Quantification of Macrophage-Driven Inflammation During Myocardial Infarction with 18F-LW223, a Novel TSPO Radiotracer with Binding Independent of the rs6971 Human Polymorphism

Visual Abstract


Organic Chemistry
All reagents and starting materials were obtained from commercial sources and used as received. All dry solvents were purified using a PureSolv 500 MD solvent purification system. All reactions were performed under argon unless otherwise stated. Brine is defined as a saturated solution of aqueous sodium chloride. Flash column chromatography was carried out using Fisher Matrix silica 60. Macherey-Nagel aluminium-backed plates pre-coated with silica gel 60 (UV254) were used for thin layer chromatography and were visualized using UV light. 1 H NMR and 13 C NMR spectra were recorded on a Bruker DPX 400 spectrometer or Bruker 500 spectrometer with chemical shift values in ppm relative to tetramethylsilane (δH 0.00 and δC 0.0) or residual chloroform (δH 7.26 and δC 77.2) as the standard. 1 H and 13 C assignments are based on twodimensional COSY and DEPT experiments, respectively. Infrared spectra were recorded on a JASCO FTIR 410 spectrometer. Mass spectra were recorded using electron impact, chemical ionization or fast atom bombardment techniques. HRMS spectra were recorded using a dual-focusing magnetic analyzer mass spectrometer. Melting points were determined on a Gallenkamp melting point apparatus. Chiral High Performance Liquid Chromatography (HPLC) methods were calibrated with the corresponding racemic mixtures. 3-Methyl-4-phenylquinoline-2-carboxylic acid was prepared as previously reported (14).
NMR spectra showed a 1.5:1 mixture of rotamers. Only signals for the major rotamer are
Saturation assays were performed to determine the Kd of PK11195 using a similar protocol, except that 6 concentrations of 3 H-PK11195 (1.6-200 nM) were used alongside 10 µM of PK11195 to determine non-specific binding. All binding assays were performed in triplicate.
GraphPad Prism version 6 (GraphPad Software, USA) was used to fit all binding affinity curves. A comparison of a one-site and two-site models was made using the least squares algorithm and model selection was compared using an F test. The null hypothesis (one-site fitting as more suitable) was rejected if p<0.05. Normalized mean %SB of each group (HAB, MAB or LAB) and a Kd value of 14.0nM (determined based on saturation binding results, Sup. Fig.2) was used to determine the best fitting model and to calculate LAB:HAB ratios. Affinity values (Ki) were calculated by fitting individual tissue samples again with a Kd 14.0nM.

Invasive Input Function Measurement
Polyethylene catheters (PE50) filled with heparinized saline (20 IU/mL) were inserted into the left femoral artery and vein with the help of a stereomicroscope and securely fastened with ligatures (6-0 silk thread). Catheters were held in place with surgical glue.

Rat Myocardial Infarction Procedure
Briefly, the skin was incised at the level of the left third and fourth ribs where the pectoral muscles were divided and retracted. Left lateral thoracotomy was then performed. With minimal handling, the pericardium was ruptured and the heart gently exteriorized from the thorax, and a nonabsorbable 5-0 ligature was placed around the left anterior descending coronary artery just above the bifurcation of the first diagonal and maneuvered back into position. Before wound closure, a drain was inserted to assist with removal of air and fluid from the thorax. Once removed, the wound was then closed in three layers. Animals were recovered and extubated once spontaneous ventilation was established, housed at 30 °C for 24 hours and given sterile sodium chloride 0.9% 0.01 mL/g fluid therapy subcutaneously in addition to another dose of Buprenorphine. After 24 hours, normal housing conditions were resumed.

Radiometabolite Blood and Tissue Processing and Analysis
All blood samples were 1 mL each and manually collected from different animals to generate a population curve, in order to respect total blood volume limits for terminal arterial blood collections in rats. Following blood and tissue collection, all samples were kept on ice until analyzed. Radioactivity in whole blood and plasma, as well as homogenized tissue samples, was assessed using a well-type γ-counter using a 400-1400 keV window (Perkin Elmer Wizzard2, USA). Plasma samples (400 μl) and tissue samples (500 μl) were processed by acetonitrile denaturation (1:1.4 v/v) and analyzed by HPLC (Ultimate2000, ThermoFisher, UK) on a Luna C18(2) 10×250 mm, 10 μm column (Phenomenex, UK) with acetonitrile/water 70/30 and flow rate of 4 mL/min to estimate the parent fraction. The plasma fp was determined using ultrafiltration units (Centrifree® 30K, Millipore, UK).

Simulation of Changes in 18 F-LW223 BPND for varying blood flow scenarios
Simulation work was carried out using a Matlab (MathWorks, USA) 2-tissue compartment code with Ordinary Differential Equation (ODE) solvers. Six different levels of K1 values (0.9, 0.7, 0.5, 0.3, 0.1 and 0.01 mL/min/cc) were tested with fixed k2 and vB=0.05. The following BPND values were simulated: 4, 10, 16 and 20; with k3 modified and k4 fixed. All micro parameters used in the simulations are shown in Supplemental Table 5. The input function used was an average of the blood curves taken from healthy animals using the SwissTrace blood sampler (Supplemental Data File 1). The plasma to whole blood ratio used was 1.84 and the AIF was metabolite corrected using a population based curve as in the experimental data. All modelled time-activity curves were noise free. Outcome

Autoradiography
Rehydrated tissue on glass slides was incubated in assay buffer (50 mM Tris Buffer) for 30 min prior to the experiment. The slides were then incubated with 18 F-LW223 (50 nM in assay buffer) for 1 hour at room temperature. Slides were washed twice in assay buffer followed by a dip in dH2O. The slides were then dried and exposed to an autoradiography plate (BAS-IP-SR 2040 super resolution screen, Cytiva, USA) and imaged on an autoradiography imager (Amersham Typhoon IP Biomolecular Imager, Cytiva, USA).
Images were analyzed using Image J, using the ratio of signal within a ROI to the background signal on each slide to calculate the tissue to background ratio (TBR).   Figure 3. Representative examples of different models versus measured data on naive rat organs. A) Logan modelling in the heart, B) lung and C) brain. Modelling constrained to a t*=30min. D) 1T modelling in the heart, E) lung and F) brain. G) 2T modelling in the heart, H) lung and I) brain.

Supplemental Figure 4. Dissociation constant (Kd) values and maximal binding (Bmax) of PK11195 in human brain and heart. A) Kd values of PK11195 and B) Bmax
calculated from saturation binding assays. Results represent the mean±S.E.M., brain n = 6, heart n = 5, ns=not significant, *=p<0.05 using an unpaired t-test for brain versus heart.
Supplemental Figure 5. Individually calculated Ki values for TSPO ligands using human brain and heart. A) PK11195 binding affinities in brain calculated using one-site fitting, HAB n = 6, MAB n = 8 and LAB n = 4 and B) heart calculated using a one-site fitting, HAB n = 4, MAB n = 5 and LAB n = 4. C) PBR28 binding affinities in brain calculated using a one-site fitting apart from MAB where a two-site fitting was used, HAB n = 4, MAB n = 5 and LAB n = 4 and D) heart calculated using a one-site fitting, HAB n = 4, MAB n = 5 and LAB n = 4. E) AB5186 binding affinities in brain calculated using a one-site fitting apart from MAB where a two-site fitting was used, HAB n = 6, MAB n = 6 and LAB n = 5 and F) heart using a one-site fitting apart from MAB where a two-site fitting was used, HAB n = 4, MAB n = 5 and LAB n = 4. G) LW223 binding affinities in brain calculated using a one-site fitting, HAB n = 5, MAB n = 5 and LAB n = 4 and H) heart calculated using a one-site fitting, HAB n = 5, MAB n = 5 and LAB n = 4. ns=not significant, *=p<0.05, **=p≤0.01 and ***=p≤0.001 using an unpaired t-test for HAB vs. LAB. All results represent the mean±SEM.