Background Cyclooxygenase-2 (COX-2) is the inducible isoform of the cyclooxygenase enzyme

Background Cyclooxygenase-2 (COX-2) is the inducible isoform of the cyclooxygenase enzyme family. for specificity of [18F]Pyricoxib for COX-2 binding in vitro and in vivo. PET imaging, biodistribution, and radiometabolite analyses were included into radiopharmacological evaluation of [18F]Pyricoxib. Results Radiotracer uptake in COX-2 positive HCA-7 cells was significantly higher than in COX-2 unfavorable HCT-116 cells (of 0.45 to 0.50 in this solvent system. Biodistribution studies in mice NIH-III mice (body weight 21??2?g) bearing subcutaneous HCA-7 tumors were intravenously injected with 3C7?MBq of [18F]Pyricoxib in 200?L 17924-92-4 of solvent (10?% EtOH/H2O). A second group 17924-92-4 of NIH-III mice (body weight 21??2?g) bearing HCA-7 tumors were treated with 2?mg of celecoxib administered via intraperitoneal injection in 100?% DMSO 60?min prior to intravenous injection of [18F]Pyricoxib 17924-92-4 (3C7?MBq) in 200?L of solvent (10?% EtOH/H2O). Animals were sacrificed at 60?min p.i. The organs and tissues of interest were rapidly excised, weighed, and the radioactivity was decided using the automatic gamma counter 17924-92-4 (see above). Radioactivity in the selected tissues and organs was calculated as percent injected dose per gram tissue (%ID/g). Data were analyzed as means??standard deviation (mean??SD) for test and were considered significant for of 3.37. This value is in the range to allow for passive diffusion, and it is also in the same range as reported lipophilicity values of other radiolabeled COX-2 inhibitors [17C19]. Cellular uptake studies of [18F]Pyricoxib in human colorectal cell lines HCA-7 and HCT-116 exhibited significantly higher radiotracer uptake and retention in COX-2-positive HCA-7 cells. However, overall uptake of the radiotracer was also sufficiently high in COX-2-unfavorable HCT-116 cells. This finding is usually indicative of a favorable passive diffusion profile of the radiotracer in combination with COX-2-mediated uptake and retention mechanisms in the case of COX-2-expressing HCA-7 cells. In COX-2-unfavorable HCT-116 cells, several COX-2 impartial uptake and retention mechanisms are likely to be responsible for the observed radiotracer uptake. Immunohistochemical analysis confirmed a high expression of COX-2 in HCA-7 tumors with lower but noticeable COX-2 expression in HCT-116 tumors. This is consistent with the Western blot analysis of both HCA-7 and HCT-116 tumor samples (Fig.?5). Both tumors showed unfavorable staining for CD68 as marker for tumor-associated macrophages [31]. This obtaining indicates that observed positive staining for COX-2 in HCT-116 tumors is not related to the infiltration of COX-2-expressing macrophages as an inflammatory response to tumor 17924-92-4 cell inoculation and tumor growth. Reduction of radiotracer uptake in HCA-7 cells in response to pre-treatment with various COX-2 inhibitors in a concentration-dependent manner indicated that cellular uptake and retention of [18F]Pyricoxib is largely related to specific binding to COX-2. However, blocking efficacy varied among the applied COX-2 inhibitors and was most evident with celecoxib. No complete blockage of radiotracer uptake could be achieved, and the remaining radioactivity levels of >35?% even at high inhibitor concentrations of 100?M is indicative of some non-specific intracellular binding of the radiotracer. Interactions of radiotracer [18F]Pyricoxib with COX-2 and non-COX targets would explain the observed broad variety in blocking efficacy using different COX-2 inhibitors, assuming that every used compound possesses a distinct affinity and selectivity profile for both COX and non-COX targets. However, given the data determined during the present study, it is not possible to speculate about the nature of potential non-COX targets, although some secondary targets have been identified in the literature. Most of the research in this area focused on celecoxib [32C34]. COX-2 inhibitors like celecoxib do not interact with COX-2 alone; they can also interact with a variety of other molecular targets. Celecoxib was shown to directly target Ca2+ ATPase, protein-dependent kinase Thbs2 1 (PDK-1), cycline-dependent kinases (CDKs) in concert with various cyclins, and carbonic anhydrases (CA) [32]. Direct inhibition of these proteins by celecoxib allows the drug to exert anti-carcinogenic properties in a COX-2 independent manner. Although coxibs are selective for COX-2 over COX-1, the assumption that these molecules are truly selective drugs is faulty. The therapeutic efficacy of drugs like celecoxib and rofecoxib can not only be attributed to the inhibition of arachidonic acid metabolism through inhibition of COX-2 enzyme exclusively. Data on the non-COX affinity profile for Pyricoxib is not available, but since celecoxib, rofecoxib, and Pyricoxib share a number of key COX-2 pharmacophores, the possibility that they share a number of non-COX molecular targets must be considered. However, at this point, potential non-COX interactions of radiotracer [18F]Pyricoxib were not further analyzed during this study. Pre-clinical PET imaging experiments provided further evidence of COX-2-mediated uptake of [18F]Pyricoxib in COX-2-expressing HCA-7 tumors. Consistent with cellular uptake results in COX-2-expressing HCA-7 cells, this radiotracer showed.