)/(volume of medium) was determined by the hematocrit, in which the tumor cell suspension was taken into a capillary tube and centrifuged, and the (volume of packed cells)/(total volume of liquid) was measured under a microscope.
PET Study of Prostate Cancer Patients with 18F-FECh Chloride
In a preliminary study (12), we found that the biodistribution of 18F-FECh was almost the same as that of 11C-choline in normal rabbit and normal humans. The only difference was that 18F-FECh was excreted rapidly into urine, whereas 11C-choline was excreted slowly. We also found that the uptake of 18F-FECh in tumors of rabbits (VX2 tumor) was very high, and it was comparable with that of 11C-choline. Our clinical work of 18F-FECh PET began on patients with prostate cancer because we had considerable experience in studying these patients with 11C-choline PET (8,9). All of the following studies were approved by the institutional ethical board and were performed after receiving informed consent from the patients.
A single run of 18F-FECh PET, in a dynamic scan mode, was performed on 1 patient (80 y old) with untreated primary prostate cancer, after intravenous injection of 370 MBq 18F-FECh chloride, without bladder irrigation, to determine the most appropriate protocol for the study of other patients. 18F-FECh PET and 11C-choline PET were performed on 16 untreated prostate cancer patients, and the whole set of the studies was performed on 2 consecutive days.
The 18F-FECh PET study was performed according to the following protocol. Patients fasted overnight. A short intravenous catheter was placed in the forearm for intravenous infusion. A 3-way Foley catheter was placed in the bladder for irrigation, with 1 tubing connected to warm saline and another connected to a urine collection bag. After completion of the transmission scan, 18F-FECh (370 MBq) and furosemide (20 mg) were injected from the intravenous line successively, and saline (500 mL) was dripped from the same line until the end of the study. The bladder irrigation started shortly after the 18F-FECh injection and continued until the end of the study (total volume of saline, 4 L).
PET images were obtained using a PET camera (Headtome IV, 6-mm spatial resolution; Shimadzu) equipped with 3 detector rings to produce 5 slices at 13-mm intervals. When the patient, fixed on the bed, underwent transmission or emission scanning, the bed position was shifted 6 times upward from the level of the pelvis to that of the liver, with a scan time of 3 min at a single bed position. The emission scan was obtained twice, starting at 30 and 60 min, respectively. PET images were reconstructed after attenuation correction. The horizontal images were displayed sequentially, with each horizontal level indicated in a planar image, on the computer screen. Usually, the horizontal images were displayed according to the standardized uptake value (SUV), where SUV was defined as (regional radioactivity concentration)/(total injected dose/body weight). Each pixel (4 x 4 x 6 mm in real size) was painted a specified color that indicated a corresponding SUV value. Usually, red (the hottest color) indicated an SUV of 
4.0.
The 11C-choline PET study was performed according to the protocol reported previously (9).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The total time required for the synthesis of 18F-FECh chloride was 65 min after the end of bombardment.
Property of Product.
The radiochemical yield of 18F-FECh chloride was approximately 40%, with the decay corrected. After proton bombardment (20 MeV, 30 µA) of 60% enriched 18O-water for 30 min, approximately 3.7 GBq 18F-FECh chloride were obtained.
The ion chromatography analysis showed that the chemical quantities of ingredients in this preparation were N,N-dimethylethanolamine, 0.12 µmol (10.7 µg) per batch; and FECh chloride, 0.05 µmol (8.6 µg) per batch. The specific radioactivity of 18F-FECh chloride was calculated as 74 GBq/µmol.
The anion-exchange resin HPLC analysis showed that 2-naphthalenesulfonic acid was below detection (<0.1 µmol per batch) in the final 18F-FECh preparation.
The stability of 18F-FECh chloride was examined by ion-exchange HPLC. The result was as follows. If 18F-FECh chloride was left at room temperature at a high concentration (1.85 GBq/mL) for 1 h, part of it decomposed to form 18F-fluoroethylbetaine. This decomposition did not occur if it was stored in a refrigerator at a low concentration (0.37 GBq/mL) for hours.
Toxicity of Product.
The 18F-FECh chloride preparation showed no toxicity in 10 mice when it was injected intravenously after decay of radioactivity: injection dose, one fifth of a single batch from the automated synthesis, 0.01 µmol (1.7 µg) FECh per mouse (25 g).
The LD50 of FECh chloride (prepared by the fluoroethanol method) examined in 20 mice after intravenous injection was 0.13 g/kg.
Biochemical Study
Formation of Phosphoryl-18F-FECh by Choline Kinase and ATP.
No-carrier-added 18F-FECh was incubated with yeast choline kinase, ATP (15 µmol), MgCl2, and dithiothreitol at 37°C for 30 min. The reaction was stopped by addition of methanol and chloroform, and the methanol-water layer was separated. After concentrating this layer, the sample was analyzed by ion-exchange HPLC using cation-exchange resin (MCI Gel CQK 31S) and anion-exchange resin (MCI Gel CQA 35S). When HPLC was performed with unreacted 18F-FECh, the radioactivity migrated slowly on the cation-exchange HPLC (retention time, 12 min) and migrated fast on the anion-exchange HPLC (retention time, 3 min) (Fig. 3A). When HPLC was performed after 18F-FECh was incubated with choline kinase and ATP, most radioactivity migrated fast on the cation-exchange HPLC and migrated slowly on the anion-exchange HPLC, which was opposite to the behavior of unreacted 18F-FECh (Fig. 3A). When HPLC was performed after 18F-FECh was incubated with choline kinase, but without ATP, this phenomenon did not occur. These observations seemed to indicate that 18F-FECh was converted into phosphoryl-18F-FECh by choline kinase and ATP.
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Formation of 32P-Phosphoryl-18F-FECh by Choline Kinase and -32P-ATP.
In the above experiment, it was shown that 18F-FECh was converted into a new compound by the reaction with choline kinase and ATP and that the new compound could be considered tentatively as phosphoryl-18F-FECh. In the following experiment, the chemical property of this new compound was studied more precisely. No-carrier-added 18F-FECh (containing approximately 0.005 µmol FECh) was incubated with yeast choline kinase, -32P-ATP (0.01 µmol), MgCl2, and dithiothreitol. (The radioactivities of 18F-FECh and -32P-ATP added were 12.3 and 0.088 MBq, respectively.) After the reaction was stopped, the methanol-water layer was concentrated and analyzed on the HPLC using anion-exchange resin. Figure 3B shows the result of the HPLC. When the radioactivity of 18F was measured, 2 components were found: a large component that migrated fast and a small component that migrated slowly. When the radioactivity of 32P was measured, no radioactivity was in the fast component but there was distinct radioactivity in the slow component, and the ratio of 32P to 18F in the slow component was even in every fraction. It was evident from this observation that 32P and 18F were tagged by the same molecule. In other words, the new compound produced by the reaction of 18F-FECh with choline kinase and -32P-ATP was undoubtedly 32P-phosphoryl-18F-FECh.
Formation of Phosphoryl-18F-FECh in Tumor Cells.
No-carrier-added 18F-FECh chloride was incubated with Ehrlich ascites tumor cells (approximately 106 cells/mL) at 37°C for 30 min. After incubation, the cells were washed by centrifugation, and the methanol-water–soluble fraction was obtained. This fraction was divided into halves, and their radioactivities were analyzed on HPLC: one half on cation-exchange HPLC and the other half on anion-exchange HPLC. Figure 4 shows the result: The cation-exchange HPLC showed that approximately 17.1% of the original 18F-FECh (slowly migrating) was converted into a fast-migrating component (or components), ascribable to either phosphoryl-18F-FECh or 18F-fluoroethylbetaine. The anion-exchange HPLC showed that approximately 17.5% of the original 18F-FECh (fast migrating) was converted into a slowly migrating component, ascribable only to phosphoryl-18F-FECh. This observation indicated that the fast-migrating component in the cation-exchange HPLC was totally ascribable to phosphoryl-18F-FECh. It also indicated that 18F-FECh was converted into phosphoryl-18F-FECh, but not into 18F-fluoroethylbetaine, in this tumor type.
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3 spots, with most concentrated in 1 spot (Fig. 5). Natural choline-containing phospholipids (phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine) migrated to locations very different from the locations of the radioactive spots (Fig. 5, arrows). Nevertheless, we assumed that our radioactive spots corresponded to the natural phospholipids but migrated faster than the natural phospholipids. This assumption was well justified because it is known that the long alkyl group is more hydrophobic than the shorter ones and the fluorinated alkyl group is more hydrophobic than the nonfluorinated ones (13,14). In this context, it was also assumed that the major radioactive spot of our specimen was phosphatidyl-18F-FECh.
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In general, 18F-FECh PET gave slightly better images of prostate cancer than 11C-choline PET in terms of spatial resolution. This observation is explainable by the shorter positron range of 18F.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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In the in vitro experiment, we confirmed that 18F-FECh was incorporated into tumor cells by active transport (against the concentration gradient); then phosphorylated inside the cells, yielding phosphoryl-18F-FECh; and finally integrated into phospholipids, probably primarily into phosphatidyl-18F-FECh. These mechanisms constitute a kind of chemical trap and seem to explain why 18F-FECh was incorporated into prostate cancer so markedly and why the SUV in prostate cancer remained constant for a long time. When the SUV of 18F-FECh in prostate cancer of a patient was compared with the SUV of 11C-choline in prostate cancer of the same patient, these values were always close to each other. This fact seemed to indicate that the mechanisms of the membrane transport of 18F-FECh and 11C-choline are the same, and the mechanisms of the phosphorylation of 18F-FECh and 11C-choline are also the same; and the biologic activities of these mechanisms for these 2 substrates are almost the same, whereas the uptake mechanism of 11C-choline in tumors has been well discussed by us (5–7,9) and by others (16,17).
The advantages of 18F-FECh over 11C-choline were as follows. First, because of the longer half-life of 18F, 18F was more convenient for long-time storage and long-distance transportation. Second, because of the shorter positron range of 18F, 18F gave a slightly higher quality of image with higher spatial resolution.
The disadvantage of 18F-FECh was the rapid excretion of radioactivity into urine (in contrast to 11C-choline), and it was necessary to irrigate the bladder continuously using a urinary catheter to eliminate the bladder radioactivity. However, this procedure was very uncomfortable for urology patients.
DeGrado et al. (18) synthesized 18F-fluoromethylcholine and observed its biodistribution in mice with prostate cancer xenografts. They reported a high uptake of this compound in tumors, and a high radioactivity in urine that was 10 times higher than that of 11C-choline at 30 min after injection.
Recently, DeGrado et al. (19) reported on their success in obtaining clear PET images of prostate cancer in patients in whom 18F-fluoromethylcholine was used instead of 18F-FECh, the bladder irrigation was avoided, and the scanning was conducted at 3–5 min after injection (before the emergence of radioactivity in urine). We also obtained a clear PET image of prostate cancer in 1 patient (Fig. 7A) using 18F-FECh, without bladder irrigation, and conducting the scanning at 2–5 min after injection. We did not adopt this protocol in the rest of this study because we wanted to examine the whole area of pelvis in the patients (our PET machine covers only 6.5 cm longitudinally at 1 bed position).
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Toshihiko Hara, MD, PhD, Department of Radiology, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162, Japan.
E-mail: ahara{at}kt.rim.or.jp
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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