Fabien Caillé

Strategies to Inhibit ABCB1- and ABCG2-Mediated Efflux Transport of Erlotinib at the Blood–Brain Barrier: A PET Study on Nonhuman Primates

The tyrosine kinase inhibitor erlotinib poorly penetrates the blood–brain barrier (BBB) because of 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 metastases in the brain. Pharmacologic strategies to inhibit ABCB1/ABCG2-mediated efflux transport at the BBB have been successfully developed in rodents, but it remains unclear whether these can be translated to humans given the 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 nonhuman primates as a model of the human BBB. Methods: Papio anubis baboons underwent PET scans of the brain after intravenous injection of 11C-erlotinib under baseline conditions (n = 4) and during intravenous 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 levels comparable to those 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 nonhuman primates resulting in pronounced increases in brain distribution of 11C-erlotinib. For patients with brain tumors, such inhibition protocols may ultimately be applied to create more effective treatments using drugs that undergo efflux transport at the BBB.

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

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

In Vivo Evaluation of 18F-MNI698: An 18F-Labeled Radiotracer for Imaging of Serotonin 4 Receptors in Brain

Serotonin 4 receptors (5-hydroxytryptamine receptor 4 [5HT4R]) hold promise as a novel therapeutic approach to multiple brain disorders, including Alzheimer and Huntington disease. In vivo imaging of these receptors with selective 5HT4R radiotracers and PET would be valuable to investigate alterations in 5HT4R in different brain disorders and to assist drug discovery. In this study, 18F-MNI698 was evaluated as a potential PET radiotracer for imaging of 5HT4R in the brain. Methods: Eighteen PET studies were performed in 3 adult rhesus monkeys. The radiotracer was administered as a bolus intravenous injection or bolus plus constant infusion (time that would be required to inject the bolus at the infusion rate = 60 min), and arterial blood was collected for data quantification. Kinetic models were used to estimate distribution volumes and binding potentials, for which the cerebellum was used as a reference region. 18F-MNI698 test–retest variability and upper mass dose limits were determined. Preblocking studies using several doses of SB204070, a selective 5HT4R antagonist, were performed. Results: 18F-MNI698 avidly entered the monkey brain (peak percentage injected dose of ∼6.6%), and its brain distribution was consistent with known 5HT4R densities. At 120 min after bolus injection and after the start of radiotracer infusion, only less than 5% and approximately 10% parent compound was present in blood, respectively. Measured binding potentials were underestimated by 22%–36% when noninvasive methods were used for data quantification in comparison with invasive methods. A good agreement was found between test–retest measurements. The radiotracer upper mass dose limit (<5% occupancy) was determined to be 13.1 μg per 70 kg of body weight. SB204070 blocked the radiotracer binding in a dose-dependent manner. Conclusion: Data indicate that 18F-MNI698 is a promising PET radiotracer for imaging of 5HT4R in the brain, and human studies are warranted based on these study results.

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

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

Diphenhydramine as a selective probe to study H+-antiporter function at the blood-brain barrier: Application to [11C]diphenhydramine positron emission tomography imaging.

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.

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.

Distinct dynamic profiles of microglial activation are associated with progression of Alzheimer's disease

Although brain neuroinflammation may play an instrumental role in the pathophysiology of Alzheimers disease, its actual impact on disease progression remains controversial, being reported as either detrimental or protective. This work aimed at investigating the temporal relationship between microglial activation and clinical progression of Alzheimers disease. First, in a large cohort of patients with Alzheimers disease we analysed the predictive value of microglial activation assessed by 18F-DPA-714 PET imaging on functional, cognitive and MRI biomarkers outcomes after a 2-year follow-up. Second, we analysed the longitudinal progression of 18F-DPA-714 binding in patients with Alzheimers disease by comparison with controls, and assessed its influence on clinical progression. At baseline, all participants underwent a clinical assessment, brain MRI, 11C-PiB, 18F-DPA-714 PET imaging and TSPO genotyping. Participants were followed-up annually for 2 years. At the end of the study, subjects were asked to repeat a second 18F-DPA-714-PET imaging. Initial 18F-DPA-714 binding was higher in prodromal (n = 33) and in demented patients with Alzheimers disease (n = 19) compared to controls (n = 17). After classifying patients into slow and fast decliners according to functional (Clinical Dementia Rating change) or cognitive (Mini-Mental State Examination score decline) outcomes, we found a higher initial 18F-DPA-714 binding in slow than fast decliners. Negative correlations were observed between initial 18F-DPA-714 binding and the Clinical Dementia Rating Sum of Boxes score increase, the MMSE score loss and the progression of hippocampal atrophy. This suggests that higher initial 18F-DPA-714 binding is associated with better clinical prognosis. Twenty-four patients with Alzheimers disease and 15 control subjects performed a second DPA-PET. We observed an increase of 18F-DPA-714 in patients with Alzheimers disease as compared with controls (mean 13.2% per year versus 4.2%) both at the prodromal (15.8%) and at the demented stages (8.3%). The positive correlations between change in 18F-DPA-714 binding over time and the three clinical outcome measures (Clinical Dementia Rating, Mini-Mental State Examination, hippocampal atrophy) suggested a detrimental effect on clinical Alzheimers disease progression of increased neuroinflammation after the initial PET examination, without correlation with PiB-PET uptake at baseline. High initial 18F-DPA-714 binding was correlated with a low subsequent increase of microglial activation and favourable clinical evolution, whereas the opposite profile was observed when initial 18F-DPA-714 binding was low, independently of disease severity at baseline. Taken together, our results support a pathophysiological model involving two distinct profiles of microglial activation signatures with different dynamics, which differentially impact on disease progression and may vary depending on patients rather than disease stages.

P-Glycoprotein (ABCB1) Inhibits the Influx and Increases the Efflux of 11C-Metoclopramide Across the Blood–Brain Barrier: A PET Study on Nonhuman Primates

PET imaging using radiolabeled avid substrates of the ATP-binding cassette (ABC) transporter 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 11C-metoclopramide, as a model of a weak ABCB1 substrate, in nonhuman primates. Methods: The regional brain kinetics of a tracer dose of 11C-metoclopramide (298 ± 44 MBq) were assessed in baboons using PET without (n = 4) or with (n = 4) intravenous coinfusion of the ABCB1 inhibitor tariquidar (4 mg/kg/h). Metabolite-corrected arterial input functions were generated to estimate the regional volume of distribution (VT), as well as the influx (K1) and efflux (k2) rate constants, using a 1-tissue-compartment model. Modeling outcome parameters were correlated with image-derived parameters, that is, areas under the regional time–activity curves (AUCs) from 0 to 30 min and from 30 to 60 min (SUV⋅min) and the elimination slope (kE; min−1) from 30 to 60 min. Results: Tariquidar significantly increased the brain distribution of 11C-metoclopramide (VT = 4.3 ± 0.5 mL/cm3 and 8.7 ± 0.5 mL/cm3 for baseline and ABCB1 inhibition conditions, respectively, P < 0.001), with a 1.28-fold increase in K1 (P < 0.05) and a 1.64-fold decrease in k2 (P < 0.001). The effect of tariquidar was homogeneous across different brain regions. The parameters most sensitive to ABCB1 inhibition were VT (2.02-fold increase) and AUC from 30 to 60 min (2.02-fold increase). VT correlated significantly (P < 0.0001) with AUC from 30 to 60 min (r2 = 0.95), with AUC from 0 to 30 min (r2 = 0.87), and with kE (r2 = 0.62). Conclusion: 11C-metoclopramide PET imaging revealed the relative importance of both the influx hindrance and the efflux enhancement components of ABCB1 in a relevant model of the human BBB. The overall impact of ABCB1 on drug delivery to the brain can be noninvasively estimated from image-derived outcome parameters without the need for an arterial input function.

Positron Emission Tomography Imaging Reveals an Importance of Saturable Liver Uptake Transport for the Pharmacokinetics of Metoclopramide

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.

Late-Stage Isotopic Carbon Labeling of Pharmaceutically Relevant Cyclic Ureas Directly from CO2

Abstract A robust, click‐chemistry‐inspired procedure for radiolabeling of cyclic ureas was developed. This protocol, suitable for all carbon isotopes (11C, 13C, 14C), is based on the direct functionalization of carbon dioxide: the universal building block for carbon radiolabeling. The strategy is operationally simple and reproducible in different radiochemistry centers, exhibits remarkably wide substrate scope with short reaction times, and demonstrates superior reactivity as compared to previously reported systems. With this procedure, a variety of pharmaceuticals and an unprotected peptide were labeled with high radiochemical efficiency.