Neutral pH Formulation for 6-[18F]uoro- (cid:0) DOPA With High Radiochemical Stability

6-[ 18 F]Fluoro-L-DOPA(FDOPA) has always been generally produced under strong acidic conditions, i.e. as an injectable solution of pH 2~3, due to its low stability at a higher or neutral pH. This necessitates the pre-treatment neutralization of this agent with an injectable NaHCO 3 solution. We have developed a neutral pH formulation for [ 18 F]FDOPA using ethanol (EtOH) and phosphate buffer to overcome the radioactive and enantiomeric stability problems at a higher pH. Upon [ 18 F]FDOPA generation by nucleophilic substitution methods, we investigated its radiochemical and enantiomeric purity in accordance with the various pH after 6 hours. After EtOH and three kinds of buffer were added, we further examined this purity at pH 6 ~ 7 after 6 hours. The ascorbic acid did not stabilize the radiochemical purity at the higher pH. A 5% EtOH and PBS buffer matrix produced the best stability for radiochemical and enantiomeric purity at pH 6.5 at the 6 hour time point. This combination maintained a > 95% radiochemical and enantiomeric purity at 6 hours after EOS (end of synthesis). Our new formulation for [ 18 F]FDOPA thus showed a high stability at neutral pH and satised QC requirements which was listed in European Pharmacopeia. It has also been approved by the Korean Ministry of Food and Drug Safety.

Regardless of the production method, the nal [ 18 F]FDOPA injectable solution has always needed to have a very low pH of between 2.0 and 3.0 because of the instability of this radiochemical at a higher pH.
Acidic solutions lead to severe pain however when directly injected into patients. Hence, [ 18 F]FDOPA solutions must be immediately neutralized with an 84 mg/mL NaHCO 3 solution prior to injection. After this neutralization step, the [ 18 F]FDOPA injectable solution must be stored in a refrigerator and used within 3 hours [15]. A previous study has reported that conventional [ 18 F]FDOPA preparations undergo decomposition via oxidative free-radical reactions and that subsequent imaging quality will be reduced by any free [ 18 F] uoride formation that had not been completely removed during HPLC puri cation [13].
There have been no prior reports however that have offered solutions to these problems. In our present study, we describe our new formulation for [ 18 F]FDOPA in the mixture of ethanol (EtOH) and buffer Page 3/9 solution at a neutral pH but shows long-term stability without de- F]FDOPA preparation were supplied by Trasis and production procedures were as previously described [13,14]. We only added 0.1% acetic acid containing 0.2 mg/mL of ascorbic acid to the mobile phase for HPLC puri cation, and these solutions were freshly prepared prior to the synthesis reactions. The starting radioactivity was 72.3±13.5 GBq. Following the production of [ 18 F]FDOPA, the radiochemical and enantiomeric purities were checked using reverse phase-high performance liquid chromatography (RP-HPLC) and chiral-HPLC, respectively. The analytical HPLC conditions were as follows: 0.1% acetic acid: methanol = 97:3 v/v, ow rate = 0.8 mL/min, Luna C 18 column (250 × 4.6 mm), and monitoring at 280 nm and via a radioactivity detector. Enantiomeric purity was evaluated using an Astec CHIROBIOTIC column (250 × 4.6 mm) and mobile phase system, i.e. 0.01 M NaH 2 PO 4 :CH 3 CN = 20:80 v/v, ow rate = 1.0 mL/min. The [ 18 F] uoride content and pH were determined using radio thin layer chromatography (radio-TLC) and a pH meter, respectively. A pH of between 2-3 was used to maintain radiochemical purity after production.

1) Addition of EtOH
After the preparation of the puri ed [ 18 F]FDOPA from RP-HPLC, we added ethanol up to a maximum concentration of 30% and evaluated its effect as an additional radiochemical stability agent. We then neutralized the resulting solution with 8.4% NaHCO 3 to obtain a pH of 6, 7, or 8. The radiochemical purity was evaluated by RP-HPLC at 0, 2, 4, and 6 hours after production.

2) Effects of 5% EtOH and temperature
After evaluation of the ethanol concentration effects, we also evaluated temperature effects at a 5% EtOH concentration. These conditions were tested at both pH 2 and pH 7 at 25 °C. We also tested a pH 7 solution containing 5% EtOH at 4 °C to detect any temperature effects after neutralization. The stability of the [ 18 F]FDOPA under these conditions was measured through the formation of free [ 18 F] uoride detected by radio-TLC [16].

3) Effects of 5% EtOH and different buffer solutions
After evaluating the effects of the ethanol concentration and temperature at pH 7, we further investigated the effects of different buffers on the stability of the [ 18 F]FDOPA compound in 5% EtOH, pH 7 and 25 °C. We tested 5 mg/mL of concentration phosphate-buffered saline (PBS), phosphate buffer, and citrate buffer. PBS was prepared by dilution of a 10 × PBS stock solution. The phosphate buffer was prepared by adding 0.558 g of Na 2 HPO 4 and 0.603 g of NaCl to a nal volume of 100 mL of deionized water. A 100 mL citrate buffer solution was prepared by the addition of 2.016 g of sodium hydrogen citrate sesquihydrate and 11.512 g of sodium citrate tribasic dihydrate. After mixing the [ 18 F]FDOPA preparation with each buffer solution, we again monitored [ 18 F] uoride formation by radio-TLC.

4) Evaluation of the nal formulation of [ 18 F]FDOPA
After preparation of the puri ed [ 18 F]FDOPA and formulation with 5% EtOH and PBS buffer, we neutralized the solution with 8.4% NaHCO 3 to obtain a pH of 6.5-7.0. The radiochemical and enantiomeric purities were then checked using RP-HPLC and chiral-HPLC, respectively, for 0, 2, 4, and 6 h. The formation of [ 18 F] uoride was also monitored by radio-TLC (n = 3 for each condition).
High radioactivity production, quality control and stability evaluation of the neutral pH injectable [ 18 F]FDOPA solution We synthesized 13 batches of [ 18 F]FDOPA with our new formulation such as PBS containing 5% EtOH.
The starting radioactivity was 126.1±39.2 GBq. We performed quality control (QC) tests on all solutions including appearance, radionuclide identity and purity, pH, radiochemical identity and purity, radiochemical impurity, enantiomeric purity, molar activity, residual solvent and EtOH, bacterial endotoxin, and sterility. We also evaluated the long time stability of 3 batches up to 6 h after preparation.

Effects of ethanol addition at various pH levels
Two kinds of radiochemical impurities were detected by RP-HPLC, based on increases in both the pH and retention time (Figure 1). At 2 hours after the addition of the 8.4% NaHCO 3 solution, the radiochemical purity dramatically decreased at pH 6 without EtOH. Over a pH range of 6-8, solutions containing 1-5% EtOH concentrations showed 10% more stability than those without EtOH up to 6 h. For the 5% EtOH solutions, the radiochemical purity at pH 6 and 7 was above 90% up to 6 h and 4 h, respectively. The decreased radiochemical purity in the 1% EtOH solution was comparable between the pH 7 and 8 solutions. Without EtOH however, the rate of decomposition of [ 18 F]FDOPA was greater at pH 8 compared with pH 7.
Interestingly, no free [ 18 F] uoride was detected by RP-HPLC. Additionally, the retention time of free [ 18 F] uoride was below 3.0 min under our analysis conditions [13]. Hence, we considered the impurities detected by RP-HPLC at the pH range of 6-8 to be unknown radioactive substances. Furthermore, the enantiomeric purity showed no decrease, and the unknown radiochemical impurities detected RP-HPLC were not detected by chiral-HPLC.

Evaluation of [ 18 F] uoride content by radio-TLC.
We performed radio-TLC analysis because we could not measure the exact amount of free [ 18 F] uoride by RP-HPLC. [ 18 F]Fluoride formation was monitored by radio-TLC in the [ 18 F]FDOPA solution before and after neutralization according previously described method [13]. Without neutralization, the [ 18 F] uoride concentration did not increase until 6 hours. However, as soon as the solution was neutralized (pH=6-7),  (Figure 4). Both the phosphate and citrate buffers also suppressed the formation of [ 18 F] uoride, but to a lesser degree as the [ 18 F] uoride levels still continuously increased up to 5% and 10%, respectively.
High radioactivity production, quality control and stability of the neutral pH injectable [ 18 F]FDOPA solution The radiochemical yield of our newly formulated [ 18 F]FDOPA was 30.4±6.1%. All of the QC parameters for this new preparation met the EP(European Pharmacopeia) criteria. The radiochemical and enantiomeric purities were 100% and 95.8±0.8%, respectively, at EOS. The molar activity was 54.7-15679.7 TBq/mmol at EOS. The ethanol concentration was 4.0 ± 0.38%, and no residual solvents were detected. Although we used EtOH at a 5% concentration in this formulation, it was only detected at 4.0 ± 0.38% in the GC analysis after sterile ltering and delivery through the instrument tubing. The radiochemical and chiral purities at 6 hours after EOS were 99.0 ± 0.46% and 98.54 ± 0.75%, respectively. No additional radiochemical impurities formed during the 6 hour period after preparation at pH 6.5-7.0.

Discussion
We have developed and evaluated a new formulation for [ 18 F]FDOPA that achieves a high stability of this compound at a neutral pH (pH 6 ~ 7). We have found that a mixture of EtOH and PBS e ciently suppresses the formation of radiochemical impurities and [ 18 F] uoride or 6 hours at a neutral pH and 25 °C. These conditions are very amenable for the clinical administration of [ 18 F]FDOPA because no cumbersome neutralization procedures are required prior to injection. In addition, a high degree of radiochemical purity is guaranteed even at a neutral pH during the preparation and transportation of this radioactive compound.
The de-[ 18 F] uorination rate of previous formulations of [ 18 F]FDOPA was previously reported to be very rapid after neutralization, with [ 18 F] uoride levels of 10% detectable within 10 min [13]. From our own clinical experience (data not shown), freshly produced [ 18 F]FDOPA in a pH 2 solution does not show any issues when it is injected immediately after neutralization [13]. Notably however, we have occasionally obtained poor quality images after neutralization, as reported also by others. In our present study, we found that EtOH was su cient to prevent the formation of radiochemical impurities, as detected by RP-HPLC, but we could not detect free [ 18 F] uoride using RP-HPLC (Fig. 1E).
However, at pH 7 and a temperature of 25 °C (Fig. 2A), a 65% [ 18 F] uoride concentration was detectable by radio-TLC. A lower temperature more e ciently decreased the formation of [ 18 F] uoride than EtOH but at 60 min after neutralization, the [ 18 F] uoride content exceeded 5%. We also could not con rm by radio-TLC analysis whether the radiochemical impurities were free [ 18 F] uoride or a mixture of free [ 18 F] uoride and other [ 18 F]labeled impurities despite performing this modality in accordance with previously described procedures.
EtOH showed good results in terms of radiochemical stability, which was better at 10% EtOH than at 5% EtOH. A 10% concentration is too high for intravenous injection however as it may lead to severe pain [17]. Moreover, storing the solution at 4 °C and at pH 7 showed better results than 5% EtOH alone. Based on these ndings, we sought another stabilization method for [ 18 F]FDOPA and decided upon a buffer solution in combination with 5% EtOH. PBS and citrate buffer produced more stability for [ 18 F]FDOPA than phosphate buffers. Moreover, a formulation with two kinds of buffers did not show a linear increase in the concentration of [ 18 F] uoride with time, which was observed with the phosphate and citrate buffers (R 2 value: 0.99 and 0.90, respectively).
This new formulation with 5% EtOH and PBS showed very stable results for 6 hours at a pH of 6.5-7.0 and all radiochemical purities detected using RP-HPLC, chiral-HPLC, and radio-TLC were at levels under 3%.
Although the mechanism preventing the formation of [ 18 F] uoride and other radiochemical impurities in a PBS or citrate buffer and 5% EtOH mixture is uncertain, it is likely that PBS and citrate buffer reacts with [ 18 F]FDOPA and causes the formation of a more stable form of this radiochemical compound, even at neutral pH, and that EtOH prevents the formation of free radicals.
Using our newly developed formulation, we can now dispense with the neutralized [ 18 F]FDOPA solutions and prepare multi-dose vials that can be directly injected into patients. This will be particularly important advantage in terms of quality control when supplying other small medical centers with this radiochemical compound because manufacturers or supply centers cannot control the neutralization steps or storage conditions used at external injection sites.