Genetic Engineering & Biotechnology News

APR15 2017

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12 | APRIL 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News Dale Shannon Cornett, Ph.D. The situation in small-molecule pharmaceu- tical R&D has been well described in recent years—low numbers of new drugs approved and the rising cost of bringing a compound through clinical development to market. Against this unrelenting pressure on discov- ery and development, one mantra that has stuck is "fail early, fail cheap." This is perhaps not surprising, given that many drugs fail due to unexpected results late in clinical trials. Pharma companies are in- creasingly aware of the disconnection between outcomes in clinical programs and early-stage development work. As part of the industry's response, ADME/TOX (absorption, distribu- tion, metabolism, excretion/toxicity) investiga- tions are now integrated as early as possible in the pathway, in order to understand essential details of drug distribution, metabolism, and toxicology ahead of going into clinical trials. Current Tools Traditional approaches and tools available for the risk assessment of safe and efficacious drugs have been based on measuring the par- ent drug in plasma—both in animal models and humans. This often does not provide a sufficient level of information to the scientist. It is recognized that most drug targets are not in plasma, and determining the relevant tis- sue distribution of not only the parent drug but also its metabolites would provide much greater understanding of pharmacology and toxicology. The current best practice methods of quantitative whole body autoradiography (QWBA) and liquid chromatography-mass spectrometry (LC-MS) put the emphasis on tissues rather than plasma, but still fall short of providing the complete picture. Both tech- niques have challenges. QWBA is the tech- nique of choice for determining drug distri- bution, 1 and the data generated is often pri- marily provided in new drug regulatory sub- missions. The technique, however, presents a composite of the total radioactivity present and cannot distinguish parent drug from me- tabolite. Thus it has severe limitations for re- searchers looking for insight into biochemical pathways and mechanisms. LC-MS analysis is performed on extracts from tissue homogenates. This type of analy- sis does not elucidate any spatial information and, just as importantly, can be misleading. For example, if an analyte in the tissue is highly localized, the homogenization process could potentially dilute it below the limit of detection (LOD). Alternatively, when an ana- lyte is determined from tissue homogenate, a researcher could draw incorrect conclusions about toxicity, because the analyte is pre- sumed to be evenly present throughout the tissue when in fact it may be highly localized. New Thinking Required Over the past five to seven years, a new approach, taken from preexisting technology, that provides quantitative, spatially resolved data on the tissue distribution of drugs and drug metabolites has emerged. Matrix-assist- ed laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is uniquely suit- ed to this task. Figure 1 shows how the MAL- DI MSI experiment is initiated by mounting a tissue section onto a target, applying a ma- trix and rastering a laser across the surface of the tissue. At each discrete location where the laser is fired, an accurate mass spectrum is acquired. By plotting the ion intensities as a function of the x and y coordinates on the tissue, ion images are generated. Software co- registers tissue histology images with these, allowing a researcher to visualize biology and chemistry in an integrated fashion. A range of mass spectrometers can be integrated into a MALDI MSI system, de- pending on application needs. MALDI-TOF systems, for example, offer high throughput (with lower ability to distinguish molecules of similar molecular weight) while Fourier transform mass spectrometers (MALDI-FT- MS) provide the ultimate in measurement ac- curacy and mass resolving power. High MS performance features of MALDI-FTMS can often yield unique molecular formula identi- fication for ion images. Principles and Practice MALDI MSI takes a conventional tissue section and coats it with a fine aerosol of MALDI matrix solution, which extracts mole- cules from the tissue, but retains the spatial re- lationships found in the underlying tissue. Fol- lowing preparation, the sample is measured in the mass spectrometer. The result is spatially resolved, accurate mass spectra. Because the laser only probes the matrix crystals that are on the surface of the section, the underlying cellular features are not disrupted and can be taken through a standard histological staining routine, so that a high-quality histology image can be captured for co-registration. MALDI-FTMS Emerges as a Valuable Tool in Small-Molecule R&D Integrating Biology and Chemistry for Deeper Understanding Drug Discovery Tutorial Figure 2. Correlation of histology and MALDI MSI image in dog liver sections Figure 3. Bruker SolariX XR Figure 1. A MALDI MSI experimental protocol

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