Differential influence of propofol and isoflurane anesthesia in a non-human primate on the brain kinetics and binding of [(18)F]DPA-714, a positron emission tomography imaging marker of glial activation.

Translocator protein 18 kDa (TSPO) expression at the mitochondrial membrane of glial cells is related to glial activation. TSPO radioligands such as [(18)F]DPA-714 are useful for the non-invasive study of neuroimmune processes using positron emission tomography (PET). Anesthetic agents were shown to impact mitochondrial function and may influence [(18)F]DPA-714 binding parameters and PET kinetics. [(18) F]DPA-714 PET imaging was performed in Papio anubis baboons anesthetized using either intravenous propofol (n = 3) or inhaled isoflurane (n = 3). Brain kinetics and metabolite-corrected input function were measured to estimate [(18) F]DPA-714 brain distribution (VT). Displacement experiments were performed using PK11195 (1.5 mg/kg). In vitro [(18)F]DPA-714 binding experiments were performed using baboon brain tissue in the absence and presence of tested anesthetics. Brain radioactivity peaked higher in isoflurane-anesthetized animals compared with propofol (SUVmax = 2.7 ± 0.5 vs. 1.3 ± 0.2, respectively) but was not different after 30 min. Brain VT was not different under propofol and isoflurane. Displacement resulted in a 35.8 ± 8.4% decrease of brain radioactivity under propofol but not under isoflurane (0.1 ± 7.0%). In vitro, the presence of propofol increased TSPO density and dramatically reduced its affinity for [(18)F]DPA-714 compared with control. This in vitro effect was not significant with isoflurane. Exposure to propofol and isoflurane differentially influences TSPO interaction with its specific radioligand [(18)F]DPA-714 with subsequent impact on its tissue kinetics and specific binding estimated in vivo using PET. Therefore, the choice of anesthetics and their potential influence on PET data should be considered for the design of imaging studies using TSPO radioligands, especially in a translational research context.

Metabolism and quantification of [(18)F]DPA-714, a new TSPO positron emission tomography radioligand.

[(18)F]DPA-714 [N,N-diethyl-2-(2-(4-(2[(18)F]-fluoroethoxy)phenyl)5,7dimethylpyrazolo[1,5a]pyrimidin-3-yl)acetamide] is a new radioligand currently used for imaging the 18-kDa translocator protein in animal models of neuroinflammation and recently in humans. The biodistribution by positron emission tomography (PET) in baboons and the in vitro and in vivo metabolism of [(18)F]DPA-714 were investigated in rats, baboons, and humans. Whole-body PET experiments showed a high uptake of radioactivity in the kidneys, heart, liver, and gallbladder. The liver was a major route of elimination of [(18)F]DPA-714, and urine was a route of excretion for radiometabolites. In rat and baboon plasma, high-performance liquid chromatography (HPLC) metabolic profiles showed three major radiometabolites accounting for 85% and 89% of total radioactivity at 120 minutes after injection, respectively. Rat microsomal incubations and analyses by liquid chromatography-mass spectrometry (LC-MS) identified seven metabolites, characterized as O-deethyl, hydroxyl, and N-deethyl derivatives of nonradioactive DPA-714, two of them having the same retention times than those detected in rat and baboon plasma. The third plasma radiometabolite was suggested to be a carboxylic acid compound that accounted for 15% of the rat brain radioactivity. O-deethylation led to a nonradioactive compound and [(18)F]fluoroacetic acid. Human CYP3A4 and CYP2D6 were shown to be involved in the oxidation of the radioligand. Finally an easy, rapid, and accurate method--indispensable for PET quantitative clinical studies--for quantifying [(18)F]DPA-714 by solid-phase extraction was developed. In vivo, an extensive metabolism of [(18)F]DPA-714 was observed in rats and baboons, identified as [(18)F]deethyl, [(18)F]hydroxyl, and [(18)F]carboxylic acid derivatives of [(18)F]DPA-714. The main route of excretion of the unchanged radioligand in baboons was hepatobiliary while that of radiometabolites was the urinary system.

Discrepancies in the P-glycoprotein-mediated transport of (18)F-MPPF: a pharmacokinetic study in mice and non-human primates.

PURPOSE: Several in vivo studies have found that the 5-HT(1A) PET radioligand (18)F-MPPF is a substrate of rodent P-glycoprotein (P-gp). However, in vitro assays suggest that MPPF is not a substrate of human P-gp. We have now tested the influence of inhibiting P-gp on the brain kinetics of (18)F-MPPF in mice and non-human primates. METHODS: We measured the peripheral kinetics (arterial input function, metabolism, free fraction in plasma (f(P))) during (18)F-MPPF brain PET scanning in baboons with or without cyclosporine A (CsA) infusion. We measured (3)H-MPPF transport at the mouse BBB using in situ brain perfusion in P-gp/Bcrp deficient mice and after inhibiting P-gp with PSC833. RESULTS: There was an unexpected 1.9-fold increase in brain area under the curve in CsA-treated baboons (n = 4), with no change in radiometabolite-corrected arterial input. However, total volume of distribution corrected for f(P) (V(T)/f(P)) remained unchanged. In situ brain perfusion showed that P-gp restricted the permeability of the mouse BBB to (3)H-MPPF while Bcrp did not. CONCLUSION: These and previous in vitro results suggest that P-gp may not influence the permeability of human BBB to (18)F-MPPF. However, CsA treatment increased (18)F-MPPF free fraction, which is responsible for a misleading, P-gp unrelated enhanced brain uptake.

[(11)C]SL25.1188, a new reversible radioligand to study the monoamine oxidase type B with PET: preclinical characterisation in nonhuman primate.

[(11)C]SL-25.1188 [(S)-5-methoxymethyl-3-[6-(4,4,4-trifluorobutoxy)-benzo[d]isoxazol-3-yl]-oxazolidin-2-one], an oxazolidinone derivative, was characterized in baboons as a radioligand for the in vivo visualization of MAO-B using positron emission tomography (PET). After i.v. injection, [(11)C]SL25.1188 presented a rapid phase of distribution in blood (about 5 min), followed by a T(1/2) elimination of 85 +/- 14 min. Plasma metabolism analysis showed that [(11)C]SL25.1188 is stable in vivo at least for 30 min. Brain uptake was rapid with the highest one observed in the striatum and thalamus, and the lowest in the pons. Calculated distribution volumes (V(T)) were as follows: striatum = 10.3, thalamus = 10.9, hippocampus = 8.9, temporal cortex = 7.7, occipital cortex = 7.2, parietal cortex = 7.4, frontal cortex = 7.4, white matter = 7.4, and pons = 6.1. Pretreatment with deprenyl (2 mg/kg, i.v.) or lazabemide (0.5 mg/kg, i.v.) reduced V(T) values in all brain areas up to 50%. In displacement experiments, injection of SL25.1188 or deprenyl (1 and 2 mg/kg, i.v., respectively) strongly reduced the specific uptake of [(11)C]SL25.1188 in all brain areas (85-100%), while a lesser displacement was observed with lazabemide (0.5 mg/kg, i.v.) (55-70% of specific binding depending on the brain area). Therefore, [(11)C]SL25.1188 is characterized in vivo by reversible binding, high brain uptake and very slow plasma metabolism, strongly suggesting that this radioligand is a potent tool for the in vivo study of brain MAO-B.

18F-ZW-104: a new radioligand for imaging neuronal nicotinic acetylcholine receptors--in vitro binding properties and PET studies in baboons.

UNLABELLED: An extensive series of radioligands has been developed for imaging central nicotinic acetylcholine receptors (nAChRs) with PET. Two halogeno-derivatives of A-85380 are being used in humans. Nevertheless, these derivatives still display too-slow brain kinetics and low signal-to-noise ratio. METHODS: A novel nAChR radioligand, 5-(6-fluorohexyn-1-yl)-3-[2(S)-2-azetidinylmethoxy]pyridine (ZW-104), was characterized in vitro using competition binding assays (nAChR subtypes heterologously expressed in HEK 293 cells and in native alpha4beta2 nAChRs from rat brain). (18)F-ZW-104 was prepared as follows: no-carrier-added nucleophilic aliphatic radiofluorination of the corresponding N-Boc-protected tosyloxy derivative 5-(6-tosyloxyhexyn-1-yl)-3-[2(S)-(N-(tert-butoxycarbonyl))-2-azetidinylmethoxy] pyridine) with the activated 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8,8,8]hexacosane (K-(18)F-F-Kryptofix 222 [K(222)] complex), followed by quantitative trifluoroacetic acid-induced removal of the N-Boc protective group. (18)F-ZW-104 was then studied in baboons using PET. RESULTS: ZW-104 showed high binding affinities for rat alpha4beta2 nAChRs (K(i), 0.2 nM) and other subtypes containing the beta2 subunit but much lower affinities for rat alpha3beta4 nAChRs (K(i), 5,500 nM) and other subtypes containing the beta4 subunit. The regional radioactivity distribution in the baboon brain matched that of the alpha4beta2 nAChR, which was similar to that of 2-(18)F-fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-(18)F-A-85380), a radioligand used in humans. Comparison between (18)F-ZW-104 and 2-(18)F-A-85380 demonstrated better in vivo binding properties of the new radioligand: a substantially greater amount of radioactivity accumulated in the brain, and the occurrence of peak uptake in the thalamus was earlier than that of 2-(18)F-A-85380 and was followed by washout. Distribution volume values in different brain regions were 2-fold higher for (18)F-ZW-104 than for 2-(18)F-A-85380. Displacement by nicotine or unlabeled ZW-104 demonstrated a lower nonspecific binding than that of 2-F-A-85380. CONCLUSION: These results suggest that (18)F-ZW-104 is a promising PET radioligand for studying nAChRs containing the beta2 subunits in humans.

Quantification of cerebral nicotinic acetylcholine receptors by PET using 2-[18F]fluoro-A-85380 and the multiinjection approach.

The multiinjection approach was used to study in vivo interactions between alpha4beta2(*) nicotinic acetylcholine receptors and 2-[(18)F]fluoro-A-85380 in baboons. The ligand kinetics was modeled by the usual nonlinear compartment model composed of three compartments (arterial plasma, free and specifically bound ligand in tissue). Arterial blood samples were collected to generate a metabolite-corrected plasma input function. The experimental protocol, which consisted of three injections of labeled or unlabeled ligand, was aiming at identifying all parameters in one experiment. Various parameters, including B'(max) (the binding sites density) and K(d)V(R) (the apparent in vivo affinity of 2-[(18)F]fluoro-A-85380) could then be estimated in thalamus and in several receptor-poor regions. B'(max) estimate was 3.0+/-0.3 pmol/mL in thalamus, and ranged from 0.25 to 1.58 pmol/mL in extrathalamic regions. Although K(d)V(R) could be precisely estimated, the association and dissociation rate constants k(on)/V(R) and k(off) could not be identified separately. A second protocol was then used to estimate k(off) more precisely in the thalamus. Having estimated all model parameters, we performed simulations of 2-[(18)F]fluoro-A-85380 kinetics to test equilibrium hypotheses underlying simplified approaches. These showed that a pseudo-equilibrium is quickly reached between the free and bound compartments, a favorable situation to apply Logan graphical analysis. In contrast, the pseudo-equilibrium between the plasma and free compartments is only reached after several hours. The ratio of radioligand concentration in these two compartments then overestimates the true equilibrium value, an unfavorable situation to estimate distribution volumes from late images after a bolus injection.

Synthesis and radiolabeling of N-[4-[4-(2-[11C]methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-carboxamide -- a potential radiotracer for D3 receptor imaging with PET.

FAUC346 (N-[4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-carboxamide), an in vitro D(3)-selective ligand, and its normethyl derivative have been synthesized from commercially available 1-(2-substituted-phenyl)piperazines. FAUC346 has been labeled using [(11)C]methyl triflate in acetone containing aqueous NaOH (5 Eq) at -10 degrees C for 1 min, purified on semipreparative reverse-phase high-performance liquid chromatography (HPLC) and formulated as an intravenous injectable solution using a Sep-Pak Plus C(18) device. Up to 5.5 GBq of [(11)C]FAUC346 (N-[4-[4-(2-[methyl-(11)C]methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-carboxamide), with a specific radioactivity of 45-75 GBq/micromol, could be obtained in 30-35 min, including HPLC purification and formulation starting from 44.4 GBq of [(11)C]carbon dioxide. Preliminary pharmacological evaluation of [(11)C]FAUC346 in rat brain clearly demonstrated in vivo selectivity for D(3) receptors and the absence of radiolabeled metabolite within the brain. These encouraging results, however, could not be confirmed in nonhuman primates; therefore, this radioligand does not appear to have the required pharmacological profile for a positron emission tomography probe for imaging D(3) receptors.

Radiosynthesis of (E)-N-(2-[11C]methoxybenzyl)-3-phenyl-acrylamidine, a novel subnanomolar NR2B subtype-selective NMDA receptor antagonist.

Recently, a novel series of amidines has been described, exhibiting high NR2B-subtype selective N-methyl-D-aspartate (NMDA) antagonist activity with nanomolar or subnanomolar affinity. Within the styrylamidine subclass, (E)-N-(2-methoxybenzyl)-3-phenyl-acrylamidine (1), displayed the highest affinity (Ki=0.7 nM versus [(3)H]ifenprodil) and was considered an appropriate candidate for isotopic labelling with carbon-11 (T(1/2): 20.38 min) at its methoxy group for imaging of NMDA receptors with PET. Derivative 1 has been labelled from the corresponding nor-analogue using [(11)C]methyl triflate and the following experimental conditions : (1) trapping at -10 degrees C of [(11)C]methyl triflate in 300 microL of acetone containing 0.6-0.8 mg of precursor 5 (2.4-3.2 micromol) and 5 microL of a 3M solution of NaOH in water (about 5 eq.); (2) concentration to dryness of the reaction mixture (at 110 degrees C, using a helium stream for 1-2 min); (3) taking up the residue with 0.5 mL of the HPLC mobile phase and (4) purification using semi-preparative HPLC (SymmetryPrep) C-18, Waters, 300 x 7.8 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [(11)C]CO(2) production batch, 120-240 m Ci (4.44-8.88 GBq) of [(11)C]-1 (20-40% decay-corrected radiochemical yield, n=5) was obtained within a total synthesis time of 25-30 min. Specific radioactivities ranged from 0.8 to 1.2 Ci/micromol (29.6-44.4 GBq/micromol) at the end of radiosynthesis. No attempts were made to further optimise these reactions, as sufficient material was obtained to allow for preliminary pharmacological characterisation.

Imaging the impact of cyclosporin A and dipyridamole on P-glycoprotein (ABCB1) function at the blood-brain barrier: A [(11)C]-N-desmethyl-loperamide PET study in nonhuman primates.

Cyclosporin A (CsA) and dipyridamole (DPy) are potent inhibitors of the P-glycoprotein (P-gp; ABCB1) in vitro. Their efficacy at inhibiting P-gp at the blood-brain barrier (BBB) is difficult to predict. Efficient and readily available (i.e. marketed) P-gp inhibitors are needed as probes to investigate the role of P-gp at the human BBB. In this study, the P-gp inhibition potency at the BBB of therapeutic doses of CsA or DPy was evaluated in baboons using Positron Emission Tomography (PET) imaging with [(11)C]-N-desmethyl-loperamide ([(11)C]dLop), a radiolabeled P-gp substrate. The preparation of dLop as authentic standard and [(11)C]dLop as radiotracer were revisited so as to improve their production yields. [(11)C]dLop PET imaging was performed in the absence (n=3, baseline condition) and the presence of CsA (15mg/kg/h i.v., n=3). Three animals were injected with i.v. DPy at either 0.56 or 0.96 or 2mg/kg (n=1), corresponding to the usual, maximal and twice the maximal dose in patients, respectively, administered immediately before PET. [(11)C]dLop brain kinetics as well as [(11)C]dLop kinetics and radiometabolites in arterial plasma were measured to calculate [(11)C]dLop area-under the time-activity curve from 10 to 30min in the brain (AUCbrain) and in plasma (AUCplasma). [(11)C]dLop brain uptake was described by AUCR=AUCbrain/AUCplasma. CsA as well as DPy did not measurably influence [(11)C]dLop plasma kinetics and metabolism. Baseline AUCR (0.85±0.29) was significantly enhanced in the presence of CsA (AUCR=10.8±3.6). Injection of pharmacologic dose of DPy did not enhance [(11)C]dLop brain distribution with AUCR being 1.2, 0.9 and 1.1 after administration of 0.56, 0.96 and 2mg/kg DPy doses, respectively. We used [(11)C]dLop PET imaging in baboons, a relevant in vivo model of P-gp function at the BBB, to show the P-gp inhibition potency of therapeutic dose CsA. Despite in vitro P-gp inhibition potency, usual doses DPy are not likely to inhibit P-gp function at the BBB.

In vivo imaging of human cerebral nicotinic acetylcholine receptors with 2-18F-fluoro-A-85380 and PET.

UNLABELLED: 2-(18)F-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine (2-(18)F-fluoro-A-85380) is a PET radioligand that is specific for nicotinic acetylcholine receptors (nAChRs) and has a high affinity for the alpha(4)beta(2) subtype. The purpose of this study was to evaluate different strategies to quantify 2-(18)F-fluoro-A-85380 binding in healthy nonsmoking human volunteers. METHODS: After intravenous injection of 189 +/- 30 MBq (0.8-5.7 nmol) of 2-(18)F-fluoro-A-85380, the first dynamic PET scan was acquired over 150 min. The second 30-min PET scan was performed 60 min later. Time-activity curves were generated from volumes of interest. 2-(18)F-Fluoro-A-85380 volume of distribution (DV) was quantified using compartmental kinetic analysis and Logan graphical analysis. In the kinetic analysis, the 1-tissue compartment model (1TCM) and the 2-tissue (2TCM) compartment model were applied. The most appropriate kinetic model was determined using the Akaike Information Criterion. The effect of reducing the PET study duration on the reliability of the DV values computed by the kinetic and the graphical analyses was evaluated. RESULTS: Time-activity curves were better described by the 2TCM. The DV values ranged from 5.2 +/- 0.5 in the occipital cortex, 6.2 +/- 0.2 in the frontal cortex, and 7.3 +/- 0.4 in the putamen to 15.4 +/- 2.1 in the thalamus. These regional DV values were consistent with the distribution of nAChRs in the human brain. Logan graphical analysis provided slightly lower DV values than those of the 2TCM (from -3.5% in the occipital cortex to -6.6% in the thalamus). The minimal study duration required to obtain stable DV estimates in all regions was similar for the 2 methods: 140 min for the 2TCM and 150 min for the Logan analysis. DV estimates obtained with the 2TCM were more stable than those calculated by the Logan approach for the same scan duration. CONCLUSION: These results show that 2-(18)F-fluoro-A-85380 can be used to assess nAChRs binding in the human brain with PET.

Influence of P-Glycoprotein Inhibition or Deficiency at the Blood-Brain Barrier on (18)F-2-Fluoro-2-Deoxy-D-glucose ( (18)F-FDG) Brain Kinetics.

The fluorinated D-glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) is the most prevalent radiopharmaceutical for positron emission tomography (PET) imaging. P-Glycoprotein's (P-gp, MDR1, and ABCB1) function in various cancer cell lines and tumors was shown to impact (18)F-FDG incorporation, suggesting that P-gp function at the blood-brain barrier may also modulate (18)F-FDG brain kinetics. We tested the influence of P-gp inhibition using the cyclosporine analog valspodar (PSC833; 5 µM) on the uptake of (18)F-FDG in standardized human P-gp-overexpressing cells (MDCKII-MDR1). Consequences for (18)F-FDG brain kinetics were then assessed using (i) (18)F-FDG PET imaging and suitable kinetic modelling in baboons without or with P-gp inhibition by intravenous cyclosporine infusion (15 mg kg(-1) h(-1)) and (ii) in situ brain perfusion in wild-type and P-gp/Bcrp (breast cancer resistance protein) knockout mice and controlled D-glucose exposure to the brain. In vitro, the time course of (18)F-FDG uptake in MDR1 cells was influenced by the presence of valspodar in the absence of D-glucose but not in the presence of high D-glucose concentration. PET analysis revealed that P-gp inhibition had no significant impact on estimated brain kinetics parameters K 1, k 2, k 3, V T , and CMRGlc. The lack of P-gp effect on in vivo (18)F-FDG brain distribution was confirmed in P-gp/Bcrp-deficient mice. P-gp inhibition indirectly modulates (18)F-FDG uptake into P-gp-overexpressing cells, possibly through differences in the energetic cell level state. (18)F-FDG is not a P-gp substrate at the BBB and (18)F-FDG brain kinetics as well as estimated brain glucose metabolism are influenced by neither P-gp inhibition nor P-gp/Bcrp deficiencies in baboon and mice, respectively.

Biodistribution and radiation dosimetry of 18F-fluoro-A-85380 in healthy volunteers.

UNLABELLED: This study reports on the biodistribution and radiation dosimetry of 2-(18)F-Fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine ((18)F-fluoro-A-85380), a promising radioligand for the imaging of central nicotinic acetylcholine receptors (nAChRs). METHODS: Whole-body scans were performed in 3 healthy male volunteers up to 2 h after intravenous injection of 137-238 MBq (18)F-fluoro-A-85380. Transmission scans (3 min per step, 8 or 9 steps according to the height of the subject) in 2-dimensional mode were used for subsequent correction of attenuation of emission scans. Emission scans (1 min per step) were acquired over 2 h. Venous blood samples were taken up to 2 h after injection of the radiotracer. Urine was freely collected up to 2 h after injection of the radiotracer. For each subject, the percentage of injected activity measured in regions of interest over brain, intestine, stomach, bladder, kidneys, and liver were fitted to a monoexponential model, as an uptake phase followed by a monoexponential washout, or to a biexponential model to generate time-activity curves. Using the MIRD method, ten source organs were considered in estimating radiation absorbed doses for organs of the body. RESULTS: Injection of (18)F-fluoro-A-85380 was clinically well tolerated and blood and urine pharmacologic parameters did not change significantly. The primary routes of clearance were renal and intestinal. Ten minutes after injection, high activities were observed in the bladder, kidneys, and liver. Slow uptake was seen in the brain. The liver received the highest absorbed dose. The average effective dose of (18)F-fluoro-A-85380 was estimated to be 0.0194 mSv/MBq. CONCLUSION: The amount of (18)F-fluoro-A-85380 required for adequate nAChR imaging results in an acceptable effective dose equivalent to the patient.

In vivo quantification of myocardial dihydropyridine binding sites: a PET study in dogs.

UNLABELLED: Abnormalities in myocardial L-type Ca(2+) channel abundance and function have been described in cardiac hypertrophy and failure. In vivo quantification of the density of these channels using PET and an adequate ligand would provide new insights into cardiac disease. METHODS: The dihydropyridine L-type Ca(2+) channel antagonist S12968 (3-ethyl 5-methyl (-)-2-[(2-(2-aminoethoxy)ethoxy)methyl]-4-(2,3-dichlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate) was labeled with (11)C and injected in various amounts (5-23 nmol), 20 or 30 min apart, into dogs. This protocol allowed a separate evaluation of the density of binding sites (B(max)) as well as association and dissociation rate constants. The parameters were calculated using a nonlinear mathematic model. RESULTS: Using the multiinjection approach, a complete model describing interactions between S12968 and the dihydropyridine binding sites was obtained. B(max) was found to be 19.2 +/- 3.3 pmol x mL(-1) of tissue. Association and dissociation constants (estimated by K(on)/VR and K(d)VR, respectively) were found to be 0.015 +/- 0.01 mL x pmol(-1) x min(-1) and 4.2 +/- 2.2 nmol x mL(-1), respectively. CONCLUSION: The present data suggest that it is possible to measure myocardial dihydropyridine binding site density with a single radiosynthesis and a simple PET protocol that is not time consuming (75 min for the total examination, including transmission and emission scans). This methodology can be useful to investigate human cardiac disease in vivo.

Fluoro-A-85380 demonstrated no mutagenic properties in in vivo rat micronucleus and Ames tests.

The potential mutagenic properties (micronucleus and the Ames tests) of fluoro-A-85380 (2-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine) were evaluated as a mandatory pre-clinical step. No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in animals treated at any dose tested. No biologically significant increase in the mean number of revertants was noted in all the Salmonella typhimurium strains tested with fluoro-A-85380. Therefore, fluoro-A-85380 demonstrated no mutagenic properties using these two tests.

[(18)F]Fallypride: metabolism studies and quantification of the radiotracer and its radiometabolites in plasma using a simple and rapid solid-phase extraction method.

INTRODUCTION: [(18)F]Fallypride, a fluorinated and substituted benzamide with high affinity for D2/D3 receptors, is a useful PET radioligand for the study of striatal/extrastriatal areas. Since [(18)F]fallypride is extensively metabolized in vivo and since PET examinations are long lasting in humans, the rapid measurement of the unchanged radiotracer in plasma is essential for the quantification of images. The present study aims: i) to evaluate if the radiometabolites of [(18)F]fallypride cross the blood-brain barrier in rodents, ii) to identify these radiometabolites in baboon plasma and iii) to develop a rapid solid phase extraction method (SPE) suitable for human applications to quantify both [(18)F]fallypride and its radiometabolites in plasma. METHODS: The metabolites P450-dependant in rat and human liver microsomes were characterized by LC-MS-MS and compared to those detected in vivo. Sequential solvent elution on Oasis®-MCX-SPE cartridges was used to quantify [(18)F]fallypride and its radiometabolites. RESULT: In rat microsomal incubations, five metabolites generated upon N/O-dealkylation or hydroxylation at the pyrrolidine and/or at the benzamide moiety were identified. No radiometabolite was detected in the rat brain. N-dealkylated and hydroxylated derivatives were detected in human microsomal incubations as well as in baboon plasma. The use of SPE (total recovery 100.2%± 2.8%, extraction yield 95.5%± 0.3%) allowed a complete separation of [(18)F]fallypride from its radiometabolites in plasma and evaluate [(18)F]fallypride at 150 min pi to be 22%± 5% of plasma radioactivity. CONCLUSIONS: The major in vivo radiometabolites of [(18)F]fallypride were produced by N-dealkylation and hydroxylation. Allowing the rapid analysis of multiple plasma samples, SPE is a method of choice for the determination of [(18)F]fallypride until late images required for quantitative PET imaging in humans.

Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide.

Glyburide (glibenclamide, GLB) is a widely prescribed antidiabetic with potential beneficial effects in central nervous system injury and diseases. In vitro studies show that GLB is a substrate of organic anion transporting polypeptide (OATP) and ATP-binding cassette (ABC) transporter families, which may influence GLB distribution and pharmacokinetics in vivo. In the present study, we used [(11)C]GLB positron emission tomography (PET) imaging to non-invasively observe the distribution of GLB at a non-saturating tracer dose in baboons. The role of OATP and P-glycoprotein (P-gp) in [(11)C]GLB whole-body distribution, plasma kinetics, and metabolism was assessed using the OATP inhibitor rifampicin and the dual OATP/P-gp inhibitor cyclosporine. Finally, we used in situ brain perfusion in mice to pinpoint the effect of ABC transporters on GLB transport at the blood-brain barrier (BBB). PET revealed the critical role of OATP on liver [(11)C]GLB uptake and its subsequent impact on [(11)C]GLB metabolism and plasma clearance. OATP-mediated uptake also occurred in the myocardium and kidney parenchyma but not the brain. The inhibition of P-gp in addition to OATP did not further influence [(11)C]GLB tissue and plasma kinetics. At the BBB, the inhibition of both P-gp and breast cancer resistance protein (BCRP) was necessary to demonstrate the role of ABC transporters in limiting GLB brain uptake. This study demonstrates that GLB distribution, metabolism, and elimination are greatly dependent on OATP activity, the first step in GLB hepatic clearance. Conversely, P-gp, BCRP, and probably multidrug resistance protein 4 work in synergy to limit GLB brain uptake.

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling Part I: PET studies.

BACKGROUND: LBT-999 (E)-N-(4-fluorobut-2-enyl)-2ß-carbomethoxy-3ß-(4'-tolyl)nortropane is a dopamine transporter (DAT) ligand. [(18)F]LBT-999 was first labelled with carbon-11; we will now describe its in vivo behaviour in comparison to that of [(11)C]LBT-999. METHODS/RESULTS: Positron emission tomography (PET) experiments (baboons) confirmed the high affinity/specificity of [(18)F]LBT-999 for DAT. The brain regional distribution was in accordance with that of DAT. Pre-treatment with LBT-999 (1 mg/kg iv), but not with desipramine, a norepinephrine (NET) antagonist, reduced the striatum-to-cerebellum ratio by 96%, confirming the specificity for DAT vs. NET. The parent compound decreased rapidly and represented 24.3 ± 5.0% of plasma radioactivity at 30 min pi. Whole-body scans showed an important bone uptake of free fluorine following metabolism of [(18)F]LBT-999. In the cerebellum and striatum, distribution volumes increased by 30-40% between 80 and 230 min, suggesting the polluting role of a radiometabolite(s). [(11)C]LBT-999 exhibited a 40% higher standardized uptake value in the striata. This difference is likely due to N-dealkylation followed by [(18)F]fluoride release. 2ß-Carbomethoxy-3ß-(4'-tolyl) nortropane is then formed, while [(11)C]2ß-carbomethoxy-3ß-(4'-tolyl) nortropane is formed following injection of [(11)C]LBT-999. This metabolite has high affinity for the DAT. In one specific PET experiment, intravenous injection of this metabolite induced a strong displacement of [(18)F]LBT-999 in the striata, confirming that this metabolite readily crosses the blood-brain barrier (BBB) and binds to DAT. CONCLUSIONS: [(18)F]LBT-999 is N-dealkylated in vivo to yield (1) a nonradioactive metabolite that crosses the BBB and has a high affinity for the DAT and (2) a [(18)F]fluoro-alkyl chain which is further defluorinated. The temporal changes in distribution volumes are consistent with the accumulation of a radiometabolite(s) in the brain. Therefore, the quantification of DAT density with [(18)F]LBT-999 is rather difficult.

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling: part II: Metabolism studies.

INTRODUCTION: LBT-999, (E)-N-(4-fluorobut-2-enyl)-2ß-carbomethoxy-3ß-(4'-tolyl)nortropane, has been developed for PET imaging of the dopamine transporter. [(18)F]LBT-999 PET studies in baboons showed a lower brain uptake than [(11)C]LBT-999 and a high bone uptake, suggesting the presence of interfering metabolites. Therefore, in vitro and in vivo metabolism of these radiotracers was investigated. METHODS: Rat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS. RESULTS: In vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [(18)F]LBT-999 was rapidly converted into a [(18)F]hydroxylated metabolite likely oxidized in plasma into a [(18)F]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [(18)F]LBT-999 decreased rapidly in plasma and was higher than that of [(11)C]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [(18)F]LBT-999, [(18)F]hydroxylated and [(18)F]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the hydroxylated compound in the striatum. CONCLUSION: P450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [(18)F]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [(18)F]fluorobutenaldehyde (or its oxidation product) and [(18)F]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification.

Transport of selected PET radiotracers by human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2): an in vitro screening.

UNLABELLED: Radiolabeled compounds used for brain imaging with PET must readily cross the blood-brain barrier (BBB) to reach their target. Efflux transporters at the BBB-P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP)-could limit their uptake by the brain. METHODS: We developed and validated an in vitro model using MDCKII cells transfected with human multidrug resistance (MDR1) or BCRP genes and assessed the transport of selected PET ligands by the concentration equilibrium technique. The tested compounds included befloxatone, (R,S)-CGP-12177, clorgyline, R-(-)-deprenyl, diprenorphine, DPA-714, fallypride, flumazenil, 2-fluoro-A-85380, LBT-999, loperamide, p-MPPF, PE2I, Pittsburgh compound B (PIB), (R,S)-PK11195, raclopride, R-(+)-verapamil, and WAY-100635. The assays were performed using the nonradioactive form of each compound (ultraviolet high-performance liquid chromatography analysis) and, when available, the (18)F-labeled analogs (γ-counting). RESULTS: Befloxatone appeared to be transported solely by BCRP. Loperamide, verapamil, and diprenorphine were the only P-gp substrates. Other ligands were transported by neither P-gp nor BCRP. CONCLUSION: The present method can readily be used to screen new-compound transport by P-gp or BCRP, even before any radiolabeling. Compounds that were previously thought to be transported by P-gp in rodents, such as p-MPPF, WAY-100635, and flumazenil, cannot be considered substrates of human P-gp. The impact of BCRP and P-gp at the BBB on the transport of befloxatone and diprenorphine in vivo remains to be evaluated with PET.