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Hepatocyte transporters control the hepatobiliary elimination of many drugs, metabolites and endogenous substances. Hepatocyte transporter function is altered in several pathophysiological situations and can be modulated by certain drugs, with a potential impact for pharmacokinetics and drug-induced liver injury. Development of substrate probes with optimal properties for selective and quantitative imaging of hepatic transporters remains a challenge. [99mTc]mebrofenin has been used for decades for hepatobiliary scintigraphy, but the specific transporters controlling its liver kinetics have been characterized more recently. These include sinusoidal influx transporters (organic anion-transporting polypeptides, OATP) responsible for hepatic uptake of [99mTc]mebrofenin, and efflux transporters (multidrug resistance-associated proteins, MRP) mediating its canalicular (liver-to-bile) and sinusoidal (liver-to-blood) excretion. Pharmacokinetic modeling enables molecular interpretation of [99mTc]mebrofenin scintigraphy data, thus offering a widely available translational method to investigate transporter-mediated drug-drug interactions in vivo. [99mTc]mebrofenin allows for phenotyping transporter activity at the different poles of hepatocytes as a biomarker of liver function.

Purpose To assess in healthy volunteers the whole-body distribution and dosimetry of [11C]metoclopramide, a new positron emission tomography (PET) tracer to measure P-glycoprotein activity at the blood-brain barrier. Procedures Ten healthy volunteers (five women, five men) were intravenously injected with 387 ± 49 MBq of [11C]metoclopramide after low dose CT scans and were then imaged by whole-body PET scans from head to upper thigh over approximately 70 min. Ten source organs (brain, thyroid gland, right lung, myocardium, liver, gall bladder, left kidney, red bone marrow, muscle and the contents of the urinary bladder) were manually delineated on whole-body images. Absorbed doses were calculated with QDOSE (ABX-CRO) using the integrated IDAC-Dose 2.1 module. Results The majority of the administered dose of [11C]metoclopramide was taken up into the liver followed by urinary excretion and, to a smaller extent, biliary excretion of radioactivity. The mean effective dose of [11C]metoclopramide was 1.69 ± 0.26 μSv/MBq for female subjects and 1.55 ± 0.07 μSv/MBq for male subjects. The two organs receiving the highest radiation doses were the urinary bladder (10.81 ± 0.23 μGy/MBq and 8.78 ± 0.89 μGy/MBq) and the liver (6.80 ± 0.78 μGy/MBq and 4.91 ± 0.74 μGy/MBq) for female and male subjects, respectively. Conclusions [11C]Metoclopramide showed predominantly renal excretion, and is safe and well tolerated in healthy adults. The effective dose of [11C]metoclopramide was comparable to other 11C-labeled PET tracers.

International audience; Epidermal growth factor receptor (EGFR), involved in cell proliferation and migration, is overexpressed in ~50% of glioblastomas. Anti-EGFR based strategies using monoclonal antibodies (mAb) such as cetuximab (CTX) have been proposed for central nervous system (CNS) cancer therapy. However, the blood-brain barrier (BBB) drastically restricts their brain penetration which limits their efficacy for the treatment of glioblastomas. Herein, a longitudinal PET imaging study was performed to assess the relevance and the impact of focused ultrasound (FUS)-mediated BBB permeabilization on the brain exposure to the anti-EGFR mAb CTX over time. For this purpose, FUS permeabilization process with microbubbles was applied on intact BBB mouse brain before the injection of 89 Zr-labeled CTX for longitudinal imaging monitoring. FUS induced a dramatic increase in mAb penetration to the brain, 2 times higher compared to the intact BBB. The transfer of 89 Zr-CTX from blood to the brain was rendered significant by FUS (k uptake = 1.3 ± 0.23 min −1 with FUS versus k uptake = 0 ± 0.006 min −1 without FUS). FUS allowed significant and prolonged exposure to mAb in the brain parenchyma. This study confirms the potential of FUS as a target delivery method for mAb in CNS.

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict at the blood–brain barrier (BBB) the brain distribution of the majority of currently known molecularly targeted anticancer drugs. To improve brain delivery of dual ABCB1/ABCG2 substrates, both ABCB1 and ABCG2 need to be inhibited simultaneously at the BBB. We examined the feasibility of simultaneous ABCB1/ABCG2 inhibition with i.v. co-infusion of erlotinib and tariquidar by studying brain distribution of the model ABCB1/ABCG2 substrate [11C]erlotinib in mice and rhesus macaques with PET. Tolerability of the erlotinib/tariquidar combination was assessed in human embryonic stem cell-derived cerebral organoids. In mice and macaques, baseline brain distribution of [11C]erlotinib was low (brain distribution volume, VT,brain 3). Co-infusion of erlotinib and tariquidar increased VT,brain in mice by 3.0-fold and in macaques by 3.4- to 5.0-fold, while infusion of erlotinib alone or tariquidar alone led to less pronounced VT,brain increases in both species. Treatment of cerebral organoids with erlotinib/tariquidar led to an induction of Caspase-3-dependent apoptosis. Co-infusion of erlotinib/tariquidar may potentially allow for complete ABCB1/ABCG2 inhibition at the BBB, while simultaneously achieving brain-targeted EGFR inhibition. Our protocol may be applicable to enhance brain delivery of molecularly targeted anticancer drugs for a more effective treatment of brain tumors.

P-glycoprotein (ABC subfamily B member 1, ABCB1) plays an important role at the blood–brain barrier (BBB) in promoting clearance of neurotoxic β-amyloid (Aβ) peptides from the brain into the blood. ABCB1 expression and activity were found to be decreased in the brains of Alzheimer disease patients. Treatment with drugs that induce cerebral ABCB1 activity may be a promising approach to delay the build-up of Aβ deposits in the brain by enhancing clearance of Aβ peptides from the brain. The aim of this study was to investigate whether PET with the weak ABCB1 substrate radiotracer 11C-metoclopramide can measure ABCB1 induction at the BBB in a β-amyloidosis mouse model (APP/PS1-21 mice) and in wild-type mice. Methods: Groups of wild-type and APP/PS1-21 mice aged 50 or 170 d underwent 11C-metoclopramide baseline PET scans or scans after intraperitoneal treatment with the rodent pregnane X receptor activator 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN, 25 mg/kg) or its vehicle over 7 d. At the end of the PET scans, brains were harvested for immunohistochemical analysis of ABCB1 and Aβ levels. In separate groups of mice, radiolabeled metabolites of 11C-metoclopramide were determined in plasma and brain at 15 min after radiotracer injection. As an outcome parameter of cerebral ABCB1 activity, the elimination slope of radioactivity washout from the brain (kE,brain) was calculated. Results: PCN treatment resulted in an increased clearance of radioactivity from the brain as reflected by significant increases in kE,brain (from +26% to +54% relative to baseline). Immunohistochemical analysis confirmed ABCB1 induction in the brains of PCN-treated APP/PS1-21 mice with a concomitant decrease in Aβ levels. There was a significant positive correlation between kE,brain and ABCB1 levels in the brain. In wild-type mice, a significant age-related decrease in kE,brain was found. Metabolite analysis showed that most radioactivity in the brain comprised unmetabolized 11C-metoclopramide in all animal groups. Conclusion: 11C-metoclopramide can measure ABCB1 induction in the mouse brain without the need to consider an arterial input function and may find potential application in Alzheimer disease patients to noninvasively evaluate strategies to enhance the clearance properties of the BBB.

Focused ultrasound in combination with microbubbles (FUS) provides an effective means to locally enhance the delivery of therapeutics to the brain. Translational and quantitative imaging techniques are needed to noninvasively monitor and optimize the impact of FUS on blood–brain barrier (BBB) permeability in vivo. Positron-emission tomography (PET) imaging using [18F]2-fluoro-2-deoxy-sorbitol ([18F]FDS) was evaluated as a small-molecule (paracellular) marker of blood–brain barrier (BBB) integrity. [18F]FDS was straightforwardly produced from chemical reduction of commercial [18F]2-deoxy-2-fluoro-D-glucose. [18F]FDS and the invasive BBB integrity marker Evan’s blue (EB) were i.v. injected in mice after an optimized FUS protocol designed to generate controlled hemispheric BBB disruption. Quantitative determination of the impact of FUS on the BBB permeability was determined using kinetic modeling. A 2.2 ± 0.5-fold higher PET signal (n = 5, p &lt, 0.01) was obtained in the sonicated hemisphere and colocalized with EB staining observed post mortem. FUS significantly increased the blood-to-brain distribution of [18F]FDS by 2.4 ± 0.8-fold (VT, 0.01). Low variability (=10.1%) of VT values in the sonicated hemisphere suggests reproducibility of the estimation of BBB permeability and FUS method. [18F]FDS PET provides a readily available, sensitive and reproducible marker of BBB permeability to noninvasively monitor the extent of BBB disruption induced by FUS in vivo.

Drugs promoting myelin repair represent a promising therapeutic approach in multiple sclerosis and several candidate molecules are currently being evaluated, fostering the need of a quantitative method to specifically measure myelin content in vivo. PET using the benzothiazole derivative 11C-PiB has been successfully used to quantify myelin content changes in humans. Stilbene derivatives, such as 11C-MeDAS, have also been shown to bind to myelin in animals and are considered a promising radiopharmaceutical class for myelin imaging. Fluorinated compounds from both classes are now commercially available and thus should constitute clinically useful myelin radiotracers. The aim of this study is to provide a head-to-head comparison of 18F-florbetaben, 18F-florbetapir, 18F-flutemetamol, 11C-MeDAS, and 11C-PiB with regard to brain kinetics and binding in white matter (WM). Four baboons underwent a 90-min dynamic PET scan for each radioligand. Arterial blood samples were collected during the exam for each radiotracer, except for 18F-florbetapir, to obtain a radiometabolite-corrected input function. Standardized uptake value ratio between 75 at 90 min (SUVR75–90), binding potential (BP) estimated with Logan method with input function, and distribution volume ratio (DVR) estimated with Logan reference method (using cerebellar gray matter as reference region) were calculated in WM and compared between tracers using mixed effect models. In WM, 18F-florbetapir had the highest SUVR75–90 (1.38 ± 0.03), followed by 18F-flutemetamol (1.34 ± 0.02), 18F-florbetaben (1.32 ± 0.07), 11C-MeDAS (1.27 ± 0.04), and 11C-PiB (1.25 ± 0.07). With regard to BP, 18F-florbetaben had the highest value (0.32 ± 0.06) compared with 18F-flutemetamol (0.20 ± 0.03), 11C-MeDAS (0.17 ± 0.03), and 11C-PiB (0.16 ± 0.03). No difference in DVR was detected between 18F-florbetaben (1.26 ± 0.06) and 18F-florbetapir (1.27 ± 0.03), but both were significantly higher in DVR than 18F-flutemetamol (1.17 ± 0.02), 11C-MeDAS (1.16 ± 0.03), and 11C-PiB (1.14 ± 0.02). Given their higher binding and longer half-life, our study indicates that 18F-florbetapir and 18F-florbetaben are promising tracers for myelin imaging which are readily available for clinical application in demyelinating diseases.

PET with avid substrates of P-glycoprotein (ABCB1) provided evidence of the role of this efflux transporter in effectively restricting the brain penetration of its substrates across the human blood–brain barrier (BBB). This may not reflect the situation for weak ABCB1 substrates including several antidepressants, antiepileptic drugs, and neuroleptics, which exert central nervous system effects despite being transported by ABCB1. We performed PET with the weak ABCB1 substrate 11C-metoclopramide in humans to elucidate the impact of ABCB1 function on its brain kinetics. Methods: Ten healthy male subjects underwent 2 consecutive 11C-metoclopramide PET scans without and with ABCB1 inhibition using cyclosporine A (CsA). Pharmacokinetic modeling was performed to estimate the total volume of distribution (VT) and the influx (K1) and efflux (k2) rate constants between plasma and selected brain regions. Furthermore, 11C-metoclopramide washout from the brain was estimated by determining the elimination slope (kE,brain) of the brain time–activity curves. Results: In baseline scans, 11C-metoclopramide showed appreciable brain distribution (VT = 2.11 ± 0.33 mL/cm3). During CsA infusion, whole-brain gray matter VT and K1 were increased by 29% ± 17% and 9% ± 12%, respectively. K2 was decreased by 15% ± 5%, consistent with a decrease in kE,brain (−32% ± 18%). The impact of CsA on outcome parameters was significant and similar across brain regions except for the pituitary gland, which is not protected by the BBB. Conclusion: Our results show for the first time that ABCB1 does not solely account for the “barrier” property of the BBB but also acts as a detoxifying system to limit the overall brain exposure to its substrates at the human blood–brain interface.

Only partial deficiency/inhibition of P-glycoprotein (P-gp, ABCB1) function at the blood-brain barrier (BBB) is likely to occur in pathophysiological situations or drug-drug interactions. This raises questions regarding the sensitivity of available PET imaging probes to detect moderate changes in P-gp function at the living BBB. In vitro, the half-maximum inhibitory concentration (IC50) of the potent P-gp inhibitor tariquidar in P-gp-overexpressing cells was significantly different using either [11C]verapamil (44 nM), [11C] N-desmethyl-loperamide (19 nM) or [11C]metoclopramide (4 nM) as substrate probes. In vivo PET imaging in rats showed that the half-maximum inhibition of P-gp-mediated efflux of [11C]metoclopramide, achieved using 1 mg/kg tariquidar ( in vivo IC50 = 82 nM in plasma), increased brain exposure by 2.1-fold for [11C]metoclopramide (p 11C]verapamil (p 11C] N-desmethyl-loperamide was unaffected (p > 0.05, n = 4). This comparative study points to differences in the “vulnerability” to P-gp inhibition among radiolabeled substrates, which were apparently unrelated to their “avidity” (maximal response to P-gp inhibition). Herein, we advocate that partial inhibition of transporter function, in addition to complete inhibition, should be a primary criterion of evaluation regarding the sensitivity of radiolabeled substrates to detect moderate but physiologically-relevant changes in transporter function in vivo.

P-glycoprotein (P-gp) is a major efflux transporter at the blood-brain barrier (BBB) where it controls the brain distribution of many drugs. Clinical translation of preclinical data obtained using invasive methods in animals is difficult due to species differences in P-gp expression and function. Positron emission tomography (PET) is an imaging technique allowing for the determination of the tissue kinetics of microdose of radiolabeled compounds. Three radiolabeled substrates of the P-gp have been developed for clinical use: [11C]verapamil, [11C]-N-desmethyl-loperamide and [11C]metoclopramide. These innovative tools enabled the study of P-gp in humans in various patho-physiological conditions including neurological diseases. This approach is also useful to predict the risk for drug-drug interaction caused by P-gp inhibitors at the BBB and address its impact for neuropharmacokinetics in vivo in humans., La P-glycoprotéine (P-gp) est un transporteur d’efflux exprimé au niveau de la barrière hématoencéphalique (BHE) qui joue un rôle déterminant dans le passage cérébral de nombreux médicaments. La translation clinique des données obtenues chez l’animal par des méthodes invasives est complexe et il est difficile de prédire les répercussions neuropharmacocinétiques de la P-gp chez l’Homme. La tomographie par émission de positons (TEP) est une méthode d’imagerie permettant de mesurer la cinétique des concentrations tissulaires d’une molécule radiomarquée injectée en microdose. À ce jour, trois radioligands substrats de la P-gp ont fait l’objet d’un développement clinique : le [11C]vérapamil, [11C]-N-desmethyl-lopéramide et le [11C]métoclopramide. Cette approche innovante a permis de montrer une régulation fonctionnelle de la P-gp au niveau de la BHE chez l’Homme au cours de certaines pathologies neurologiques. Cette approche a également permis d’évaluer le risque d’interactions médicamenteuses impliquant des inhibiteurs de la P-gp au niveau de la BHE et d’en mesurer les conséquences sur le passage cérébral de médicaments substrats de ce transporteur.

The antiemetic and gastroprokinetic drug metoclopramide is a weak substrate of the blood-brain barrier (BBB) efflux transporter P-glycoprotein (P-gp) and displays central nervous system (CNS) side effects (i.e. extrapyramidal symptoms, tardive dyskinesia) caused by dopamine D2 receptor blockade in the basal ganglia. These side effects occur with a higher incidence in elderly people. We used positron emission tomography (PET) to assess the brain distribution of [11 C]metoclopramide in young (n = 11, 26 ± 3 years) and elderly (n = 7, 68 ± 9 years) healthy men both after administration of a microdose (9 ± 7 µg) and a microdose co-injected with a therapeutic dose of unlabeled metoclopramide (10 mg). For both doses, elderly subjects had a significantly higher total volume of distribution (VT ) of [11 C]metoclopramide in the basal ganglia than young subjects (microdose: +26%, therapeutic dose: +41%). Increases in VT (= K1 /k2 ) were caused by significant decreases in the transfer rate constant of [11 C]metoclopramide from brain into plasma (k2 , microdose: -18%, therapeutic dose: -30%), while the distributional clearance from plasma into brain (K1 ) remained unaltered. This reduction in the clearance of [11 C]metoclopramide (k2 ) from the brains of elderly subjects may be caused by an age-related decrease in the activity of P-gp at the BBB and may contribute to the higher incidence of CNS side effects of metoclopramide in the aged population. Our data suggest that an age-associated decrease in the clearance properties of the BBB may modulate the CNS effects or side effects of clinically used P-gp substrates.

The adenosine triphosphate-binding cassette transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are 2 efflux transporters at the blood-brain barrier (BBB) that effectively restrict brain distribution of dual ABCB1/ABCG2 substrate drugs, such as tyrosine kinase inhibitors. Pharmacologic inhibition of ABCB1/ABCG2 may improve the efficacy of dual-substrate drugs for treatment of brain tumors, but no marketed ABCB1/ABCG2 inhibitors are currently available. In the present study, we examined the potential of supratherapeutic-dose oral erlotinib to inhibit ABCB1/ABCG2 activity at the human BBB. Methods: Healthy men underwent 2 consecutive PET scans with 11C-erlotinib: a baseline scan and a second scan either with concurrent intravenous infusion of the ABCB1 inhibitor tariquidar (3.75 mg/min, n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, n = 7; 650 mg, n = 8; or 1,000 mg, n = 2). Results: Although tariquidar administration had no effect on 11C-erlotinib brain distribution, oral erlotinib led, at the 650-mg dose, to significant increases in volume of distribution (23% ± 13%, P = 0.008), influx rate constant of radioactivity from plasma into brain (58% ± 26%, P = 0.008), and area under the brain time-activity curve (78% ± 17%, P = 0.008), presumably because of combined partial saturation of ABCG2 and ABCB1 activity. Inclusion of further subjects into the 1,000-mg dose group was precluded by adverse skin events (rash). Conclusion: Supratherapeutic-dose erlotinib may be used to enhance brain delivery of ABCB1/ABCG2 substrate anticancer drugs, but its clinical applicability for continuous ABCB1/ABCG2 inhibition at the BBB may be limited by safety concerns.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high...

The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.

International audience; Background: Modafinil, a nonamphetaminic wake-promoting compound, is prescribed as first line therapy in narcolepsy, an invalidating disorder characterized by excessive daytime sleepiness and cataplexy. Although its mode of action remains incompletely known, recent studies indicated that modafinil modulates astroglial connexin-based gap junctional communication as administration of a low dose of flecainide, an astroglial connexin inhibitor, enhanced the wake-promoting and procognitive activity of modafinil in rodents and healthy volunteers. The aim of this study is to investigate changes in glucose cerebral metabolism in rodents, induced by the combination of modafinil+flecainide low dose (called THN102).Methods: The impact of THN102 on brain glucose metabolism was noninvasively investigated using 18F-2-fluoro-2-deoxy-D-glucose Positron Emission Tomography imaging in Sprague-Dawley male rats. Animals were injected with vehicle, flecainide, modafinil, or THN102 and further injected with 18F-2-fluoro-2-deoxy-D-glucose followed by 60-minute Positron Emission Tomography acquisition. 18F-2-fluoro-2-deoxy-D-glucose Positron Emission Tomography images were coregistered to a rat brain template and normalized from the total brain Positron Emission Tomography signal. Voxel-to-voxel analysis was performed using SPM8 software. Comparison of brain glucose metabolism between groups was then performed.Results: THN102 significantly increased regional brain glucose metabolism as it resulted in large clusters of 18F-2-fluoro-2-deoxy-D-glucose uptake localized in the cortex, striatum, and amygdala compared with control or drugs administered alone. These regions, highly involved in the regulation of sleep-wake cycle, emotions, and cognitive functions were hence quantitatively modulated by THN102.Conclusion: Data presented here provide the first evidence of a regional brain activation induced by THN102, currently being tested in a phase II clinical trial in narcoleptic patients.

International audience; Elacridar (GF120918) is a highly potent inhibitor of both P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), the main efflux transporters expressed at the blood-brain barrier (BBB). Elacridar shows very low aqueous solubility, which complicates its formulation for i.v. administration. An intravenous infusion protocol would be preferred to achieve high and controlled plasma concentrations of elacridar in large animals, including nonhuman primates. Formulation of elacridar for i.v. infusion was achieved using a co-solvent strategy, resulting in an aqueous dispersion with a final concentration of 5 g L-1 elacridar with tetrahydrofuran (5% w/v) in aqueous D-glucose solution (2.5%, w/v). Particle size (mean = 2.8 +/- 0.9 mu m) remained stable for 150 min. The preparation was i.v. administered as a continuous infusion (12 mg kg(-1) h(-1) for 90 min) to three baboons. Arterial and venous plasma pharmacokinetics (PK) of elacridar were monitored using a newly developed and validated HPLCUV method. Elacridar concentration increased rapidly to reach a plateau at 9.5 mu g mL(-1) within 20 min after the start of infusion. Elacridar PK in venous plasma did not differ firm arterial plasma facing the BBB, indicating the absence of an arteriovenous concentration gradient. Intravenous infusion of elacridar allows for controlled exposure of the BBB and offers a useful tool to assess the impact of ABCB1/ABCG2 on drug disposition to the brain in nonhuman primates, a relevant animal model for the study of transporter function at the BBB.

The effects of acute alcohol exposure to the central nervous system are hypothesized to involve the innate immune system. The neuroimmune response to an initial and acute alcohol exposure was investigated using translocator protein 18 kDa (TSPO) PET imaging, a non-invasive marker of glial activation, in adolescent baboons. Three different alcohol-naive adolescent baboons (3–4 years old, 9 to 14 kg) underwent 18F-DPA-714 PET experiments before, during and 7–12 months after this initial alcohol exposure (0.7–1.0 g/l). The brain distribution of 18F-DPA-714 (VT; in ml/cm3) was estimated in several brain regions using the Logan plot analysis and the metabolite-corrected arterial input function. Compared with alcohol-naive animals (VTbrain = 3.7 ± 0.7 ml/cm3), the regional VTs of 18F-DPA-714 were significantly increased during alcohol exposure (VTbrain = 7.2 ± 0.4 ml/cm3; p

Imaging probes are needed to assess the interplay between liver uptake and biliary excretion, which controls the clearance of many compounds. 99mTc-mebrofenin (MEB) is a radiopharmaceutical for hepatobiliary scintigraphy (HBS) transported through the hepatocyte by OATP transporters at the sinusoidal (blood-liver) interface, and the MRP2 transporter at the canalicular (liver-bile) interface. Our aim was to develop a simple method to separately measure the activity of these two transporter systems in vivo. 99mTc-MEB was injected into control rats (CTL, n = 5) and after a bolus of rifampicin (RIF, 40 mg/kg IV, n = 5), an OATP/MRP2 inhibitor. Planar dynamic acquisitions were performed during 40 min. ROI were drawn over the heart, liver and intestine to obtain time-activity curves (TACs). Three quantification methods were tested: (i) calculation of the transfer rate constants between regions using integration plot analysis, (ii) estimation of the area under the TAC ratio (AUCR) between regions and (iii) compartmental modelling considering a mean volume for organs and a dual blood input function for the liver (hepatic artery and portal vein). (i) RIF significantly decreased the blood-to-liver transfer rate constant (0 to 2 min, linear part) (CTL = 1.5 ± 0.2 min−1, RIF = 0.3 ± 0.0 min−1). However, the liver-to-intestine transfer rate constant could not be accurately determined with this method. (ii) AUCRliver/blood (0 to 2 min) was significantly decreased by RIF (CTL = 2.0 ± 0.2, RIF = 0.8 ± 0.1). A significant decrease was also observed for AUCRintestine/liver (10 to 25 min, CTL = 1.4 ± 0.2, RIF = 0.6 ± 0.2). (iii) These results were confirmed by compartmental modeling, which identified a significant decrease for RIF-treated rats in k1 (blood to hepatocyte transport, CTL = 7.4 ± 1.6 min−1, RIF = 0.9 ± 0.1 min−1), k2 (hepatocyte to blood backflux, CTL = 0.15 ± 0.08 min−1, RIF = 0.01 ± 0.01 min−1) and k3 (hepatocyte to intrahepatic bile-duct transport, CTL = 0.15 ± 0.08 min−1, RIF = 0.05 ± 0.02 min−1) but not for k5 (intrahepatic bile-duct to intestine, CTL = 0.12 ± 0.03 min−1, RIF = 0.64 ± 0.88 min−1). 99mTc-MEB HBS can study the interplay between sinusoidal and canalicular transporters in mediating hepatobiliary clearance in vivo. AUCR is a simple parameter to describe the respective activity of both transporter systems. Compartmental modeling enriched the interpretation of the data and revealed the importance of an additional transport system responsible for the backflux of 99mTc-MEB from liver to blood, most likely MRP3.

Background Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. Methods To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. Results One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. Conclusions In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax., The enzymatically active components of B anthracis toxins and bacteria can diffuse very early in the blood during inhalational anthrax model, paving the way for an ultra-precocious diagnosis and the setup of an early and effective treatment.

International audience; Overcoming the efflux mediated by ATP–binding cassette (ABC) transporters at the blood-brain barrier (BBB)remains a challenge for the delivery of small molecule tyrosine kinase inhibitors (TKIs) such as erlotinib to thebrain. Inhibition of ABCB1 and ABCG2 at the mouse BBB improved the BBB permeation of erlotinib but could notbe achieved in humans. BBB disruption induced by focused ultrasound (FUS) was investigated as a strategy toovercome the efflux transport of erlotinib $in\ vivo$.In rats, FUS combined with microbubbles allowed for a large and spatially controlled disruption of the BBB inthe left hemisphere. ABCB1/ABCG2 inhibition was performed using elacridar (10 mg/kg i.v). The brain kineticsof erlotinib was studied using $^{11}$C-erlotinib Positron Emission Tomography (PET) imaging in 5 groups (n = 4–5rats per group) including a baseline group, immediately after sonication (FUS), 48 h after FUS (FUS + 48 h),elacridar (ELA) and their combination (FUS + ELA). BBB integrity was assessed using the Evan's Blue (EB)extravasation test. Brain exposure to $^{11}$C-erlotinib was measured as the area under the curve (AUC) of the brainkinetics (% injected dose (%ID) versus time (min)) in volumes corresponding to the disrupted (left) and the intact(right) hemispheres, respectively.EB extravasation highlighted BBB disruption in the left hemisphere of animals of the FUS and FUS + ELAgroups but not in the control and ELA groups. EB extravasation was not observed 48 h after FUS suggestingrecovery of BBB integrity. Compared with the control group (AUC$_{Baseline}$ = 1.4 $\pm$ 0.5%ID.min), physical BBBdisruption did not impact the brain kinetics of $^{11}$C-erlotinib in the left hemisphere (p > .05) either immediately(AUC$_{FUS}$ = 1.2 $\pm$ 0.1%ID.min) or 48 h after FUS (AUC$_{FUS}$+48h = 1.1 $\pm$ 0.3%ID.min). Elacridar similarly increased $^{11}$C-erlotinib brain exposure to the left hemisphere in the absence (AUC$_{ELA}$ = 2.2 $\pm$ 0.5%ID.min,$p$ < .001) and in the presence of BBB disruption (AUC$_{FUS+ELA}$ = 2.1 $\pm$ 0.5%ID.min, p < .001). AUC$_{left}$ wasnever significantly different from AUC$_{right}$ ($p$ > .05), in any of the tested conditions.BBB integrity is not the rate limiting step for erlotinib delivery to the brain which is mainly governed by ABCmediated efflux. Efflux transport of erlotinib persisted despite BBB disruption.

P-glycoprotein (ABCB1) is expressed at the blood-retina barrier (BRB), where it may control distribution of drugs from blood to the retina and thereby influence drug efficacy and toxicity. Methods: We performed PET scans with the ABCB1 substrate (R)-11C-verapamil on 5 healthy male volunteers without and with concurrent infusion of the ABCB1 inhibitor tariquidar. We estimated the rate constants for radiotracer transfer across the BRB (K1, k2) and total retinal distribution volume VTResults: During ABCB1 inhibition, retinal VT and influx rate constant K1 were significantly, by 1.4 ± 0.5-fold and 1.5 ± 0.3-fold, increased compared with baseline. Retinal efflux rate constant k2 was significantly decreased by 2.8 ± 1.0-fold. Conclusion: We found a significant increase in (R)-11C-verapamil distribution to the retina during ABCB1 inhibition, which provides first in vivo evidence for ABCB1 transport activity at the human BRB. The increase in retinal distribution was approximately 2.5-fold less pronounced than previously reported for the blood-brain barrier.

Diphenhydramine, a sedative histamine H1-receptor (H1R) antagonist, was evaluated as a probe to measure drug/H+-antiporter function at the blood–brain barrier. In situ brain perfusion experiments in mice and rats showed that diphenhydramine transport at the blood–brain barrier was saturable, following Michaelis–Menten kinetics with a Km = 2.99 mM and Vmax = 179.5 nmol s−1 g−1. In the pharmacological plasma concentration range the carrier-mediated component accounted for 77% of diphenhydramine influx while passive diffusion accounted for only 23%. [14C]Diphenhydramine blood–brain barrier transport was proton and clonidine sensitive but was influenced by neither tetraethylammonium, a MATE1 (SLC47A1), and OCT/OCTN (SLC22A1-5) modulator, nor P-gp/Bcrp (ABCB1a/1b/ABCG2) deficiency. Brain and plasma kinetics of [11C]diphenhydramine were measured by positron emission tomography imaging in rats. [11C]Diphenhydramine kinetics in different brain regions were not influenced by displacement with 1 mg kg−1 unlabeled diphenhydramine, indicating the specificity of the brain positron emission tomography signal for blood–brain barrier transport activity over binding to any central nervous system target in vivo. [11C]Diphenhydramine radiometabolites were not detected in the brain 15 min after injection, allowing for the reliable calculation of [11C]diphenhydramine brain uptake clearance (Clup = 0.99 ± 0.18 mL min−1 cm−3). Diphenhydramine is a selective and specific H+-antiporter substrate. [11C]Diphenhydramine positron emission tomography imaging offers a reliable and noninvasive method to evaluate H+-antiporter function at the blood–brain barrier.

Background and Purpose The respective impact and interplay between ABC (P-glycoprotein/P-gp/Abcb1a, BCRP/ABCG2, MRP/ABCC) and SLC transporter functions at the blood–brain barrier (BBB) and blood–retinal barriers (BRB) are incompletely understood. Experimental Approach We measured the initial cerebral and retinal distribution of selected ABC substrates by in situ carotid perfusion using P-gp/Bcrp knockout mice and chemical ABC/SLC modulation strategies. P-gp, Bcrp, Mrp1 and Mrp4 were studied by confocal retina imaging. Key Results Chemical or physical disruption of P-gp increased [3H]-verapamil transport by ~10-fold at the BBB and ~1.5-fold at the BRB. [3H]-Verapamil transport involved influx-mediated by an organic cation clonidine-sensitive/diphenhydramine-sensitive proton antiporter at both barriers; this effect was unmasked when P-gp was partially or fully inhibited/disrupted at the BBB. Studies of [3H]-mitoxantrone and [3H]-zidovudine transport suggested, respectively, that Bcrp efflux was less involved at the BRB than BBB, whereas Mrps were significantly and similarly involved at both barriers. Confocal imaging showed that P-gp and Bcrp were expressed in intra-retinal vessels (inner BRB/iBRB) but absent from the blood/basal membrane of cells of the retinal pigment epithelium (outer BRB/oBRB/RPE) where, in contrast, Mrp1 and Mrp4 were localized. Conclusions and Implications P-gp, Bcrp, Mrp1 and Mrp4 are differentially expressed at the outer and inner BRB, resulting in an altered ability to limit substrate distribution at the retina as compared with the BBB. [3H]-Verapamil distribution is not P-gp-specific and involves a proton antiporter at both the BBB and BRB. However, this transport is concealed by P-gp at the BBB, but not at the BRB, where P-gp activity is reduced.

In the clinical practice, complete repression of P-glycoprotein (P-gp) function at blood-brain barrier (BBB) is not likely to occur. However, partial inhibition/deficit is suspected in many pathophysiological situations. PET imaging using radiolabelled substrates of P-glycoprotein (P-gp) is the most advanced technique to study P-gp function at the human BBB. However, the sensitivity of current radiolabelled P-gp substrates to detect moderate changes in P-gp function at the BBB remained to be compared. In vitro and in vivo studies were conducted to compare 11C-verapamil (11C-V), 11C-N-desmethyl-loperamide (11C-N) and 11C-metoclopramide (11C-M). In vitro uptake assays were performed in MDCKII-hMDR1 cells to compare the transport capacity and the 50% inhibitory concentration (IC50) toward the P-gp inhibitors tariquidar (TQD) and cyclosporine A (CsA) for each radiolabelled substrate. In vivo, increasing I.V. doses of TQD on the brain distribution of 11C-M were first studied by microPET imaging in rats. In vitro modelling results suggest that the transport capacity and IC50 are highly dependent of the substrate/inhibitor couples. With both inhibitors, 11C-M showed the best sensitivity (1.4 to 6 fold lower IC50) and the lowest transport capacity. In vivo, IC50 was reached for the 1 mg/kg TQD dose. In vivo experiments using 11C-V and 11C-N in the same conditions are ongoing. Compared with 11C-M, avid P-gp substrates (11C-V and 11C-N) may lack sensitivity to detect partial P-gp inhibition/deficit and may underestimate the impact of fine regulation of P-gp function at the human BBB.

Background Erlotinib is an epidermal growth factor receptor (EGFR)-targeting tyrosine kinase inhibitor approved for treatment of non-small cell lung cancer. The wide inter-individual pharmacokinetic (PK) variability of erlotinib may impact treatment outcome and/or toxicity. Recent in vivo studies reported a nonlinear uptake transport of erlotinib into the liver, suggesting carrier-mediated system(s) to mediate its hepatobiliary clearance. Erlotinib has been identified in vitro as a substrate of organic anion-transporting polypeptide (OATP) transporters which expression does not restrict to hepatocytes and may impact the tissue uptake of erlotinib in vivo. Results The impact of rifampicin (40 mg/kg), a potent OATP inhibitor, on the liver uptake and exposure to tissues of 11C-erlotinib was investigated in rats (4 animals per group) using positron emission tomography (PET) imaging. Tissue pharmacokinetics (PK) and corresponding exposure (area under the curve, AUC) were assessed in the liver, kidney cortex, abdominal aorta (blood pool) and the lungs. The plasma PK of parent 11C-erlotinib was also measured using arterial blood sampling to estimate the transfer rate constant (k uptake) of 11C-erlotinib from plasma into different tissues. PET images unveiled the predominant distribution of 11C-erlotinib-associated radioactivity to the liver, which gradually moved to the intestine, thus highlighting hepatobiliary clearance. 11C-erlotinib also accumulated in the kidney cortex. Rifampicin did not impact AUCaorta but reduced k uptake, liver (p

International audience; Rationale: PET imaging using radiolabeled high-affinity substrates of P-glycoprotein (ABCB1) has convincingly revealed the role of this major efflux transporter in limiting the influx of its substrates from blood into the brain across the blood-brain barrier (BBB). Many drugs, such as metoclopramide, are weak ABCB1 substrates and distribute into the brain even when ABCB1 is fully functional. In this study, we used kinetic modeling and validated simplified methods to highlight and quantify the impact of ABCB1 on the BBB influx and efflux of $^{11}$C-metoclopramide, as a model weak ABCB1 substrate, in non-human primates. Methods: The regional brain kinetics of a tracer dose of $^{11}$C-metoclopramide (298 ± 44 MBq) were assessed in baboons using PET without (n = 4) or with intravenous co-infusion of the ABCB1 inhibitor tariquidar (4 mg/kg/h, n = 4). Metabolite-corrected arterial input functions were generated to estimate the regional volume of distribution (V$_T$) as well as the influx (K$_1$) and efflux (k$_2$) rate constants, using a one-tissue compartment model. Modeling outcome parameters were correlated with image-derived parameters, i.e. area under the curve AUC$_{0-30}$$_{min}$ and AUC$_{30-60min}$ (SUV.min) as well as the elimination slope (k$_E$; min$^{-1}$) from 30 to 60 min of the regional time-activity curves. Results: Tariquidar significantly increased the brain distribution of $^{11}$C-metoclopramide (V$_T$ = 4.3 ± 0.5 mL/cm$^3$ and 8.7 ± 0.5 mL/cm$^3$ for baseline and ABCB1 inhibition conditions, respectively, P

Positron emission tomography (PET) imaging using [11C]metoclopramide, a P-glycoprotein (P-gp) substrate, was used to investigate the contribution of transport processes to metoclopramide liver clearance. The liver kinetics obtained after injection of [11C]metoclopramide were measured using PET in rats (n=4‐5) in the absence (tracer dose) and the presence of a pharmacologic dose of metoclopramide (3 mg/kg), with or without P-gp inhibition using i.v. tariquidar (8 mg/kg). Corresponding [11C]metoclopramide kinetics and metabolism in plasma (n=3) were measured using radio-HPLC analysis. [11C]metoclopramide exposure to the liver and plasma was described by the area under the time-activity curve (AUC) of the radioactivity kinetics in the liver and parent [11C]metoclopramide kinetics in plasma, respectively. The pharmacologic dose of metoclopramide resulted in a ∼2.2-fold increase in [11C]metoclopramide AUCplasma, while P-gp inhibition did not. AUCliver was lower using the pharmacologic dose (42.9 ± 13.8 SUV·min) compared with the tracer dose (210.0 ± 32.4 SUV·min). P-gp inhibition enhanced the liver exposure in the pharmacologic condition only (81.0 ± 3.1 SUV·min). [11C]metoclopramide PET imaging suggests an unpredicted role for hepatocyte uptake transporter(s) in controlling metoclopramide pharmacokinetics in addition to the known contribution of the metabolic enzymes and the P-gp.

The tyrosine kinase inhibitor erlotinib poorly penetrates the blood-brain barrier (BBB) due to efflux transport by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) thereby limiting its utility in the treatment of non-small cell lung cancer brain metastases. Pharmacological strategies to inhibit ABCB1/ABCG2-mediated efflux transport at the BBB have been successfully developed in rodents, but it remains unclear if these can be translated to humans given pronounced species differences in ABCG2/ABCB1 expression ratios at the BBB. We assessed the efficacy of two different ABCB1/ABCG2 inhibitors to enhance brain distribution of 11C-erlotinib in non-human primates as a model of the human BBB. Methods: Papio anubis baboons underwent PET scans of the brain following i.v. injection of 11C-erlotinib under baseline conditions (n = 4) and during i.v. infusion of high-dose erlotinib (10 mg/kg/h, n = 4) or elacridar (12 mg/kg/h, n = 3). Results: Under baseline conditions, 11C-erlotinib distribution to the brain (total volume of distribution, VT = 0.22 ± 0.015 mL/cm3) was markedly lower than its distribution to muscle tissue surrounding the skull (VT = 0.86 ± 0.10 mL/cm3). Elacridar infusion resulted in a 3.5 ± 0.9-fold increase in 11C-erlotinib distribution to the brain (VT = 0.81 ± 0.21 mL/cm3, P < 0.01), reaching comparable levels as in muscle tissue, without changing 11C-erlotinib plasma pharmacokinetics. During high-dose erlotinib infusion 11C-erlotinib brain distribution was also significantly (1.7 ± 0.2-fold) increased (VT = 0.38 ± 0.033 mL/cm3, P < 0.05) with a concomitant increase in 11C-erlotinib plasma exposure. Conclusion: We successfully implemented ABCB1/ABCG2 inhibition protocols in non-human primates resulting in pronounced increases in brain distribution of 11C-erlotinib. Such inhibition protocols may ultimately find application for a more effective treatment of patients with brain tumors using drugs that undergo efflux transport at the BBB.

Introduction A growing area of research suggests that neuroimmunity may impact the pharmacology of opioids. Microglia is a key component of the brain immunity. Preclinical and clinical studies have demonstrated that microglial modulators may improve morphine-induced analgesia and prevent the development of tolerance and dependence. Positron emission tomography (PET) using translocator protein 18 kDa (TSPO) radioligand is a clinically validated strategy for the non-invasive detection of microglial activation. We hypothesized that TSPO PET imaging may be used to study the neuroimmune component of opioid tolerance and withdrawal. Methods Healthy rats (n = 6 in each group) received either saline or escalating doses of morphine (10–40 mg/kg) on five days to achieve tolerance and a withdrawal syndrome after morphine discontinuation. MicroPET imaging with [ 18 F]DPA-714 was performed 60 h after morphine withdrawal. Kinetic modeling was performed to estimate [ 18 F]DPA-714 volume of distribution (V T ) in several brain regions using dynamic PET images and corresponding metabolite-corrected input functions. Immunohistochemistry (IHC) experiments on striatal brain slices were performed to assess the expression of glial markers (Iba1, GFAP and CD68) during 14 days after morphine discontinuation. Results The baseline binding of [ 18 F]DPA-714 to the brain (V T = 0.086 ± 0.009 mL cm −3 ) was not increased by morphine exposure and withdrawal (V T = 0.079 ± 0.010 mL cm −3 ) indicating the absence of TSPO overexpression, even at the regional level. Accordingly, expression of glial markers did not increase after morphine discontinuation. Conclusions Morphine tolerance and withdrawal did not detectably activate microglia and had no impact on [ 18 F]DPA-714 brain kinetics in vivo.

Knockout (KO) animals are useful tools with which to assess the interplay between P-glycoprotein (P-gp; Abcb1) and the breast cancer resistance protein (Bcrp, Abcg2), two major ABC-transporters expressed at the blood-brain barrier (BBB). However, one major drawback of such deficient models is the possible involvement of compensation between transporters. In the present study, P-gp and Bcrp distribution in the brain as well as P-gp expression levels at the BBB were compared between the Bcrp TGEM KO rat model and the wild-type (WT) strain. Therefore, we used confocal microscopy of brain slices and western blot analysis of the isolated brain microvessels forming the BBB. This deficient rat model was used to assess the influence of Bcrp on the brain and peripheral kinetics of its substrate [(11)C]befloxatone using positron emission tomography (PET). The influence of additional P-gp inhibition was tested using elacridar (GF120918) 2 mg/kg in Bcrp KO rats. The distribution pattern of P-gp in the brain as well as P-gp expression levels at the BBB was similar in Bcrp-deficient and WT rats. Brain and peripheral kinetics of [(11)C]befloxatone were not influenced by the lack of Bcrp. Neither was the brain uptake of [(11)C]befloxatone in Bcrp-deficient rats influenced by the inhibition of P-gp. In conclusion, the Bcrp-deficient rat strain, in which we detected no compensatory mechanism or modification of P-gp expression as compared to WT rats, is a suitable model to study Bcrp function separately from that of P-gp at the BBB. However, although selectively transported by BCRP in vitro, our results suggest that [(11)C]befloxatone PET imaging might not be biased by impaired function of this transporter in vivo.

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.

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.

The roles of interleukin-17 (IL-17) and neutrophils in the lung have been described as those of two intricate but independent players. Here we identify neutrophils as the primary IL-17-secreting subset of cells in a model of inhalation anthrax using A/J and C57BL/6 mice. With IL-17 receptor A knockout (IL-17RA −/− ) mice, we confirmed that IL-17A/F signaling is instrumental in the self-recruitment of this population. We also show that the IL-17A/F axis is critical for surviving pulmonary infection, as IL-17RA −/− mice become susceptible to intranasal infection by Bacillus anthracis Sterne spores. Strikingly, infection with a fully virulent strain did not affect IL-17RA −/− mouse survival. Eventually, by depleting neutrophils in wild-type and IL-17RA −/− mice, we demonstrated the crucial role of IL-17-secreting neutrophils in mouse survival of infection by fully virulent B. anthracis . This work demonstrates the important roles of both IL-17 signaling and neutrophils in clearing this pathogen and surviving pulmonary B. anthracis infection.

Lung immune responses control pathogens while avoiding detrimental inflammation. The dynamics of lung homeostasis are not fully understood. Primary antimicrobial defenses are orchestrated by epithelial cells (ECs), interacting in close association with dendritic cells (DCs) and other innate immune cells. However, microbes, such as Bacillus anthracis use host cellular machinery, including DC-migratory capacity to reach the draining lymph nodes where they germinate and produce toxins that disrupt protective immunity leading to death. Here, we describe the immunobiology of major lung cell subsets and their cellular synapses, to reveal the dynamics of B. anthracis infection and its effects on lung immune mechanisms. Vaccines strategies that mobilize protective mucosal and systemic immunity against this deadly microbe are highlighted.

Cationic (99m)Tc-agents like (99m)Tc-hexakis-2-methoxyisobutyl isonitrile ((99m)Tc-MIBI) cannot be used for brain imaging because they do not enter the brain as readily as some uncharged (99m)Tc-compounds. The mechanism by which cationic (99m)Tc-agents are transported across the blood-brain barrier (BBB) remains unclear. We explored (99m)Tc-MIBI transport by in situ mouse brain perfusion to determine the influence of BBB features like the ATP-binding cassette transporters (Abcb1/P-glycoprotein (P-gp), Abcc1/Mrp1, and Abcg2/Bcrp), organic cation transporters (Slc22a1-3/Oct1-3), the transmembrane potential and the dipole membrane potential. P-gp reduced (99m)Tc-MIBI transport across the BBB of P-gp-deficient mice 2.2-fold, as confirmed by PSC833 and GF120918 inhibition. Paradoxically verapamil decreased its transport '0.6-fold'. Reducing the BBB dipole membrane potential with tetraphenylborate or phloretin increased (99m)Tc-MIBI transport about 12- and 20-fold, respectively. Guanidine, diphenhydramine, and carnitine significantly decreased (99m)Tc-MIBI transport, but tetraethylammonium did not. (99m)Tc-MIBI transport at the BBB is restricted by P-gp but not by Mrp1 or Bcrp. Some organic cations reduced the influx of (99m)Tc-MIBI into the brain independently of Oct1, 2 and 3, but this could be due to their effect on another cation transporter. The membrane dipole potential of the luminal BBB membrane appeared to be the main factor restricting (99m)Tc-MIBI permeability.

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.

Summary Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially as a potential bioterrorism agent. Progress is gradually being made towards understanding the mechanisms used by Bacillus anthracis to escape the immune system and to induce severe septicaemia associated with toxaemia and leading to death. Recent advances in fundamental research have revealed previously unsuspected roles for toxins in various cell types. We summarize here pathological data for animal models and macroscopic histological examination data from recent clinical records, which we link to the effects of toxins. We describe three major steps in infection: (i) an invasion phase in the lung, during which toxins have short-distance effects on lung phagocytes; (ii) a phase of bacillus proliferation in the mediastinal lymph nodes, with local effects of toxins; and (iii) a terminal, diffusion phase, characterized by a high blood bacterial load and by long-distance effects of toxins, leading to host death. The pathophysiology of inhalational anthrax thus involves interactions between toxins and various cell partners, throughout the course of infection.

Mice are exquisitely sensitive to infection with encapsulated B. anthracis strains, even in the absence of toxins. Bioluminescence imaging shows that the lungs are the terminals targets before death, whatever the route of infection: cutaneous, inhalational or digestive. In this study, we addressed the pathophysiological mechanisms of this phenomenon during a cutaneous infection in the absence of toxins. Ultrastructural analysis before the appearance of any clinical signs shows a high density of encapsulated bacteria colonizing the alveolar capillary network. Histological analysis when clinical signs were patent, just before death, showed lung tissue damage, with bacteria localized in alveolar capillaries and spaces along alveolar cells. Bronchiolar epithelium injury and intra-alveolar hemorrhages were also observed. Using functional approaches, we detected hypoxia of blood vessel endothelial cells in the lung, and multifocal hypoxic zones in the brain parenchyma, associated with neurological signs at the time of death. During the bacteriemic phase of infection, we determined that the bacteria circulating in blood presented as chains of circa 13 μm in length with a mean of 4 bacteria per chain. To address the mechanisms of lung colonization, we mimicked this phase of infection by intravenous inoculation of bioluminescent bacteria of a similar chain length; the bacteria were immediately trapped within the lung capillary network. Encapsulated bacteria of shorter length were not sequestered and trapping was decreased in the absence of the capsule surrounding the bacilli. In conclusion, we show that the high pathogenicity of B. anthracis in the absence of toxins is most probably linked to physical sequestration of blood-circulating encapsulated bacilli of a given length. Clearly, in the case of anthrax, the toxins produced by the trapped encapsulated B. anthracis will add locally their deleterious effects. Controlling the lung-targeted pathology would be beneficial for the prognosis of anthrax infection.

In vivo microscopy has recently become a gold standard in lung immunology studies involving small animals, largely benefiting from the democratization of multiphoton microscopy allowing for deep tissue imaging. This technology represents currently our only way of exploring the lungs and inferring what happens in human respiratory medicine. The interest of lung in vivo microscopy essentially relies upon its relevance as a study model, fulfilling physiological requirements in comparison with in vitro and ex vivo experiments. However, strategies developed in order to overcome movements of the thorax caused by breathing and heartbeats remain the chief drawback of the technique and a major source of invasiveness. In this context, minimizing invasiveness is an unavoidable prerequisite for any improvement of lung in vivo microscopy. This review puts into perspective the main techniques enabling lung in vivo microscopy, providing pros and cons regarding invasiveness.

Bacillus anthracis secretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.

Summary Chronic stress is known to induce immunological disorders. In the present study we examined the consequences of chronic restraint stress on the immune response to tetanus toxin in mice. We investigated the repartition of subsets of lymphoid cells in blood and spleen, the functional ability of lymphocytes to proliferate and to produce cytokines, and antibody titres against tetanus toxin following stress. We report discordance of the stimulation index of lymphocytes in the restraint group: the proliferating rate severely decreased following stimulation with a relevant antigen, whereas it increased with mitogen. Thus, we report a decrease in cytokine production with relevant antigen (interferon-γ and interleukin-10), without a T helper type 1 and 2 secretion imbalance. Moreover, we observed an alteration in the humoral response, including a delay in isotype maturation and an immunoglobulin G1/G2a imbalance.

The dynamics of the lung immune system at the microscopic level are largely unknown because of inefficient methods of restraining chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by the second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis , the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process.

Lung tissue motion arising from breathing and heart beating has been described as the largest annoyance of in vivo imaging. Consequently, infected lung tissue has never been imaged in vivo thus far, and little is known concerning the kinetics of the mucosal immune system at the cellular level. We have developed an optimized post-processing strategy to overcome tissue motion, based upon two-photon and second harmonic generation (SHG) microscopy. In contrast to previously published data, we have freed the lung parenchyma from any strain and depression in order to maintain the lungs under optimal physiological parameters. Excitation beams swept the sample throughout normal breathing and heart movements, allowing the collection of many images. Given that tissue motion is unpredictably, it was essential to sort images of interest. This step was enhanced by using SHG signal from collagen as a reference for sampling and realignment phases. A normalized cross-correlation criterion was used between a manually chosen reference image and rigid transformations of all others. Using CX3CR1 +/gfp mice this process allowed the collection of high resolution images of pulmonary dendritic cells (DCs) interacting with Bacillus anthracis spores, a Gram-positive bacteria responsible for anthrax disease. We imaged lung tissue for up to one hour, without interrupting normal lung physiology. Interestingly, our data revealed unexpected interactions between DCs and macrophages, two specialized phagocytes. These contacts may participate in a better coordinate immune response. Our results not only demonstrate the phagocytizing task of lung DCs but also infer a cooperative role of alveolar macrophages and DCs.

While most deaths from asphyxia related to buprenorphine (BUP) overdose have been reported in males, higher plasma concentrations of BUP and its toxic metabolite norbuprenorphine (NBUP) have been observed in females. We previously demonstrated that P-glycoprotein (P-gp) modulation at the blood-brain barrier (BBB) contributes highly to BUP-related respiratory toxicity, by limiting NBUP entrance into the brain. In this work, we sought to investigate the role of P-gp-mediated transport at the BBB in gender and strain-related variability of BUP and NBUP-induced respiratory effects in mice. Ventilation was studied using plethysmography, P-gp expression using western blot, and transport at the BBB using in situ cerebral perfusion. In male Fvb and Swiss mice, BUP was responsible for ceiling respiratory effects. NBUP-related reduction in minute volume was dose-dependent but more marked in Fvb (p0.01 at 1mg/kg NBUP and p0.001 at 3 and 9mg/kg NBUP) than in Swiss mice (p0.001 at 9mg/kg NBUP). Female Fvb mice were more susceptible to BUP than males with significantly increased inspiratory time (p0.05) and to NBUP with significantly increased expiratory time (p0.01). Following BUP administration, plasma BUP concentrations were significantly higher (p0.01) and plasma NBUP concentrations significantly lower (p0.001) in Fvb mice compared to Swiss mice. Plasma BUP concentrations were significantly higher (p0.05) and plasma NBUP concentrations significantly lower (p0.01) in male compared to female Fvb mice. In contrast, following NBUP administration, comparable plasma NBUP concentrations were observed in both genders and strains. No differences in P-gp expression or BUP and NBUP transport across the BBB were observed between male and female Fvb mice as well as between Swiss and Fvb mice. Our results suggest that P-gp-mediated transport across the BBB does not play a key-role in gender and strain-related variability in BUP and NBUP-induced respiratory toxicity in mice. Both gender- and strain-related differences in respiratory effects of BUP could be attributed to BUP itself rather than to its metabolite, NBUP.

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.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.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.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.

Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.

Lung efficiency as gas exchanger organ is based on the delicate balance of its associated mucosal immune system between inflammation and sterility. In this study, we developed a dynamic imaging protocol using confocal and two-photon excitation fluorescence (2PEF) on freshly harvested infected lungs. This modus operandi allowed the collection of important information about CX3CR1+ pulmonary cells. This major immune cell subset turned out to be distributed in an anisotropic way in the lungs: subpleural, parenchymal and bronchial CX3CR1+ cells have then been described. The way parenchymal CX3CR1+ cells react against LPS activation has been considered using Matlab software, demonstrating a dramatic increase of average cell speed. Then, interactions between Bacillus anthracis spores and CX3CR1+ dendritic cells have been investigated, providing not only evidences of CX3CR1+ cells involvement in pathogen uptake but also details about the capture mechanisms.

Bacillus anthracis is a nonmotile, facultative anaerobe that occasionally infects humans. However, it more frequently causes disease in animals, including field-grazed herbivores in particular (Mock and Fouet, 2001). The ecological cycle of this gram-positive bacterium involves the proliferation of vegetative bacilli in the host, leading to the generation of dormant spores, which may persist in the soil in a viable state for decades. Spores ingested by grazing herbivores typically constitute the infectious form of this pathogen. B. anthracis has two main virulence factors, which have been described in detail in previous studies: the poly-γ-glutamate (PGA) capsule (encoded by pXO2; Candela and Fouet, 2006) and protein toxins (encoded by a temperature-sensitive pXO1 plasmid; Young and Collier, 2007). The capsule plays an important role in establishing disease, by protecting the bacillus against complement fixation and phagocytes. Toxins essentially provide the coup de grace in infected hosts, killing the host and triggering the sporulation of the bacilli responsible for perpetuating this deadly cycle. The combination of an enzymatically active component—lethal factor (LF) or edema factor (EF)—with protective antigen (PA) as a cell surface-binding component results in the production of lethal toxin (LT) and edema toxin (ET), respectively (Young and Collier, 2007). There are three main clinical presentations of anthrax disease in humans (Inglesby et al., 2002): a cutaneous form, the most frequent in natural conditions and often benign, and the more aggressive intestinal and pneumonic forms, involving sepsis and resulting in death. Interest in the pneumonic form was fuelled by the bioterrorists attacks in 2001 in the United States. Cutaneous anthrax has been shown to occur after the deposition of spores on the skin. Cuts or abrasion of the skin may favor infection. The areas of skin exposed to the air, as on the arms, hands, face, and neck, are the most frequently affected. The spores are thought to germinate in the skin and to proliferate locally, giving rise to a black eschar surrounded by edema (Inglesby et al., 2002). The intestinal form of the disease is linked to the ingestion of contaminated meat from the carcasses of animals that died from anthrax. The lesions of intestinal anthrax occur mostly in the terminal ileum and cecum, in the lower intestinal tract. A recent study with a bioluminescent capsulated

The (99m)Tc-tricarbonyl core may be used as an ideal tool for gamma-labeling ligands in noninvasive SPECT imaging. However, most (99m)Tc-tricarbonyl-labeled agents have difficulty crossing the blood-brain barrier (BBB). We radiolabeled the neuroactive indole ibogaine with (99m)Tc-tricarbonyl and measured its transport into the mouse brain by in situ brain perfusion. We measured the interactions of [(99m)Tc(CO)(3)-ibogaine](+) and (99m)Tc-tricarbonyl with the main BBB efflux transporters P-gp and BCRP in vitro and in vivo. Ibogaine was radiolabeled (yield: over 95%). [(99m)Tc(CO)(3)-ibogaine](+) entered the brain (K(in)) poorly (0.18 microL/g/s), at about the same rate as (99m)Tc-tricarbonyl (0.16 microL/g/s) and [(99m)Tc-sestamibi](+) (0.10 microL/g/s). The CNS tracer [(99m)Tc-HMPAO](0) entered the brain approximately 70-times higher than [(99m)Tc(CO)(3)-ibogaine](+). In vitro studies revealed that neither [(99m)Tc(CO)(3)-ibogaine](+) nor (99m)Tc-tricarbonyl ion were substrates for P-gp or BCRP. But lowering the membrane dipole potential barrier with phloretin enhanced the brain transport of [(99m)Tc(OH(2))(3)(CO)(3)](+) approximately 3-fold. Thus, ibogaine directly labeled with (99m)Tc-tricarbonyl is not suitable for CNS imaging because of its poor uptake. Brain transport is not restricted by efflux transporters but is reduced by its lipophilicity and interaction with the membrane-positive dipole potential.