Wadad Saba

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.

In vivo Quantification of Monoamine Oxidase A in Baboon Brain: A PET Study Using [11C]befloxatone and the Multi-Injection Approach

[11C]befloxatone is a high-affinity, reversible, and selective radioligand for the in vivo visualization of the monoamine oxidase A (MAO-A) binding sites using positron emission tomography (PET). The multi-injection approach was used to study in baboons the interactions between the MAO-A binding sites and [11C]befloxatone. The model included four compartments and seven parameters. The arterial plasma concentration, corrected for metabolites, was used as input function. The experimental protocol—three injections of labeled and/or unlabeled befloxatone—allowed the evaluation of all the model parameters from a single PET experiment. In particular, the brain regional concentrations of the MAO-A binding sites (B′max) and the apparent in vivo befloxatone affinity (Kd) were estimated in vivo for the first time. A high binding site density was found in almost all the brain structures (170±39 and 194±26 pmol/mL in the frontal cortex and striata, respectively, n=5). The cerebellum presented the lowest binding site density (66±13 pmol/mL). Apparent affinity was found to be similar in all structures (KdVR=6.4±1.5 nmol/L). This study is the first PET-based estimation of the Bmax of an enzyme.

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.

Biopersistence and translocation to extrapulmonary organs of titanium dioxide nanoparticles after subacute inhalation exposure to aerosol in adult and elderly rats.

The increasing industrial use of nanoparticles (NPs) has raised concerns about their impact on human health. Since aging and exposure to environmental factors are linked to the risk for developing pathologies, we address the question of TiO2 NPs toxicokinetics in the context of a realistic occupational exposure. We report the biodistribution of titanium in healthy young adults (12-13-week-old) and in elderly rats (19-month-old) exposed to 10mg/m3 of a TiO2 nanostructured aerosol 6h/day, 5days/week for 4 weeks. We measured Ti content in major organs using inductively coupled plasma mass spectrometry immediately and up to 180days after the end of exposure. Large amounts of titanium were initially found in lung which were slowly cleared during the post-exposure period. From day 28, a small increase of Ti was found in the spleen and liver of exposed young adult rats. Such an increase was however never found in their blood, kidneys or brain. In the elderly group, translocation to extra-pulmonary organs was significant at day 90. Ti recovered from the spleen and liver of exposed elderly rats was higher than in exposed young adults. These data suggest that TiO2 NPs may translocate from the lung to extra-pulmonary organs where they could possibly promote systemic health effects.

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.

Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient

In clinical practice, rifampicin exposure is estimated from its concentration in venous blood samples. In this study, we hypothesized that differences in rifampicin concentration may exist between arterial and venous plasma. An HPLC-UV method for determining rifampicin concentration in plasma using rifapentine as an internal standard was validated. The method, which requires a simple protein precipitation procedure as sample preparation, was performed to compare venous and arterial plasma kinetics after a single therapeutic dose of rifampicin (8.6 mg/kg i.v, infused over 30 min) in baboons (n=3). The method was linear from 0.1 to 40 μg mL(-1) and all validation parameters fulfilled the international requirements. In baboons, rifampicin concentration in arterial plasma was higher than in venous plasma. Arterial Cmax was 2.1±0.2 fold higher than venous Cmax. The area under the curve (AUC) from 0 to 120 min was ∼80% higher in arterial plasma, indicating a significant arteriovenous concentration gradient in early rifampicin pharmacokinetics. Arterial and venous plasma concentrations obtained 6h after rifampicin injection were not different. An important arteriovenous equilibration delay for rifampicin pharmacokinetics is reported. Determination in venous plasma concentrations may considerably underestimate rifampicin exposure to organs during the distribution phase.

Acute morphine exposure increases the brain distribution of [18F]DPA-714, a PET biomarker of glial activation in nonhuman primates.

Abstract Background: The neuroinflammatory response to morphine exposure modulates its antinociceptive effects, tolerance, and dependence. Positron emission tomography radioligands for translocator protein-18kDa such as [18F]DPA-714 are noninvasive biomarkers of glial activation, a hallmark of neuroinflammation. Methods: [18F]DPA-714 positron emission tomography imaging was performed in 5 baboons at baseline and 2 hours after i.m. morphine injection (1 mg/kg). Brain kinetics and metabolite-corrected input function were measured to estimate [18F]DPA-714 brain distribution. Results: Morphine significantly increased [18F]DPA-714 brain distribution by a 1.3 factor (P<.05; paired t test). The effect was not restricted to opioid receptor-rich regions. Differences in baseline [18F]DPA-714 binding were observed among baboons. The response to morphine predominated in animals with the highest baseline uptake. Conclusions: [18F]DPA-714 positron emission tomography imaging may be useful to noninvasively investigate the brain immune component of morphine pharmacology. Correlation between baseline brain distribution and subsequent response to morphine exposure suggest a role for priming parameters in controlling the neuroinflammatory properties of opioids.