Charge-solvated versus protonated salt forms of cyclodepsipeptide toxins in electrospray: Dissociation of alkali-cationized forms enables straightforward sequencing of cereulide.

Bacillus cereus is responsible for foodborne outbreaks worldwide. Among the produced toxins, cereulide induces nausea and vomiting after 30 min to 6 h following the consumption of contaminated foods. Cereulide, a cyclodepsipeptide, is an ionophore selective to K+ in solution. In electrospray, the selectivity is reduced as [M + Li]+; [M + Na]+ and [M + NH4]+ can also be detected without adding corresponding salts. Two forms are possible for alkali-cationized ions: charge-solvated (CS) that exclusively dissociates by releasing a bare alkali ion and protonated salt (PS), yielding alkali product ions by covalent bond cleavages (CBC) promoted by mobile proton. Based on a modified peptide cleavage nomenclature, the PS product ion series (b, a, [b + H2O] and [b + CnH2nO] [n = 4, 5]) are produced by Na+/Li+/K+-cationized cereulide species that specifically open at ester linkages followed by proton mobilization promoting competitive ester CBC as evidenced under resonant collision activation. What is more, unlike the sodiated or lithiated cereulide, which regenerates little or no alkali cation, the potassiated forms lead to an abundant K+ regeneration. This occurs by splitting of (i) the potassiated CS forms with an appearance threshold close to that of the PS first fragment ion generation and (ii) eight to four potassiated residue product ions from the PS forms. Since from Na+/Li+-cationized cereulide, (i) the negligible Na+/Li+ regeneration results in a higher sensibility than that of potassiated forms that abundantly releasing K+, and (ii) a better sequence recovering, the use of Na+ (or Li+) should be more pertinent to sequence isocereulides and other cyclodepsipeptides.

Energy Resolved Mass Spectrometry for Interoperable Non-resonant Collisional Spectra in Metabolomics.

In untargeted metabolomics, the unambiguous identification of metabolites remains a major challenge. This requires high-quality spectral libraries for reliable metabolite identification, which is essential for translating metabolomics data into meaningful biological information. Several attempts have been made to generate reproducible product ion spectra (PIS) under a low collision energy (ELab) regime and nonresonant collisional conditions but have not fully succeeded. We examined the ERMS (energy-resolved mass spectrometry) breakdown curves of two lipo-amino acids and showed the possibility to highlight "singular points", called descriptors hereafter (linked to respective ELab depending on the instrument), for each of the monomodal product ion profiles. Using several instruments based on different technologies, the PIS recorded at these specific ELab sites shows remarkable similarities. The descriptors appeared as being independent of the fragmentation mechanisms and can be used to overcome the main instrumental effects that limit the interoperability of spectral libraries. This proof-of-concept study, performed on two particular lipo-amino acids, demonstrates the high potential of ERMS-derived information to determine the instrument-specific ELab at which PIS recorded in nonresonant conditions become highly similar and instrument-independent, thus comparable across platforms. This innovative but straightforward approach could help remove some of the obstacles to metabolite identification in nontargeted metabolomics, putting an end to a challenging chimera.

Hydrogen/deuterium exchange mass spectrometry in the world of small molecules.

The combined use of hydrogen/deuterium exchange (HDX) and mass spectrometry (MS), referred to as HDX-MS, is a powerful tool for exploring molecular edifices and has been used for over 60 years. Initially for structural and mechanistic investigation of low-molecular weight organic compounds, then to study protein structure and dynamics, then, the craze to study small molecules by HDX-MS accelerated and has not stopped yet. The purpose of this review is to present its different facets with particular emphasis on recent developments and applications. Reversible H/D exchanges of mobilizable protons as well as stable exchanges of non-labile hydrogen are considered whether they are taking place in solution or in the gas phase, or enzymatically in a biological media. Some fundamental principles are restated, especially for gas-phase processes, and an overview of recent applications, ranging from identification to quantification through the study of metabolic pathways, is given.

PeakForest: a multi-platform digital infrastructure for interoperable metabolite spectral data and metadata management.

INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .

Direct Comparison of [18F]F-DPA with [18F]DPA-714 and [11C]PBR28 for Neuroinflammation Imaging in the same Alzheimer's Disease Model Mice and Healthy Controls.

PURPOSE: In this study we compared the recently developed TSPO tracer [18F]F-DPA, with [18F]DPA-714 and [11C]PBR28 by performing in vivo PET imaging on the same Alzheimer's disease mouse model APP/PS1-21 (TG) and wild-type (WT) mice with all three radiotracers. PROCEDURES: To compare the radiotracer uptake, percentage of injected dose/mL (%ID/mL), standardized uptake value ratios to cerebellum (SUVRCB), and voxel-wise analyses were performed. RESULTS: The peak uptake of [18F]F-DPA was higher than 4.3% ID/mL, while [18F]DPA-714 reached just over 3% ID/mL, and [11C]PBR28 was over 4% ID/mL in only one brain region in the WT mice. The peak/60-min uptake ratios of [18F]F-DPA were significantly higher (p < 0.001) than those of [18F]DPA-714 and [11C]PBR28. The differences in [18F]F-DPA SUVRCB between WT and TG mice were highly significant (p < 0.001) in the three studied time periods after injection. [18F]DPA-714 uptake was significantly higher in TG mice starting in the 20-40-min timeframe and increased thereafter, whereas [11C]PBR28 uptake became significant at 10-20 min (p < 0.05). The voxel-wise analysis confirmed the differences between the radiotracers. CONCLUSIONS: [18F]F-DPA displays higher brain uptake, higher TG-to-WT SUVRCB ratios, and faster clearance than [18F]DPA-714 and [11C]PBR28, and could prove useful for detecting low levels of inflammation and allow for shorter dynamic PET scans.

Combining Chemical Knowledge and Quantum Calculation for Interpreting Low-Energy Product Ion Spectra of Metabolite Adduct Ions: Sodiated Diterpene Diester Species as a Case Study.

We investigated the product ion spectra of [M + Na]+ from diterpene diester species and low molecular mass metabolites analyzed by electrospray ionization (ESI). Mainly, the formation of protonated salt structures was proposed to explain the observed neutral losses of carboxylic acids. It also facilitates understanding sodium retention on product ions or on neutral losses. In addition, the occurrence of consecutive carboxylic acid losses is rather unexpected under resonant excitation conditions. Quantum calculation demonstrated that the exothermic character of such neutral losses can represent a relevant explanation. There is no doubt that the formation and role of the protonated salt structures will be helpful for a better understanding and software-assisted interpretation of tandem mass spectra from small molecules, especially in the ever-growing metabolomics field.

First Direct Evidence of Interpartner Hydride/Deuteride Exchanges for Stored Sodiated Arginine/Fructose-6-phosphate Complex Anions within Salt-Solvated Structures.

Mass spectrometric investigations of noncovalent binding between low molecular weight compounds revealed the existence of gas-phase (GP) noncovalent complex (NCC) ions involving zwitterionic structures. ESI MS is used to prove the formation of stable sodiated NCC anions between fructose (F6P) and arginine (R) moieties. Theoretical calculations indicate a folded solvated salt (i.e., sodiated carboxylate interacting with phosphate) rather than a charge-solvated form. Under standard CID conditions, [(F6P+R-H+Na)-H]- competitively forms two major product ions (PIs) through partner splitting [(R-H+Na) loss] and charge-induced cross-ring cleavage while preserving the noncovalent interactions (noncovalent product ions (NCPIs)). MS/MS experiments combined with in-solution proton/deuteron exchanges (HDXs) demonstrated an unexpected labeling of PIs, i.e., a correlated D-enrichment/D-depletion. An increase in activation time up to 3000 ms favors such processes when limited to two H/D exchanges. These results are rationalized by interpartner hydride/deuteride exchanges (⟨HDX⟩) through stepwise isomerization/dissociation of sodiated NCC-d11 anions. In addition, the D-enrichment/D-depletion discrepancy is further explained by back HDX with residual water in LTQ (selective for the isotopologue NCPIs as shown by PI relaxation experiments). Each isotopologue leads to only one back HDX unlike multiple HDXs generally observed in GP. This behavior shows that NCPIs are zwitterions with charges solvated by a single water molecule, thus generating a back HDX through a relay mechanism, which quenches the charges and prevents further back HDX. By estimating back HDX impact on D-depletion, the interpartner ⟨HDX⟩ complementarity was thus illustrated. This is the first description of interpartner ⟨HDX⟩ and selective back HDX validating salt-solvated structures.

Evaluation of erythropoietin biosimilars Epotin™, Hemax® and Jimaixin™ by electrophoretic methods used for doping control analysis and specific N-glycan analysis revealed structural differences from original epoetin alfa drug Eprex®.

Recombinant human erythropoietin (rEPO) biosimilars are copies of epoetin drugs developed after the first patents ended. However differences in the process of production can result in small structural differences when compared to the reference product. Differences in N-glycosylation profiles are of particular importance for rEPOs, because they can drastically impact the half-life in circulation and activity. Changes of structure can also impact electrophoretic profiles that are used to reveal the presence of a rEPO in a doping control sample. In this study three not well characterized biosimilars were evaluated (Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria). As these products could be used for doping, first their EPO profiles were determined using the antidoping methods (electrophoretic separation by the charge (isolectric focusing, IEF-PAGE) or the molecular weight (SDS-PAGE) and specific EPO immunodetection). Compared to the original epoetin alfa Eprex®, it revealed more basic isoforms for Epotin™ and Jimaixin™ after IEF-PAGE and a slightly lower molecular weight after SDS-PAGE in particular for Hemax®. To better understand the reason for these differences, EPO specific N-glycans were evaluated using two complementary approaches: MALDI-TOF mass spectrometry (MS) and hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. All three biosimilars presented a significant decrease in the major glycan forms of Eprex® along with an increase in less complex forms. Jimaixin™ and Epotin™ presented also a lower amount of fully sialylated forms. HILIC method also showed that O-acetylation level of sialic acid residues might vary from one rEPO to the other.

Suspect and non-targeted screening of chemicals of emerging concern for human biomonitoring, environmental health studies and support to risk assessment: From promises to challenges and harmonisation issues.

Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017-2021) are described, with an emphasis on harmonisation measures.

Comparative Evaluation of Data Dependent and Data Independent Acquisition Workflows Implemented on an Orbitrap Fusion for Untargeted Metabolomics.

Constant improvements to the Orbitrap mass analyzer, such as acquisition speed, resolution, dynamic range and sensitivity have strengthened its value for the large-scale identification and quantification of metabolites in complex biological matrices. Here, we report the development and optimization of Data Dependent Acquisition (DDA) and Sequential Window Acquisition of all THeoretical fragment ions (SWATH-type) Data Independent Acquisition (DIA) workflows on a high-field Orbitrap FusionTM TribridTM instrument for the robust identification and quantification of metabolites in human plasma. By using a set of 47 exogenous and 72 endogenous molecules, we compared the efficiency and complementarity of both approaches. We exploited the versatility of this mass spectrometer to collect meaningful MS/MS spectra at both high- and low-mass resolution and various low-energy collision-induced dissociation conditions under optimized DDA conditions. We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

Proposal for a chemically consistent way to annotate ions arising from the analysis of reference compounds under ESI conditions: A prerequisite to proper mass spectral database constitution in metabolomics.

Nowadays, high-resolution mass spectrometry is widely used for metabolomic studies. Thanks to its high sensitivity, it enables the detection of a large range of metabolites. In metabolomics, the continuous quest for a metabolite identification as complete and accurate as possible has led during the last decade to an ever increasing development of public MS databases (including LC-MS data) concomitantly with bioinformatic tool expansion. To facilitate the annotation process of MS profiles obtained from biological samples, but also to ease data sharing, exchange, and exploitation, the standardization and harmonization of the way to describe and annotate mass spectra seemed crucial to us. Indeed, under electrospray (ESI) conditions, a single metabolite does not produce a unique ion corresponding to its protonated or deprotonated form but could lead to a complex mixture of signals. These MS signals result from the existence of different natural isotopologues of the same compound and also to the potential formation of adduct ions, homomultimeric and heteromultimeric ions, fragment ions resulting from "prompt" in-source dissociations. As a joint reflection process within the French Infrastructure for Metabolomics and Fluxomics (MetaboHUB) and with the purpose of developing a robust and exchangeable annotated MS database made from pure reference compounds (chemical standards) analysis, it appeared to us that giving the metabolomics community some clues to standardize and unambiguously annotate each MS feature was a prerequisite to data entry and further efficient querying of the mass spectral database. The use of a harmonized notation is also mandatory for interlaboratory MS data exchange. Additionally, thorough description of the variety of MS signals arising from the analysis of a unique metabolite might provide greater confidence on its annotation.

Experimental evidence that electrospray-produced sodiated lysophosphatidyl ester structures exist essentially as protonated salts.

Sodiated lysoglycerophosphatidylethanolamine (LGPE) and lysoglycerophosphatidylcholine (LGPC) species dissociate under low collision energy by covalent bond cleavage resulting in product ions with either sodium retention or without sodium retention. For explaining these fragmentations, sodium chelation by heteroatoms (as charge-solvated structures) is often considered, and consequently, under keV collision conditions, sodium is "spectator" of cleavages (charge remote fragmentation). However, cleavage of such charge-solvated forms under low-energy conditions should result in sodium desolvation rather than covalent bond cleavage. In the present study, protonated salts are proposed as the main representative structures of the sodiated LGPE and LGPC forms. These structures are generated from sodiation of zwitterionic and betaine forms of LGPE and LGPC molecules, respectively. Experimental evidence to determine which structure is involved in the dissociations is provided, especially by comparing the dissociation of LGPL sodiated forms with that of sodiated polyethylene glycols. Energy-resolved mass spectrometry breakdown experiments were performed on a quadrupole time-of-flight instrument to demonstrate that both LGPE and LGPC sodiated forms exist as protonated salt structures. From such structures, proton migration by prototropy can result in different bond cleavages whereas the salt moiety remains spectator of these processes.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

[18F]F-DPA for the detection of activated microglia in a mouse model of Alzheimer's disease.

INTRODUCTION: Neuroinflammation is associated with several neurological disorders, including Alzheimer's disease (AD). The translocator protein 18 kDa (TSPO), due to its overexpression during microglial activation and relatively low levels in the brain under normal neurophysiological conditions, is commonly used as an in vivo biomarker for neuroinflammation. Reported here is the preclinical evaluation of [18F]F-DPA, a promising new TSPO-specific radioligand, as a tool for the detection of activated microglia at different ages in the APP/PS1-21 mouse model of AD and a blocking study to determine the specificity of the tracer. METHODS: [18F]F-DPA was synthesised by the previously reported electrophilic 18F-fluorination methodology. In vivo PET and ex vivo brain autoradiography were used to observe the tracer distribution in the brains of APP/PS1-21 and wildtype mice at different ages (4.5-24 months). The biodistribution and degree of metabolism of [18F]F-DPA were analysed and the specificity of [18F]F-DPA for its target was determined by pre-treatment with PK11195. RESULTS: The in vivo PET imaging and ex vivo brain autoradiography data showed that [18F]F-DPA uptake in the brains of the transgenic animals increased with age, however, there was a drop in the tracer uptake at 19 mo. Despite the slight increase in [18F]F-DPA uptake with age in healthy animal brains, significant differences between wildtype and transgenic animals were seen in vivo at 9 months and ex vivo already at 4.5 months. The specificity study demonstrated that PK11195 can be used to significantly block [18F]F-DPA uptake in all the brain regions studied. CONCLUSIONS: In vivo time activity curves plateaued at approximately 20-40 min suggesting that this is the optimal imaging time. Significant differences in vivo are seen at 9 and 12 mo. Due to the higher resolution, ex vivo autoradiography with [18F]F-DPA can be used to visualise activated microglia at an early stage of AD pathology.

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 Alzheimer's 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.

Avidin/Biotin Bioinspired Platform for Dual In Vivo 18F-PET/NIRF Molecular Imaging.

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of innovative multimodal PET/NIRF probes a very exciting prospect. Herein, the bioinspired design of novel platform exploiting the strength and specificity of interactions between radioactive and fluorescent biotin derivatives and an avidin core is reported. The combination of an original [18F]fluoropyridinylated-biotin derivative and commercially available fluorescent biotin derivatives (Atto-425 and Atto-680) is investigated. The in vivo distribution of such a customized platform is also reported, for the first time, in healthy rodent using PET and ex vivo fluorescence imaging.

Neuroprotective effect of the alpha 7 nicotinic receptor agonist PHA 543613 in an in vivo excitotoxic adult rat model.

Neuroinflammation is a key component of the pathophysiology of neurodegenerative diseases. The link between nicotine intake and positive outcome has been established, suggesting a role played by nicotinic receptors (nAChRs), especially α7nAChRs. The objective of this study was to evaluate the potential dose effects of PHA 543613 on neuron survival and striatal microglial activation in a rat model of brain excitotoxicity. A preliminary study was performed in vitro to confirm PHA 543613 agonist properties on α7nAChRs. Rats were lesioned in the right striatum with quinolinic acid (QA) and received either vehicle or PHA 543613 at 6 or 12mg/kg twice a day until sacrifice at Day 4 post-lesion. We first compared the translocator protein quantitative autoradiography in QA-lesioned brains with [3H]DPA-714 and [3H]PK-11195. The effects of PHA 543613 on microglial activation and neuronal survival were then evaluated through [3H]DPA-714 binding and immunofluorescence staining (Ox-42, NeuN) on adjacent brain sections. We demonstrated that [3H]DPA-714 provides a better signal-to-noise ratio than [3H]PK-11195. Furthermore, we showed that repeated PHA 543613 administration at a dose of 12mg/kg to QA-lesioned rats significantly protected neurons and reduced the intensity of microglial activation. This study reinforces the hypothesis that α7nAChR agonists can provide beneficial effects in the treatment of neurodegenerative diseases through potential modulation of microglial activation.

Radiosynthesis and Preclinical Evaluation of [18F]F-DPA, A Novel Pyrazolo[1,5a]pyrimidine Acetamide TSPO Radioligand, in Healthy Sprague Dawley Rats.

PURPOSE: Many neurological conditions result in the overexpression of the translocator protein 18 kDa (TSPO), today recognized as a biomarker for microglial activation and neuroinflammation imaging. The pyrazolo[1,5-a]pyrimidine acetamides are a particularly attractive class of TSPO-specific ligands, prompting the development of several positron emission tomography (PET) radiotracers. This includes F-DPA, a recently reported fluorinated ligand (K i = 1.7 nM), wherein the fluorine atom is directly linked to the phenyl moiety without the presence of an alkyl or alkoxy spacer chain. Reported here is the preparation of [18F]F-DPA using [18F]Selectfluor bis(triflate) and the preliminary evaluation of [18F]F-DPA in healthy rats. Its metabolic profile and biodistribution in rats are compared with that of [18F]DPA-714, a closely related structure. PROCEDURES: [18F]F-DPA was synthesized by electrophilic fluorination using [18F]Selectfluor bis(triflate), [18F]DPA-714 was synthesized by conventional nucleophilic fluorination. The biodistribution of both radiotracers was compared in Sprague Dawley rats. Radiometabolites of both radiotracers in plasma and brain homogenates were analyzed by radioTLC. RESULTS: The radiochemical yield of [18F]F-DPA was 15 ± 3 % and the specific activity was 7.8 ± 0.4 GBq/µmol. The radiochemical purity exceeded 99 %. The in vivo time activity curves of [18F]F-DPA demonstrate rapid entry into the brain and a concentration equilibrium at 20-30 min after injection. The metabolic profiles at 90 min after radiotracer injection in the plasma show that unchanged [18F]F-DPA and [18F]DPA-714 account for 28.3 ± 6.4 and 11.1 ± 2.6 % of the remaining radioactivity, respectively. In the brain, unchanged [18F]F-DPA accounts for 93.5 ± 2.8 % of the radioactivity; whereas for [18F]DPA-714, this value is 53.6 ± 1.6 %. CONCLUSIONS: [18F]Selectfluor bis(triflate) was successfully used to label F-DPA with fluorine-18. The labeling position on the aromatic moiety imparts a higher stability compared to [18F]DPA-714 with regard to in vivo metabolism. [18F]F-DPA is a promising new radiotracer and warrants further investigation in animal models of disease.

Synthesis and in vitro characterization of novel fluorinated derivatives of the translocator protein 18 kDa ligand CfO-DPA-714.

The translocator protein 18 kDa (TSPO) is today a validated target for a number of therapeutic applications, but also a well-recognized diagnostic/imaging biomarker for the evaluation of inflammatory related-disease state and progression, prompting the development of specific and dedicated TSPO ligands worldwide. For this purpose, pyrazolo[1,5-a]pyrimidine acetamides constitute a unique class of high affinity and selectivity TSPO ligands; it includes DPA-714, a fluorine-containing derivative that has also been labelled with the positron-emitter fluorine-18, and is nowadays widely used as a Positron Emission Tomography imaging probe. Recently, to prevent defluorination issues encountered in vivo with this tracer, a first series of analogues was reported where the oxygen atom bridging the phenyl ring of the core structure and the fluorinated moiety was replaced with a more robust linkage. Among this new series, CfO-DPA-714 was discovered as a highly promising TSPO ligand. Herein, a novel series of fluorinated analogues of the latter molecule were synthesized and in vitro characterized, where the pharmacomodulation at the amide position of the molecule was explored. Thirteen compounds were thus prepared from a common key-ester intermediate (synthesized in 7 steps from 4-iodobenzoate - 11% overall yield) and a set of commercially available amines and obtained with moderate to good yields (23-81%) and high purities (>95%). With one exception, all derivatives displayed nanomolar to subnanomolar affinity for the TSPO and also high selectivity versus the CBR (Ki (CBR)/Ki (TSPO) > 103). Within this series, three compounds showed better Ki values (0.25, 0.26 and 0.30 nM) than that of DPA-714 (0.91 nM) and CfO-DPA-714 (0.37 nM), and favorable lipophilicity for brain penetration (3.6 < logD7.4 < 4.4). Among these three compounds, the N-methyl-N-propyl amide analogue (9) exhibited similar metabolic stability when compared to CfO-DPA-714 in mouse, rat and human microsomes. Therefore, the latter compound stands out as a promising candidate for drug development or for use as a PET probe, once fluorine-18-labelled, for in vivo neuroinflammation imaging.