In vivo and in vitro blood cellular partitioning kinetics may affect plasma input function determination of the COX-1 radioligand 11C-PS13

1616 Background: 11C-PS131 is selective for cyclooxygenase-1 (COX-1), an enzyme that mediates the inflammatory response. COX-1 is constitutively expressed in most blood cells including platelets2. Despite extensive extracellular binding of 11C-PS13 to plasma protein3, the concentration of radioactivity in whole blood is consistently greater than that in plasma. This is in part due to the fact that binding to plasma protein is weak and rapidly reversible, whereas binding to intracellular COX-1 is of high affinity3. The cellular uptake of 11C-PS13 involves passive diffusion across membranes and specific binding to enzyme receptor. The aim of this study was to identify and determine the in vitro kinetics of cellular uptake and release of radioactivity to accurately define the plasma input function and reduce variability. Methods: Blood was obtained from human (N=2) and nonhuman (N =8) primates part of ongoing studies. Hematocrits (Hct) were determined by capillary centrifugation. Whole blood and plasma radioactivities were quantified via γ-counter and plasma parent fraction was determined by radio-HPLC4. The in vivo cellular partitioning of 11C-PS13 was performed in nonhuman primates on blood samples that were drawn periodically after injection. Cell saturation uptake of 11C-PS13 was achieved by varying molar activities of administered doses. Specificity of cell uptake was evaluated during preblocked imaging studies with COX-1 inhibitor ketoprofen. Cellular blood volume (VBC) and its time-stability were determined by dividing AUC of cells by that of the free plasma parent. In vitro cellular blood uptake was performed at room temperature and at 37°C. After adding 11C-PS13 to blood and mixing well, blood samples were periodically removed for γ-counting followed by obtaining concomitant plasma by centrifugations. Cellular radioactivity concentration was calculated by: Cells(cpm/mL) = [Cwhole blood(cpm/mL) - Cplasma(cpm/mL) x (1-Hct)] / Hct. The experiment on release of radioactivity from human cells was done in vitro at room temperature. At equilibrium (after 40 min incubation) blood cells were harvested by centrifugation and the radioactive plasma was replaced by the same volume of nonradioactive plasma. Redistribution of radioactivity was then periodically monitored. Results: We assumed no radiometabolites entered blood cells based on in vivo time-stability analysis VBC. In vivo blood cells were saturated with PS13 between 1-9 nM. This blood cellular uptake was specific to COX-1 with a 78% blockade using ketoprofen. in vitro cell uptake was instantaneous and faster at 37°C than that at room temperature. In vitro release rate of 11C-PS13 from pre-equilibrated cells was 0.12%/min. Conclusion: Our in vivo-in vitro results indicated that most reliable recommendations for plasma input function descriptors were those if obtained from blood samples that were immediately (within 1 minute) radio-analyzed after their draw and centrifuged with enough gravitational force to remove all platelets from plasma. Delay in processing leads to in vitro redistribution of radioactivity and significant fluctuations in estimating plasma input function via adding noise to later time points of the input function. References: 1. Kim et al.JNM (2018) 59: 1907. 2. Boilard et al.J Immunol (2011) 186: 4361. 3. Shrestha et al.ACS Chem Neurosci (2018) 9: 2620. 4. Zoghbi et al.J Nucl Med (2006) 47: 520.