From molecules to visuals: Empowering drug discovery and development with mass spectrometry imaging.

Over the past three decades, mass spectrometry imaging (MSI) has emerged as a valuable tool for the spatial localization of drugs and metabolites directly from tissue surfaces without the need for labels. MSI offers molecular specificity, making it increasingly popular in the pharmaceutical industry compared to conventional imaging techniques like quantitative whole-body autoradiography (QWBA) and immunohistochemistry, which are unable to distinguish parent drugs from metabolites. Across the industry, there has been a consistent uptake in the utilization of MSI to investigate drug and metabolite distribution patterns, and the integration of MSI with omics technologies in preclinical investigations. To continue the further adoption of MSI in drug discovery and development, we believe there are two key areas that need to be addressed. First, there is a need for accurate quantification of analytes from MSI distribution studies. Second, there is a need for increased interactions with regulatory agencies for guidance on the utility and incorporation of MSI techniques in regulatory filings. Ongoing efforts are being made to address these areas, and it is hoped that MSI will gain broader utilization within the industry, thereby becoming a critical ingredient in driving drug discovery and development.

Applying Automation and High-Throughput MALDI Mass Spectrometry for Peptide Metabolic Stability Screening.

The discovery of peptide therapeutics represents a fast-growing segment of pharmaceutical research. During the early discovery process, a large number of peptide candidates needs to be rapidly screened for metabolic stability in relevant biological matrices. In most cases, peptide stability assays are quantified using LC-MS/MS, which may take hours to analyze 384 samples and generates liters of solvent waste. Herein, we introduce a high-throughput screening (HTS) platform for peptide stability assessment founded on Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS). Full automation has been implemented for sample preparation with minimal manual intervention. The limit of detection, linearity, and reproducibility of the platform were evaluated, and metabolic stabilities have been determined for a number of peptide candidates. The MALDI-MS-based HTS workflow is able to analyze 384 samples in less than 1 h while only using 115 µL of total solvent. Although this process allows for very rapid assessment of peptide stability, given the nature of the MALDI process, it is noteworthy that spot-to-spot variations and ionization bias are observed. Therefore, LC-MS/MS may still be needed for confident, quantitative measurements and/or when the ionization efficiency of certain peptides is inadequate using MALDI.

Mass spectrometry imaging of diclofenac and its metabolites in tissues using nanospray desorption electrospray ionization.

Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, previous attempts using traditional mass spectrometry imaging (MSI) techniques have completely or partially failed and therefore little is known about their localization in tissues. Herein, we use nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI), an ambient liquid extraction-based ionization technique, as a viable alternative to other MSI techniques to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. MSI data acquired over a broad m/z range showed low signals of the drug and its metabolites resulting from the low ionization efficiency and substantial signal suppression on the tissue. Significant improvements in the signal-to-noise were obtained using selected ion monitoring (SIM) with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide and hydroxydiclofenac are localized to the inner medulla and cortex of the kidney, respectively, which is consistent with the previously reported localization of enzymes that process diclofenac into its respective metabolites. In contrast, a uniform distribution of diclofenac and its metabolites was observed in the liver tissue. Concentration ratios of diclofenac and hydroxydiclofenac calculated from nano-DESI MSI data are generally in agreement to those obtained using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collectively, our results demonstrate that nano-DESI MSI can be successfully used to image diclofenac and its primary metabolites and derive relative quantitative data from different tissue regions. Our approach will enable a better understanding of metabolic processes associated with diclofenac and other drugs that are difficult to analyze using commercially available MSI platforms.

Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation.

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

Intermediate human activities maximize dryland ecosystem services in the long-term land-use change: Evidence from the Sangong River watershed, northwest China.

Human activities cause widespread changes in landscape composition, which can affect ecosystem services produced by these landscapes. It is usually believed that ecosystem services can be maximized only when we eliminate all human activities. However, this belief is not the case, at least in dryland ecosystems. Here, a gradient of human activity intensity was used to investigate changes in the value of ecosystem services over 30-years of land-use change between 1990 and 2020 in the arid Sangong River watershed of northwest China. Spatial analyses were performed to determine how the value of dryland ecosystem services changed with human activity intensity. Stepwise regressions and linear programming models were also performed to examine how to optimize the value of ecosystem services (i.e., regulating services, provisioning services, supporting services, and cultural services). We found that landscapes of the Sangong River watershed became increasingly fragmented and that human activities gradually intensified, but the value of ecosystem services fluctuated rather than linearly decreasing over the past 30 years. Specifically, a unimodal relationship was observed between human activities and ecosystem services. The peak value of ecosystem services was 5799 USD ha-1 yr-1 under intermediate human activity intensity (i.e., human footprint index ranged from 0.2 to 0.4 at a scale of one). Gross domestic product (GDP) per capita, population, and water consumption were the three most important driving factors of human activities and ecosystem services. Our results suggest that intermediate human activities may maximize dryland ecosystem services in long-term land-use change at the watershed scale, and highlight the importance of regulating economic development, population, and water consumption for the management of dryland ecosystem services.

Development and Application of DropletProbe Mass Spectrometry for Examining Biodistribution of Therapeutics.

dropletProbe mass spectrometry is a novel technique for molecular characterization of surfaces. It can be used for rapid ex vivo analysis of therapeutics from thin animal tissue sections and has been shown to improve understanding of a drug's absorption, distribution, metabolism and excretion (ADME) properties. Here, we describe the tissue distribution analysis of diclofenac from a dosed whole-body mouse thin tissue section using a dropletProbe mass spectrometry system.

Simultaneous multielement imaging of liver tissue using laser ablation inductively coupled plasma mass spectrometry.

Analysis of the spatial distribution of metals, metalloids, and non-metals in biological tissues is of significant interest in the life sciences, helping to illuminate the function and roles these elements play within various biological pathways. Chemical imaging methods are commonly employed to address biological questions and reveal individual spatial distributions of analytes of interest. Elucidation of these spatial distributions can help determine key elemental and molecular information within the respective biological specimens. However, traditionally utilized imaging methods prove challenging for certain biological tissue analysis, especially with respect to applications that require high spatial resolution or depth profiling. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been shown to be effective for direct elemental analysis of solid materials with high levels of precision. In this work, chemical imaging using LA-ICP-MS has been applied as a powerful analytical methodology for the analysis of liver tissue samples. The proposed analytical methodology successfully produced both qualitative and quantitative information regarding specific elemental distributions within images of thin tissue sections with high levels of sensitivity and spatial resolution. The spatial resolution of the analytical methodology was innovatively enhanced, helping to broaden applicability of this technique to applications requiring significantly high spatial resolutions. This information can be used to further understand the role these elements play within biological systems and impacts dysregulation may have.

Integrated laser ablation-dropletProbe-mass spectrometry for absolute drug quantitation, metabolite detection, and distribution in tissue.

RATIONALE: Spatially resolved and accurate quantitation of drug-related compounds in tissue is a much-needed capability in drug discovery research. Here, application of an integrated laser ablation-dropletProbe-mass spectrometry surface sampling system (LADP-MS) is reported, which achieved absolute quantitation of propranolol measured from <500 × 500 µm thin tissue samples. METHODS: Mouse liver and kidney thin tissue sections were coated with parylene C and analyzed for propranolol by a laser ablation/liquid extraction workflow. Non-coated adjacent sections were microdissected for validation and processed using standard bulk tissue extraction protocols. High-performance liquid chromatography with positive ion mode electrospray ionization tandem mass spectrometry was applied to detect the drug and its metabolites. RESULTS: Absolute propranolol concentration in ~500 × 500 µm tissue regions measured by the two methods agreed within ±8% and had a relative standard deviation within ±17%. Quantitation down to ~400 × 400 µm tissue regions was shown, and this resolution was also used for automated mapping of propranolol and phase II hydroxypropranolol glucuronide metabolites in kidney tissue. CONCLUSIONS: This study exemplifies the capabilities of integrated laser ablation-dropletProbe-mass spectrometry (LADP-MS) for high resolution absolute drug quantitation analysis of thin tissue sections. This capability will be valuable for applications needing to quantitatively understand the spatial distribution of small molecules in tissue.

Absolute quantitation of propranolol from 200-µm regions of mouse brain and liver thin tissues using laser ablation-dropletProbe-mass spectrometry.

RATIONALE: The ability to quantify drugs and metabolites in tissue with sub-mm resolution is a challenging but much needed capability in pharmaceutical research. To fill this void, a novel surface sampling approach combining laser ablation with the commercial dropletProbe automated liquid surface sampling system (LA-dropletProbe) was developed and is presented here. METHODS: Parylene C-coated 200 × 200 µm tissue regions of mouse brain and kidney thin tissue sections were analyzed for propranolol by laser ablation of tissue directly into a preformed liquid junction. Propranolol was detected by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) in positive electrospray ionization mode. Quantitation was achieved via application of a stable-isotope-labeled internal standard and an external calibration curve. RESULTS: The absolute concentrations of propranolol determined from 200 × 200 µm tissue regions were compared with the propranolol concentrations obtained from 2.3-mm-diameter tissue punches of adjacent, non-coated sections using standard bulk tissue extraction protocols followed by regular HPLC/MS/MS analysis. The average concentration of propranolol in both organs determined by the two employed methods agreed to within ±12%. Furthermore, the relative abundances of phase II hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous results. CONCLUSIONS: This work illustrates that depositing a thin layer of parylene C onto thin tissue prior to analysis, which seals the surface and prevents direct liquid extraction of the drug from the tissue, coupled to the novel LA-dropletProbe surface sampling system is a viable approach for sub-mm resolution quantitative drug distribution analysis.

Mapping Lipogenic Flux: A Gold LDI-MS Approach for Imaging Neutral Lipid Kinetics.

Spatial characterization of triglyceride metabolism is an area of significant interest which can be enabled by mass spectrometry imaging via recent advances in neutral lipid laser desorption analytical approaches. Here, we extend recent advancements in gold-assisted neutral lipid imaging and demonstrate the potential to map lipid flux in rodents. We address here critical issues surrounding the analytical configuration and interpretation of the data for a group of select triglycerides. Specifically, we examined how the signal intensity and spatial resolution would impact the apparent isotope ratio in a given analyte (which is an important consideration when performing MS based kinetics studies of this kind) with attention given to molecular ions and not fragments. We evaluated the analytics by contrasting lipid flux in well characterized mouse models, including fed vs fed states and different dietary perturbations. In total, the experimental paradigm described here should enable studies of hepatic lipogenesis; presumably, this logic can be enhanced via the inclusion of ion mobility and/or fragmentation. Although this study was carried out in robust models of liver lipogenesis, we expect that the model system could be expanded to a variety of tissues where zonated (or heterogeneous) lipid synthesis may occur, including solid tumor metabolism.

Combining MALDI mass spectrometry imaging and droplet-base surface sampling analysis for tissue distribution, metabolite profiling, and relative quantification of cyclic peptide melanotan II.

Peptides have become a fast-growing segment of the pharmaceutical industry over the past few decades. It is essential to develop cutting edge analytical techniques to support the discovery and development of peptide therapeutics, especially to examine their absorption, distribution, metabolism and excretion (ADME) properties. Herein, we utilized two label-free mass spectrometry (MS) based techniques to investigate representative challenges in developing therapeutic peptides, such as tissue distribution, metabolic stability and clearance. A tool proof-of-concept cyclic peptide, melanotan II, was used in this study. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which is a well-developed label-free imaging technique, was used to map the detailed molecular distribution of melanotan II and its metabolites. Droplet-based liquid microjunction surface sampling liquid chromatography-high resolution mass spectrometry (LMJ-SSP-LC-HRMS) was used in combination with MALDI-MSI to rapidly profile molecular information and provide structural insights on drug and metabolites. Using both techniques in parallel allowed a more comprehensive and complementary data set than using either technique independently. We envision MALDI-MSI and droplet-based LMJ-SSP-LC-HRMS, which can be used in combination or as standalone techniques, to become valuable tools for assessing the in vivo fate of peptide therapeutics in support of drug discovery and development.

Optimization of dropletProbe-Mass Spectrometry for Whole-Body Tissue Distribution Analysis of Drug-Like Molecules.

dropletProbe mass spectrometry (MS) is an emerging tool for the rapid ex vivo analysis of drugs in tissues and whole-body sections. Its use has been demonstrated to better understand a drug's absorption, distribution, metabolism, and excretion (ADME) properties. To further optimize the overall utility of this technique, it is important to characterize and understand the various tissue matrix effects and extraction solvents on the overall performance of dropletProbe MS analyses. Herein, we systematically evaluated the impact of extraction solvents and various tissues on the relative detected signal intensities of a test set of diverse drugs. It was observed that the tissue matrix had a minimal effect on the performance of dropletProbe MS for the limited set of tested compounds once an optimized extraction solvent was identified. A general starting extraction solvent of 1:1 acetonitrile/water (v:v) was identified to efficiently extract the test set of compounds from various tissues. Next, the optimized conditions were used to map the distribution of the drug diclofenac and its metabolites in whole-body mouse sections. The relative tissue distribution of diclofenac and its metabolites, including the phase II acyl-glucuronide metabolite, were successfully determined with the technique. It is recommended these conditions are used as a general guideline when initiating dropletProbe MS studies of therapeutic drug-like compounds.

Isobaric Labeling Strategy Utilizing 4-Plex N,N-Dimethyl Leucine (DiLeu) Tags Reveals Proteomic Changes Induced by Chemotherapy in Cerebrospinal Fluid of Children with B-Cell Acute Lymphoblastic Leukemia.

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

Multiplex Quantitative Glycomics Enabled by Periodate Oxidation and Triplex Mass Defect Isobaric Multiplex Reagents for Carbonyl-Containing Compound Tags.

Glycosylation is one of the most important post-translational modifications (PTMs) with essential physiological functions, including protein folding, cell signaling, and immune response. Thus, various qualitative and quantitative glycomics analysis strategies have been developed. Recently, the isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tag was developed for quantitative glycomics with multiplexing capacity and increased reporter ion yield. To further improve quantification efficiency and enable quantifying low-abundance species, the mass defect based triplex SUGAR (mdSUGAR) tag has been designed. In addition, we also introduce additional reaction sites for mdSUGAR at the terminal sialic acid by periodate oxidation of the polyhydroxy chain to extend the mass difference and lower the requirement for resolving power. As a result, mdSUGAR tags show complete labeling efficiency, improved fragmentation pattern, and accurate quantification. Moreover, the quantitative performance of the mdSUGAR tags in a complex system has been systematically evaluated and demonstrated reliable results.

Mass Spectrometric Imaging Reveals Temporal and Spatial Dynamics of Bioactive Lipids in Arteries Undergoing Restenosis.

Restenosis, or renarrowing of the arterial lumen, is a common recurrent disease following balloon angioplasty and stenting treatments for cardiovascular disease. A major technical barrier for deciphering restenotic mechanisms is the dynamic, spatial profiling of bioactive lipids in the arterial wall, especially in small animals. Here, applying matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), we conducted the first lipidomic study of temporal-spatial profiling in a small animal model of angioplasty-induced restenosis. Cross sections were collected 3, 7, and 14 days after balloon angioplasty of rat carotid arteries. MALDI-MSI analyses showed that diacylglycerols (DAGs), signaling lipids associated with restenosis, and lysophosphatidylcholines (LysoPCs), whose function was uncharacterized in restenosis, dramatically increased at postangioplasty day 7 and day 14 in the neointimal layer of balloon-injured arteries compared to uninjured controls. In contrast, sphingomyelins (SMs) did not increase, but rather decreased at day 3, day 7, and day 14 in injured arteries versus the uninjured control arteries. These results revealed previously unexplored distinct temporal-spatial lipid dynamics in the restenotic arterial wall. Additionally, we employed time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem MS imaging for both molecular identification and imaging at high spatial resolution. These imaging modalities provide powerful tools for unraveling novel mechanisms of restenosis involving lipids or small signaling molecules.

Visualization and Identification of Neurotransmitters in Crustacean Brain via Multifaceted Mass Spectrometric Approaches.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has emerged as a label-free analytical tool for fast biomolecule profiling on tissue sections. Among various functional molecules, mapping neurotransmitters and related metabolites is of tremendous significance, as these compounds are critical to signaling in the central nervous system. Here, we demonstrated the use of both derivatization and reaction-free approaches that greatly reduced signal complexity and thus enabled complementary signaling molecule visualization on crab brain sections via MALDI-LTQ-Orbitrap XL platform. Pyrylium salt served as a primary amine derivatization reagent and produced prominent signal enhancement of multiple neurotransmitters, including dopamine, serotonin, γ-aminobutyric acid, and histamine that were not detected in underivatized tissues. Molecules with other functional groups, such as acetylcholine and phosphocholine, were directly imaged after matrix application. The identities of discovered neurotransmitters were verified by standards using LC-MS/MS. This study broadens our understanding of metabolic signaling in the crustacean nervous system and highlights potential of multifaceted MS techniques for unambiguous neurotransmitter characterization.

Identification of Double Bond Position Isomers in Unsaturated Lipids by m-CPBA Epoxidation and Mass Spectrometry Fragmentation.

Lipids are highly diverse biomolecules associated with several biological functions including structural constituent, energy storage, and signal transduction. It is essential to characterize lipid structural isomers and further understand their biological roles. Unsaturated lipids contain one or multiple carbon-carbon double bonds. Identifying double bond position presents a major challenge in unsaturated lipid characterization. Recently, several advancements have been made for double bond localization by mass spectrometry (MS) analysis. However, many of these studies require complex chemical reactions or advanced mass spectrometers with special fragmentation techniques, which limits the application in lipidomics study. Here, an innovative meta-chloroperoxybenzoic acid ( m-CPBA) epoxidation reaction coupling with collision-induced dissociation (CID)-MS/MS strategy provides a new tool for unsaturated lipidomics analysis. The rapid epoxidation reaction was carried out by m-CPBA with high specificity. Complete derivatization was achieved in minutes without overoxidized byproduct. Moreover, diagnostic ion pair with 16 Da mass difference indicated localization of carbon-carbon double bond in MS/MS spectra. Multiple lipid classes were evaluated with this strategy and generated abundant fragments for structural analysis. Unsaturated lipid analysis of yeast extract using this strategy took less than 30 min, demonstrating the potential for high-throughput lipidomics analysis by this approach. This study opens a door for high throughput unsaturated lipid analysis with minimal requirement for instrumentation, which could be widely applied in lipidomics analysis.

Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags: A Probe for Quantitative Glycomic Analysis.

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry.

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Clemastine effects in rat models of a myelination disorder.

BackgroundPelizaeus Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system caused by impaired differentiation of oligodendrocytes. This study was prompted by findings that antimuscarinic compounds enhance oligodendrocyte differentiation and remyelination in vitro. One of these compounds, clemastine fumarate, is licensed for treatment of allergic conditions. We tested whether clemastine fumarate can promote myelination in two rodent PMD models, the myelin-deficient and the PLP transgenic rat.MethodsPups were treated with daily injections of clemastine (10-30 mg/kg/day) on postnatal days 1-21. Neurologic phenotypes and myelination patterns in the brain, optic nerves, and spinal cords were assessed using histological techniques.ResultsNo changes in neurological phenotype or survival were observed even at the highest dose of clemastine. Postmortem staining with Luxol fast blue and myelin basic protein immunohistochemistry revealed no evidence for improved myelination in the CNS of treated rats compared to vehicle-treated littermates. Populations of mature oligodendrocytes were unaffected by the treatment.ConclusionThese results demonstrate lack of therapeutic effect of clemastine in two rat PMD models. Both models have rapid disease progression consistent with the connatal form of the disease. Further studies are necessary to determine whether clemastine bears a therapeutic potential in milder forms of PMD.