Chemistry and radiochemistry
In detail description of synthesis of precursors, reference analogs (1 to 5) and production of the secondary radiolabeling agents 11CH3I and 18FEtBr are summarized in Additional file 1.
4-(2-[18F]fluoroethoxy)benzoyl aminoethylbenzenesulfonamide [18F]-(3) and 4-[11C]methoxybenzoyl aminoethylbenzenesulfonamide [11C]-(4)
The synthons 11CH3I or 18FEtBr were bubbled into a solution consisting of the phenolic precursor, 4-hydroxy-N-[2-(4-sulfamoylphenyl)ethyl]benzamide (2, 0.2 mg) and Cs2CO3 (1 to 2 mg) in anhydrous dimethylformamide (DMF). The reaction mixture was heated at 90°C for 5 min (11CH3I) or 15 min (18FEtBr). The reaction mixture was diluted with water (1 mL) and injected onto a high-performance liquid chromatography (HPLC) system (XBridge C18 column, 5 μm, 4.6 × 150 mm; Waters Corporation, Milford, MA, USA) eluted with a mixture of 0.05 M NaOAc (pH 5.5) and EtOH (80:20 v/v) at a flow rate of 1 mL/min. UV detection of the HPLC eluate was performed at 254 nm. The radiolabeled product [18F]-(3) was collected after 15 min and [11C]-(4) eluted 11 min after injection on the HPLC system. The collected peak corresponding to the desired radioligand was then diluted with saline (Mini Plasco®, Braun, Melsungen, Germany) to obtain a final EtOH concentration of ≤5%, and the solution was filtered through a sterile 0.22-μm membrane filter (Millex®-GV, Millipore Co., Billerica, MA, USA). Quality control was performed on an analytical HPLC system consisting of an XBridge C18 column (3.5 μm, 3 × 100 mm; Waters Corporation) eluted with a mixture of 0.05 M NaOAc buffer (pH 5.5) and acetonitrile (80:20 v/v) at a flow rate of 0.8 mL/min. UV detection was performed at 254 nm. The tracers [18F]-(3) and [11C]-(4) were eluted at 10 and 5 min, respectively, and their identity was confirmed by co-elution with authentic nonradioactive reference solutions. The tracers [18F]-(3) and [11C]-(4) were synthesized with a decay-corrected radiochemical yield of 45% and 30% (n = 3), respectively (relative to the starting radioactivity of 18FEtBr and 11CH3I), and with a radiochemical purity of ≥98%. Starting from 18FEtBr and 11CH3I, the synthesis time to obtain the pure product was 50 ± 10 min for [18F]-(3) and 40 ± 5 min for [11C]-(4). The average specific activity was found to be in the range of 37 to 71 GBq/μmol at the end of synthesis (EOS).
4-(2-[18F]fluoroethoxy)benzenesulfonamide [18F]-(5) and 4-[11C]methoxybenzene sulfonamide [11C]-(6)
The synthons 18FEtBr or 11CH3I were bubbled into a solution of the phenolic precursor 4-hydroxybenzene-1-sulfonamide (0.8 mg) in a mixture of 1 M NaOH (2.5 μL) and DMF (0.3 mL). The mixture was heated at 90°C for 5 min (11CH3I) or 15 min (18FEtBr). The crude mixture was diluted with water (1 mL) and injected onto an HPLC column (XTerra C18, 5 μm, 7.8 × 150 mm; Waters Corporation) eluted with a mixture of 0.05 M NH4OAc (pH 6.8) and EtOH (90:10 v/v) at a flow rate of 2 mL/min. UV detection of the HPLC eluate was performed at 254 nm. The radiolabeled product [18F]-(5) was collected after 16 min, and [11C]-(6) was eluted after 15 min on the HPLC system. The collected peak corresponding to the desired radioligand was then diluted with saline (Mini Plasco®, Braun, Melsungen, Germany) to obtain a final EtOH concentration of ≤5%, and the solution was sterile filtered through a 0.22-μm membrane filter (Millex®-GV, Millipore Co.). QC was performed on an analytical HPLC system consisting of an XTerra C18 column (5 μm, 4.6 × 250 mm; Waters Corporation). For [18F]-(5), the mobile phase was a mixture of 0.05 M NH4OAc (pH 6.8) and EtOH (80:20 v/v); for [11C]-(6), a mixture of 0.05 M NH4OAc (pH 6.8) and acetonitrile (80:20 v/v) was used. The flow rate was 0.9 mL/min. UV detection was performed at 254 nm. The tracers [18F]-(5) and [11C]-(6) were eluted at 11 and 9 min, respectively, and the identity of the tracers was confirmed by co-elution with authentic nonradioactive reference solutions. The tracers [18F]-(5) and [11C]-(6) were synthesized with a decay-corrected radiochemical yield of 65% (n = 3) and with a radiochemical purity of ≥98%. Starting from 18FEtBr and 11CH3I, the synthesis time to obtain the pure product was 55 ± 10 min for [18F]-(5) and 50 ± 5 min for [11C]-(6). The average specific activity for both tracers was found to be 90 GBq/μmol at the EOS.
In vitro studies
Log D (1-octanol/phosphate buffer pH 7.4)
Determination of the distribution coefficient (log D1-octanol/phosphate buffer pH 7.4), was carried out by a shake flask method [15]. An aliquot (25 μL) of the tracer agents 18F]-(3), 11C]-(4), 18F]-(5), or 11C]-(6) (185 to 555 kBq/mL) was added to a polypropylene tube (5 mL; Sarstedt, Nümbrecht, Germany) containing 2 mL of 0.025 M sodium phosphate buffer pH 7.4 and 2 mL of 1-octanol. The tube was vortexed for 2 min at room temperature followed by centrifugation at 3,000 rpm for 10 min (Eppendorf centrifuge 5810, Eppendorf, Westbury, NY, USA). Aliquots of 50 μL of the 1-octanol phase and 500 μL of the phosphate buffer phase were pipetted into separate tared Eppendorf tubes with adequate care to avoid cross contamination between the two phases. The samples were weighed, and radioactivity was quantified using an automated gamma counter. The experiments were carried out sixfold.
Determination of inhibition constant
The inhibition constants (K
i) of the reference analogs (3), (4), (5), and (6) against human CA (hCA) I and II isozymes were determined by assaying the CA-catalyzed CO2 hydration activity [16], using Applied Photophysics' (Leatherhead, UK) stopped-flow instrument. Phenol red (0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes as buffer (pH 7.5) and 0.1 M Na2SO4 (for maintaining constant ionic strength) at 25°C following the CA-catalyzed CO2 hydration reaction for a period of 10 to 100 s (the uncatalyzed reaction needs around 60 to 100 s under assay conditions, whereas the catalyzed reactions take around 6 to 10 s). The CO2 concentrations ranged from 1.7 to 17 mM for the determination of kinetic parameters. Each compound was tested in the concentration range between 0.01 nM to 100 μM. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of the compounds (0.1 mM) were prepared in distilled water with 10% to 20% (v/v) DMSO (which is non-inhibitory at these concentrations), and dilutions up to 0.01 nM were made with distilled water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay, in order to allow the formation of enzyme-inhibitor complex. The inhibition constants were obtained by nonlinear least square methods using PRISM 3 (GraphPad Software, La Jolla, CA, USA), and they represent the mean from at least three different determinations.
Whole blood analysis
Blood samples from a healthy human volunteer were collected in a BD vacutainer™ (4.5 mL; containing lithium heparin; BD, Franklin Lakes, NJ, USA). Aliquots of whole blood (0.4 mL) were incubated with [11C]-(4) or [11C]-(6) (370 kBq/0.1 mL) for 10 min and with [18F]-(3) or [18F]-(5) (37 kBq/0.1 mL) for 60 min at room temperature. A different incubation time with 11C and 18F tracer agents was used considering the radionuclides' half-life (11C, 20 min; 18F, 110 min). After incubation for 10 or 60 min, the plasma was separated from the blood cells by centrifugation at 3,000 rpm (1,837×g) for 5 min (Eppendorf centrifuge 5810). To remove residual plasma and unbound tracer from the RBCs, phosphate-buffered saline (PBS) pH 7.4 (0.4 mL) was added to the cell fraction. After incubation for 2 min, the PBS was separated from the RBCs using centrifugation. This rinsing procedure was carried out twice. For competition studies with acetazolamide (AAZ), the same procedure was followed as mentioned afore. To the whole blood and tracer agent mixture, a solution of AAZ (0.1 mL) was added to result final concentrations of 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 mM, and as a control, PBS (0.1 mL, PBS pH 7.4) was added. The radioactivity associated with the RBCs and plasma was quantified using an automated gamma counter.
Distribution of activity within the blood
Human blood (12 mL) was collected in a syringe containing 2.4 mL ACD (citrate-dextrose solution), and 3.7 MBq was added and gently mixed. Two milliliters of a 2% methylcellulose was added, gently mixed, and allowed to stand for 60 min so that the RBCs sediment by gravity. The supernatant was carefully drawn and centrifuged at 1,000 rpm (140×g) for 5 min. The plasma-rich supernatant and white blood cell (WBC)-rich pellet were separated [17]. The activity in the three fractions was counted using an automated gamma counter. The procedure was carried out in triplicate.
In vivo studies
Biodistribution studies
The biodistribution studies were performed in wild-type NMRI mice with body weights ranging from 30 to 40 g. Mice were intravenously injected with 0.1 MBq of 18F]-(3) or 18F]-(5) and 5.5 MBq of 11C]-(4) or 11C]-(6) under anesthesia (2% isoflurane in O2 at a flow rate of 1 L/min). The animals were killed by decapitation at 2 min or 60 min p.i. (n = 4/time point for 18F]-(3), 11C]-(4), and 11C]-(6); n = 6 for 18F]-(5)). Blood was collected, and all major organs were dissected and collected in tarred tubes and were weighed. The radioactivity in each organ was counted using an automated gamma counter, corrected for background radioactivity and expressed as follows: percentage of the injected dose (% ID) or as standardized uptake value (SUV) (SUV = (Counts in tissue per gram of tissue) / (Injected counts per total body mass (g)). For the calculation of total radioactivity in the blood, blood mass was assumed to be 7% of the body mass [18].
Small animal imaging studies
PET images were acquired on a FOCUS 220 tomograph (Siemens/Concorde Microsystems, Knoxville, TN, USA). During all scan sessions, rats were anesthetized (2.5% isoflurane in O2 at a flow rate of 1 L/min) and scanned in prone position. [18F]-(5) (37 MBq) was administered via the tail vein, and a 1-h dynamic scan was acquired. The images were acquired in list mode and binned in sinograms using a 21-dynamic frame protocol (4 × 15, 4 × 60, 5 × 180, 8 × 300 s). Reconstruction was performed with the Focus 220 software (using Fourier rebinning, followed by two-dimensional (2D) OSEM algorithm), and data were analyzed using PMOD (version 2.65; PMOD, Zurich, Switzerland). The radioactivity concentration in the heart region was expressed as SUVs as a function of time after injection of the radiotracer.
An electrocardiogram (ECG)-gated micro-PET scan was performed with anesthetized (2.5% isoflurane in O2 at a flow rate of 1 L/min) Wistar rats (n = 2) after administration of 48 MBq/0.7 mL of [18F]-(5) via the tail vein. The acquisition was carried out at rest (0.5 h) and stress (0.5 h) by infusion of dobutamine at 10 μg/kg/min. The acquired data were reconstructed into a series of 12 ECG-gated images. To reduce noise, images were filtered with a 3D Gaussian filter (FWHM 1 mm) along the spatial dimensions and with a low pass filter (preserving only the mean and the first four harmonics) along the time dimension. A volume of interest containing the LV cavity in all 12 images was manually defined. For each image, the cavity volume was determined by applying a threshold of 50% of the maximum value inside that volume of interest.
Anesthetized rats also underwent cMRI with a 9.4-T 20-cm bore horizontal magnet using a linear resonator for excitation combined with a 2 × 2-phased array coil for detection (BRUKER Biospin, Ettlingen, Germany) and a retrospectively gated FLASH sequence (INTRAGATE®, BRUKER; repetition time (TR)/echo time (TE) = 7.6/1.8 ms, flip angle = 17°, matrix = 256 × 256, field of view (FOV) = 6 × 6 cm, 10 to 12 1-mm-thick short axis slices covering the LV, 15 frames reconstructed). To ensure stable and reproducible results, physiological parameters such as body temperature and respiratory and heart rates were carefully monitored throughout the imaging session, and it is noteworthy to mention that there was an interval of 7 to 10 days between the micro-PET and cMRI scan. A manual delineation of the endocardium, ignoring the papillary muscles, was carried out using a homemade software for cMRI [19]. The LVEF values were computed as follows: LVEF = (EDV − ESV/EDV) × 100, where EDV and ESV are end-diastolic volume and end-systolic volumes, respectively.
Pig study
Myocardial infarction was induced in 25- to 30-kg domestic pigs as previously described [20]. In brief, the left anterior descending coronary artery was temporarily occluded by a 90-min balloon inflation of a bare metal stent distal to the first diagonal branch. Continuous ECG and invasive pressure monitoring was registered during the whole procedure. Eight weeks later, gated blood pool PET and MRI were performed. 18F]-(5) (185 MBq) was administered via a venous catheter, and a 60-min ECG-gated PET scan was acquired in the list mode (HiRez Biograph 16, Siemens, Knoxville, TN, USA). A low-dose CT scan was conducted for attenuation correction. Based on the simultaneously recorded ECG signal, the cardiac cycle was divided in eight frames, and a PET sinogram was created from the list mode data for each frame. From these sinograms, eight images were reconstructed using 2D OSEM (five iterations and eight subsets) after Fourier rebinning. LV volumes and EF were determined in the same way as for the small animal study, i.e., by applying a threshold of 50% of the maximum in a manually defined volume of interest containing the cavity. The person who analyzed the PET data was blinded from the MRI results.
The MRI images were obtained in supine position on a 3-T unit (TRIO, Siemens, Erlangen, Germany) with ECG gating and during suspended respiration. A contiguous stack of short-axis images covering the entire ventricle was acquired with a 2D FLASH (fast low-angle shot) sequence using retrospective gating and the following imaging parameters: TR/TE = 25.45/2.39 ms, flip angle = 14°, matrix = 170 × 208, FOV = 310 × 380 mm, slice thickness = 6 mm, 40 cardiac phases, bandwidth 445 Hz/pixel). Volumes were determined by manual contouring in the same manner as described for the rat studies above.
All animal studies were approved by the Ethics Committee for Animal Experimentation (KU Leuven, Belgium) and were performed in accordance with the Guide for Care and Use of Laboratory Animals (NIH).