Highly efficient production of [18F]fallypride using small amounts of base concentration

Applied Radiation and Isotopes 68 (2010) 2279–2284

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Highly efficient production of [18F]fallypride using small amounts of base concentration Byung Seok Moon a,b, Jun Hyung Park a, Hong Jin Lee a, Ji Sun Kim a, Hee Sup Kil c, Byoung Se Lee c, Dae Yoon Chi c, Byung Chul Lee a,b,n, Yu Kyeong Kim a, Sang Eun Kim a,b a

Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam 463-707, Republic of Korea Institute of Radiation Medicine, Seoul National University Medical Research Center, Seoul National University, Seoul 110-744, Republic of Korea c Department of Chemistry, Sogang University, Seoul 140-742, Republic of Korea b

a r t i c l e in fo

abstract

Article history: Received 23 December 2009 Received in revised form 3 June 2010 Accepted 16 June 2010

To minimize the base concentration of a phase-transfer catalyst, [18F]fluoride was extracted from 18 O-enriched water trapped on an activated ion exchange cartridge (Chromafixs PS-HCO3) using different concentrations of tetrabutylammonium bicarbonate (TBAHCO3) or Kryptofix 2.2.2./K2CO3 in organic solvents such as CH3CN/H2O or MeOH/H2O. The optimal labeling condition for [18F]fallypride with automated synthesis was that 2 mg of tosyl-fallypride in acetonitrile (1 mL) was heated at 100 1C for 10 min using 40% TBAHCO3 (10 mL). [18F]Fallypride was obtained with a high radiochemical yield of approximately 68 71.6% (decay-corrected, n ¼ 42) with a total synthesis time of 51 7 1.2 min, including HPLC purification and solid-phase purification for the final formulation. & 2010 Elsevier Ltd. All rights reserved.

Keywords: [18F]Fallypride Automated radiosynthesis PET Dopamine D2/D3 receptor

1. Introduction The dopamine receptor antagonist [18F]fallypride, ((S)-N-[(1-allyl2-pyrrolidinyl)methyl]-5-(3-[18F]-fluoropropyl)-2,3-dimethoxybenzamide), which has an extremely high selectivity and high affinity (Ki ¼ 30 pM for D2 receptor sites), is currently being used as a potential dopamine D2/D3 receptor imaging agent for positron emission tomography (PET). Since the importance of cortical dopamine functions related to cognition in neuropsychiatric illnesses has been increasingly considered, [18F]fallypride has attracted attention for its ability to visualize extrastriatal as well as striatal D2 receptors (Elsinga et al., 2006; Mukherjee et al., 1999, 1995, 2002; Siessmeier et al., 2005; Vandehey et al., 2009). In addition, half life of the radionuclide (fluorine-18, t1/2 ¼110 min) in [18F]fallypride offers good prospects for assessing low concentrations of extrastriatal dopamine D2/D3 receptors. Therefore, the efficient radiochemical synthesis of [18F]fallypride with high radiochemical yield and specific activity is important for routine clinical studies. Mukherjee and coworkers, performing manual synthesis, reported moderate yields of about 20–40% with 33–63 GBq/mmol of specific activity and a relatively long labeling time of fluorine-18 incorporation (approximately 30 min) as well as a

n Corresponding author at: Department of Nuclear Medicine, Seoul National University Bundang Hospital, 300 Gumidong, Bundanggu, Seongnam 463-707, Republic of Korea. Tel.: + 82 31 787 2956; fax: + 82 31 787 4072. E-mail address: [email protected] (B.C. Lee).

0969-8043/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2010.06.016

long purification time. Moreover, the lengthy purification process of high-performance liquid chromatography (HPLC) had to be used to avoid mass peaks due to significant thermal degradation of the precursor, the formation of the corresponding alcohol or olefin resulting from elimination reactions, as well as other unidentified peaks is approximately 2 mg of precursor scales. In some cases, further purification of [18F]fallypride appears to be necessary in order to obtain higher specific activities (Christian et al., 2009; Mukherjee et al., 1999, 1995). Although it has been shown using automated synthesis that [18F]fallypride can be prepared by one-step radiochemical synthesis with a tosylate precursor, the radiochemical yields are somewhat low (5–20%) for the same reason described above for manual synthesis (Ansari et al., 2006; Shi et al., 2002). Recently, microfluidic devices with milder reaction conditions have emerged to produce radiotracers for molecular imaging by microPET (Gillies et al., 2005; Jeffery et al., 2004; Lee et al., 2005; Liow et al., 2005; Lu et al., 2009; Lucignani, 2006; Steel et al., 2007). These devices have several advantages, including the use of lower amounts of precursor and base, easier and more efficient purification, relatively high conversion yield, and short preparation time. Recently, the successful production of [18F]fallypride with microreaction devices in small doses of approximately 0.5–1.5 mCi was reported (Lu et al., 2009), although high-dose-scale production was not addressed. More recently, some groups have reported moderate radiochemical yields in automated [18F]fallypride production of about 30–40% (Ansari et al., 2009; Kuhnast et al., 2009; Lukic et al., 2009; Mock et al., 2009; Vuong et al., 2009). In these studies, however, production was carried out at high

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temperatures (150–165 1C) or involved the use of an unusual microwave system in automatic devices. Moreover, various byproducts were produced during HPLC purification, as described above. The use of high base concentrations in the fluorine-18 labeling step is likely the main reason why various side products were generated, providing low radiochemical yield particularly in the case of base-sensitive tosyl-fallypride. In automatic synthesis, QMAs or Chromafixs PS-HCO3 cartridges are frequently used in a capture-andrelease manner to extract fluorine-18 ion because fluorine-18 is largely produced in diluted 18O-enriched water. During this process, fluorine-18 can be released from the polymer cartridge by elution with relatively large amounts of K2CO3/K2.2.2., CsCO3/K2.2.2., TBAOH, TBAHCO3, etc. Such large amounts of base lead to the production of more than the expected amounts of various side products and hence require more quantity of precursor. The radiochemical yield and purity also diminishes. Therefore, the incorporation of fluorine-18 into base-sensitive [18F]fallypride has not been achieved in high radiochemical yields, thus far. For the routine clinical use of [18F]fallypride, an automated preparation with a high radiochemical yield is required. Here, we demonstrate the minimization of base concentration when fluorine-18 is eluted from an ion-exchange cartridge in radiosynthesis using a commercial TracerLab FXFN chemistry module. We also describe the production of [18F]fallypride in relatively high doses with high specific activity, purity, and shortened preparation time for routine clinical use.

2. Materials and methods 2.1. Precursor and chemicals All commercial reagents and solvents were used without further purification unless otherwise specified. Reagents and solvents were commercially purchased from Sigma-Aldrich (US). The tosylate precursor ((S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-toluenesulfonyloxypropyl)-2,3-dimethoxybenzamide) for [18F]fallypride and standard [19F]fallypride were obtained from FutureChem Co., Ltd. (Korea). H18 2 O was purchased from Taiyo Nippon Sanso Corporation (Japan). 18F-fluoride was produced at Seoul National University Bundang Hospital by 18O(p,n)18F reaction through proton irradiation using a KIRAMS-13 cyclotron (Samyoung Unitech Co., Ltd.). Chromafixs PS-HCO3 (45 mg) cartridges were purchased from Macherey-Nagel Ins. (Germany, Cat. no.: 731876). Sep-Paks Plus tC18 cartridges were purchased from Waters Corp. (US, Part no.: WAT036810). All radiochemical processes, including the separation of fluorine-18 from 16/18O-enriched water (diluted with H16 2 O), the incorporation of fluorine-18 with tosyl-fallypride, HPLC purification [a preparative column (Nucleosil C18, 7 mm, 16  250 mm, Machery-Nagel, Germany) and guard column (Nucleosil C18, 7 mm, 16  50 mm)], and reformulation of the end-product for clinically injectable EtOH/saline solutions, were performed in the TracerLab FXFN (GE Healthcare). HPLC-grade solvents were used for HPLC (J.T. Baker, US) after membrane filtering (0.22 mm, Whatman). All radioactivities were measured using a VDC-505 activity calibrator from Veenstra Instruments (Netherlands) and are presented as decay-corrected values unless otherwise noted. 2.2. Separation of fluorine-18 The separation of fluorine-18 from a Chromafixs PS-HCO3 cartridge was performed with various concentrations of phasetransfer catalysts such as K2CO3/Krptofix2.2.2. or TBAHCO3. MeOH (1.0 mL)/H2O (0.2 mL) (Lee et al., 2007) or CH3CN (1.0 mL)/H2O (0.2 mL) were used as the elution solvents for eluting trapped fluorine-18 from a Chromafixs PS-HCO3 cartridge.

2.3. [18F]Fluorination with tosylate-fallypride precursor The automatic synthesis of [18F]fallypride was evaluated under a separate condition in which most of the trapped radioactivity of the fluorine-18 ( 495%) was extracted from the Chromafixs PS-HCO3 cartridge by a phase-transfer catalyst such as K2CO3/Krptofix2.2.2. or TBAHCO3. To measure the radioactivity accurately, the remaining radioactivity in the PS-HCO3 cartridge and the activity extracted by a phase-transfer catalyst after being taken out of the automatic device were measured using an activity calibrator. After determining the optimal extraction conditions for fluorine-18, the aqueous fluorine-18 aqueous catalyst was completely dried. The tosylate precursor (2 mg) dissolved in anhydrous CH3CN (1 mL) was added to carbon reaction vessel containing the dried 18F  /phase-transfer catalyst, and experiments were conducted at 100 1C for 10 or 30 min (Fig. 1). The crude product was cooled using compressed air and then diluted with 3 mL of HPLC solvent to rinse the reaction vessel. The mixture was automatically injected into the HPLC system for separation. 2.4. HPLC purification, reformulation, quality control, and specific activity The HPLC conditions for purification were 70% CH3CN/H2O containing 0.6% triethylamine (TEA) at a flow rate of 7 mL/min, using a UV detector at 254 nm or 280 nm and a g-ray detector with a Nucleosil C18 column. The collected [18F]fallypride (7.0–10.0 mL) fraction was directly transferred to the replacement unit filled with 100 mL of water for solid-phase extraction. The diluted solution was passed through a tC18 Sep-Pak cartridge to remove the HPLC solvent in the collected fraction. After the trapped [18F]fallypride on Sep-Pak cartridge was washed with 10 mL of water, [18F]fallypride was eluted to the sterile vial with 1 mL of EtOH, followed by 10 mL of saline. Quality control was performed by HPLC and radio-TLC (Radio-thin layer chromatography). The specific activity was obtained by integrating the UV area after the collected elute was re-injected and then performing a conversion to activity/mol from the calibration curve. 2.5. Automation For the automatic production of [18F]fallypride, we used the TracerLab FXFN module. The profile of operating system is presented in Fig. 2. The present study consists of nine reagent supply vials (vials 1–9, V1–V9) at the top from left to right and vial 10 (V10) for reformulation on the lower right. The reagents or solvents in each vial are summarized in Table 1.

3. Results and discussion The preparation of [18F]fallypride is well established and can be easily synthesized with a single-step reaction. Although the labeling of [18F]fallypride was reported by Mukherjee and coworkers with 20–40% radiochemical yield in 1995 (Mukherjee et al., 1995), subsequent work using automated production systems did not obtain sufficient yields (Ansari et al., 2006; Shi et al., 2002). In the case of [18F]fallypride, the concentration of the base is an important factor for efficient fluorine-18 labeling because its precursor has a high sensitivity to excess amounts of base, resulting in the formation of the corresponding alcohol, olefins from elimination reactions, and other unidentified compounds. In this context, we directed our optimization efforts toward objectives such as: (a) determining the minimum base

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OMe

OMe OMe H N

TsO

2281

O

OMe H N

phase-transfer catalyst N

18

CH3CN, 100 °C

F

N

O [18F]Fallypride

Fig. 1. Radiosynthesis of [18F]fallypride from its tosylate precursor in a single-step reaction with fluorine-18.

Fig. 2. A schematic of the TracerLab FXFN chemistry module.

Table 1 Reagents in each vial.

Table 2 Recovery rate of fluorine-18 from the Chromafixs PS-HCO3 cartridge with different phase-transfer catalysts, base concentrations, and solvents.

Vial

Reagent

Vial

Reagent

V1 V2 V3 V4 V5

phase-transfer catalyst not used precursor (2 mg) in CH3CN (1 mL) not used HPLC solvent (1.5 mL)

V6 V7 V8 V9 V10

HPLC solvent (1.5 mL) saline (10 mL) ethanol (1 mL) DIa water (10 mL) DI water (100 mL)

a

DI water: Deionized water.

concentration that can elute over 95% of fluorine-18 trapped by the Chromafixs PS-HCO3 cartridge and (b) developing a setup with minimum reaction time. The automated nucleophilic fluorination of [18F]fallypride with the radiosynthesis module resulted in different radiochemical yields depending on the concentrations of phase-transfer catalysts. Consistent with the former objective, fluorine-18 trapped in the Chromafixs PS-HCO3 cartridge was eluted by various base concentrations, solvents, and phase-transfer catalysts, resulting in rates of recovery ranging from 18 to over 95%, as summarized in Table 2. Over 95% of the fluorine-18 was extracted from the PS-HCO3 cartridge in entries 3, 5, 8, and 10 (Table 2). The fully automated synthesis of [18F]fallypride, therefore, was performed in these

1 2 3 4 5 6 7 8 9 10

phase-transfer catalyst

solvent (1.0/0.2 mL)

recovery %a

K2.2.2./K2CO3 (5.5/0.5 mg) K2.2.2./K2CO3 (5.5/1.5 mg) K2.2.2./K2CO3 (13/3.0 mg) K2.2.2./K2CO3 (11/0.5 mg) K2.2.2./K2CO3 (11/0.8 mg) 40% TBAHCO3 (10 mL) 40% TBAHCO3 (20 mL) 40% TBAHCO3 (30 mL) 40% TBAHCO3 (5 mL) 40% TBAHCO3 (10 mL)

CH3CN/H2O CH3CN/H2O CH3CN/H2O MeOH/H2O MeOH/H2O CH3CN/H2O CH3CN/H2O CH3CN/H2O MeOH/H2O MeOH/H2O

 18  57 495  88 495  52  74 495  80 495

a The starting radioactivity (8.1–26 GBq), residual radioactivity in the PS-HCO3 cartridge, and recovered radioactivity with phase-transfer catalysts were measured using an activity calibrator after removal from the automatic device (n¼2 or 3).

fluorine-18 eluting conditions in which over 95% of the radioactivity was extracted from the cartridge. Other conditions (entries 1, 2, 4, 6, 7, and 9) with lower base concentrations do not meet clinical production standards even with high radiolabeling yield and low side-product levels due to the high loss of radioactivity when separated from the ion-exchange cartridge. Using the optimized fluorine-18 extraction condition (Table 2,

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Table 3 The radiochemical yields of [18F]fallypride under various conditionsa.

Mock et al., 2009 Kuhnast et al., 2009 Vuong et al., 2009 Lukic et al., 2009 Shi et al., 2002 1 2 3 4 5

Phase-transfer catalyst

Extraction solvent

Time and temperature

RCY (%)b

Total time (min)

K2.2.2./K2CO3 K2.2.2./K2CO3 TBAHCO3 K2.2.2./K2CO3 K2.2.2./K2CO3 K2.2.2./K2CO3 (13/3.0 mg) K2.2.2./K2CO3 (11/0.8 mg) 40% TBAHCO3 (30 mL) 40% TBAHCO3 (10 mL) 40% TBAHCO3 (10 mL)

CH3CN/H2O CH3CN/H2O CH3CN/H2O CH3CN/H2O CH3CN/H2O CH3CN/H2O MeOH/H2O CH3CN/H2O MeOH/H2O MeOH/H2O

N.D.c, 120 1C 5 min, 165 1C 15 min, 105 1C 20 min, 85 1C N.D. 30 min, 100 1C 30 min, 100 1C 30 min, 100 1C 30 min, 100 1C 10 min, 100 1C

30–50 40 32 32 15 26 7 4.9 71 7 1.9 44 7 4.1 71 7 1.2 68 7 1.6d

60–70 45 60 70 45 747 1.2 747 1.2 747 1.2 747 1.2 517 1.2

a Reaction conditions: Starting radioactivity (8.1–52 GBq), 2 mg of tosylate precursor in CH3CN (1 mL), a reaction temperature of 100 1C in the carbon reaction vessel. b Decay-corrected yield (n ¼2 or 3). c Not described. d n¼42.

entries 3, 5, 8, and 10), the automatic production of [18F]fallypride was evaluated and the results are summarized in Table 3. The use of K2.2.2./K2CO3 (13/3.0 mg, entry 3) at 100 1C for 30 min led to a lower radiochemical yield of about 26%. In addition, HPLC purification revealed unidentified radiopeaks and overlapped with those of [18F]fallypride as well as peaks of organic impurities such as olefin and other unidentified compounds (entry 1 in Table 3, data not shown). The use of 40% TBAHCO3 (30 mL, entry 3) under the same conditions also led to a relatively low radiochemical yield of about 44%. However, radiopeaks were completely separated with no overlapping organic impurities despite isocratic HPLC conditions. The optimal conditions for [18F]fallypride production were K2.2.2./K2CO3 (11/0.8 mg, entry 2) and 10 mL of 40% TBAHCO3 (entry 4), each of which gave a 71% radiochemical yield and required a total preparation time of approximately 74 min. HPLC purification, however, revealed slightly more impurities in the K2.2.2./K2CO3 (11/0.8 mg, entry 2) condition than in the 40% TBAHCO3 condition (entry 4). In regards to the latter objective, shortening the labeling time from 30 to 10 min in the 40% TBAHCO3 condition (10 mL) still gave a high radiochemical yield of 68% within a total elapsed time of 51 min (entry 5), with a high specific activity of approximately 140–192 GBq/mmol. Though the result of entry 4 showed a slightly higher radiochemical yield than that of entry 5 by taking decay-corrected factor, both automatic synthesis conditions showed similar radiochemical yields of about 46–50% without decay correction. Consequently, shortening the incorporation time of fluorine-18 (10 min, entry 5) is useful in practical clinical use. This was mainly due to the fact that 10 min was sufficient for labeling at the optimal temperature without another driving force such as high temperature (165 1C) and microwave. Fig. 3 shows a raw HPLC chromatogram from the FXFN module for entry 5, which was confirmed by co-injecting standard [19F]fallypride (Fig. 4). Some research groups have performed HPLC purification at 254 nm of the UV detector. Therefore, we compared UV mass patterns between 254 and 280 nm under the same radiosynthetic condition for [18F]fallypride (entry 5). As shown in Fig. 5, the HPLC profile of UV (254 nm) showed a slight increase at 1 min that remained until the time of [18F]fallypride separation. The total volume of the collected [18F]fallypride was re-injected in another HPLC system (Gilson Co., US). However, as shown in Fig. 6, the various mass peaks were not observed in the other HPLC system (UV: 254 nm). Overall, the [18F]fluoride (8.1–26 GBq in H18 2 O) from the target was directly delivered to the fluorine-18 reservoir (lower left in

Fig. 2) and transferred to the Chromafixs PS-HCO3 cartridge over 160 s for trapping. The trapped fluorine-18 and TBAHCO3 (10 mL) in MeOH/H2O (1.0/0.2 mL) from vial 1 were moved to the carbon reaction vessel. After complete drying for 315 s at 65–95 1C by vacuum and He gas flow, the tosylate precursor (2 mg) in CH3CN (1 mL) from vial 3 was added to the reactor and incubated at 100 1C for 10 min. After cooling to 40 1C by air flow, the HPLC solvent from vial 5 (1.5 mL) was added to the reactor to dilute the reaction mixture, which in turn was transferred to the HPLC injector vial (upper middle), after which the reactor was rinsed again with the HPLC solvent from vial 6 (1.5 mL). The combined solution in the HPLC injector vial was automatically injected into the HPLC system. The purification of HPLC was performed in 70% CH3CN/H2O containing 0.6% TEA at a flow rate of 7 mL/min, using a UV detector at 280 nm and a g-ray detector in the FXFN module. The fraction of [18F]fallypride collected from HPLC at around 12.5–13.5 min was transferred to vial 10 (lower right), which was filled with 100 mL of H2O. The diluted solution was moved to a tC18 Sep-Pak cartridge to trap only [18F]fallypride and to remove the clinically unavailable HPLC solvent. The cartridge was rinsed with 10 mL of H2O from vial 9 and then collected by 1 mL of EtOH from vial 8. Finally, 10 mL of saline from vial 7 was eluted to the sterile vial for adjustment of 10% ethanol/saline. Consequently, [18F]fallypride was obtained with a high radiochemical yield of about 68 71.6% (decay-corrected, n ¼42) within 51 71.2 min, the total synthesis time of which included HPLC purification and solid-phase extraction (for final formulation).

4. Conclusion In conclusion, we successfully obtained [18F]fallypride with high radiochemical yield (6871.6%, decay-corrected), purity (497%), and specific activity (140–192 GBq/mmol) using a commercial chemistry module without major modifications. The total synthesis time of [18F]fallypride, including HPLC purification and solid-phase purification, was much shorter than that of other procedures. This automated procedure for [18F]fallypride production could facilitate its routine clinical use in dopamine D2/D3 studies. The fluorine-18 extraction method using small amounts of base concentration in [18F]fallypride production is a useful example of an automated procedure on such base-sensitive precursors. The application of this method should encourage for fluorine-18 labeling in other automated syntheses.

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10,000

2283

HPLC Gamma Detector

8,000 6,000 4,000 2,000 0 HPLC UV Detector / AU

1,000 800 600 400 200 0 1

2

3

4

5

6

7

8

9 10 11 Time / min

12

13

14

15

16

17

18

19

20

Fig. 3. Raw HPLC profile from the FXFN module (upper: g-ray and bottom: UV—280 nm).

Fig. 4. The HPLC profile during co-injection of standard [19F]fallypride (Xterra RP-18; 50% CH3CN/H2O contained 0.6% TEA; flow rate: 3 mL/min and red: g-ray, black: UV—280 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

14,000 12,000 10,000 8,000 6,000 4,000 2,000 0

HPLC Gamma Detector

HPLC UV Detector / AU

1,000 800 600 400 200 0 1

2

3

4

5

6

7

8

9 10 11 Time / min

12

13

14

15

16

17

Fig. 5. Raw HPLC profile from the FXFN module (upper: g-ray and bottom: UV—254 nm).

18

19

20

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Fig. 6. The HPLC profile during re-injection of total collected [18F]fallypride (Gilson Co. HPLC system, Xterra RP-18; 50% CH3CN/H2O contained 0.6% TEA; flow rate: 3 mL/min and red: g-ray, black: UV—254 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

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