Involvement of CB1 and CB2 receptors in neuroprotective effects of cannabinoids in experimental TDP-43 related frontotemporal dementia using male mice.

BACKGROUND: The elevation of endocannabinoid levels through inhibiting their degradation afforded neuroprotection in CaMKIIα-TDP-43 mice, a conditional transgenic model of frontotemporal dementia. However, which cannabinoid receptors are mediating these benefits is still pending to be elucidated. METHODS: We have investigated the involvement of the CB1 and the CB2 receptor using chronic treatments with selective ligands in CaMKIIα-TDP-43 mice, analysis of their cognitive deterioration with the Novel Object Recognition test, and immunostaining for neuronal and glial markers in two areas of interest in frontotemporal dementia. RESULTS: Our results confirmed the therapeutic value of activating either the CB1 or the CB2 receptor, with improvements in the animal performance in the Novel Object Recognition test, preservation of pyramidal neurons, in particular in the medial prefrontal cortex, and attenuation of glial reactivity, in particular in the hippocampus. In addition, the activation of both CB1 and CB2 receptors reduced the elevated levels of TDP-43 in the medial prefrontal cortex of CaMKIIα-TDP-43 mice, an effect exerted by mechanisms that are currently under investigation. CONCLUSIONS: These data reinforce the notion that the activation of CB1 and CB2 receptors may represent a promising therapy against TDP-43-induced neuropathology in frontotemporal dementia. Future studies will have to confirm these benefits, in particular with one of the selective CB2 agonists used here, which has been thoroughly characterized for clinical development.

Functional characterization of Francisella tularensis subspecies holarctica genotypes during tick cell and macrophage infections using a proteogenomic approach.

Tularemia is a vector-borne disease caused by the Gram-negative bacterium Francisella tularensis. Known hosts and vectors in Europe are hare and ticks. F. tularensis is transmitted from ticks and animals, but also from the hydrotelluric environment and the consumption of contaminated water or food. A changing climate expands the range in which ticks can live and consequently might contribute to increasing case numbers of tularemia. Two subspecies of F. tularensis are human pathogenic. Francisella tularensis tularensis (Ftt) is endemic in North America, while Francisella tularensis holarctica (Fth) is the only subspecies causing tularemia in Europe. Ft is classified as a category A bioterrorism agent due to its low infectious dose, multiple modes of transmission, high infectivity and potential for airborne transmission and has become a global public health concern. In line with the European survey and previous phylogenetic studies, Switzerland shows the co-distribution of B.6 and B.12 strains with different geographical distribution and prevalence within the country. To establish itself in different host environments of ticks and mammals, F. tularensis presumably undergoes substantial changes on the transcriptomics and proteomic level. Here we investigate the transcriptomic and proteomic differences of five strains of Fth upon infection of rabbit macrophages and tick cells.

Identification of (R)-[18F]YH134 for Monoacylglycerol Lipase Neuroimaging and Exploration of Its Use for Central Nervous System and Peripheral Drug Development.

This study aimed to evaluate (R)-[18F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on (R)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, (R)-[18F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of (R)-[18F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume (V T) of (R)-[18F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using (R)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of (R)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Phylogeography of Francisella tularensis subspecies holarctica and epidemiology of tularemia in Switzerland.

Tularemia, an endemic disease that mainly affects wild animals and humans, is caused by Francisella tularensis subsp. holarctica (Fth) in Switzerland. The Swiss Fth population consist of multiple different subclades which are distributed throughout the country. The aim of this study is to characterize the genetic diversity of Fth in Switzerland and to describe the phylogeographic relationship of isolates by single nucleotide polymorphism (SNP) analysis. This analysis is combined with human surveillance data from reported cases over the last 10 years and in vitro and in silico antibiotic resistance tests to provide insight into the epidemiology of tularemia in Switzerland. We sequenced the whole genomes of 52 Fth strains of human or tick origin collected in Switzerland between 2009 and 2022 and analyzed together with all publicly available sequencing data of Swiss and European Fth. Next, we performed a preliminary classification with the established canonical single nucleotide polymorphism nomenclature. Furthermore, we tested 20 isolates from all main Swiss clades for antimicrobial susceptibility against a panel of antimicrobial agents. All 52 sequenced isolates from Switzerland belong to major clade B.6, specifically subclades B.45 and B.46, previously described in Western Europe. We were able to accurately reconstruct the population structure according to the global phylogenetic framework. No resistance to clinically recommended antibiotics could be identified in vitro or in silico in the western B.6 strains.

Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth.

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods.

Influence of Chlorinating Agents on the Formation of Stable Biomarkers in Hair for the Retrospective Verification of Exposure.

Chlorine, as a dual-use chemical, is an essential industrial chemical which has been used as a chemical weapon in the past due to its toxicity and availability. The retrospective verification of chlorine intoxication is often especially challenging, and unambiguous markers are still missing. In this study, the effects of different chlorinating and oxidizing agents on human hair were investigated. Samples were exposed to a variety of chlorinating chemicals for a short time and then completely hydrolyzed by a HBr solution to break down their keratin proteins into individual amino acids. After derivatization and targeted liquid chromatography-mass spectrometry analysis, 3-chlorotyrosine and 3,5-dichlorotyrosine were unambiguously identified from human hair exposed to chlorine, hypochlorite, and sulfuryl chloride. Our results show long-term stability of these markers in the biological matrix, as the chlorotyrosines can still be found 10 months post-exposure at the same levels. Finally, an untargeted analysis was able to discriminate between some of the different intoxicants.

A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature.

Chronic inflammation and blood-brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood-brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood-brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood-brain barrier dysfunction caused by inflammatory insults.

Multi-parameter optimization: Development of a morpholin-3-one derivative with an improved kinetic profile for imaging monoacylglycerol lipase in the brain.

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [11C]7 was synthesized via direct 11CO2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [11C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [11C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [11C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [11C]7 ([11C]RO7284390) showed promising results warranting further clinical evaluation.

Considerations and recommendations for assessment of plasma protein binding and drug-drug interactions for siRNA therapeutics.

At the time of writing, although siRNA therapeutics are approved for human use, no official regulatory guidance specific to this modality is available. In the absence of guidance, preclinical development for siRNA followed a hybrid of the small molecule and biologics guidance documents. However, siRNA differs significantly from small molecules and protein-based biologics in its physicochemical, absorption, distribution, metabolism and excretion properties, and its mechanism of action. Consequently, certain reports typically included in filing packages for small molecule or biologics may benefit from adaption, or even omission, from an siRNA filing. In this white paper, members of the 'siRNA working group' in the IQ Consortium compile a list of reports included in approved siRNA filing packages and discuss the relevance of two in vitro reports-the plasma protein binding evaluation and the drug-drug interaction risk assessment-to support siRNA regulatory filings. Publicly available siRNA approval packages and the literature were systematically reviewed to examine the role of siRNA plasma protein binding and drug-drug interactions in understanding pharmacokinetic/pharmacodynamic relationships, safety and translation. The findings are summarized into two decision trees to help guide industry decide when in vitro siRNA plasma protein binding and drug-drug interaction studies are warranted.

Transporters in Drug Development: International Transporter Consortium Update on Emerging Transporters of Clinical Importance.

During its fourth transporter workshop in 2021, the International Transporter Consortium (ITC) provided updates on emerging clinically relevant transporters for drug development. Previously highlighted and new transporters were considered based on up-to-date clinical evidence of their importance in drug-drug interactions and potential for altered drug efficacy and safety, including drug-nutrient interactions leading to nutrient deficiencies. For the first time, folate transport pathways (PCFT, RFC, and FRα) were examined in-depth as a potential mechanism of drug-induced folate deficiency and related toxicities (e.g., neural tube defects and megaloblastic anemia). However, routine toxicology studies conducted in support of drug development appear sufficient to flag such folate deficiency toxicities, whereas prospective prediction from in vitro folate metabolism and transport inhibition is not well enough established to inform drug development. Previous suggestion of a retrospective study of intestinal OATP2B1 inhibition to explain unexpected decreases in drug exposure were updated. Furthermore, when the absorption of a new molecular entity is more rapid and extensive than can be explained by passive permeability, evaluation of the OATP2B1 transport may be considered. Emerging research on hepatic and renal OAT2 is summarized, but current understanding of the importance of OAT2 was deemed insufficient to justify specific consideration for drug development. Hepatic, renal, and intestinal MRPs (MRP2, MRP3, and MRP4) were revisited. MRPs may be considered when they are suspected to be the major determinant of drug disposition (e.g., direct glucuronide conjugates); MRP2 inhibition as a mechanistic explanation for drug-induced hyperbilirubinemia remains justified. There were no major changes in recommendations from previous ITC whitepapers.

Discovery, synthesis and evaluation of novel reversible monoacylglycerol lipase radioligands bearing a morpholine-3-one scaffold.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the endocannabinoid degradation in the brain. It has recently emerged as a promising therapeutic target in the treatment of neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Development of MAGL-specific radioligands for non-invasive imaging by positron-emission tomography (PET) would deepen our knowledge on the relevant pathological changes in diseased states and accelerate drug discovery. In this study, we report the selection and synthesis of two morpholine-3-one derivatives as potential reversible MAGL PET tracer candidates based on their multiparameter optimization scores. Both compounds ([11C]1, [11C]2) were radiolabeled by direct [11C]CO2 fixation and the in vitro autoradiographic studies demonstrated their specificity and selectivity towards MAGL. Dynamic PET imaging using MAGL knockout and wild-type mice confirmed the in vivo specificity of [11C]2. Our preliminary results indicate that morpholine-3-one derivative [11C]2 ([11C]RO7279991) binds to MAGL in vivo, and this molecular scaffold could serve as an alternative lead structure to image MAGL in the central nervous system.

Development of High Brain-Penetrant and Reversible Monoacylglycerol Lipase PET Tracers for Neuroimaging.

Monoacylglycerol lipase (MAGL) is one of the key enzymes in the endocannabinoid system. Inhibition of MAGL has been proposed as an attractive approach for the treatment of various diseases. In this study, we designed and successfully synthesized two series of piperazinyl pyrrolidin-2-one derivatives as novel reversible MAGL inhibitors. (R)-[18F]13 was identified through the preliminary evaluation of two carbon-11-labeled racemic structures [11C]11 and [11C]16. In dynamic positron-emission tomography (PET) scans, (R)-[18F]13 showed a heterogeneous distribution and matched the MAGL expression pattern in the mouse brain. High brain uptake and brain-to-blood ratio were achieved by (R)-[18F]13 in comparison with previously reported reversible MAGL PET radiotracers. Target occupancy studies with a therapeutic MAGL inhibitor revealed a dose-dependent reduction of (R)-[18F]13 accumulation in the mouse brain. These findings indicate that (R)-[18F]13 ([18F]YH149) is a highly promising PET probe for visualizing MAGL non-invasively in vivo and holds great potential to support drug development.

Whole-Genome Assemblies of 16 Burkholderia pseudomallei Isolates from Rivers in Laos.

We report 16 Burkholderia pseudomallei genomes, including 5 new multilocus sequence types, isolated from rivers in Laos. The environmental bacterium B. pseudomallei causes melioidosis, a serious infectious disease in tropical and subtropical regions. The isolates are geographically clustered in one clade from around Vientiane, Laos, and one clade from further south.

Associations of the intestinal microbiome with the complement system in neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the aged population. The etiology of AMD is multifactorial including nutritional factors, genetic variants mainly in the complement pathway, environmental risk factors and alterations in the intestinal microbiome. However, it remains unexplored whether there is an interdependency of these factors leading to the development of AMD. To investigate this issue, a shotgun metagenomics analysis of 57 neovascular AMD and 58 healthy controls as well as of 16 complement C3-deficient mice and 16 wildtypes was performed. Whereas the class Negativicutes was more abundant in patients, the genus Oscillibacter and species Bacteroides had a significantly higher prevalence in persons without AMD. Similar taxonomic features were identified that distinguished wildtype mice from C3-deficient mice. Moreover, several purine signaling pathways were associated with both, neovascular AMD and C3 deficiency. While SNPs within the complement factor B gene were more abundant in controls, SNPs within the high temperature requirement A serine peptidase 1 and complement factor H (CFH) genes were associated with neovascular AMD. Using a classification model, Negativicutes was identified as a potential biomarker for AMD and furthermore, it positively correlated with CFH. This study suggests an association between the intestinal microbiome and the complement system in neovascular AMD.

Application of the Extended Clearance Classification System (ECCS) in Drug Discovery and Development: Selection of Appropriate In Vitro Tools and Clearance Prediction.

In vitro to in vivo extrapolation (IVIVE) to predict human hepatic clearance, including metabolism and transport, requires extensive experimental resources. In addition, there may be technical challenges to measure low clearance values. Therefore, prospective identification of rate-determining step(s) in hepatic clearance through application of the Extended Clearance Classification System (ECCS) could be beneficial for optimal compound characterization. IVIVE for hepatic intrinsic clearance (CLint,h) prediction is conducted for a set of 36 marketed drugs with low-to-high in vivo clearance, which are substrates of metabolic enzymes and active uptake transporters in the liver. The compounds were assigned to the ECCS classes, and CLint,h, estimated with HepatoPac (a micropatterned hepatocyte coculture system), was compared with values calculated based on suspended hepatocyte incubates. An apparent permeability threshold (apical to basal) of 50 nm/s in LLC-PK1 cells proved optimal for ECCS classification. A reasonable performance of the IVIVE for compounds across multiple classes using HepatoPac was achieved (with 2-3-fold error), except for substrates of uptake transporters (class 3b), for which scaling of uptake clearance using plated hepatocytes is more appropriate. Irrespective of the ECCS assignment, metabolic clearance can be estimated well using HepatoPac. The validation and approach elaborated in the present study can result in proposed decision trees for the selection of the optimal in vitro assays guided by ECCS class assignment, to support compound optimization and candidate selection. SIGNIFICANCE STATEMENT: Characterization of the rate-determining step(s) in hepatic elimination could be on the critical path of compound optimization during drug discovery. This study demonstrated that HepatoPac and plated hepatocytes are suitable tools for the estimation of metabolic and active uptake clearance, respectively, for a larger set of marketed drugs, supporting a comprehensive strategy to select optimal in vitro tools and to achieve Extended Clearance Classification System-dependent in vitro to in vivo extrapolation for human clearance prediction.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Structure-Permeability Relationship of Semipeptidic Macrocycles-Understanding and Optimizing Passive Permeability and Efflux Ratio.

We herein report the first thorough analysis of the structure-permeability relationship of semipeptidic macrocycles. In total, 47 macrocycles were synthesized using a hybrid solid-phase/solution strategy, and then their passive and cellular permeability was assessed using the parallel artificial membrane permeability assay (PAMPA) and Caco-2 assay, respectively. The results indicate that semipeptidic macrocycles generally possess high passive permeability based on the PAMPA, yet their cellular permeability is governed by efflux, as reported in the Caco-2 assay. Structural variations led to tractable structure-permeability and structure-efflux relationships, wherein the linker length, stereoinversion, N-methylation, and peptoids site-specifically impact the permeability and efflux. Extensive nuclear magnetic resonance, molecular dynamics, and ensemble-based three-dimensional polar surface area (3D-PSA) studies showed that ensemble-based 3D-PSA is a good predictor of passive permeability.