(1) Biomolecular Mass Spectrometry Imaging, National Resource for Mass Spectrometry Imaging, Department of Pharmaceutical Biosciences, (2) Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala Biomedical Center, Uppsala University, Uppsala University, Box 591, SE-75124 Uppsala, Sweden, (3) Center for Molecular Medicine, Department of Neurology and Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden, and (4) Bruker Daltonik GmbH, 28359 Bremen, Germany
Small-molecule neurotransmitters, their precursors and metabolites are involved in the brain chemical network and transmit signals between neurons. Changes in their concentrations are associated with numerous normal neuronal processes, such as sleep and aging, but also in several disease states, including neurological disorders (e.g., Parkinson’s and Alzheimer’s disease), depression and drug addiction. Knowledge about their relative abundance and spatial distribution would provide insights into these complex neurological processes and disorders. At present, researchers rely on indirect histochemical, immunohistochemical, and ligand-based assays to detect these small-molecule transmitter substances or tissue homogenates analyzed by HPLC. Current neuroimaging techniques have very limited abilities to directly identify and quantify neurotransmitters from brain sections.
By performing matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) analysis directly on the surface of a tissue section, the technique has quickly been established as a powerful in situ visualization tool for measuring abundance and spatial distribution of endogenous and pharmaceutical compounds, lipids, peptides and small proteins. We have recently introduced a reactive MALDI matrix, which selectively targets the primary amine group on neurotransmitters, metabolites and neuroactive substances but also function as a matrix for the ionization (Neuron, 2014, 84:697-707). However, the limitation of using such reactive matrix to study the full molecular pathways of for example dopaminergic or serotonergic biosynthesis and metabolism is its limitation to target all downstream dopamine metabolites derived from monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT) enzymes. The majority of small molecule neurotransmitters such as catecholamines, amino acids and trace amines possess phenolic hydroxyl and/or primary or secondary amines, which are proper nucleophilic groups. We therefore developed a new reactive matrix that can selectively target and charge-tag both phenolic and primary amine groups, thus enabling MALDI-MSI of both MAO and COMT downstream metabolites. We have focused on a nucleophilic aromatic substitution reaction with such functional groups. By this developed reactive matrix, we were able to detect and map the localization of most of the neurotransmitters and metabolites involved in the dopaminergic and serotonergic network in a single brain tissue section and it represents a novel methodology, which assists their identification through the selectivity of the reaction. The sensitivity and specificity of the imaging approach of neurochemicals has a great potential in many diverse applications in fields such as neuroscience, pharmacology, drug discovery, neurochemistry, and medicine.
We have employed IMS in studies of a variety of biologically and medically relevant research projects, several of which will be presented including studies in diabetic nephropathy involving both proteins and lipids and the differentiation of benign skin lesions from melanomas. In addition, IMS has been applied to drug targeting and metabolic studies both in specific organs and also in intact whole animal sections following drug administration.
This presentation describes recent technological advances both in sample preparation and instrumental performance to achieve images at high spatial resolution (1-10 microns) and at high speeds so that a typical sample tissue once prepared can be imaged in minutes. Instrumentation used in these studies includes both MALDI FTICR MS and MALDI TOF mass spectrometers. Applications utilize MS/MS, ultra-high mass resolution, and ion accumulation devices for IMS studies. Finally, new biocomputational approaches will be discussed that deals with the high data dimensionality of IMS and our implementation of ‘image fusion’ in terms of predictive integration of MS images with microscopy and other imaging modalities.
Delivering safe and efficacious drugs is tied to our ability to understand the complex mechanistic relationships between molecular initiation events of pharmacologically active compounds and the cascade of biological consequences. MALDI imaging MS (IMS) technology has taken us beyond “plasma centric” studies and allows us to directly map the tissue disposition and molecular changes associated with drug pharmacology. With this approach, we can achieve greater insight into the mechanisms of drug absorption, improve and innovate delivery strategies, observe drugs reaching their intended targets, as well as unintended targets and correlate these events with PK/PD data.
We have explored the application MALDI IMS to investigate the distribution of drugs and their metabolites as well as endogenous compounds in tissue samples without the need for labeling techniques. This methodology allows for the correlation of analyte tissue distributions with histology images, thereby integrating chemical structures with tissue morphology. MALDI IMS provides high spatial resolution (10µm), is highly sensitive, and can be quantitative. Furthermore, this imaging modality offers the potential to further our mechanistic understanding of drug disposition, disease progression and pharmacology (including toxicology) by providing snap shots of temporal and causal changes. While MALDI IMS is primarily being employed to determine the tissue distribution of drugs and their metabolites, it is becoming evident that more detailed understanding of biological system can be gained by including the changes in endogenous compound distribution as a function of disease and pharmacology.
This presentation will focus on our efforts to couple MALDI IMS and histology in drug development to better understand tissue disposition and gain mechanistic insights into drug correlated toxicities and efficacy. Case studies from early and late stage drug development, where MALDI IMS was employed to investigate the mechanisms of adverse events, provide insights into disposition, and PK/PD relationships will be presented. The potential of MALDI IMS beyond just studying drugs and their metabolites will also be presented. In addition, the dependence of realizing the full potential of MALDI IMS on enhanced software tools and improved data handling will be presented.