Examples of progress made during the prior year are summarized below. 1) Efflux transporters at the blood-brain barrier can decrease the entry of drugs and increase the removal of those molecules able to bypass the transporter. This issue is important because overexpression of efflux transporters can cause resistance to anti-epileptic medications, as occurs after chronic treatment in about one-third of patients. We previously hypothesized that 18F-FCWAY, a radioligand for the serotonin 5-HT1A receptor, is a weak substrate for permeability glycoprotein (P-gp) based on its very early peak and rapid washout from human brain. To determine whether 18F-FCWAY is a substrate for P-gp in humans, we examined the effect of tariquidar, a potent and selective P-gp inhibitor, using human cells transcribed with transporter expressed gene, transporter knockout mice, and in vivo human PET imaging following 18F-FCWAY injection. Because many substrates can be transported by either P-gp, breast cancer resistance protein (BCRP), or multidrug resistant protein (MRP1) the three most prevalent ABC transporters at the human blood-brain barrier we also examined the effect of BCRP and MRP1 in human cell culture and transgenic mice. Towards this end, we performed three sets of experiments. In vitro, we conducted fluorescence-activated cell sorting (FACS) flow cytometry studies in cells over-expressing P-gp, BCRP, and MRP1 treated with inhibitors specific to each transporter and with FCWAY. Ex vivo, we measured 18F-FCWAY concentrations in plasma and brain homogenate of transporter knockout mice using -counter and radio-HPLC. In vivo, we conducted PET studies to assess changes in eight humans who received 18F-FCWAY during an infusion of tariquidar (2-4mg/kg iv), a potent and selective P-gp inhibitor. In vitro studies showed that FCWAY allowed fluorescent substrates to get into the cell by competitive inhibition of all three transporters at the cell membrane. Ex vivo measurements in knockout mice indicated that 18F-FCWAY was a substrate only for P-gp and not BCRP. In vivo, tariquidar increased 18F-FCWAY brain uptake in seven of eight subjects by 60-100% compared to each person's baseline. Tariquidar did not increase brain uptake via some peripheral mechanism, given that it did not significantly alter concentrations in plasma of the parent radioligand 18F-FCWAY or its brain-penetrant radiometabolite 18F-FC. These results demonstrate that 18F-FCWAY is a weak substrate for efflux transport at the blood-brain barrier; some radioligand can enter brain, but its removal is hastened by P-gp. Although 18F-FCWAY is not ideal for measuring serotonergic receptors, the results suggest that weak substrate radioligands can be useful for measuring both increased and decreased function of efflux transporters, which is not possible with currently available radioligands such as 11C-loperamide and 11C-verapamil, which are avid substrates for transporters. 2) The cyclooxygenase (COX) system is implicated in the pathophysiology of brain diseases, including Alzheimers disease and depression, and is a potential biomarker for neuroinflammation. We sought to develop a PET radioligand for COX-1, which is almost exclusively localized in microglia and whose expression is increased by neuroinflammation. We also sought to develop a radioligand selective for COX-2, which is expressed in both neurons and glia. Although COX-2 is known to play a much larger role than COX-1 in peripheral inflammation, the relative role of these two enzymes in the brain is controversial. We synthesized and screened more than 50 compounds for inhibitory potency using in vitro enzymatic assays in whole blood from monkey and human. The leading candidates for development as PET radioligandsPS13 for COX-1 and MC1 for COX-2were radiolabeled with a short half-lived radioisotope, 11C (half-life = 20.4 min). About 180 MBq of each radioligand was injected intravenously into rhesus monkeys. Dynamic PET scans of brain and whole body were acquired for two hours, concurrent with frequent sampling of blood to measure the concentration of parent radioligand separated from radiometabolites. As determined by whole blood enzymatic assay, our results showed that PS13 was a potent and preferential inhibitor of COX-1 and that MC1 was a potent and preferential inhibitor of COX-2. With regard to brain, 11CPS13 showed specific uptake in healthy monkeys under baseline conditions; 11CMC1 showed specific uptake only after inducing neuroinflammation by administering a pyrogen (specifically, lipopolysaccharide). In addition, in the periphery, 11CPS13 showed specific uptake in organs with high expression of COX-1, such as spleen and gastrointestinal tract. No specific uptake of the COX-2 radioligand was noted in a healthy monkey brain. That is, COX-2 is minimally present in healthy brain without inflammation. In sum, these two PET radioligands displayed excellent imaging properties for selectively measuring COX-1 or COX-2 in monkeys. The percentage of total brain uptake specifically bound to each target was moderate for the COX-1 radioligand in a healthy monkey brain, and increased in a monkey model of neuroinflammation for the COX-2 radioligand. Our in vivo brain and peripheral imaging data show that COX-1 is present at baseline and increased by inflammation. In contrast, COX-2 is minimally present at baseline, but markedly increased after inflammation i.e., COX-2 is inducible. The results suggest that these two radioligands can be used as biomarkers of disease (e.g. for Alzheimers disease or depression), as well as to measure target engagement of anti-inflammatory drugs and to monitor therapeutic response.
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