ARTICLE IN PRESS
Applied Radiation and Isotopes 65 (2007) 676–681 www.elsevier.com/locate/apradiso
The automatic production of 16a-[18F]fluoroestradiol using a conventional [18F]FDG module with a disposable cassette system Seung Jun Oha,, Dae Yoon Chib, Christoph Mosdzianowskic, Hee Seup Kilb, Jin Sook Ryua, Dae Hyuk Moona a
Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea b Department of Chemistry, Inha University, Inchon 402-751, Korea c GE Healthcare Technologies, Liege, Rue Marie Curie 78, B-4431 Loncin (Liege), Belgium Received 14 February 2006; received in revised form 17 April 2006; accepted 12 June 2006
Abstract We have developed a fully automatic method for the synthesis of 16a-[18F]fluoroestradiol ([18F]FES) using a disposable cassette system and conventional [18F]FDG module. [18F]FES was synthesized using a GE TracerLab MX module and a modified module control program. Following [18F]fluorination, we hydrolyzed the product three times with a mixture of 2 N HCl and CH3CN. After HPLC purification, the decay corrected radiochemical yield of [18F]FES was 45.372.8%, which was stable to 98.270.2% at 6 h after synthesis. This new automated synthesis method provides high and reproducible yields with the advantage of a disposable cassette system. r 2006 Elsevier Ltd. All rights reserved. Keywords: [18F]Fluoroestradiol; Automatic synthesis; Breast cancer; [18F]Fluoride; Disposable cassette system
1. Introduction 16a-[18F]Fluoroestradiol ([18F]FES) is a radiopharmaceutical used in positron emission tomography for imaging of specific estrogen receptors (Eckelman, 1994). Compared with 16b-[18F]FES, 16a-[18F]FES has a 2.5-fold higher relative binding affinity to these receptors (Katzenellenbogen, 2001). Using this radiopharmaceutical, clear images of primary and metastatic breast tumors can be obtained. Tumor uptake of [18F]FES has been found to correlate closely with receptor levels on biopsy samples (Dehdashti et al., 1995; McGuire et al., 1991). In addition, reduced tumor uptake of [18F]FES has been demonstrated in breast cancer patients who have started anti-estrogen hormone therapy (Mortimer et al., 1996, 2001). [18F]FES is manually synthesized from the precursor 3-methoxymethyl-16b,17b-epiestriol-O-cyclic sulfone (1), resulting in a high radiochemical yield and low by-product production (Berridge et al., 1990; Lim et al., 1996). [18F]FES has also been automatically synthesized using a Corresponding author. Tel.: +82 2 3010 4595; fax: +82 2 3010 4588.
E-mail address:
[email protected] (S.J. Oh). 0969-8043/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2006.06.016
conventional [18F]FDG synthetic module, but without a cassette (Romer et al., 1999). Use of a disposable cassette would provide stable radiochemical yields without the need for cleaning procedures. Addition of an automatic rinsing process, by a modification of the control program, would reduce the residual activity on the cassette, and would reduce exposure to radiation during back-to-back runs. Thus, [18F]FES production using a disposable cassette with conventional [18F]FDG module would be more suitable for routine daily production for clinical purposes. In the present study, we developed a [18F]FES production method using a conventional [18F]FDG module with a disposable cassette. This method was subjected to quality control procedures and stability tests for routine clinical purposes. 2. Materials and methods 2.1. Chemicals The precursor, 3-methoxymethyl-16b,17b-epiestriol-Ocyclic sulfone, was obtained from FutureChem (Seoul,
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Korea). Solvents and reagents (reagent grade) were purchased from Sigma-Aldrich (Milwaukee WI, USA), Lancaster (Lancashire, UK), and Acros Chemicals (Geel, Belgium), and were used without further purification.
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3-methoxymethyl-16b,17b-epiestriol-O-cyclic sulfone in 2 ml of CH3CN. [18F]fluorination was allowed to proceed for 8 min at 105 1C and for 2 min at 85 1C. The solvent was removed at 85 1C for 2 min under vacuum pressure and nitrogen purging. Hydrolysis was performed by multiple azeotropic evaporations using a mixture of 2 N HCl and CH3CN. After delivery of 2.2 ml of this mixture to the reactor, the mixture was heated under nitrogen purging, while maintaining negative pressure by vacuum, for 2 min at 85 1C and for 5 min at 95 1C. The hydrolysis procedure was repeated three times to prevent the production of side products and to maintain acid concentration (Romer et al., 1999). After hydrolysis, the reaction mixture was diluted with the ethanolic solution from the yellow vial, and the mixture was injected automatically into the HPLC. Following HPLC purification, using the conditions described in Section 2.3, the [18F]FES collected was diluted with 100 ml of H2O and trapped on an additional C18 cartridge to remove excess ethanol. The trapped [18F]FES was eluted with 1 ml of ethanol and diluted with 4 ml of 0.9% saline. Fig. 3
2.2. Chemistry module and synthesis of [18F]FES We used a GE TracerLab MX (Liege, Belgium) [18F]FDG module and made modifications of the [18F]FES sequence program and the disposable cassette used for [18F]FDG synthesis. All Sep-Pak cartridges in the disposable cassette were removed and manifolds 2 and 3 were connected with silicone tubing (Fig. 1). The design of the disposable cassette is shown in Fig. 1. This cassette had four reagent supply vials. Acetonitrile (7 ml) was placed in vial 1 (blue); the precursor (1–10 mg) with 2 ml CH3CN in vial 2 (red); 5.4 ml of EtOH:H2O (7:3) solution in vial 3 (yellow); and a mixture of 0.69 ml 2 N HCl and 6.21 ml CH3CN in vial 4 (green). Two 30 ml disposable syringes were installed. [18F]FES was prepared by fluorination and hydrolysis (Fig. 2). [18F]Fluoride (3.7 GBq) delivered from the cyclotron was trapped on a QMA cartridge and eluted to the reactor with a mixture of 7 mg of K2CO3 in 300 ml H2O and 22 mg of K222 in 300 ml of CH3CN. Following complete drying, performed under vacuum pressure and nitrogen purging at 95 1C, we added 1–10 mg of the precursor,
2.3. HPLC Purification We used a Breeze HPLC pump (Waters, Milford, MA, USA) with a UV 2457 (Waters) and a NaI PIN Diode detector system (Bioscan, Washington, DC, USA) for
1mL Ethanol Syringe Vacuum Pump
HPLC Injector Eluant
HPLC Column
Radioactive Detector UV Detector
Sample Reservoir
Pneumatic 3-way valve 1
Syringe Pump 2
Syringe Pump 1 18
Waste Bottle
Finlet
QMA Cartridge
V1
V2
V3
V4 Pneumatic Pneumatic 3-way valve 2 C18 3-way valve 3 Sep-Pak
Manifold 1
Manifold 2 Sillion tubing Manifold 3
Waste Bottle
Reactor
O-18 water Reservoir
N2gas
Venting needle
Product Vial
Waste Bottle Vacuum Pump
100mL H2O Hot-cell
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Fig. 1. Diagram of the disposable cassette for the radiochemical synthesis of [ F]FES. Silicon tubing connects manifolds 2 and 3. Vials 1–4 contain acetonitrile, the precursor, EtOH:H2O (7:3), and 0.69 ml 2 N HCl plus 6.21 ml CH3CN, respectively. The outlet of manifold 3 is connected to the HPLC automatic injector.
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O O S O O [18F]F -
OSO318
F
OH 0.2 N HCl
18
F
O
O O
1
HO O
[18F]FES
Fig. 2. Automatic synthetic route for [18F]FES with a cyclic sulfonate precursor.
Fig. 3. Crude HPLC chromatogram of the automatic synthesis of [18F]FES. The upper panel shows a UV chromatogram at 280 nm and the lower panel shows a chromatogram of radioactivity.
HPLC purification. For sample injection from a chemistry module, we used a 10 ml HPLC loop and an automatic injector (Rheodyne, Rohnert Park, CA, USA).
For this injection, we used a Rheodyne autoinjector that has 6-ports in the injector. Each connection was labeled as follows: port 1, a connection from HPLC pump; port 2,
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10 mL loop; port 3, sample reservoir; port 4, waste port; port 5, 10 mL loop; port 6, to HPLC column. We have a 10 mL sample reservoir at the injector port of 3 and the waste port of the autoinjector was connected to a vacuum pump. We also have a 0.22 mm vented sterile filter (MillexGS, Millipore, Billerica, MA, USA) between the sample reservoir and injector port 3 to remove insoluble compounds and air from the reaction mixture (Gee and Bender, 1997). By vacuum from the pump, the reaction mixtures moved to the sample loop from the sample reservoir; they were automatically injected to the HPLC column by rotation of the injector after complete moving of sample to loop. A Nucleosil 100-7 C18 (Machery-Nagel, 10 mm, 10 250 mm) HPLC column was used; this was eluted with ethanol:water (65:35) at 3.5 ml/min, and monitored at 280 nm with a UV detector. We used three pneumatic 3-way valves (Alltech, USA), operated by compressed air. The valve position was in Fig. 1. Valve 1 was used to collect purified [18F]FES from HPLC column and valves 2 and 3 were used to remove EtOH and concentrate [18F]FES on solid phase extraction cartridge. The purified [18F]FES was diluted with 100 ml of H2O in the 200 ml collection bottle and passed through a C18 Sep-Pak Plus (360 mg sorbent) or an Oasis HLB (30 and 200 mg of sorbent) cartridge (Waters, USA) to remove excess EtOH and to trap the [18F]FES. Before synthesis, we added 100 mL of H2O to this 200 mL bottle and made a connection of 1 mL of EtOH syringe to 3-way valve 2 from outside of the hot-cell. We describe this connection in Fig. 1. The trapped [18F]FES was eluted to the vial which has 20 mg/mL ascorbic acid with 1 ml EtOH passed via a sterile 0.22 mm filter. 2.4. Evaluation of automatic synthesis with high radioactivity We used 37 GBq/1 ml and 74 GBq/2 ml of [18F]fluoride for high radioactivity [18F]FES synthesis and reproducibility tests (n ¼ 10 and 5, respectively) with 2 mg precursor for these syntheses.
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All radiochemical yields were decay-corrected and endof-synthesis (EOS) yields were calculated. 2.5. Quality control and stability checking Following synthesis of [18F]FES from 37 GBq/mL [ F]fluoride (n ¼ 10) or from 74 GBq/mL [18F]fluoride (n ¼ 5), we measured radiochemical purity by radio TLC (developing solvent: acetone; Merck 60 F254, Germany) and analytical HPLC (Thermo Separation Products, USA). The residual organic solvents in the final product were analyzed by gas chromatography (Acme-2000, YoungLin Instrument, Korea). Supelcowax-10 (SigmaAldrich) fused silica capillary columns (60 m 0.25 mm 0.25 mm film thickness) were used for analysis. The temperatures of the oven, injector and detector were 75, 250 and 250 1C, respectively. We used a flame ionization detector system with a flow rate of 105 ml/min. Gas chromatography data were analyzed with an Autochro2000 (Younglin Instrument, Korea). Tests for pyrogen, Kryptofix2.2.2 and sterility were performed as described previously (Hung, 2002). The pH of the final solution was measured using a pH meter (Corning, USA). Long-term radiochemical stability for up to 6 h at room temperature was determined on each 10 ml solution (9 ml saline+370 MBq/mL [18F]FES solution) using a radioTLC and an analytical HPLC (n ¼ 3), with acceleration stability tests performed at pH values of 1, 4, 7, 10, and 13. 18
3. Results Radiochemical yields depended on the amount of precursor (Table 1). Starting with 2 mg precursor, the HPLC purified radiochemical yield after [18F]fluorination for 8 min at 105 1C and for 2 min at 85 1C was 45.372.8%. Although the radiochemical yield increased as the amount of precursor increased (68.475.2% from 10 mg precursor), we could not completely separate the organic impurities from [18F]FES when we started with higher amounts of precursor. We could not analyze them and only considered that they were cold [18F]FES and hydrolyzed precursor
Table 1 Yield and specific activity of synthesized [18F]Fluoroestradiol (n ¼ 3) Precursor amount (mg) Yield of radiochemical (%) Measured specific activity (GBq/mmol)
1 20.473.7 57.971.7
2 45.372.8 57.773.8
*Reaction conditions: 1. 2. 3. 4. 5. 6.
Precursor: 3-methoxymethyl-16b,17b-epiestriol-O-cyclic sulfone. 3.7 GBq/mL [18F]F as starting radioactivity. [18F]fluorination for 8 min at 105 1C and for 2 min at 85 1C. 3 azeotropic hydrolysis with 2 N HCl and CH3CN. HPLC purification. Radiochemical yield: decay-corrected radiochemical yield after HPLC purification.
5 55.778.4 32.979.4
10 68.475.2 20.271.8
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impurities. The measured specific activity of the product was 57.773.8 GBq/mmol when we started with 2 mg of precursor, but only 20.271.8 GBq/mmol when we started with 10 mg of precursor. (n ¼ 3 for each condition) Starting with 37 GBq of [18F]F (n ¼ 10), the radiochemical yield was 48.372.7%, whereas starting with 74 GBq of [18F]F (n ¼ 5), the yield was 45.273.8%. Total synthesis time, including HPLC purification and formulation, was 75.875.2 min. After concentrating [18F]FES on the C18 cartridge, we obtained an injectable solution containing 10% ethanol. The C18 plus cartridge showed a trapping efficiency 495%, whereas the 30 mg sorbent HLB Oasis cartridge had a trapping efficiency of 60–70%. Although the 200 mg sorbent HLB cartridge showed 495% of trapping efficiency, we only obtained 50–60% [18F]FES from total trapped radioactivity with 1 mL of EtOH elution. At the time of synthesis, the purity of the radiochemical was 98.770.8%. The results of quality control procedures showed that the synthesized [18F]FES was suitable for routine clinical use. Peak retention times of cold FES and [18F]FES were the same in the co-injection HPLC procedures. Radiochemical purity was 98.270.2% (n ¼ 3) after 6 h, and remained 495% after several acceleration tests. 4. Discussion Starting with 74 GBq of 18F and using a conventional [ F]FDG synthetic module with a disposable cassette, we obtained a high radiochemical yield of [18F]FES (45.273.8%). In this procedure, we modified only sequence software and the disposable cassette, without any hardware changes. We used a two-stage heating protocol for [18F]fluorination. A reaction temperature of 105 1C was not high and there was a low possibility of the reaction vessel cap rupture due to high internal pressure generated by the high temperature. However, because our TracerLab MX module did not have a cooling system comparable to that reported previously (Romer et al., 1999), it was necessary to reduce the reaction temperature in order to decrease the internal pressure in the reactor. Without this temperature reduction, we may have lost much of the radioactivity when we opened the valve connected to the reactor. Therefore, after 8 min of heating at 105 1C, we reduced the [18F]fluorination temperature to 85 1C for 2 min to reduce the internal pressure of the reactor. We also subjected the reaction product to repeated azeotropic hydrolysis. Although this procedure requires a long preparation time, it has several advantages, including low contamination with side-products and efficient removal of unreacted [18F]F (Lim et al., 1996; Romer et al., 1999). The hydrolysis step included nitrogen purging and application of negative pressure. Under these conditions, and in the presence of 0.2 N HCl, the unreacted [18F]F changed to H[18F]F, which could then be evaporated to the 18
waste bottle. As a result, our radioactive HPLC chromatogram was very clean. After HPLC purification, the final product had a high concentration of ethanol. Previously, the final product was diluted with saline or evaporated to remove excess ethanol (Lim et al., 1996; Romer et al., 1999). We used a solidphase extraction method to reduce the quantity of ethanol in the final [18F]FES injectable solution, obviating the need for cumbersome procedures such as heating for evaporation. In this study, we used a commercial disposable cassette system. This cassette could be replaced for each run, ensuring clean and reproducible conditions for each production, preventing cross-contamination, and resulting in high, reproducible yields. Although cassette replacement entails higher costs, the addition of an automatic rinsing process with a modification of the control program may reduce the residual activity on the cassette, as well as reducing radiation exposure in cases of back-to-back runs and lowering replacement costs. 5. Conclusion We have developed an automated method of [18F]FES production using a commercial FDG module and a modified disposable cassette system, resulting in consistently high radiochemical yields. Automated synthesis of [18F]FES may enhance the clinical applications of this radiochemical. Acknowledgment This study was supported by the Korean Ministry of Science & Technology (MOST), through its real-time molecular imaging research program. References Berridge, M.S., Franceschini, M.P., Rosenfeld, E., Tewson, T.J., 1990. Cyclic sulfates: useful substrates for selective nucleophilic substitution. J. Org. Chem. 55, 1211–1217. Dehdashti, F., Mortimer, J.E., Siegel, B.A., Griffeth, L.K., Bonasera, T.J., Fusselman, M.J., Detert, D.D., Cutler, P.D., Katzenellenbogen, J.A., Welch, M.J., 1995. Positron tomographic assessment of estrogen receptors in breast cancer: comparison with FDG-PET and in vitro receptor assays. J. Nucl. Med. 36, 1766–1774. Eckelman, W.C., 1994. The application of receptor theory to receptorbinding and enzyme-binding oncologic radiopharmaceuticals. Nucl. Med. Biol. 21, 759–769. Gee, A.D., Bender, D., 1997. The use of vented sterile filters in the automation of preparative HPLC injection. XIIth International Symposium on Radiopharmaceutical Chemistry Abstract 304. Hung, J.C., 2002. Comparison of various requirements of the quality assurance procedures for 18F-FDG injection. J. Nucl. Med. 43, 1495–1506. Katzenellenbogen, J.A., 2001. Steroids labeled with 18F for imaging tumors by positron emission tomography. J. Fluorine Chem. 109, 49–54. Lim, J.L., Zheng, L., Berridge, M.S., Tewson, T.J., 1996. The use of 3-methoxymethyl-16b, 17b-epiestriol-O-cyclic sulfone as the precursor
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