Improved Stability and Practicality for Synthesis of 4-Borono-2-[18F]fluoro-l-phenylalanine by Combination of [18O]O2 Single-Use and [18F]CH3COOF Labeling Agents

Purpose 4-Borono-2-[18F]fluoro-l-phenylalanine ([18F]FBPA) synthesized with [18F]F2, produced using the 18O(p, n)18F reaction, has been reported for increasing radioactivity. However, a dedicated system and complex procedure is required to reuse the costly [18O]O2 gas; also, the use of [18F]F2 as a labeling agent reduces the labeling rate and radiochemical purity. We developed a stable and practical method for [18F]FBPA synthesis by combining [18F]F2, produced using a [18O]O2 single-use system, and a [18F]CH3COOF labeling agent. Methods The produced [18F]F2 was optimized, and then [18F]FBPA was synthesized. For passivation of the target box, 0.5% F2 was pre-irradiated in argon. Gaseous products were discarded; the target box was filled with [18O]O2 gas, and then irradiated (first irradiation). Then, the [18O]O2 gas was discarded, 0.05–0.08% F2 in argon was fed into the target box, and it was again irradiated (second irradiation). The [18F]F2 obtained after this was passed through a CH3COONa column, converting it into the [18F]CH3COOF labeling agent, which was then used for [18F]FBPA synthesis. Results The mean amount of as-obtained [18F]F2 was 55.0 ± 3.3 GBq and that of as-obtained [18F]CH3COOF was 21.6 ± 1.4 GBq after the bombardment. The radioactivity and the radiochemical yield based on [18F]F2 of [18F]FBPA were 4.72 ± 0.34 GBq and 12.2 ± 0.1%, respectively. The radiochemical purity and molar activity were 99.3 ± 0.1% and 231 ± 22 GBq/mmol, respectively. Conclusion We developed a method for [18F]FBPA production, which is more stable and practical compared with the method using [18O]O2 gas-recycling and [18F]F2 labeling agent.


Introduction
Boron neutron capture therapy (BNCT) is an anti-cancer treatment that is based on the 10 B(n, α) 7 Li reaction in tumors, and the high linear energy transfer of the α and 7 Li particles generated upon the irradiation of 10 B [1][2][3][4]. Recently, the BNCT system was approved as a medical device for neutron irradiation, and 10 B-4-borono-L-phenylalanine ( 10 BPA) was approved as a 10 B-carrier-drug for cancer cells, by the Ministry of Health, Labour and Welfare in Japan. Before neutron irradiation, the amount of 10 B present in tumors and the surrounding normal tissues was typically evaluated using positron emission tomography (PET). Subsequently, 4-borono-2-[ 18 F]fluoro-L-phenylalanine ([ 18 F]FBPA) [5][6][7][8] was chosen because the distribution of [ 18 F]FBPA in a tracer dose was correlated with that of a therapeutic dose of 10 BPA in both rats [9,10] and humans [7]. The availability of [ 18 F]FBPA is critical to the successful determination of the cellular 10 B levels to ensure treatment efficacy, and several approaches have been reported to address its synthesis. The [ 18 F]FBPA synthesis developed by Ishiwata et al. [11] involves an electrophilic substitution reaction using [ 18 F]CH 3 COOF or [ 18 F]F 2 for the direct 18 F-labeling of the aromatic ring. In their process, Ishiwata et al. employed the 20 Ne(d, α) 18 F nuclear reaction involving a small cross-section. By employing 20 Ne(d, α) 18 F as the nuclear reaction, and [ 18 F]CH 3 COOF as the labeling agent, the amount of radioactivity of the generated [ 18 F]FBPA was 1200 ± 160 MBq by irradiation for 120 min [12]. When using an alternate method, [ 18 F]F 2 gas was produced by using two-step proton beam irradiation and the nuclear reaction of 18 O(p, n) 18 [13,14]. The use of this method allowed the production of large amounts of [ 18 F]F 2 (102 ± 27 GBq) and [ 18 F]FBPA (5.3 ± 1.2 GBq) [15], though it required a dedicated recycling system to recover the costly [ 18 O]O 2 gas and a complicated procedure [13,14]. In addition, [ 18 F]F 2 was used as a labeling agent in a previous study [15]; however, it was reported that the direct electrophilic substitution of aromatic ring in BPA with extremely reactive [ 18 F]F 2 resulted in a lower 18 [11,12].
The purpose of this study was to combine the advantages of these two methods and establish a more practical and stable synthesis process. That is, it involved the development of an efficient and economical 18

Cyclotron and Target Material
An energy proton beam of 18 MeV was obtained using CYPRIS-HM-18 (Sumitomo Heavy Industries, Tokyo, Japan). An aluminum (Al) target, originally used for the production of [ 11 C]CO 2 , was employed as is for the [ 18 F]F 2 production. This target was conical in shape with a length of 154 mm, a front and back diameter of 20 and 30 mm, respectively, and a volume of 75 mL. The 18-MeV proton beam was decelerated to 14.4 MeV by passing it through a vacuum foil made of 10 μm thickness Havar and a target foil made of 600 μm thickness aluminum. Enriched oxygen-[ 18 O]O 2 gas (>98 atom%) was used as the target gas. Argon gas (>99.99995% pure) and argon gas mixed with 2% F 2 gas were used for the passivation and recovery of the adsorbed [ 18 F]F 2 gas, respectively (Taiyo Nippon Sanso Corporation, Tokyo, Japan).

Target System
The target box was connected via three gas supply lines to the gas cylinders, with each line incorporating switching valves (Fig. 1) (3) argon gas for 2% F 2 gas dilution and target system purging. The stainless-steel piping of the inlet and outlet lines, solenoid valves, and pressure gauges were used to construct this system. The valves were switched manually. The filling gas pressure, irradiation, and gas transfer to the synthesizer were controlled using an automated synthesis control system (Cupid system, Sumitomo Heavy Industries, Tokyo, Japan).

Pre-irradiation of Target for Passivation
In this study, we employed a two-step irradiation method for the production of [ 18 F]F 2 [13,14]. First, preirradiation was carried out for 10 min to passivate the surface of the target box with 0.5% fluorine in argon at a set pressure of 14.8 kg/cm 2 and a proton beam of 17 μA. This operation is important not only for the passivation of the Al target but also for removing any contamination from the target [14,16,17]. Gaseous products from the pre-irradiation sequence were passed through a synthesizer installed in a hot-cell and then discarded. After the argon purge, the target pressure was reduced to atmospheric pressure.

Confirmation of [ 18 F]F 2 Amount at Each Irradiation Step After Passivation
After pre-irradiation with 0.5% F 2 mixed with argon, the first irradiation with [ 18 O]O 2 gas at 25 μA for 10 min and the second irradiation at 17 μA for 10 min with 0.03-0.9% F 2 mixed with argon were carried out. The second irradiation was repeated twice (total three times), which were the third and fourth irradiations in the sequence according to the calculation of the total amount of [ 18 F]F 2 in the target box. The radioactivity of the gaseous product was preserved using a charcoal and soda-lime column attached to the waste line and both columns were measured at the same time using a dose-calibrator. The recovery rate of [ 18 F]F 2 from the target in each irradiation step was calculated as the sum of the radioactivity obtained at each irradiation step.

Optimization of Proton Beam Current, Irradiation Time, and Fluorine Gas Concentration
A target box with a larger volume than that used in previous studies for [ 18 F]F 2 production [14,16,17] was selected; therefore, the irradiation conditions and F 2 gas concentration for the recovery of [ 18 F]F 2 were optimized accordingly. During the second irradiation, the proton beam irradiation for 10 min at 5 μA or 17 μA and for 2, 10, or 20 min at 17 μA was used for the irradiation of argon with 0.05-0.08% F 2 at 14.8 kg/ cm 2 . In addition, variations in the F 2 gas concentration in the 0-0.9% range (amounts in the range 0-448 μmol) were also investigated by irradiation at 17 μA for 10 min.

Production of [ 18 F]F 2 Gas for [ 18 F]FBPA Synthesis
After the pre-irradiation, the target box was filled with [ 18 O]O 2 gas at a pressure of 15.0 kg/cm 2 and then irradiated with a proton beam current of 30 μA for 150 min (first irradiation). The resulting initially irradiated [ 18 O]O 2 gas was discarded via the same route used during the pre-irradiation step without any recycling. The target box was then filled with a 0.05-0.08% mixture of F 2 gas in argon at a pressure of 14.8 kg/cm 2 and was irradiated at a proton beam current of 17 μA for 20 min (second irradiation). Thereafter, the irradiated gas was collected and used to synthesize [ 18 F]FBPA.

Synthesis of [ 18 F]FBPA Solution
The [ 18 F]F 2 -containing gas obtained after the second irradiation was passed through a CH 3 COONa column, resulting in the conversion of [ 18 F]F 2 to [ 18 F]CH 3 COOF and introduced into the reactor at a constant flow rate of 300 mL/min. The [ 18 F]FBPA was synthesized with a cassette-type synthesizer CFN-MPS200 (Sumitomo Heavy Industries, Tokyo, Japan), as described in previous reports [11,12]. The [ 18 F]CH 3 COOF was introduced into a solution of 30 mg of 4-borono-L-phenylalanine (>97%, Matrix Scientific, Columbia, USA) dissolved in 4 mL of trifluoroacetic acid (TFA) at room temperature (Fig. 2). Subsequently, TFA was removed from the reactor under reduced pressure, while maintaining a N 2 gas flow rate of 200 mL/min at 120°C. The resulting residue was dissolved in 2 mL of 0.1% aqueous acetic acid and injected into a high-performance liquid chromatography (HPLC) column (YMC-Pack ODS-A 20 mm × 150 mm HPLC column  [18], and were as follows: volume per batch, radioactivity, half-life, appearance (color and particles), endotoxin levels, sterility, pH, radionuclidic identity, radionuclidic purity, radiochemical purity, and residual solvent amounts (ethanol, acetic acid, and TFA). For the carrier amount of FBPA, the maximum dose per patient was set to <5 mg which is sufficiently safe, based on the doses in precious reports [19], and the administrable injection volume was calculated. HPLC for analysis was performed with YMC-Pack ODS AQ 4.6 mm × 150.0 mm column (YMC, Kyoto, Japan) and 50 mmol/L NaH 2 PO 4 solution as eluent. The radiochemical purity and carrier amount of FBPA were measured using a radioactivity detector and UV detector at 280 nm, respectively, with a flow rate of 1.5 mL/ min. The FBPA standard used for the measurement of the carrier amount was provided by Osaka Prefecture University. In the residual solvent test, acetic acid and TFA were used under the same conditions as the carrier amount of FBPA was measured at UV 210 nm and a flow rate of 0.5 mL/ min. Residual ethanol was measured using gas chromatography with TSG-1 15% SHINCARBON A 60/80 (3.1 m × 3.2 mm I.D., Shimadzu, Kyoto, Japan). The injection port, column, and flame ionization detector temperature were set to 180°C, 90°C, and 180°C, respectively. The carrier gas was nitrogen, while the flow rate was maintained at 30 mL/ min. The pH value was determined by potentiometry using a F-72 pH/ion meter calibrated with a standard pH solution (Horiba, Kyoto, Japan), and the endotoxin test was carried out using a Toxinometer® ET-6000 (FUJIFILM Wako Pure Chemical). In addition, the enantiomeric purity of [ 18 F]FBPA was evaluated by chiral HPLC with a Crownpak CR (-) 4.0 mm × 150 mm column (Daicel, Tokyo, Japan) and a perchloric acid aqueous solution (pH 2.0) at 1.0 mL/min at 25°C.

Results
After the surface passivation in the target box with pre-irradiation, the recovery rate of [ 18 F]F 2 after the first, second, third, and fourth irradiations was 3%, 75%, 16%, and 6%, respectively, calculated from the total radioactivity. Figure 3 depicts the relationship between the radioactivity and proton irradiation time as well as that between the radioactivity and proton beam current in the second irradiation. The radioactivity of the collected [ 18 F]F 2 increased with increase in the proton irradiation time and the proton beam current. Figure 4 depicts the relationship between the radioactivity of [ 18 F]F 2 and the concentration of the F 2 added in the argon gas. When only argon gas (without F 2 gas) was used, a negligible amount of [ 18 F]F 2 was collected. However, the radioactivity of the [ 18 F]F 2 was constant when the concentration of the added F 2 gas was increased up to 0.9%.
In  Figure 5). The nonradioactive FBPA content was 0.3-0.4 mg/mL, and its molar activity was 231 ± 22 GBq/mmol. The ethanol, acetic acid and TFA as the residual solvent were <15 ppm, 38 ± 15 ppm, and < 15 ppm, respectively. All other quality control parameters satisfied the specification criteria. In addition, the optical purity of the resulting [ 18 F]FBPA solution with the same synthesis protocol was >99 % (Fig. 6).

Discussion
We developed a method for producing [  gas was mostly recycled. Therefore, a large amount of installation cost and continuous maintenance was required; thus, the production of [ 18 F]F 2 using 18 O 2 gas has been possible only in limited facilities. On the other hand, we have constructed that a new one-way gas filling system using [ 18 ]O 2 gas, similar to the irradiation system for C-11 production, was possible to obtain stable radioactivity with high radiochemical purity by combining this system with [ 18 F]CH 3 COOF. In addition, as an improvement of practicality, we have elucidated that by using a target box for [ 11 C]CO 2 , the synthesis using [ 18 F]F 2 including [ 18 F]FBPA can be performed for clinical use at many PET cyclotron facilities where proton beam can be irradiated.
In the method originally developed by Ishiwata et al., a radioactivity of 0.85 ± 0.20 GBq, a molar activity of 54 ± 7 GBq/mmol, and a radiochemical purity of 99.5 ± 0.4% (at EOS) were obtained for [ 18 F]FBPA, based on data obtained from the daily production at Osaka University Hospital (n = 21) (unpublished data). The proposed method improved the radioactivity five-fold, the molar activity 4-fold, and obtains equivalent highly radiochemical purity of [ 18   method adopted in this study, allows the study of 7-8 patients using one PET/CT scanner. Furthermore, in this study, we employed 0.1% aqueous acetic acid, as reported by Ishiwata et al., as the eluent for HPLC [11]. However, when 1 mM phosphate-buffered saline (PBS), pH = 6.7, was used as an eluent for the same purification, which does not require the additional eluent-evaporation step, and affords a 32 min synthesis time for the final [ 18 F]FBPA solution [12]. The application of the PBS eluent to the present study could further increase the radioactivity by 16% due to the reduced synthesis time.
The key bottleneck in synthesizing a large amount of [ 18 F]FBPA was the lack of a suitable method for producing large amounts of [ 18 F]F 2 . A number of focused studies have been reported on the irradiation methods, target box materials, and the constitution of the target gas required for [ 18 F]F 2 production [13,14,16,17]. In their [ 18 F]F 2 production process with 18 O(p, n) 18 F, Bishop et al. reported that approximately 13% of the generated [ 18 F]F 2 was wasted after the first irradiation and 87% remained in the target system [14]. After the second, third, and fourth irradiations, the amounts of [ 18 F]F 2 were 54%, 23%, and 10% of the total [ 18 F]F 2 , respectively. In this study, 3% of [ 18 F]F 2 was collected after the first irradiation, such that 97% of the [ 18 F]F 2 remained in the target system before the second irradiation. After the second, third, and fourth irradiations, the collected amounts of [ 18 F]F 2 were 75%, 16%, and 6%, respectively. In both studies, the second irradiation process consistently provided the highest fraction of [ 18 F]F 2 . The reason for the difference observed between Bishop's (54%) and our study (75%) may be attributed to the difference in the passivation conditions for the preirradiation (concentration of F 2 gas in argon; 100 μmol vs. 224 μmol, proton beam current; 10 μA vs. 17 μA, respectively) or contamination of F 2 into [ 18  In the second irradiation, the concentration of the F 2 carrier gas was also important to the release of the adherent [ 18 F]F 2 . Hess et al. reported that a reduction in the F 2 carrier gas concentration was associated with a decrease in the [ 18 F]F 2 production and an increase in molar activity [18]. In this study,  [11]. This means that the production of radioactive byproducts in the [ 18 F]FBPA produced using [ 18 F]CH 3 COOF is decreased and could be almost separated from the radioactive byproducts (the separation HPLC chromatogram is presented in Fig. 7),  [12]. Similarly, our study showed that the same result was obtained (unpublished data). The higher radiochemical purity obtained in this study (99.3 ± 0.1%) than that obtained by using [ 18 F]F 2 as the labeling agent (98 ± 1%) [15] may be attributed to the differences in the variety and quantity of radioactive byproducts obtained using the two processes.
Furthermore, it has been reported that the synthesis of [ 18 F]FBPA was performed by using [ 18 F]F 2 produced from [ 18 F]fluoride [21][22][23]. This method has the advantage of providing high molar activity (0.9-1.5 GBq/μmol); however, it is not suitable for regular use because of the low labeling rate and the need for complex manufacturing systems and radioactivity detector for measurement of radio-gases [22].
In a [ 18 F]FBPA synthesis, the radio-optical purity and its mass production were important, and our group had previously reported on the superiority of L-[ 18    in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.