Marie-Anne Peyronneau

Metabolism and Quantification of [18F]DPA-714, a New TSPO Positron Emission Tomography Radioligand

[18F]DPA-714 [N,N-diethyl-2-(2-(4-(2[18F]-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 [18F]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 [18F]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 [18F]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 [18F]DPA-714 by solid-phase extraction was developed. In vivo, an extensive metabolism of [18F]DPA-714 was observed in rats and baboons, identified as [18F]deethyl, [18F]hydroxyl, and [18F]carboxylic acid derivatives of [18F]DPA-714. The main route of excretion of the unchanged radioligand in baboons was hepatobiliary while that of radiometabolites was the urinary system.

Transport of Selected PET Radiotracers by Human P-Glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2): An In Vitro Screening

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 18F-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.

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 [11C]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 [11C]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 [11C]GLB uptake and its subsequent impact on [11C]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 [11C]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.

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

[11C]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, [11C]SL25.1188 presented a rapid phase of distribution in blood (about 5 min), followed by a T1/2 elimination of 85 ± 14 min. Plasma metabolism analysis showed that [11C]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 (VT) 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 VT 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 [11C]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, [11C]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. Synapse 64:61–69, 2010.

Discrepancies in the P-glycoprotein-Mediated Transport of 18F-MPPF: A Pharmacokinetic Study in Mice and Non-human Primates

PurposeSeveral in vivo studies have found that the 5-HT1A PET radioligand 18F-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 18F-MPPF in mice and non-human primates.MethodsWe measured the peripheral kinetics (arterial input function, metabolism, free fraction in plasma (fP)) during 18F-MPPF brain PET scanning in baboons with or without cyclosporine A (CsA) infusion. We measured 3H-MPPF transport at the mouse BBB using in situ brain perfusion in P-gp/Bcrp deficient mice and after inhibiting P-gp with PSC833.ResultsThere 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 fP (VT/fP) remained unchanged. In situ brain perfusion showed that P-gp restricted the permeability of the mouse BBB to 3H-MPPF while Bcrp did not.ConclusionThese and previous in vitro results suggest that P-gp may not influence the permeability of human BBB to 18F-MPPF. However, CsA treatment increased 18F-MPPF free fraction, which is responsible for a misleading, P-gp unrelated enhanced brain uptake.

Imaging the impact of the P-glycoprotein (ABCB1) function on the brain kinetics of metoclopramide

The effects of metoclopramide on the central nervous system (CNS) in patients suggest substantial brain distribution. Previous data suggest that metoclopramide brain kinetics may nonetheless be controlled by ATP-binding cassette (ABC) transporters expressed at the blood–brain barrier. We used 11C-metoclopramide PET imaging to elucidate the kinetic impact of transporter function on metoclopramide exposure to the brain. Methods: 11C-metoclopramide transport by P-glycoprotein (P-gp; ABCB1) and the breast cancer resistance protein (BCRP; ABCG2) was tested using uptake assays in cells overexpressing P-gp and BCRP. 11C-metoclopramide brain kinetics were compared using PET in rats (n = 4–5) in the absence and presence of a pharmacologic dose of metoclopramide (3 mg/kg), with or without P-gp inhibition using intravenous tariquidar (8 mg/kg). The 11C-metoclopramide brain distribution (VT based on Logan plot analysis) and brain kinetics (2-tissue-compartment model) were characterized with either a measured or an imaged-derived input function. Plasma and brain radiometabolites were studied using radio–high-performance liquid chromatography analysis. Results: 11C-metoclopramide transport was selective for P-gp over BCRP. Pharmacologic dose did not affect baseline 11C-metoclopramide brain kinetics (VT = 2.28 ± 0.32 and 2.04 ± 0.19 mL⋅cm−3 using microdose and pharmacologic dose, respectively). Tariquidar significantly enhanced microdose 11C-metoclopramide VT (7.80 ± 1.43 mL⋅cm−3) with a 4.4-fold increase in K1 (influx rate constant) and a 2.3-fold increase in binding potential (k3/k4) in the 2-tissue-compartment model. In the pharmacologic situation, P-gp inhibition significantly increased metoclopramide brain distribution (VT = 6.28 ± 0.48 mL⋅cm−3) with a 2.0-fold increase in K1 and a 2.2-fold decrease in k2 (efflux rate), with no significant impact on binding potential. In this situation, only parent 11C-metoclopramide could be detected in the brains of P-gp–inhibited rats. Conclusion: 11C-metoclopramide benefits from favorable pharmacokinetic properties that offer reliable quantification of P-gp function at the blood–brain barrier in a pharmacologic situation. Using metoclopramide as a model of CNS drug, we demonstrated that P-gp function not only reduces influx but also mediates the efflux from the brain back to the blood compartment, with additional impact on brain distribution. This PET-based strategy of P-gp function investigation may provide new insight on the contribution of P-gp to the variability of response to CNS drugs between patients.

Preparation and evaluation of novel pyrazolo[1,5- a ]pyrimidine acetamides, closely related to DPA-714, as potent ligands for imaging the TSPO 18kDa with PET

A series of four novel analogues of DPA-714, bearing a fluoroalkynyl side chain (with a length ranging from three to six carbon atoms) in replacement of the fluoroethoxy motif, have been synthetized in six steps from commercially available methyl 4-iodobenzoate. The synthetic strategy for the preparation of these N,N-diethyl-2-(2-(4-(ω-fluoroalk-1-ynyl)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamides (7a-d) consisted in derivatizing a key iodinated building block featuring the pyrazolopyrimidine acetamide backbone of DPA-714, by Sonogashira couplings with various alkynyl reagents. The resulting alkynols were subsequently fluorinated, yielding the expected target derivatives. All four analogues exhibited slightly higher affinity and selectivity towards the TSPO 18kDa (Ki vs [(3)H]PK11195: 0.35-0.79nM; Ki vs [(3)H]flunitrazepam: >1000nM) when compared to DPA-714 (Ki vs [(3)H]PK11195: 0.91nM; Ki vs [(3)H]flunitrazepam: >1000nM). Lipophilicities (HPLC, logD7.4) increased with the chain length (from 3.6 to 4.3) and were significantly higher than the one determined for DPA-714 (2.9). Preliminary in vitro metabolism evaluation using rat microsomal incubations and LC-MS analyses showed, for all four novel analogues, the absence of defluorinated metabolites. Among them, the fluoropentynyl compound, DPA-C5yne (7c), was selected, labelled in one single step with fluorine-18 from the corresponding tosylate and in vivo evaluated with PET on our in-house-developed rat model of acute local neuroinflammation.

[18F]DPA-C5yne, a novel fluorine-18-labelled analogue of DPA-714: radiosynthesis and preliminary evaluation as a radiotracer for imaging neuroinflammation with PET.

DPA-C5yne, the lead compound of a novel series of DPA-714 derivatives in which the fluoroethoxy chain linked to the phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn-1-yl moiety, is a high affinity (Ki : 0.35 nM) and selective ligand targeting the translocator protein 18 kDa. In the present work, DPA-C5yne was labelled with no-carrier-added [(18)F]fluoride based on a one-step tosyloxy-for-fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3-5.2 GBq of [(18)F]DPA-C5yne, ready-to-use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to 110 GBq/µmol within 50-60 min, starting from a 30 GBq [(18)F]fluoride batch (14-17%). LogP and LogD of [(18)F]DPA-C5yne were measured using the shake-flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies performed on slices of ((R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA)-lesioned rat brains showed a high target-to-background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated using DPA-C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore, DPA-C5yne proved to be stable in plasma at 37°C for at least 90 min.

Differential influence of propofol and isoflurane anesthesia in a non-human primate on the brain kinetics and binding of [18F]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 [18F]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 [18F]DPA‐714 binding parameters and PET kinetics. [18F]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 [18F]DPA‐714 brain distribution (VT). Displacement experiments were performed using PK11195 (1.5 mg/kg). In vitro [18F]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 [18F]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 [18F]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.

From Structure–Activity Relationships on Thiazole Derivatives to the In Vivo Evaluation of a New Radiotracer for Cannabinoid Subtype 2 PET Imaging

Upregulation of the cannabinoid type 2 receptors (CB2R) unveils inflammation processes of pathological disorders, such as cancer, pain, or neurodegenerative diseases. Among others, CB2R agonist A-836339 has been labeled with carbon-11 for PET imaging of the CB2R and displayed promising results in a mouse model of Alzheimers disease. The aim of the present work was to develop fluorinated analogs of A-836339 for labeling with fluorine-18 to design a new PET tracer for CB2R imaging. Seven fluorinated analogs of A-836339 were synthesized in two to three steps and their binding affinities and selectivities for both the human and the mouse CB2R were measured as well as their early ADME profiles. Among them, compound 2f (KihCB2R = 0.1 nM, KihCB1R/KihCB2R = 300) displayed high affinity and selectivity for CB2R but also promising lipophilicity, kinetic solubility, and membrane permeation properties and was further selected for in vitro metabolism studies. Incubation of 2f with human or rat liver microsomes followed by LC/MS analysis revealed the presence of six different metabolites mainly resulting from oxidation reactions. A tosylated precursor of 2f was synthesized in two steps and radiolabeled with fluorine-18 to afford [18F]2f in 15 ± 5% radiochemical yield and a molar activity of 110 ± 30 GBq/μmol. Autoradiographies of rat spleen and biodistribution studies in healthy rats including pretreatments with either CB2R or CB1R-specific compounds suggested that [18F]2f is a specific tracer for the CB2R in vivo. We have therefore demonstrated here that [18F]2f is a promising novel tracer for imaging CB2R in vivo using PET. Further investigation in animal models of inflammation will follow.