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Mass spectrometry technique may speed drug discovery and development

BioPhotonics
Oct 2006
Correlation of drug distribution and protein changes sheds light on drug efficacy

Gary Boas

Pharmaceutical companies spend an average of eight years and roughly $500 million to get a new drug to market. They could avoid much of this time and expense if there were a way to identify unsuccessful drugs before they reach the late stage of clinical trials. This would require further development of the early stages of drug discovery — to gain better understanding of drug distributions, for example, or of the relationship between a drug’s distribution and its pharmacological or toxicological effects.

A group with Vanderbilt University in Nashville, Tenn., has developed a technique that may help. In an Analytical Chemistry paper published online on Aug. 3, the researchers described use of matrix-assisted laser desorption/ionization (Maldi) imaging mass spectrometry to analyze whole-organ and whole-body animal tissue sections, successfully measuring the concentrations of drugs as well as the associated protein changes in tissue — the latter representing the therapeutic response to the drugs. Correlating these can reveal important information about the efficacy of a drug much earlier than otherwise would be possible.


The technique also enables measurement of protein changes in tissue, reflecting a therapeutic response to drugs. Correlating these with the drug and metabolite distributions can tell researchers a great deal about drug efficacy, much earlier than would be possible using conventional methods. Shown here are optical (left) and protein (right) images of rat liver (blue in the protein image), kidney (pink) and brain tissue (red; with white and green representing substructures of the brain).

Perhaps the most important aspect of the method is that it allows direct monitoring of the drug. “No [other] technique allows you to visualize the molecular distribution of a compound,” said Sheerin Khatib-Shahidi, the first author of the paper. “You can only follow a label.” Because other methods track the distribution of a label, researchers can never be entirely certain that they are looking at the original drug: The label may have been maintained by a metabolite encountered along the way or simply cleaved away from the drug. For this reason, she added, the techniques offer only limited ability to connect the signal with the molecular identity.


Researchers used an imaging mass spectrometry technique to track drug (top; organs are outlined in red) and metabolite (bottom) distributions in animal models, which could contribute to the drug-discovery process. Unlike other techniques used for a similar purpose, this method allows direct monitoring of the drug, and not of the label associated with it.


This applies also to the current gold standard for characterization of new drugs. Whole-body autoradiography, use of which is required for FDA approval of pharmaceuticals, provides spatial and quantitative information about the absorption and distribution of radiolabeled drugs by tracking the radioactive substance as it diffuses through tissue. However, it cannot distinguish between the originally labeled compound and any part of the radiolabel that may have separated from it. The Maldi technique described by the researchers can, but does not yet offer quantitative information. For this reason, Khatib-Shahidi said, it could serve as an important complement to autoradiography.

The technique images these distributions using Maldi mass spectrometry to desorb and detect compounds, relying on a matrix that co-crystallizes with the analytes in question. The matrix and analytes alike desorb from the sample surface when irradiated with ultraviolet light. This produces ions that can be analyzed by a time-of-flight mass spectrometer. Researchers can then reconstruct a molecular image of the tissue using an ordered array of the mass spectra.

To demonstrate the method’s potential for drug discovery and development, the investigators used it to measure the relative concentrations of drugs in tissue as well as the distribution of several metabolites, label-free. They also used it to detect protein changes in tissue, enabling evaluation of the drugs’ efficacy. For the former experiments, they analyzed whole-body tissue sections from male Fischer 344 rats, which had been given oral doses of Olanzapine, a drug doctors use to treat mood disorders, including schizophrenia and acute mania in bipolar patients. They used a time-of-flight mass spectroscopy system made by MDS Sciex of Concord, Ontario, Canada, outfitted with a 337-nm nitrogen laser operating at 20 Hz. They acquired spectra by irradiating the sample in 400-μm steps.

Imaging whole-body tissue sections with Maldi imaging mass spectrometry was not easy. “We’re taking a very large sample size, having to break it down into sections that we can analyze and then reconstructing it,” Khatib-Shahidi said. “It’s at times laborious, but the payoff is very big.”

Indeed, the investigators were able to directly image drug and metabolites throughout the rats two and six hours after administration. At two hours, they could see the drug in almost all tissues, with especially high concentrations in certain organs.

To measure the protein changes, they imaged tissue sections from the liver, kidney and brain, using either a Maldi-Tof/Tof-MS system made by Bruker Daltonics of Billerica, Mass., outfitted with a solid-state Smartbeam laser operating at 200 Hz, or a Maldi-Tof-MS system made by Applied Biosystems of Foster City, Calif., equipped with a 337-nm nitrogen laser operating at 20 Hz. Here, the technique detected protein signals from each of the tissue types, in some cases enabling organ- or region-specific images.

By imaging the distributions of drugs and metabolites and providing for correlations of these and protein changes, the imaging mass spectrometry method offers a broad overview of the many complex interactions following administration of pharmaceutical compounds. It can, therefore, advance drug discovery and development, providing insight into drug efficacy as well as pharmacological and toxicological effects, long before it reaches the late stage of clinical trials.

The researchers are monitoring drug accumulation to target tissues — to determine how well a cancer therapy drug localizes to a brain tumor, for example. In addition, they are exploring whether they can identify early protein biomarkers indicative of early drug efficacy or toxicity.

For validation, they are following conventional quantitative methods used by the pharmaceutical industry. They also are comparing the relative information obtained from the imaging mass spectrometry images to quantitative information provided by whole-body autoradiography.

“Ultimately, we would like to develop the technique to allow for absolute quantitation of each analyte directly from the organs analyzed,” Khatib-Shahidi said.


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