N-desmethyl-loperamide is selective for P-glycoprotein among three ATP-binding cassette transporters at the blood-brain barrier.

[(11)C]N-desmethyl-Loperamide ([(11)C]dLop) is used in positron emission tomography (PET) to measure the in vivo activity of efflux transporters that block the passage of drugs across the blood-brain barrier. The three most prevalent ATP-binding cassette efflux transporters at the blood-brain barrier are P-glycoprotein (P-gp), multidrug resistance protein 1 (Mrp1), and breast cancer resistance protein (BCRP). We sought to measure the selectivity of dLop among these three transporters. The selectivity of dLop at low concentrations (< or =1 nM) was measured both as the accumulation of [(3)H]dLop in human cells that overexpress each transporter and as the uptake of [(11)C]dLop in brains of mice that lack genes encoding P-gp, Mrp1, or BCRP. The selectivity of dLop at high concentrations (> or =20 microM) was measured as the inhibition of uptake of a fluorescent substrate and the change in cytotoxicity of drugs effluxed at each transporter. Accumulation of [(3)H]dLop was lowest in cells overexpressing P-gp, and the uptake of [(11)C]dLop was highest in brains of mice lacking P-gp. At high concentrations, dLop selectively inhibited P-gp function and also decreased the resistance of only the P-gp-expressing cells to cytotoxic agents. dLop is selective for P-gp among these three transporters, but its activity is dependent on concentration. At low concentrations (< or =1 nM), dLop acts only as a substrate; at high concentrations (> or =20 microM), it acts as both a substrate and an inhibitor (i.e., a competitive substrate). Because low concentrations of radiotracer are used for PET imaging, [(11)C]dLop acts selectively and only as a substrate for P-gp.

Effects of ketoconazole on the biodistribution and metabolism of [11C]loperamide and [11C]N-desmethyl-loperamide in wild-type and P-gp knockout mice.

INTRODUCTION: [(11)C]Loperamide and [(11)C]N-desmethyl-loperamide ([(11)C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [(11)C]loperamide metabolism is N-demethylation to [(11)C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [(11)C]loperamide and [(11)C]dLop in mice, and thereby improve the quality of these radiotracers. METHODS: Studies were performed in wild-type and P-gp knockout (mdr-1a/b -/-) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, was pretreated with ketoconazole (50 mg/kg, ip), while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [(11)C]loperamide or [(11)C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-high-performance liquid chromatography. RESULTS: Ketoconazole increased the plasma concentrations of [(11)C]loperamide and its main radiometabolite, [(11)C]dLop, by about twofold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased threefold. Furthermore, ketoconazole increased the brain concentrations of [(11)C]loperamide and the radiometabolite [(11)C]dLop by about twofold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82% and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [(11)C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [(11)C]dLop concentration. The least polar radiometabolite of [(11)C]dLop was identified with LC-MS(n) as the N-hydroxymethyl analog of [(11)C]dLop and this also behaved as a P-gp substrate. CONCLUSION: In this study, ketoconazole (50 mg/kg, ip) proved partially effective for inhibiting the N-demethylation of [(11)C]loperamide in mouse in vivo but had relatively smaller or no effect on [(11)C]dLop.

Quantification of translocator protein (18 kDa) in the human brain with PET and a novel radioligand, (18)F-PBR06.

UNLABELLED: Translocator protein (TSPO) (18 kDa), formerly called the peripheral benzodiazepine receptor, is upregulated on activated microglia and macrophages and is, thus, a biomarker of inflammation. We previously reported that an (11)C-labeled aryloxyanilide (half-life, 20 min) was able to quantify TSPOs in the healthy human brain. Because many PET centers would benefit from a longer-lived (18)F-labeled radioligand (half-life, 110 min), the objective of this study was to evaluate the ability of a closely related aryloxyanilide ((18)F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline [(18)F-PBR06]) to quantify TSPOs in the healthy human brain. METHODS: A total of 9 human subjects were injected with (18)F-PBR06 (approximately 185 MBq) and scanned for 5 h, with rest periods outside the camera. The concentrations of (18)F-PBR06, separated from radiometabolites, were measured in arterial plasma. RESULTS: Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model. Even if data for all time points were used for the fitting, concentrations of brain activity measured with PET were consistently greater than the modeled values at late (280-300 min) but not at early time points. The greater values may have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume (V(T)), which is the summation of receptor binding and nondisplaceable activity, we investigated which scan duration would be associated with maximal or near-maximal identifiability. We found that a scan of 120 min provided the best identifiability of V(T) (approximately 2%). The images showed no significant defluorination. CONCLUSION: (18)F-PBR06 can quantify TSPOs in the healthy human brain using 120 min of image acquisition and concurrent measurements of radioligand in plasma. Although brain activity is likely contaminated with radiometabolites, the percentage contamination is thought to be small (<10%), because values of distribution volume are stable during 60-120 min and vary by less than 10%. (18)F-PBR06 is a longer-lived and promising alternative to (11)C-labeled radioligands to measure TSPOs as a biomarker of inflammation in the brain.

Human brain imaging and radiation dosimetry of 11C-N-desmethyl-loperamide, a PET radiotracer to measure the function of P-glycoprotein.

UNLABELLED: P-glycoprotein (P-gp) is a membrane-bound efflux pump that limits the distribution of drugs to several organs of the body. At the blood-brain barrier, P-gp blocks the entry of both loperamide and its metabolite, N-desmethyl-loperamide (N-dLop), and thereby prevents central opiate effects. Animal studies have shown that (11)C-dLop, compared with (11)C-loperamide, is an especially promising radiotracer because it generates negligible radiometabolites that enter the brain. The purposes of this study were to determine whether (11)C-dLop is a substrate for P-gp at the blood-brain barrier in humans and to measure the distribution of radioactivity in the entire body to estimate radiation exposure. METHODS: Brain PET scans were acquired in 4 healthy subjects for 90 min and included concurrent measurements of the plasma concentration of unchanged radiotracer. Time-activity data from the whole brain were quantified using a 1-tissue-compartment model to estimate the rate of entry (K(1)) of radiotracer into the brain. Whole-body PET scans were acquired in 8 healthy subjects for 120 min. RESULTS: For brain imaging, after the injection of (11)C-dLop the concentration of radioactivity in the brain was low (standardized uptake value, approximately 15%) and stable after approximately 20 min. In contrast, uptake of radioactivity in the pituitary was about 50-fold higher than that in the brain. The plasma concentration of (11)C-dLop declined rapidly, but the percentage composition of plasma was unusually stable, with the parent radiotracer constituting 85% of total radioactivity after approximately 5 min. The rate of brain entry was low (K(1) = 0.009 +/- 0.002 mL.cm(-3).min(-1); n = 4). For whole-body imaging, as a measure of radiation exposure to the entire body the effective dose of (11)C-dLop was 7.8 +/- 0.6 muSv/MBq (n = 8). CONCLUSION: The low brain uptake of radioactivity is consistent with (11)C-dLop being a substrate for P-gp in humans and confirms that this radiotracer generates negligible quantities of brain-penetrant radiometabolites. In addition, the low rate of K(1) is consistent with P-gp rapidly effluxing substrates while they transit through the lipid bilayer. The radiation exposure of (11)C-dLop is similar to that of many other (11)C-radiotracers. Thus, (11)C-dLop is a promising radiotracer to study the function of P-gp at the blood-brain barrier, at which impaired function would allow increased uptake into the brain.

P-glycoprotein function at the blood-brain barrier imaged using 11C-N-desmethyl-loperamide in monkeys.

UNLABELLED: 11C-Loperamide is an avid substrate for P-glycoprotein (P-gp), but it is rapidly metabolized to 11C-N-desmethyl-loperamide (11C-dLop), which is also a substrate for P-gp and thereby contaminates the radioactive signal in the brain. Should further demethylation of 11C-dLop occur, radiometabolites with low entry into the brain are generated. Therefore, we evaluated the ability of 11C-dLop to quantify the function of P-gp at the blood-brain barrier in monkeys. METHODS: Six monkeys underwent 12 PET scans of the brain, 5 at baseline and 7 after pharmacologic blockade of P-gp. A subset of monkeys also underwent PET scans with 15O-water to measure cerebral blood flow. To determine whether P-gp blockade affected peripheral distribution of 11C-dLop, we measured whole-body biodistribution in 4 monkeys at baseline and after P-gp blockade. RESULTS: The concentration of 11C-dLop in the brain was low under baseline conditions and increased 5-fold after P-gp blockade. This increase was primarily caused by an increased rate of entry into the brain rather than a decreased rate of removal from the brain. With P-gp blockade, uptake of radioactivity among brain regions correlated linearly with blood flow, suggesting a high single-pass extraction. After correction for cerebral blood flow, the uptake of 11C-dLop was fairly uniform among brain regions, suggesting that the function of P-gp is fairly uniformly distributed in the brain. On whole-body imaging, P-gp blockade significantly affected distribution of radioactivity only to the brain and not to other visually identified source organs. The effective dose estimated for humans was approximately 9 microSv/MBq. CONCLUSION: PET with 11C-dLop can quantify P-gp function at the blood-brain barrier in monkeys. The single-pass extraction of 11C-dLop is high and requires correction for blood flow to accurately measure the function of this efflux transporter. The low uptake at baseline and markedly increased uptake after P-gp blockade suggest that 11C-dLop will be useful to measure a wide range of P-gp functions at the blood-brain barrier in humans.

Metabotropic glutamate subtype 5 receptors are quantified in the human brain with a novel radioligand for PET.

UNLABELLED: We developed a radioligand, 3-fluoro-5-(2-(2-(18)F-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ((18)F-SP203), for metabotropic glutamate subtype 5 (mGluR5) receptors that showed both promising (high specific binding) and problematic (defluorination) imaging characteristics in animals. The purposes of this initial evaluation in human subjects were to determine whether (18)F-SP203 is defluorinated in vivo (as measured by uptake of radioactivity in the skull) and to determine whether the uptake in the brain can be quantified as distribution volume relative to concentrations of (18)F-SP203 in plasma. METHODS: Seven healthy subjects were injected with (18)F-SP203 (323 +/- 87 MBq) and scanned over 5 h, with rest periods outside the camera. The concentrations of (18)F-SP203, separated from radiometabolites, were measured in arterial plasma. RESULTS: The skull was difficult to visualize on PET images in the initial 2 h, because of high radioactivity in the brain. Although radioactivity in the skull and adjacent cortex showed some cross-contamination, the concentration of radioactivity in the skull was less than half of that in the adjacent cortex during the initial 2 h. Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model, consistent with measurable amounts of both receptor-specific and nonspecific binding. The concentrations of activity in the brain measured with PET were consistently greater than the modeled values at late but not early time points and may well have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume, we selected a scan duration (i.e., 2 h) associated with maximal or near-maximal identifiability. Distribution volume was well identified ( approximately 2%) by only 2 h (and even just 1) of image acquisition. CONCLUSION: This initial evaluation of (18)F-SP203 in healthy human subjects showed that defluorination is relatively small and that brain uptake can be robustly calculated as distribution volume. The values of distribution volume were well identified and had relatively small variation in this group of 7 subjects. These results suggest that (18)F-SP203 will have good sensitivity to measure mGluR5 receptors for both within-subject studies (e.g., receptor occupancy) and between-subject studies (e.g., patients vs. healthy subjects).

Synthesis and evaluation of [N-methyl-11C]N-desmethyl-loperamide as a new and improved PET radiotracer for imaging P-gp function.

[(11)C]Loperamide has been proposed for imaging P-glycoprotein (P-gp) function with positron emission tomography (PET), but its metabolism to [N-methyl-(11)C] N-desmethyl-loperamide ([(11)C]dLop; [(11)C]3) precludes quantification. We considered that [(11)C]3 might itself be a superior radiotracer for imaging brain P-gp function and therefore aimed to prepare [(11)C]3 and characterize its efficacy. An amide precursor (2) was synthesized and methylated with [(11)C]iodomethane to give [(11)C]3. After administration of [(11)C]3 to wild-type mice, brain radioactivity uptake was very low. In P-gp (mdr-1a(-/-)) knockout mice, brain uptake of radioactivity at 30 min increased about 3.5-fold by PET measures, and over 7-fold by ex vivo measures. In knockout mice, brain radioactivity was predominantly (90%) unchanged radiotracer. In monkey PET experiments, brain radioactivity uptake was also very low but after P-gp blockade increased more than 7-fold. [(11)C]3 is an effective new radiotracer for imaging brain P-gp function and, in favor of future successful quantification, appears free of extensive brain-penetrant radiometabolites.