Bertrand Kuhnast

Comparative Evaluation of the Translocator Protein Radioligands 11C-DPA-713, 18F-DPA-714, and 11C-PK11195 in a Rat Model of Acute Neuroinflammation

Overexpression of the translocator protein, TSPO (18 kDa), formerly known as the peripheral benzodiazepine receptor, is a hallmark of activation of cells of monocytic lineage (microglia and macrophages) during neuroinflammation. Radiolabeling of TSPO ligands enables the detection of neuroinflammatory lesions by PET. Two new radioligands, 11C-labeled N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-yl]acetamide (DPA-713) and 18F-labeled N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-yl)acetamide (DPA-714), both belonging to the pyrazolopyrimidine class, were compared in vivo and in vitro using a rodent model of neuroinflammation. Methods: 11C-DPA-713 and 18F-DPA-714, as well as the classic radioligand 11C-labeled (R)-N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)isoquinoline-3-carboxamide (PK11195), were used in the same rat model, in which intrastriatal injection of (R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique gave rise to a strong neuroinflammatory response. Comparative endpoints included in vitro autoradiography and in vivo imaging on a dedicated small-animal PET scanner under identical conditions. Results: 11C-DPA-713 and 18F-DPA-714 could specifically localize the neuroinflammatory site with a similar signal-to-noise ratio in vitro. In vivo, 18F-DPA-714 performed better than 11C-DPA-713 and 11C-PK11195, with the highest ratio of ipsilateral to contralateral uptake and the highest binding potential. Conclusion: 18F-DPA-714 appears to be an attractive alternative to 11C-PK11195 because of its increased bioavailability in brain tissue and its reduced nonspecific binding. Moreover, its labeling with 18F, the preferred PET isotope for radiopharmaceutical chemistry, favors its dissemination and wide clinical use. 18F-DPA-714 will be further evaluated in longitudinal studies of neuroinflammatory conditions such as are encountered in stroke or neurodegenerative diseases.

Evaluation of the PBR/TSPO radioligand [18F]DPA-714 in a rat model of focal cerebral ischemia

Focal cerebral ischemia leads to an inflammatory reaction involving an overexpression of the peripheral benzodiazepine receptor (PBR)/18-kDa translocator protein (TSPO) in the cerebral monocytic lineage (microglia and monocyte) and in astrocytes. Imaging of PBR/TSPO by positron emission tomography (PET) using radiolabeled ligands can document inflammatory processes induced by cerebral ischemia. We performed in vivo PET imaging with [18F]DPA-714 to determine the time course of PBR/TSPO expression over several days after induction of cerebral ischemia in rats. In vivo PET imaging showed significant increase in DPA (N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) uptake on the injured side compared with that in the contralateral area on days 7, 11, 15, and 21 after ischemia; the maximal binding value was reached 11 days after ischemia. In vitro autoradiography confirmed these in vivo results. In vivo and in vitro [18F]DPA-714 binding was displaced from the lesion by PK11195 and DPA-714. Immunohistochemistry showed increased PBR/TSPO expression, peaking at day 11 in cells expressing microglia/macrophage antigens in the ischemic area. At later times, a centripetal migration of astrocytes toward the lesion was observed, promoting the formation of an astrocytic scar. These results show that [18F]DPA-714 provides accurate quantitative information of the time course of PBR/TSPO expression in experimental stroke.

Fluorine-18-labeled phospholipid quantum dot micelles for in vivo multimodal imaging from whole body to cellular scales.

We have designed new nanoprobes applicable for both positron emission tomography (PET) and optical fluorescence in vivo imaging. Fluorine-18, which is commonly used for clinical imaging, has been coupled to phospholipid quantum dot (QD) micelles. This probe was injected in mice and we demonstrated that its dynamic quantitative whole body biodistribution and pharmacokinetics could be monitored using PET as well as the kinetics of their cellular uptake using in vivo fibered confocal fluorescence imaging. Phospholipid micelle encapsulation of QDs provides a highly versatile surface chemistry to conjugate multiple chemicals and biomolecules with controlled QD:molecule valency. Here, we show that, in contrast with several previous studies using other QD polymer coatings, these phospholipid QD micelles exhibit long circulation half-time in the bloodstream (on the order of 2 h) and slow uptake by reticulo-endothelial system.

Current paradigm of the 18-kDa translocator protein (TSPO) as a molecular target for PET imaging in neuroinflammation and neurodegenerative diseases

Neuroinflammation is a process characterised by drastic changes in microglial morphology and by marked upregulation of the 18-kDa translocator protein (TSPO) on the mitochondria. The continual increase in incidence of neuroinflammation and neurodegenerative diseases poses a major health issue in many countries, requiring more innovative diagnostic and monitoring tools. TSPO expression may constitute a biomarker for brain inflammation that could be monitored by using TSPO tracers as neuroimaging agents. From medical imaging perspectives, this review focuses on the current concepts related to the TSPO, and discusses briefly on the status of its PET imaging related to neuroinflammation and neurodegenerative diseases in humans.

In vivo imaging of brain lesions with [11C]CLINME, a new PET radioligand of peripheral benzodiazepine receptors

The peripheral benzodiazepine receptor (PBR) is expressed by microglial cells in many neuropathologies involving neuroinflammation. PK11195, the reference compound for PBR, is used for positron emission tomography (PET) imaging but has a limited capacity to quantify PBR expression. Here we describe the new PBR ligand CLINME as an alternative to PK11195. In vitro and in vivo imaging properties of [11C]CLINME were studied in a rat model of local acute neuroinflammation, and compared with the reference compound [11C]PK11195, using autoradiography and PET imaging. Immunohistochemistry study was performed to validate the imaging data. [11C]CLINME exhibited a higher contrast between the PBR‐expressing lesion site and the intact side of the same rat brain than [11C]PK11195 (2.14 ± 0.09 vs. 1.62 ± 0.05 fold increase, respectively). The difference was due to a lower uptake for [11C]CLINME than for [11C]PK11195 in the non‐inflammatory part of the brain in which PBR was not expressed, while uptake levels in the lesion were similar for both tracers. Tracer localization correlated well with that of activated microglial cells, demonstrated by immunohistochemistry and PBR expression detected by autoradiography. Modeling using the simplified tissue reference model showed that R1 was similar for both ligands (R1 ∼ 1), with [11C]CLINME exhibiting a higher binding potential than [11C]PK11195 (1.07 ± 0.30 vs. 0.66 ± 0.15). The results show that [11C]CLINME performs better than [11C]PK11195 in this model. Further studies of this new compound should be carried out to better define its capacity to overcome the limitations of [11C]PK11195 for PBR PET imaging.

[18F]DPA-714, [18F]PBR111 and [18F]FEDAA1106-selective radioligands for imaging TSPO 18 kDa with PET: automated radiosynthesis on a TRACERLAb FX-FN synthesizer and quality controls.

Imaging of TSPO 18 kDa with PET is more and more considered as a relevant biomarker of inflammation in numerous diseases. Development of new radiotracers for TSPO 18 kDa has seen acceleration in the last years and the challenge today is to make available large amounts of such a radiotracer in compliance with GMP standards for application in humans. We present in this technical note automated productions of [(18)F]DPA-714, [(18)F]PBR111 and [(18)F]FEDAA1106, three promising radiotracers for TSPO 18 kDa imaging, using a TRACERlab FX-FN synthesizer. This note also includes the quality control data of the validation batches for the manufacturing qualification of clinical production of [(18)F]DPA-714.

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

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

Effects of Selected OATP and/or ABC Transporter Inhibitors on the Brain and Whole-Body Distribution of Glyburide

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

In vivo validation of free-space fluorescence tomography using nuclear imaging

The performance of small animal photonic imaging has been considerably improved since the development of fluorescence diffuse optical tomography (fDOT), which can reconstruct fluorescent probe distribution inside tissue. However, the quantification capabilities of this new technology are still a topic of debate, especially in comparison to classical nuclear imaging techniques. Here, we present a method to in vivo calibrate the quantity and localization of a probe provided by free-space fDOT (where no plate is compressing the mouse) with positron emission tomography (PET) and x-ray computed tomography, respectively. This methodology allowed us to demonstrate a strong linear correlation (R(2)=0.95) between fDOT and PET for probe concentrations ranging from 3 nM to 1 μM in a deep-seated organ.

Fluorine‐18 Chemistry for Molecular Imaging with Positron Emission Tomography

Publisher Summary Positron emission tomography (PET) is a high-resolution, sensitive, functional-imaging technique in nuclear medicine that permits repeated, non-invasive assessment, and quantification of specific biological and pharmacological processes at the molecular level in humans and animals. It is the most advanced technology currently available for studying in vivo molecular interactions in terms of distribution, pharmacokinetics, and pharmacodynamics. Molecular PET imaging requires the preparation of a positron-emitting radiolabeled probe or radiotracer. For this purpose, fluorine-18 is becoming increasingly the radionuclide of choice not only due to its adequate physical and nuclear characteristics but also due to the successful use in clinical oncology of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), currently the most widely used PET radiopharmaceutical and manifestly a motor behind the growing availability and interest for this positron emitter in radiopharmaceutical chemistry. This chapter addresses this complex interdisciplinary and rapidly growing field from a radiochemist point of view, focusing on the synthesis of fluorine-18-labelled radiopharmaceuticals. The successful use in clinical oncology of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), currently the most widely used PET radiopharmaceutical, is manifestly also the motor behind the growing availability and interest for this positron emitter in radiopharmaceutical chemistry. The use of fluorine-18, however, presents some drawbacks in particular the limited options in labeling strategies. The synthesis of complex structures labeled with fluorine-18 remains a challenge but undoubtedly, fluorine-18 is already, and will continue to be, a royal gateway to success in molecular imaging with PET.