Examples of progress made during the prior year are summarized below. 1) In an important study, we demonstrated that the function of permeability glycoprotein (P-gp) could be selectively measured using a combination of tariquidar (a P-gp inhibitor) and the radiotracer 11C-N-desmethyl-loperamide (dLop) (Kannan et al, 2011). This initial study, in addition to being an important addition to the literature, was a key step in our continued exploration of P-gp. The radiotracer 11C-dLop images the in vivo function of P-gp, a transporter that blocks the entry of drugs that are substrates into brain. When P-gp is inhibited, 11C-dLop, a potent opiate agonist, enters the brain and becomes trapped there. From an imaging perspective, this trapping is beneficial because it amplifies the PET signal, essentially by accumulating radioactivity over time. In a previous study, we had demonstrated that this trapping was not caused by binding to opiate receptors. The present study examined whether 11C-dLop, a weak base, is ionically trapped in acidic lysosomes. To test this hypothesis, we used lysosomotropics to measure 3H-dLop accumulation in human cells. Because the in vivo trapping of dLop was seen after P-gp inhibition, we also measured 3H-dLop uptake in P-gp-expressing cells treated with the P-gp inhibitor tariquidar. All lysosomotropics decreased 3H-dLop accumulation by at least 50%. Surprisingly, we found that in P-gp-expressing cells, tariquidaras well as another P-gp inhibitordecreased 3H-dLop uptake. Consequently, we measured 11C-dLop uptake before and after pre-administration of tariquidar in lysosome-rich organs of P-gp knockout (KO) mice and humans. After tariquidar pre-treatment in both species, radioactivity uptake in these organs decreased by 35% to 40%. Our results indicate that dLop is trapped in lysosomes and that tariquidar competes with dLop for lysosomal accumulation in vitro and in vivo. It is important to note that although tariquidar and dLop compete for lysosomal trapping in the periphery, such competition does not occur in brain because tariquidar has negligible entry into brain. In summary, tariquidar and 11C-dLop can be used in combination to selectively measure the function of P-gp at the blood-brain barrier. 2) Our laboratory developed 11C-NOP-1A, a new radioligand for the nociceptin/orphanin FQ peptide (NOP) receptor. We found that 11C-NOP-1A is a useful radioligand for quantifying NOP receptors in monkey brain, and that its radiation dose is similar to that of other 11C-labeled ligands for neuroreceptors (Kimura et al, in press). We then extended this work to humans. In a second study, we assessed the ability of 11C-NOP-1A to quantify NOP receptors in human brain and estimated its radiation safety profile (Lohith et al, 2012). We determined that 11C-NOP-1A is a promising radioligand that reliably quantifies NOP receptors in human brain. The effective dose in humans is low and similar to that of other 11C-labeled radioligands, allowing multiple scans in one subject. The NOP receptor is a new class of opioid receptor that may play a pathophysiologic role in anxiety and drug abuse and is a potential therapeutic target in these disorders. In our first study, we assessed the utility of 11C-NOP-1A for quantifying NOP receptors in monkey brain and estimated the radiation safety profile of this novel radioligand based on its biodistribution in monkeys;11C-NOP-1A has high affinity (Ki = 0.15 nM) and appropriate lipophilicity (measured logD = 3.4) for PET brain imaging. Baseline and blocking PET scans were acquired from head to thigh on three rhesus monkeys for approximately 120 minutes after injection of 11C-NOP-1A. These six PET scans were used to quantify NOP receptors in brain and to estimate radiation exposure to organs of the body. In the blocked scans, a selective nonradioactive NOP receptor antagonist (SB-612111;1 mg/kg i.v.) was administered before 11C-NOP-1A. In all scans, arterial blood was sampled to measure the parent radioligand 11C-NOP-1A. Distribution volume (VT;a measure of receptor density) was calculated with a compartment model using brain and arterial plasma data. Radiation-absorbed doses were calculated using the Medical Internal Radiation Dose Committee scheme. After 11C-NOP-1A injection, peak uptake of radioactivity in brain had a high concentration (5 SUV), occurred early (12 minutes), and thereafter washed out quickly. VT (mL E cm-3) was highest in neocortex (20) and lowest in hypothalamus and cerebellum (13). SB-612111 blocked 50-70% of uptake and reduced VT in all brain regions to 7 mL E cm-3. Distribution was well identified within 60 minutes of injection and stable for the remaining 60 minutes, consistent with only parent radioligand and not radiometabolites entering brain. Whole body scans confirmed that the brain had specific (i.e., displaceable) binding but could not detect specific binding in peripheral organs. The effective dose for humans estimated from the baseline scans in monkeys was 5.0 Sv/MBq. This preliminary study established that 11C-NOP-1A is a useful radioligand for quantifying NOP receptors in monkey brain, and that its radiation dose is similar to that of other 11C-labeled ligands for neuroreceptors. Thus, we conducted a second study to assess the ability of 11C-NOP-1A to quantify NOP receptors in human brain and estimated its radiation safety profile. After intravenous injection of 11C-NOP-1A, seven healthy subjects underwent brain PET for two hours and serial sampling of radial arterial blood to measure parent radioligand concentrations. VT was determined by compartmental (one- and two-tissue) and noncompartmental methods. A separate group of nine healthy subjects underwent whole-body PET to estimate whole-body radiation exposure (effective dose). We found that after 11C-NOP-1A injection, the peak concentration of radioactivity in brain was high (57 standardized uptake values), occurred early (10 minutes), and then washed out quickly. The unconstrained two-tissue-compartment model gave excellent VT identifiability (1.1% SE) and fitted the data better than a one-tissue-compartment model. Regional VT values (mL_cm23) ranged from 10.1 in temporal cortex to 5.6 in cerebellum. VT was well identified in the initial 70 minutes of imaging and remained stable for the remaining 50 minutes, suggesting that brain radioactivity was most likely parent radioligand, as supported by the fact that all plasma radiometabolites of 11C-NOP-1A were less lipophilic than the parent radioligand. Voxel-based MA1 VT values correlated well with results from the two-tissue-compartment model, showing that parametric methods can be used to compare populations. Whole-body scans showed radioactivity in brain and in peripheral organs expressing NOP receptors, such as heart, pancreas, and spleen. 11C-NOP-1A was significantly metabolized and excreted via the hepatobiliary route. Gall bladder had the highest radiation exposure (21 mSv/MBq), and the effective dose was 4.3 mSv/MBq. This study demonstrated that 11C-NOP-1A is a promising radioligand that reliably quantifies NOP receptors in human brain. The effective dose in humans is low and similar to that of other 11C-labeled radioligands, allowing multiple scans in one subject.
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