Automated synthesis of radiopharmaceuticals for positron emission tomography: an apparatus for [1-11C] labeled carboxylic acid

A fully automated apparatus for the synthesis and formulation of short-lived 11C (t1/2 = 20 min)-labeled carboxylic acids for positron emission tomograpy (PET) has been developed. Injectable solutions of [1-11C]acetic acid, [1-11C]octanoic acid and [1-11C]palmitic acid wilh radioactivities of 6.36-8.29 GBq, 0.070-1.43 GBq and 0.42-0.89 GBq were obtained. The preparation time was under 40 min after the end of bombardment. An automatic washing function means that labeled compound of the same or different kinds can be produced several times a day without any maintenance of the system. The control system is sited away from the ‘hot laboratory’, so operator exposure to radiation is minimized.


Introduction
Positron emission tomography (PET) is a non-invasive diagnostic imaging technique in which short-lived radioactive tracers are used to measure the anatomical distribution and kinetics of in vivo biochemical processes [1]. [1-C]Labeled carboxylic acids are useful radiotracers in PET studies; [1-C]labeled acetate and palmitate have been used extensively as tracers tbr monitoring cardiovascular fianctions [2][3][4]. Although a number of" semior fully-automated systems fbr the production of lC-labeled carboxylic acids have been developed [5][6][7][8][9][10][11], they have drawbacks in that they need manual washing befbre re-use and/or require the attendance of specialist operators. Thus, a reliable system, which would reproducibly deliver radiopharmaceuticals on a routine and repetitive basis, is required. 'l'he design and construction of a fully automated apparatus fbr producing [1-11C]labeled carboxylic acids is described in this paper. The apparatus has been applied to the repetitive production of [1-11C] acetic  Automated synthesis apparatus of [1-C]labeled carboxylic acid ltardware The automated apparatus consists of a synthesis system and a control system. The synthesis system, an automanual switch box, and I/O boards are placed in a Reagent supply unils The reagent supply unit has seven reservoirs (1-7 in figure 1) tbr reagent solutions and solvents. Each solution or solvent in the reservoirs is stored under argon atmosphere, and can be transferred to the flasks (8-11 in figure 1) in one or two steps. In the latter case, the liquid is allowed to flow fiom the reservoir into a volumetric tube by argon gas pressure. When a photosensor (31-37 in figure 1) on the tube detects the liquid, the valve at the lower end of the reservoir is switched to stop the flow of the liquid. The contents of the tube are transferred into the reaction flask by argon gas pressure. By the use of this volumetric device, the same volume of liquid may be repeatedly measured out and added to the flask; even moistureor air-sensitive liquids can be handled. After a synthetic run, all of the main flow lines and the flasks (8,9 and 13 in figure 1) can be washed by passing a washing solvent stored in reservoir 4, and then dried with dry nitrogen. In this way radiopharmaceuticals of the same or different kind can be produced during a day without interruption due to maintenance.

Reaction unit
The reaction unit has two flasks (8 and 9 in figure 1). Flask (8 in figure 1) carries out the Grignard coupling, and is placed in a dry box (45 in figure 1) to keep off any moisture. Flask 2 (9 in figure 1) quenches the reaction and evaporates the solvent. Both flasks are about 2 ml in volume and have jackets through which the heating/cooling fluid is circulated under temperature control by a thermostat (23 in figure 1). The mixing of" the reaction mixture in flask is accomplished by bubbling

Purification unit
The purification unit is composed of two systems: a degassor system which also serves as a part of an injecting system into the high performance liquid chromatography (HPLC) column and an HPLC system. The degassor system consists of a degassing flask (13 in figure 1; about 2 ml in volume), a photosensor (38 in figure 1), a rotary six-way valve (17 in figure 1) and a sample loop (18 in figure 1). The degassing flask has a jacket through which a heating/cooling fluid is circulated. The HPLC system consists of a pump (16 in figure 1), an HPLC column (19 in figure 1), a UV detector (20 in figure 1) and a radiation detector (22 in figure 1). The concentrate obtained in flask 2 is transtirred to the degassing flask, degassed and then sent to the sample loop of an HPLC apparatus, through the photosensor (38) and a rotary six-way valve. When the photosensor (38) detects the end of the solution, the rotary six-way valve rotates its position automatically and the mixture in the sample loop is then loaded to the HPLC column by the HPLC pump (16). The fraction which contains the product is detected by the radiation detector (22) and collected in a flask (10 in figure 1).

Pharmacological formulation unit
The pharmacological formulation unit has two flasks (10 and 11 in figure 1) and one vial. Flask 3 (10 in figure 1) has a jacket which is thermostated by circulating the heating/cooling fluid through it. The contents of flask 3 are concentrated under reduced pressure stirred with a magnetic stirrer (27 in figure 1) and heated in order to remove any organic solvent. The residual solution in flask 3 is transferred to flask 4 (11 in figure 1), which is equipped with a magnetic stirrer (28 in figure 1), a pH sensor (29 in figure 1) a.nd a level sensor (30 in figure 1). The pH and volume of the solution can be adjusted in flask 4 to the desired values by adding an alkaline solution, an albumin solution or saline. The resultant solution is filtered through a 0.22 gm membrane filter (41 in figure 1) into a product vial (12 in figure 1) to give a solution ofa 11C-labeled compound in a ready-to-use form for a PET study after an appropriate quality assurance procedure.

Control system
Computer and interface hardware The apparatus is controlled with a personal computer (PC-9821Ap, NEC); an adaptor (RS422) in the computer controls I/O boards, Optomux (Opto22, USA). Six Optomux digital and two analogue brain boards, each with 16 I/O channels, are used. Each board has a unique address to help identify every device in the system, for example five digital output boards give 80 switches for operating valves and relays. One digital input board reads the status of 16 indicator lamps that are used for monitoring photosensors, the level sensor, and the position of the rotary six-way valve. The analogue boards are for reading status information from the system, including radioactivity, pH, UV absorbance, the rates of the mass flow controllers and pressure, and for writing information to set rates of the mass flow controllers.

Soflware
The software was developed with a development program called 'Hyakuninriki' (Asahi Electronics Co. Ltd, Japan), which operates under MS-DOS. The software has functions tbr control logic and graphic displays. The program consists of a series of connected control blocks with the flow depending on the control logic sequence. More than 140 control blocks of 16 different types are used and each block is programmed to perform a function, such as controlling a switching sequence or reading the status of sensors. The program consists of five processes, for example preparation of the apparatus, Grignard reaction, purification, formulation and washing processes (see figure 2). The preparation of the apparatus process is controlled by the method of close loop and a time sequential method. The Grignard reaction process and the purification process are performe d by sequential control using a signal from the photosensor and a time sequential method. The formulation process is controlled by the method of close loop, and the washing process is controlled by the method of close loop, sequential control using the signal of photosensors and a time sequential method.
Operating procedure Layout of the hot experiment zone The layout of the hot-experiment zone is shown in figure 3; the four rooms used (cyclotron room, hot laboratory, pass room and control room) are isolated from each other. The values of the radioisotope calibrators can be monitored with two video cameras in the control room.

Preparation for synthesis
The reservoirs (1-7 in figure 1) of the apparatus are filled as follows: reservoir 1-3 (0"2M solution of Grignard reagents in anhydrous THF), reservoir 4 (anhydrous THF), reservoir 5 (a mixture of 1N HC1 and the eluent for HPLC 1:1 (v/v)), reservoir 6 (7 NaHCO 3 solution in the case of acetic and octanoic acid or an albumin solution in the case of palmitic acid), reservoir 7 (saline).

Quenching and evaporalion
The power switches of the vacuum pump and the magnetic stirrer (26 in figure 1) are turned on. Then valves V30 and V40 are switched, and the reaction mixture is concentrated under reduced pressure. After evaporation, the vacuum pump is stopped, and in order to break the vacuum, valve V29 is ;witched. The residue in flask 2 is dissolved in 2"0 ml of a solution from reservoir 5 in a similar manner (flow line: 46-43-5-V15-35-9-V29) as above.

Purijicalion and formulalion
The product of the previous step is purified and separated via the degassor system and HPLC system as follows" The pH value is adjusted, if necessary, to the range from 8"5 to 9"0 by the addition of the NaHCO a solution from reservoir 6 and the resulting solution in flask 4 is then diluted with saline from reservoir 7 by adding it to the solution until the level sensor, which is set beforehand to a volume of 10 ml, is activated. Finally the product is filtered through a 0"22 gm sterile filter by applying argon gas pressure (flow line: 46-43-V19-V21-V34-V35-Vll-41-12) into a sealed vial placed in a radioisotope calibrator measuring the radioactivity of the final product in passroom (figure 2). The product is then analysed by an analytical HPLC to confirm the quality.  The apparatus washes automatically, so labeled compounds of the same or different kinds can be produced several times during a day without maintenance of the system. Table 2  The reservoirs of the apparatus were filled as follows (the quantities in parentheses are those used for one synthesis)--reservoir 1:0"2 M CH3MgBr/THF (0"5 ml), reservoir 4: THF (1"0 ml), reservoir 5: a mixture of 1N HC1 and 0.025 M NaC1 (1:1 (v/v)) (0"5 ml), reservoir 6: 7 NaHCO3a q. (2"0 ml), reservoir 7: saline (5"0 ml). The fluid in the thermostat (23 in figure 1) was cooled to the temperature of-26C, and valves V44 and V45 were switched in order to cool flask only. The HPLC conditions used were for preparation, column: Waters protein pack G-QA 20 I.D.*100mm, eluent: 0"025M NaC1, flow rate: 5"0 ml/min, detector (UV): 214 nm, temperature: room temperature, retention time: c. 11 min and radiodetector: Aloka Positron Monitor TCS-R81-3454 and for analysis, column: YMC-Pack ODS-AQ, AQ-303 4"6 I.D.*250mm, eluent: 20mM H3PO4-NaH2PO4 (pH2"8), flow rate: 0"7 ml/min, detector (UV): 214 nm, temperature: room temperature, retention time: c. 8"5 min, and radiodetector: Aloka Gamma Detector FGD-102 and Radio Analyser RLC700.

Production of [l_11C]palmitic acid
The reservoirs of the apparatus were filled as follows, (the quantities in parentheses are those used in one synthesis)reservoir 3:0"2 M C15H31MgBr/THF (0"5 ml), reservoir 4: THF (1"0 ml), reservoir 5: a mixture of 1N HC1 and CH3CN: H20 95:7 (v/v) (0"5 ml), reservoir 6: Albumin solution (2"0 ml), reservoir 7: saline (5"0 ml). The fluid in the thermostat (23 in figure 1) was warmed to the temperature of 56C, and the outlet of it was opened by switching valve V45. Flask 1, 2 and the degas flask were warmed to the same temperature. The HPLC conditions All the conditions are the same as above for the production of [1-lC]octanoic acid. The apparatus is washed between sequential production of[ 1-1 C-] octanoic acids.