Application of a Rapid and Integrated Analysis System (RIAS) as a High-Throughput Processing Tool for In Vitro ADME Samples by Liquid Chromatography/Tandem Mass Spectrometry

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Over the past decade, drug discovery programs have started to address the optimization of key ADME properties already at an early stage of the process. Hence, analytical chemists have been confronted with tremendously rising sample numbers and have had to develop methodologies accelerating quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS). This article focuses on the application of a generic and fully automated LC/MS/MS, named Rapid and Integrated Analysis System (RIAS), as a high-throughput platform for the rapid quantification of drug-like compounds in various in vitro ADME assays. Previous efforts were dedicated to the setup and feasibility study of a workflow-integrated platform combining a modified high-throughput liquid handling LC/MS/MS system controlled by a customized software interface and a customized data-processing and reporting tool. Herein the authors present an extension of this previously developed basic application to a broad set of ADME screening campaigns, covering CYP inhibition, Caco-2, and PAMPA assays. The platform is capable of switching automatically between various ADME assays, performs MS compound optimization if required, and provides a speed of 8 s from sample to sample, independently of the type of ADME assay. Quantification and peak review are adopted to the high-throughput environment and tested against a standard HPLC-ESI technology

An Ultrafast and Flexible LC-MS/MS System Paves the Way for Machine Learning Driven Sample Processing and Data Evaluation in Early Drug Discovery

Rationale: Low speed and flexibility of most LC-MS/MS approaches in early drug discovery delays sample analysis from routine in vivo studies within the same day of measurements. A highthroughput platform for the rapid quantification of drug compounds in various in vivo assays was developed and established in routine bioanalysis. Methods: Automated selection of an efficient and adequate LC method was realized by autonomous sample qualification for ultrafast batch gradients (9 s/sample) or for fast linear gradients (45 s/sample) if samples require chromatography. The hardware and software components of our Rapid and Integrated Analysis System (RIAS) were streamlined for increased analytical throughput via state-of-the-art automation while keeping high analytical quality. Results: Online decision-making was based on a quick assay suitability test (AST) based on a small and dedicated sample set evaluated by two different strategies. 84% of the acquired data points were within ±30% accuracy and 93% of the deviations between the lower limit of quantitation (LLOQ) values were ≤2-fold compared to standard LC-MS/MS systems while speed, flexibility and overall automation was significantly improved. Conclusions: The developed platform provided an analysis time of only 10 min (batch-mode) and 50 min (gradient-mode) per standard pharmacokinetic (PK) study (62 injections). Automation, data evaluation and results handling were optimized to pave the way for machine learning based decision-making regarding the evaluation strategy of the AS

Ultrahigh-Throughput ESI-MS: Sampling Pushed to Six Samples per Second by Acoustic Ejection Mass Spectrometry

We present an acoustic ejection mass spectrometry (AEMS) setup for ESI-MS based sample injection at a sampling rate faster than current ESI and MALDI techniques. A modified acoustic droplet ejection system was combined with an open port interface and a modified ESI source. To simulate applications of drug metabolism and pharmacokinetics analysis and high-throughput screening campaigns, two stress tests were performed regarding ion suppression and system endurance in combination with minor sample preparation. Maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at FWHM of 105 ms and %RSD of 7.7%/7.5% without internal standard correction. Enzyme assay buffer and crude dog plasma caused signal suppression of 51%/73% at %RSD of 5.7%/6.7% (n = 120) and stable OPI performance during 1100 injections. An endurance buffer revealed minor OPI pollution and constant signals for >25.000 injections (%RSD = 8.5%, n = 10,557). </div

CD40-TNF activation in mice induces extended sickness behavior syndrome co-incident with but not dependent on activation of the kynurenine pathway

The similarity between sickness behavior syndrome (SBS) in infection and autoimmune disorders and certain symptoms in major depressive disorder (MDD), and the high co-morbidity of autoimmune disorders and MDD, constitutes some of the major evidence for the immune-inflammation hypothesis of MDD. CD40 ligand-CD40 immune-activation is important in host response to infection and in development of autoimmunity. Mice given a single intra-peritoneal injection of CD40 agonist antibody (CD40AB) develop SBS for 2-3days characterized by weight loss and increased sleep, effects that are dependent on the cytokine, tumor necrosis factor (TNF). Here we report that CD40AB also induces behavioral effects that extend beyond acute SBS and co-occur with but are not mediated by kynurenine pathway activation and recovery. CD40AB led to decreased saccharin drinking (days 1-7) and decreased Pavlovian fear conditioning (days 5-6), and was without effect on physical fatigue (day 5). These behavioral effects co-occurred with increased plasma and brain levels of kynurenine and its metabolites (days 1-7/8). Co-injection of TNF blocker etanercept with CD40AB prevented each of SBS, reduced saccharin drinking, and kynurenine pathway activation in plasma and brain. Repeated oral administration of a selective indoleamine 2,3-dioxygenase (IDO) inhibitor blocked activation of the kynurenine pathway but was without effect on SBS and saccharin drinking. This study provides novel evidence that CD40-TNF activation induces deficits in saccharin drinking and Pavlovian fear learning and activates the kynurenine pathway, and that CD40-TNF activation of the kynurenine pathway is not necessary for induction of the acute or extended SBS effects

Mouse chronic social stress increases blood and brain kynurenine pathway activity and fear behaviour: both effects are reversed by inhibition of indoleamine 2,3-dioxygenase

Psychosocial stress is a major risk factor for mood and anxiety disorders, in which excessive reactivity to aversive events/stimuli is a major psychopathology. In terms of pathophysiology, immune-inflammation is an important candidate, including high blood and brain levels of metabolites belonging to the kynurenine pathway. Animal models are needed to study causality between psychosocial stress, immune-inflammation and hyper-reactivity to aversive stimuli. The present mouse study investigated effects of psychosocial stress as chronic social defeat (CSD) versus control-handling (CON) on: Pavlovian tone-shock fear conditioning, activation of the kynurenine pathway, and efficacy of a specific inhibitor (IDOInh) of the tryptophan-kynurenine catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1), in reversing CSD effects on the kynurenine pathway and fear. CSD led to excessive fear learning and memory, whilst repeated oral escitalopram (antidepressant and anxiolytic) reversed excessive fear memory, indicating predictive validity of the model. CSD led to higher blood levels of TNF-α, IFN-γ, kynurenine (KYN), 3-hydroxykynurenine (3-HK) and kynurenic acid, higher KYN and 3-HK in amygdala and hippocampus. However, CSD was without effect on IDO1 gene or protein expression in spleen, ileum and liver, whilst increasing liver TDO2 gene expression. Nonetheless, oral IDOInh reduced blood and brain levels of KYN and 3-HK in CSD mice to CON levels, and we therefore infer that CSD increases IDO1 activity by increasing its post-translational activation. Furthermore, repeated oral IDOInh reversed excessive fear memory in CSD mice to CON levels. IDOInh reversal of CSD-induced hyper-activity in the kynurenine pathway and fear system contributes significantly to the evidence for a causal pathway between psychosocial stress, immune-inflammation and the excessive fearfulness that is a major psychopathology in stress-related neuropsychiatric disorders

Effects of a single dose of amisulpride on functional brain changes during reward- and motivation-related processing using task-based fMRI in healthy subjects and patients with major depressive disorder — study protocol for a randomized clinical trial

Abstract Background Anhedonia and other deficits in reward- and motivation-related processing in psychiatric patients, including patients with major depressive disorder (MDD), represent a high unmet medical need. Neurobiologically, these deficits in MDD patients are mainly associated with low dopamine function in a frontostriatal network. In this study, alterations in brain activation changes during reward processing and at rest in MDD patients compared with healthy subjects are explored and the effects of a single low dose of the dopamine D2 receptor antagonist amisulpride are investigated. Methods This is a randomized, controlled, double-blind, single-dose, single-center parallel-group clinical trial to assess the effects of a single dose of amisulpride (100 mg) on blood-oxygenation-level-dependent (BOLD) responses during reward- and motivation-related processing in healthy subjects (n = 60) and MDD patients (n = 60). Using functional magnetic resonance imaging (fMRI), BOLD responses are assessed during the monetary incentive delay (MID) task (primary outcome). Exploratory outcomes include BOLD responses and behavioral measures during the MID task, instrumental learning task, effort-based decision-making task, social incentive delay task, and probabilistic reward task as well as changes in resting state functional connectivity and cerebral blood flow. Discussion This study broadly covers all aspects of reward- and motivation-related processing as categorized by the National Institute of Mental Health Research Domain Criteria and is thereby an important step towards precision psychiatry. Results regarding the immediate effects of a dopaminergic drug on deficits in reward- and motivation-related processing not only have the potential to significantly broaden our understanding of underlying neurobiological processes but might eventually also pave the way for new treatment options. Trial registration ClinicalTrials.gov NCT05347199. April 12, 2022

Supplemental_Material – Supplemental material for Automated MALDI Target Preparation Concept

<p>Supplemental material, Supplemental_Material for Automated MALDI Target Preparation Concept by Martin Winter, Robert Ries, Carola Kleiner, Daniel Bischoff, Andreas H. Luippold, Tom Bretschneider and Frank H. Büttner in SLAS Technology</p

Supplemental_Table_S1 – Supplemental material for Automated MALDI Target Preparation Concept

<p>Supplemental material, Supplemental_Table_S1 for Automated MALDI Target Preparation Concept by Martin Winter, Robert Ries, Carola Kleiner, Daniel Bischoff, Andreas H. Luippold, Tom Bretschneider and Frank H. Büttner in SLAS Technology</p