LRRK2 Inhibition by BIIB122 in Healthy Participants and Patients with Parkinson's Disease.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising therapeutic approach for the treatment of Parkinson's disease (PD). OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the potent, selective, CNS-penetrant LRRK2 inhibitor BIIB122 (DNL151) in healthy participants and patients with PD. METHODS: Two randomized, double-blind, placebo-controlled studies were completed. The phase 1 study (DNLI-C-0001) evaluated single and multiple doses of BIIB122 for up to 28 days in healthy participants. The phase 1b study (DNLI-C-0003) evaluated BIIB122 for 28 days in patients with mild to moderate PD. The primary objectives were to investigate the safety, tolerability, and plasma pharmacokinetics of BIIB122. Pharmacodynamic outcomes included peripheral and central target inhibition and lysosomal pathway engagement biomarkers. RESULTS: A total of 186/184 healthy participants (146/145 BIIB122, 40/39 placebo) and 36/36 patients (26/26 BIIB122, 10/10 placebo) were randomized/treated in the phase 1 and phase 1b studies, respectively. In both studies, BIIB122 was generally well tolerated; no serious adverse events were reported, and the majority of treatment-emergent adverse events were mild. BIIB122 cerebrospinal fluid/unbound plasma concentration ratio was ~1 (range, 0.7-1.8). Dose-dependent median reductions from baseline were observed in whole-blood phosphorylated serine 935 LRRK2 (≤98%), peripheral blood mononuclear cell phosphorylated threonine 73 pRab10 (≤93%), cerebrospinal fluid total LRRK2 (≤50%), and urine bis (monoacylglycerol) phosphate (≤74%). CONCLUSIONS: At generally safe and well-tolerated doses, BIIB122 achieved substantial peripheral LRRK2 kinase inhibition and modulation of lysosomal pathways downstream of LRRK2, with evidence of CNS distribution and target inhibition. These studies support continued investigation of LRRK2 inhibition with BIIB122 for the treatment of PD. © 2023 Denali Therapeutics Inc and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Glucocerebrosidase Activity is not Associated with Parkinson's Disease Risk or Severity.

BACKGROUND: Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are risk factors for Parkinson's disease (PD). OBJECTIVE: To explore the association between GCase activity, PD phenotype, and probability for prodromal PD among carriers of mutations in the GBA and LRRK2 genes. METHODS: Participants were genotyped for the G2019S-LRRK2 and nine GBA mutations common in Ashkenazi Jews. Performance-based measures enabling the calculation of the Movement Disorder Society (MDS) prodromal probability score were collected. RESULTS: One hundred and seventy PD patients (102 GBA-PD, 38 LRRK2-PD, and 30 idiopathic PD) and 221 non-manifesting carriers (NMC) (129 GBA-NMC, 45 LRRK2-NMC, 15 GBA-LRRK2-NMC, and 32 healthy controls) participated in this study. GCase activity was lower among GBA-PD (3.15 ± 0.85 µmol/L/h), GBA-NMC (3.23 ± 0.91 µmol/L/h), and GBA-LRRK2-NMC (3.20 ± 0.93 µmol/L/h) compared to the other groups of participants, with no correlation to clinical phenotype. CONCLUSIONS: Low GCase activity does not explain the clinical phenotype or risk for prodromal PD in this cohort. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

SAR towards indoline and 3-azaindoline classes of IDO1 inhibitors.

A novel series of IDO1 inhibitors have been identified with good IDO1 Hela cell and human whole blood activity. These inhibitors contain an indoline or a 3-azaindoline scaffold. Their structure-activity-relationship studies have been explored. Compounds 37 and 41 stood out as leads due to their good potency in IDO1 Hela assay, good IDO1 unbound hWB IC50s, reasonable unbound clearance, and good MRT in rat and dog PK studies.

The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia.

In previous work we evaluated an opioid glycopeptide with mixed µ/δ-opioid receptor agonism that was a congener of leu-enkephalin, MMP-2200. The glycopeptide analogue showed penetration of the blood-brain barrier (BBB) after systemic administration to rats, as well as profound central effects in models of Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesia (LID). In the present study, we tested the glycopeptide BBI-11008 with selective δ-opioid receptor agonism, an analogue of deltorphin, a peptide secreted from the skin of frogs (genus Phyllomedusa). We tested BBI-11008 for BBB-penetration after intraperitoneal (i.p.) injection and evaluated effects in LID rats. BBI-11008 (10 mg/kg) demonstrated good CNS-penetrance as shown by microdialysis and mass spectrometric analysis, with peak concentration levels of 150 pM in the striatum. While BBI-11008 at both 10 and 20 mg/kg produced no effect on levodopa-induced limb, axial and oral (LAO) abnormal involuntary movements (AIMs), it reduced the levodopa-induced locomotor AIMs by 50% after systemic injection. The N-methyl-D-aspartate receptor antagonist MK-801 reduced levodopa-induced LAO AIMs, but worsened PD symptoms in this model. Co-administration of MMP-2200 had been shown prior to block the MK-801-induced pro-Parkinsonian activity. Interestingly, BBI-11008 was not able to block the pro-Parkinsonian effect of MK-801 in the LID model, further indicating that a balance of mu- and delta-opioid agonism is required for this modulation. In summary, this study illustrates another example of meaningful BBB-penetration of a glycopeptide analogue of a peptide to achieve a central behavioral effect, providing additional evidence for the glycosylation technique as a method to harness therapeutic potential of peptides.

Varying the rate of intravenous cocaine infusion influences the temporal dynamics of both drug and dopamine concentrations in the striatum.

The faster drugs of abuse reach the brain, the greater is the risk of addiction. Even small differences in the rate of drug delivery can influence outcome. Infusing cocaine intravenously over 5 vs. 90-100 s promotes sensitization to the psychomotor and incentive motivational effects of the drug and preferentially recruits mesocorticolimbic regions. It remains unclear whether these effects are due to differences in how fast and/or how much drug reaches the brain. Here, we predicted that varying the rate of intravenous cocaine infusion between 5 and 90 s produces different rates of rise of brain drug concentrations, while producing similar peak concentrations. Freely moving male Wistar rats received acute intravenous cocaine infusions (2.0 mg/kg/infusion) over 5, 45 and 90 s. We measured cocaine concentrations in the dorsal striatum using rapid-sampling microdialysis (1 sample/min) and high-performance liquid chromatography-tandem mass spectrometry. We also measured extracellular concentrations of dopamine and other neurochemicals. Regardless of infusion rate, acute cocaine did not change concentrations of non-dopaminergic neurochemicals. Infusion rate did not significantly influence peak concentrations of cocaine or dopamine, but concentrations increased faster following 5-s infusions. We also assessed psychomotor activity as a function of cocaine infusion rate. Infusion rate did not significantly influence total locomotion, but locomotion increased earlier following 5-s infusions. Thus, small differences in the rate of cocaine delivery influence both the rate of rise of drug and dopamine concentrations, and psychomotor activity. A faster rate of rise of drug and dopamine concentrations might be an important issue in making rapidly delivered cocaine more addictive.

Delta Opioid Pharmacology in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that compromises multiple neurochemical substrates including dopamine, norepinephrine, serotonin, acetylcholine, and glutamate systems. Loss of these transmitter systems initiates a cascade of neurological deficits beginning with motor function and ending with dementia. Current therapies primarily address the motor symptoms of the disease via dopamine replacement therapy. Exogenous dopamine replacement brings about additional challenges since after years of treatment it almost invariably gives rise to dyskinesia as a side effect. Therefore there is a clear unmet clinical need for improved PD therapeutics. Opioid receptors and their respective peptides are expressed throughout the basal ganglia and cortex where monoaminergic denervation strongly contributes to PD pathology. Delta opioid receptors are of particular interest because of their dense localization in basal ganglia and because activating this system is known to enhance locomotor activity under a variety of conditions. This chapter will outline much of the work that has demonstrated the effectiveness of delta opioid receptor activation in models of PD and its neuroprotective properties. It also discusses some of the challenges that must be addressed before moving delta opioid receptor agonists into a clinical setting.

Asphyxia-activated corticocardiac signaling accelerates onset of cardiac arrest.

The mechanism by which the healthy heart and brain die rapidly in the absence of oxygen is not well understood. We performed continuous electrocardiography and electroencephalography in rats undergoing experimental asphyxia and analyzed cortical release of core neurotransmitters, changes in brain and heart electrical activity, and brain-heart connectivity. Asphyxia stimulates a robust and sustained increase of functional and effective cortical connectivity, an immediate increase in cortical release of a large set of neurotransmitters, and a delayed activation of corticocardiac functional and effective connectivity that persists until the onset of ventricular fibrillation. Blocking the brain's autonomic outflow significantly delayed terminal ventricular fibrillation and lengthened the duration of detectable cortical activities despite the continued absence of oxygen. These results demonstrate that asphyxia activates a brainstorm, which accelerates premature death of the heart and the brain.

Microfabrication and in Vivo Performance of a Microdialysis Probe with Embedded Membrane.

Microdialysis sampling is an essential tool for in vivo neurochemical monitoring. Conventional dialysis probes are over 220 µm in diameter and have limited flexibility in design because they are made by assembly using preformed membranes. The probe size constrains spatial resolution and governs the amount of tissue damaged caused by probe insertion. To overcome these limitations, we have developed a method to microfabricate probes in Si that are 45 µm thick × 180 µm wide. The probes contain a buried, U-shaped channel that is 30 µm deep × 60 µm wide and terminates in ports for external connection. A 4 mm length of the probe is covered with a 5 µm thick nanoporous membrane. The membrane was microfabricated by deep reactive ion etching through a porous aluminum oxide layer. The microfabricated probe has cross-sectional area that is 79% less than that of the smallest conventional microdialysis probes. The probes yield 2-20% relative recovery at 100 nL/min perfusion rate for a variety of small molecules. The probe was successfully tested in vivo by sampling from the striatum of live rats. Fractions were collected at 20 min intervals (2 µL) before and after an intraperitoneal injection of 5 mg/kg amphetamine. Analysis of fractions by liquid chromatography-mass spectrometry revealed reliable detection of 14 neurochemicals, including dopamine and acetylcholine, at basal conditions. Amphetamine evoked a 43-fold rise in dopamine, a result nearly identical to a conventional dialysis probe in the same animal. The microfabricated probes have potential for sampling with higher spatial resolution and less tissue disruption than conventional probes. It may also be possible to add functionality to the probes by integrating other components, such as electrodes, optics, and additional channels.

Stimulation of δ opioid receptor and blockade of nociceptin/orphanin FQ receptor synergistically attenuate parkinsonism.

δ opioid peptide (DOP) receptors are considered a therapeutic target in Parkinson's disease, although the use of DOP agonists may be limited by side effects, including convulsions. To circumvent this issue, we evaluated whether blockade of nociceptin/orphanin FQ (N/OFQ) tone potentiated the antiparkinsonian effects of DOP agonists, thus allowing for reduction of their dosage. Systemic administration of the N/OFQ receptor (NOP) antagonist J-113397 [(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one] and the DOP receptor agonist SNC-80 [(+)-4-[(αR)-α-(2S,5R)-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N-N-diethylbenzamide] revealed synergistic attenuation of motor deficits in 6-hydroxydopamine hemilesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. In this model, repeated administration of the combination produced reproducible antiparkinsonian effects and was not associated with rescued striatal dopamine terminals. Microdialysis studies revealed that either systemic administration or local intranigral perfusion of J-113397 and SNC-80 led to the enhancement of nigral GABA, reduction of nigral Glu, and reduction of thalamic GABA levels, consistent with the view that NOP receptor blockade and DOP receptor stimulation caused synergistic overinhibition of nigro-thalamic GABA neurons. Whole-cell recording of GABA neurons in nigral slices confirmed that NOP receptor blockade enhanced the DOP receptor-induced effect on IPSCs via presynaptic mechanisms. Finally, SNC-80 more potently stimulated stepping activity in mice lacking the NOP receptor than wild-type controls, confirming the in vivo occurrence of an NOP-DOP receptor interaction. We conclude that endogenous N/OFQ functionally opposes DOP transmission in substantia nigra reticulata and that NOP receptor antagonists might be used in combination with DOP receptor agonists to reduce their dosage while maintaining their full therapeutic efficacy.

Reducing adsorption to improve recovery and in vivo detection of neuropeptides by microdialysis with LC-MS.

Neuropeptides are an important class of neurochemicals; however, measuring their concentration in vivo by using microdialysis sampling is challenging due to their low concentration and the small samples generated. Capillary liquid chromatography with mass spectrometry (cLC-MS) can yield attomole limits of detection (LOD); however, low recovery and loss of sample to adsorptive surfaces can still hinder detection of neuropeptides. We have evaluated recovery during sampling and transfer to the cLC column for a selection of 10 neuropeptides. Adding acetonitrile to sample eliminated carryover and improved LOD by 1.4- to 60-fold. The amount of acetonitrile required was found to have an optimal value that correlated with peptide molecular weight and retention time on a reversed phase LC column. Treating AN69 dialysis membrane, which bears negative charge due to incorporated sulfonate groups, with polyethylenimine (PEI) improved recovery by 1.2- to 80-fold. The effect appeared to be due to reducing electrostatic interaction between peptides and the microdialysis probe because modification increased recovery only for peptides that carried net positive charge. The combined effects improved LOD of the entire method by 1.3- to 800-fold for the different peptides. We conclude that peptides with both charged and hydrophobic regions require combined strategies to prevent adsorption and yield the best possible detection. The method was demonstrated by determining orexin A, orexin B, and a novel isoform of rat ß-endorphin in the arcuate nucleus. Dialysate concentrations were below 10 pM for these peptides. A standard addition study on dialysates revealed that while some peptides can be accurately quantified, some are affected by the matrix.

Amphetamine stimulates movement through thalamocortical glutamate release.

The ventrolateral thalamus (VL) is a primary relay point between the basal ganglia and the primary motor cortex (M1). Using dual probe microdialysis and locomotor behavior monitoring, we investigated the contribution of VL input into M1 during amphetamine (AMPH)-stimulated monoamine release and hyperlocomotion in rats. Tetrodotoxin (10 µM) perfusion into the VL significantly lowered hyperactivity induced by AMPH (1 mg/kg i.p.). This behavioral response corresponded to reduced cortical glutamate and monoamine release. To determine which glutamate receptors the thalamocortical projections acted upon, we perfused either the α-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) (10 µM) or the N-methyl-D-aspartic acid (NMDA) receptor antagonist (MK-801) intracortically followed by systemic AMPH. The results show that AMPA/kainate, and to a lesser extent NMDA receptors, mediated the observed effects. As glutamate-monoamine interactions could possibly occur through local or circuit-based mechanisms, we isolated and perfused M1 tissue ex vivo to determine the extent of local glutamate-dopamine interactions. Taken together, these results demonstrate that AMPH generates hyperlocomotive states via thalamocortical signaling and that cortical AMPA receptors are an important mediator of these effects. This study utilizes dual probe microdialysis sampling and comprehensive LC-MS analysis to determine the effects of amphetamine (1 mg/kg i.p.) on thalamocortical neurotransmission. Using pharmacological tools such as local thalamic tetrodotoxin (TTX) perfusion and glutamate antagonist at the cortical level, we demonstrate that thalamocortical glutamate (acting primarily through cortical AMPA receptors) is an essential component in amphetamine-induced hyperlocomotion.

Rapid dopamine transmission within the nucleus accumbens: dramatic difference between morphine and oxycodone delivery.

While most drugs of abuse increase dopamine neurotransmission, rapid neurochemical measurements show that different drugs evoke distinct dopamine release patterns within the nucleus accumbens. Rapid changes in dopamine concentration following psychostimulant administration have been well studied; however, such changes have never been examined following opioid delivery. Here, we provide novel measures of rapid dopamine release following intravenous infusion of two opioids, morphine and oxycodone, in drug-naïve rats using fast-scan cyclic voltammetry and rapid (1 min) microdialysis coupled with high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS). In addition to measuring rapid dopamine transmission, microdialysis HPLC-MS measures changes in GABA, glutamate, monoamines, monoamine metabolites and several other neurotransmitters. Although both opioids increased dopamine release in the nucleus accumbens, their patterns of drug-evoked dopamine transmission differed dramatically. Oxycodone evoked a robust and stable increase in dopamine concentration and a robust increase in the frequency and amplitude of phasic dopamine release events. Conversely, morphine evoked a brief (~ 1 min) increase in dopamine that was coincident with a surge in GABA concentration and then both transmitters returned to baseline levels. Thus, by providing rapid measures of neurotransmission, this study reveals previously unknown differences in opioid-induced neurotransmitter signaling. Investigating these differences may be essential for understanding how these two drugs of abuse could differentially usurp motivational circuitry and powerfully influence behavior.

Ultrasound imaging of core muscles activity in multiparous women with vaginal laxity: a cross-sectional study.

Vaginal laxity (VL) is a common condition among multiparous women, especially those who have delivered vaginally. Since pelvic floor muscles (PFMs) work synergistically with other core muscles, physical therapy protocols that aim to treat VL should train the PFMs in combination with other core muscles. To investigate the activity of core muscles in multiparous women with and without VL, and its relation to sexual function. An observational, cross-sectional study. The study included 100 multiparous women, who were divided into two groups according to their scores on the vaginal laxity questionnaire (VLQ). Women who scored between 1 and 3 on the VLQ were categorized as having VL (n = 48), while those who scored between 5 and 7 were placed in the control group (n = 52). The primary outcomes were PFM displacement, diaphragmatic excursion, transversus abdominis activation ratio, and lumbar multifidus thickness measured by ultrasound imaging. The secondary outcome was sexual functioning, evaluated using the Arabic female sexual function index (ArFSFI). The VL group had significantly lower PFM displacement (mean difference (MD) - 0.42; 95% confidence interval (CI) - 0.49 to - 0.33; p = 0.001), diaphragmatic excursion (MD - 2.75; 95% CI - 2.95 to - 2.55; p = 0.001), lumbar multifidus thickness (MD - 10.08; 95% CI - 14.32 to - 5.82; p = 0.02), and ArFSFI scores (MD - 9.2; 95% CI - 10.59 to - 7.81; p = 0.001) in comparison to the control group (p < 0.05). Nevertheless, the transversus abdominis activation ratio demonstrated no significant difference between the two groups (MD 0.06; 95% CI - 0.05 to 0.17; p = 0.33). Multiparous women with VL had significantly lower PFM displacement, diaphragmatic excursion, lumbar multifidus thickness, and sexual function index scores than women in the control group. The only exception was transversus abdominis activation, which did not differ significantly between the VL and control groups.

Employing machine learning for enhanced abdominal fat prediction in cavitation post-treatment.

This study investigates the application of cavitation in non-invasive abdominal fat reduction and body contouring, a topic of considerable interest in the medical and aesthetic fields. We explore the potential of cavitation to alter abdominal fat composition and delve into the optimization of fat prediction models using advanced hyperparameter optimization techniques, Hyperopt and Optuna. Our objective is to enhance the predictive accuracy of abdominal fat dynamics post-cavitation treatment. Employing a robust dataset with abdominal fat measurements and cavitation treatment parameters, we evaluate the efficacy of our approach through regression analysis. The performance of Hyperopt and Optuna regression models is assessed using metrics such as mean squared error, mean absolute error, and R-squared score. Our results reveal that both models exhibit strong predictive capabilities, with R-squared scores reaching 94.12% and 94.11% for post-treatment visceral fat, and 71.15% and 70.48% for post-treatment subcutaneous fat predictions, respectively. Additionally, we investigate feature selection techniques to pinpoint critical predictors within the fat prediction models. Techniques including F-value selection, mutual information, recursive feature elimination with logistic regression and random forests, variance thresholding, and feature importance evaluation are utilized. The analysis identifies key features such as BMI, waist circumference, and pretreatment fat levels as significant predictors of post-treatment fat outcomes. Our findings underscore the effectiveness of hyperparameter optimization in refining fat prediction models and offer valuable insights for the advancement of non-invasive fat reduction methods. This research holds important implications for both the scientific community and clinical practitioners, paving the way for improved treatment strategies in the realm of body contouring.

Distinctive immunoregulatory effects of adenosine on T cells of older humans.

A role for adenosine in immunosenescence was investigated in T cells from older (≥65 yr) and younger (24-45 yr) healthy humans. Adenosine concentrations in cultures of activated T cells were significantly higher (P<0.0001) for older (145±47 nM, mean±sd) than younger (58±5.5 nM) subjects. Expression of the activation coreceptor CD28 was suppressed significantly by 0.1 to 1 µM exogenous adenosine, with greater effects of 1 µM (P<0.01) on T cells of younger (mean suppression of 67 and 65% for CD4 and CD8 T cells, respectively) than older (means of 42 and 46%) subjects. T-cell chemotaxis to CCL21 was suppressed significantly by 0.3 and 1 µM exogenous adenosine, with mean maximum decreases of 39 and 49%, respectively, for younger subjects and 28 and 31% for older subjects. Generation of IL-2 and IFN-γ by T cells of younger and older subjects was suppressed substantially only at adenosine levels of 3 µM or higher. Lower baseline expression of CD28 and chemotaxis to CCL21 and S1P for T cells from older subjects attributable to endogenous adenosine were reversed completely by two different A(2A) adenosine receptor antagonists without affecting T cells of younger subjects. Adenosine is an endogenous T-cell immunosuppressor in older humans, and A(2A) antagonists reverse adenosine-induced T-cell deficiencies of aging.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

In vivo neurochemical monitoring using benzoyl chloride derivatization and liquid chromatography-mass spectrometry.

In vivo neurochemical monitoring using microdialysis sampling is important in neuroscience because it allows correlation of neurotransmission with behavior, disease state, and drug concentrations in the intact brain. A significant limitation of current practice is that different assays are utilized for measuring each class of neurotransmitter. We present a high performance liquid chromatography (HPLC)-tandem mass spectrometry method that utilizes benzoyl chloride for determination of the most common low molecular weight neurotransmitters and metabolites. In this method, 17 analytes were separated in 8 min. The limit of detection was 0.03-0.2 nM for monoamine neurotransmitters, 0.05-11 nM for monoamine metabolites, 2-250 nM for amino acids, 0.5 nM for acetylcholine, 2 nM for histamine, and 25 nM for adenosine at sample volume of 5 µL. Relative standard deviation for repeated analysis at concentrations expected in vivo averaged 7% (n = 3). Commercially available (13)C benzoyl chloride was used to generate isotope-labeled internal standards for improved quantification. To demonstrate utility of the method for study of small brain regions, the GABA(A) receptor antagonist bicuculline (50 µM) was infused into a rat ventral tegmental area while recording neurotransmitter concentration locally and in nucleus accumbens, revealing complex GABAergic control over mesolimbic processes. To demonstrate high temporal resolution monitoring, samples were collected every 60 s while neostigmine, an acetylcholine esterase inhibitor, was infused into the medial prefrontal cortex. This experiment revealed selective positive control of acetylcholine over cortical glutamate.

Mass spectrometry "sensor" for in vivo acetylcholine monitoring.

Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. The use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS "sensor" was applied to monitor acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetylcholinesterase, that evoked rapid increases in acetycholine and tetrodotoxin, a blocker of Na(+) channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs.

2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.