[11C]Carbon dioxide was generated from a nitrogen/oxygen (1000 ppm) target (14N(p,α)11C) using an EBCO TR 19 cyclotron (Advanced Cyclotron Systems INC. Richmond, Canada). High performance liquid chromatography (HPLC) purification was performed by a Knauer HPLC system (Sonntek Inc., Woodcliff Lake, NJ, USA) with a model K-5000 pump, a Rheodyne 7125 injector, a model 87 variable wavelength monitor, and a NaI radioactivity detector.
Specific activity was determined by measuring the radioactivity and the mass; the latter was derived from a standard curve (UV absorbance at 254 nm by peak area) after HPLC injection of different quantities of the authentic reference compound. Radiochemical purity was determined by HPLC and verified by thin-layer chromatography (TLC) using and measuring radioactivity distribution on Macherey–Nagel polygram sil G/UV254 plastic-backed TLC plates with a Bioscan system 200 imaging scanner (Bioscan Inc., Washington, DC). 11C-Radioactivity was measured by a MINAXI γ 5000 automated gamma counter (Packard Instrument, Meriden, CT). All measurements were decay corrected. All chemicals were purchased from Sigma-Aldrich, with the exception of anhydrous DMF, which was obtained from Acros Organics (USA).
4.2.1. Synthesis of Labeling Precursor (2)
Metergoline (300 mg, 0.74 mmol) was dissolved in 15 ml of MeOH and 50 mg of palladium on activated charcoal (10% wt) was added. While stirring, the mixture was sparged with a slow stream of hydrogen gas for 20 minutes. The reaction flask was sealed under an atmosphere of H2 (using a H2 filled balloon) and stirred at room temperature. Reaction progress was monitored by TLC (MeOH as the eluent) until no metergoline remained (typically 12–16h). At this point, the reaction mixture was filtered through celite to remove the activated charcoal and the solvent was removed under reduced pressure. The resulting residue was dissolved in a small volume of ethyl acetate and concentrated under reduced pressure. This process was repeated until a white solid remained (60 mg, 0.22 mmol, 30%), which required no additional purification. 1H NMR (400 MHz, CDCl3), δ: 7.20 (t, J = 8 Hz, 1H), 7.12 (d, J = 8 Hz, 1H), 6.93 (d, J = 4 Hz, 1H), 6.74 (s, 1H), 3.76 (s, 3H), 3.43–3.38 (dd, J =16 Hz, 4 Hz, 1H), 3.13 (d, J = 8 Hz, 1H), 2.98 (m, 1H), 2.76–2.66 (m, 4H), 2.49 (s, 3H), 2.18–2.12 (m, 1H), 1.96–1.91 (m, 2H), 1.15–1.06 (q, J = 12 Hz, 1H). 13C NMR (125 MHz, CDCl3), δ: 134.6, 133.8, 126.7, 122.9, 122.7, 112.8, 111.0, 106.9, 67.8, 62.0, 46.8, 43.6, 40.8, 39.6, 33.0, 32.4, 27.2.
4.2.2. Synthesis of [11C]Metergoline (3)
Labeling precursor 2 (1 mg) dissolved in 100 μL of DMF was combined with 100 μL of a solution of DBU (300 mM in DMF) and then treated with a 100 μL aliquot of benzyl chloride (300 mM in DMF) in a cone-bottom 5 mL reaction vessel equipped with a septum and screw cap. The resulting solution was sparged with helium for 15 min. At the end-of-bombardment, the target gas from the cyclotron was passed through the solution and 11CO2 was captured. The expansion of the target gas through the reaction solution required ~2.5 min. During some syntheses,11CO2 from the target was captured on Alltech 4Å, 50–80 mesh crushed mol. sieves (330 mg packed in a ¼” ID SS tube) using a custom built trap and release module. The 11CO2 was released from the mol. sieves at 250–300°C in a stream of helium (~50 mL/min) and captured in the precursor/DBU solution as detailed above. This process required 5–6 min.
After 11CO2 was captured in the reaction solution (using either method), the vessel was then heated to 75°C for 7.5 min at which time 100 μL of trifluoroacetic acid was added. The volatile activity was removed by sparging the solution with He. (This required ~0.5 min). The solution was diluted with 1.0 ml of water and purified by HPLC (Luna C18(2) column (250 × 10 mm, 5 μm particles) using a two solvent (A:B) gradient elution at 5mL/min: Solvent A: 0.1M (aq) ammonium formate, Solvent B: MeCN; 80:20 for 3 min, changing to 10:90 linearly from 3–23 min and holding until 30 min). The fraction containing [11C]metergoline was collected (retention time 15.5–16.5 min), and the solvent was removed on a rotary evaporator. Following purification and concentration, [11C]metergoline was dissolved in saline (4.0 mL) and the resulting solution was filtered through an Acrodisc 13-mm Syringe Filter with 0.2 μm HT Tuffryn Membrane (Pall Corporation, Ann Arbor, MI) into a sterile vial for delivery to the imaging facility. The radiochemical yield based on the loss of volatile 11CO2 and HPLC was typically 35% (decay corrected to EOB). Specific activity for the imaging studies was 3.2–5.1Ci/μmol (calculated at EOB). Typical radiosynthesis and purification time was 45 min.
For quality control (i.e. identity verification and radiochemical purity), analytical TLC and HPLC were performed. [11C]Metergoline was cospotted with unlabelled (i.e. non-radioactive) standard and analyzed by radioTLC (MeOH with 0.1% TEA, Rf = 0.5). Analytical HPLC was accomplished using a Phenomenex Gemini C18 column (250 × 4.6 mm, 5 μm particles) using an isocratic elution (1.0 ml/min, 60% aqueous mobile phase to 40% MeCN). Using this method, [11C]metergoline eluted at 8.5 min. Radiochemical purity exceeded 99% as determined by both radioHPLC and radioTLC and chemical purity was >95% as determined from analytical HPLC with 254 nm detection.
4.3 LogD Determination
An aliquot (~50 μl) of the formulated [11C]metergoline was added to a test tube containing 2.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The test tube was mixed by vortex for 2 min and then centrifuged for 2 min to fully separate the aqueous and organic phase. A sample taken from the octanol layer (0.1 mL) and the aqueous layer (1.0 mL) were saved for radioactivity measurement. An additional aliquot of the octanol layer (2.0 mL) was carefully transferred to a new test tube containing 0.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The previous procedure (vortex mixing, centrifugation, sampling, and transfer to the next test tube) was repeated until six sets of aliquot samples had been prepared. The radioactivity of each sample was measured in a well counter (Picker, Cleveland, OH). The logD of each set of sample was derived by the following equation: logD = log (decay-corrected radioactivity in octanol sample × 10/decay-corrected radioactivity in phosphate buffer sample).
4.4 Plasma Protein Binding Assay
An aliquot of [11C]metergoline in saline (10 μl) was added to a sample of baboon plasma (0.8-mL, pooled from at least 4 separate animals). The mixture was gently mixed by repeated inversion and incubated for 10 min at room temperature. Following incubation a small sample (20 μl) was removed to determine the total radioactivity in the plasma sample (AT; AT=Abound+Aunbound). An additional 0.2 mL of the plasma sample was placed in the upper compartment of a Centrifree® tube (Amicon, Inc., Beverly, MA) and then centrifuged for 10 min. The upper part of the Centrifree tube was discarded and an aliquot (20 μl) from the bottom part of the tube was removed to determine the amount of radioactivity that passed through the membrane (Aunbound). Plasma protein binding was derived by the following equation: % unbound=Aunbound×100/AT.
4.5 PET imaging and arterial plasma sampling
All experiments with animals were approved by the Brookhaven Institutional Animal Care and Use Committee. Four female Papio anubis baboons were used for the 15 PET scans performed for this study. Anesthesia was accomplished by an intramuscular injection of ketamine hydrochloride (10 mg/kg) and then maintained with oxygen (800 mL/min), nitrous oxide (1500 mL/min) and isoflurane (Forane, 1–4%) during scanning. [11C]Metergoline was injected through a catheter placed in a radial arm vein, and arterial blood was sampled through a catheter in the popliteal artery at the following time intervals: every 5 s for 2 min, then 2, 5, 10, 20, 30, 45, 60 and 90 min. Heart rate, respiration rate, pO2 and body temperature were checked during the PET scanning. Dynamic PET imaging was performed by Siemens HR+ (Siemens high-resolution, whole-body PET scanner with 4.5×4.5×4.8 mm resolution at the center of field of view) with the brain or torso in the field of view, for a total of 90 min with the following 55 time frames in 3D mode: 12×5, 12×10, 6×20, 6×30, 8×60, 2×120, 4×300, 5×600 s. Prior to each emission scan, a transmission scan was obtained by rotating a 68Ge rod source to correct for attenuation. A total of 15 studies in the baboon were conducted (3 × brain, 1 × torso kinetics, 1 × whole body following dynamic brain scan), with an average injected dose of 3.72 ± 0.90 mCi (n = 15).
Six of the studies were performed (as detailed above) following intravenous pretreatment with one of the following drugs: metergoline (n = 2, brain; n = 1 torso), citalopram (n = 2), and altanserin (n = 1). Metergoline (1 mg) was dissolved in a solution of 0.5 mL EtOH and 0.5 mL 0.1 M HCl and passed through a sterilizing filter into a multi-injection vial containing 9 mL of sterile saline. The 1mg (10 mL) metergoline pretreatment was administered as a bolus 10 min prior to injection of [11C]metergoline. Citalopram-HBr (5.0 mg/kg based on the free base) was dissolved in 10 mL of saline and passed through a sterilizing filter into a multi-injection vial. The citalopram solution was administered as a bolus 20 min prior to injection of [11C]metergoline. Altanserin (0.5 mg/kg) was dissolved in 1.5 mL of 2% HCl in EtOH, diluted to 30 mL with saline, and passed through a sterilizing filter (final pH ~5.5). Altanserin was administered 15 min prior to injection of [11C]metergoline.
4.6 Plasma Metabolite Analysis
The percent of unmetabolized radiotracer in the sampled baboon plasma was determined using a robot solid phase extraction (SPE) method and validated using manual HPLC analysis.15
For the robot SPE method baboon plasma (~0.2 ml), sampled at given time points during the PET study (typically 1, 5, 10, 30, 60 and 90 min), was added to water (3 ml) at room temperature and mixed by vortex. The mixture was then applied onto Varian BondElut CN cartridges (500 mg, preconditioned with 5 ml methanol followed 5 ml water then preloaded with 2 ml of deionized water). Polar metabolites were removed by two 5 ml deionized water rinses. The percentage radioactivity remaining on the cartridge was reported as the % of unmetabolized radiotracer. A control consisting of baboon plasma at time zero was mixed with radiotracer and analyzed to confirm that unmetabolized radiotracer did not elute under these conditions. For validation by HPLC, baboon plasma (0.2–0.5 ml) sampled at various time points during the PET study was counted, added to a solution of unlabeled standard (20 μl of a 1 mg/ml solution) in methanol (.5 ml), subjected to an ultrasonic cell disruptor and centrifuged. Both supernatant and pellet were counted to determine the percent extracted. The supernatant was analyzed by HPLC (Phenonenx Ultramex 5 C18 5u 250 × 5.6 mm column using 80:20 MeOH: 0.1 M ammonium formate with 0.1% triethylamine at 1.2 mL/min). The fraction of radioactivity coeluting with the unlabeled standard, relative to the total radioactivity collected from the HPLC column was reported as the % of unmetabolized radiotracer, . Total activity recovered from the HPLC column was compared to a counting standard; generally recoveries from the column were 92–110%.
Evaluation of [11C]metergoline metabolism using plasma samples.
4.7 Image analysis
Emission data from the dynamic scans (PET) were corrected for attenuation and reconstructed using filtered back projection. After ECAT7 files were converted to ANALYZE format and time frames 5–30 were summed for each file, images were coregistered with published template images16 (H215O PET and MR) manually and then normalized (12 nonlinear iterations) using PMOD® (PMOD Technologies, Ltd). Regions of interest (ROIs) on the baboon and rodent brain were drawn on summed images and then projected to the dynamic images to obtain time activity curves (TACs) expressed as %injected dose/cm3 (decay corrected) versus time. Regions of interest (ROIs) on the baboon brain were drawn on summed images and then projected to the dynamic images to obtain time activity curves (TACs) expressed as %injected dose/cm3 (decay corrected) versus time. A common ROI file was used to generate TACs each study. Torso data we were analyzed using Amide Software and manually drawn 3-D ROIs.
4.8 Image analysis
Logan graphical analysis derived from the TACs and metabolite-correct plasma data was used to determine distribution volume (VT
This analysis was accomplished with General Kinetic Modeling Tool
(PKIN) in PMOD. Binding potential (BPND
) was calculated by taking the ratio of a given VT to a reference region (cerebellum) and subtracting 1 (the presumed non-specific contribution).