September 2, 2008

Research creates tool to quickly analyze where drugs go in the body

A team led by R. Graham Cooks, Purdue's Henry Bohn Hass Distinguished Professor of Chemistry, has developed a new analysis tool that can provide a map of the distribution of a drug within the body. The new tool requires no chemical pretreatment.

Knowing where a drug is circulated and where it accumulates in the body is critical to evaluate its effectiveness and potential for toxicity, said Cooks, who also is co-founder of Purdue's Center for Analytical Instrumentation Development.

"This is the first analysis that works on an unadulterated sample to show where a drug and its metabolites travel in the body," he said "The analysis method is very fast and can be done on the spot, which could save pharmaceutical researchers valuable time and resources."

The speed presents another advantage in its ability to track a drug's course over time, he said.

"The amount of a drug present in specific organs or areas of the body drops off quickly over time," Cooks said. "It is important to know where a drug is at different points in its time course. This tool allows an almost instant analysis and has the potential to provide key metabolism data."

The new analysis tool is based on a mass spectrometry method invented in Cooks' lab. Mass spectrometry is widely used in drug discovery and development because it tells researchers the chemical makeup of a sample and can detail the amounts of specific molecules. Most spectrometry methods require expensive markers to illuminate the molecule of interest and pretreatment of the sample with chemicals. Cooks' method is the only mass spectrometry technique that can evaluate an untreated sample, he said.

In addition, the method offers an "open search" of the molecules present in a sample, he said.

"Analysis that requires a chemical marker to highlight a molecule limits what can be detected," Cooks said. "You have to know ahead of time what you want to highlight in order to choose the appropriate marker, and all you can see is what has been marked.  An open search eliminates this need and shows the full spectrum of molecules present. A drug can break down into many different molecules as it is metabolized by the body, and you don't always know what you will find."

A paper detailing the research was published in the Aug. 15 edition of the Proceedings of the National Academies of Science. Co-authors include Justin M. Wiseman of Prosolia Inc.; Demian R. Ifa and Nicolas E. Manicke, researchers in Purdue's chemistry department; Yongxin Zhu of Bioanalytical Systems Inc.; Candice B. Kissinger of Purdue's Chao Center for Industrial Pharmacy; and Peter T. Kissinger of Prosolia Inc. and Bioanalytical Systems Inc.

Kissinger is chairman of Prosolia, the company that has commercialized the mass spectrometry method. He said the new analysis tool using the method provides spatial information that can't be obtained through other common analyses such as blood tests.

"It is especially exciting to get an image of the different molecules in different areas of the body," Kissinger said. "If a company develops a drug intended to treat brain tissue, it is not good if it goes to the kidney or the lungs. Often a percentage of a drug treatment goes to places where it has no benefit or to places where it could produce a toxic side effect."

Cooks' method also gathers a tremendous amount of information from a single sample, he said.

"This type of molecular imaging can look at a variety of molecules at once and can create hundreds of images from a single tissue sample," said Kissinger, who also is a Purdue professor of analytical chemistry. "This maximizes a limited amount of resources and reduces the number of samples needed to complete research."

Cooks' analysis method is a type of mass spectrometry called desorption electrospray ionization, or DESI.

Mass spectrometry works by first turning molecules into ions, or electrically charged versions of themselves, so their masses can be analyzed. Conventional mass spectrometry requires chemical separations, manipulations of samples and containment in a vacuum chamber for ionization and analysis. Cooks' technology performs the ionization step in the air or directly on surfaces outside of the mass spectrometer's vacuum chamber.

The ionization is done through a stream of water sprayed in the presence of an electric field to create positively charged water droplets. Molecules within a sample hit by the charged water droplets also pick up a charge and become ions. The molecules are then vacuumed into the spectrometer to be measured and analyzed.

The spectrometer scans one thin section of the sample at a time and records the information. A software program created by the research team interprets the results to produce a two-dimensional image mapping the distribution of molecules within the entire sample.

The team examined the distribution of the drug Clozapine, which inhibits the activity of a chemical in the brain. Concentrations of the drug from 0.5 micrograms to 10.6 micrograms were detected in rat brain, lung, kidney and testis tissue. The results showed good agreement with standard evaluation techniques.

Prosolia Inc. and Purdue's prototyping program will create 10 prototypes of the equipment to be tested in other academic laboratories.

"Purdue is providing resources to help move this technology further along and speed the commercialization process," Kissinger said. "It takes time to bring new medical technology into regular use, but this technology could be available to research laboratories within a few months."

This work shows how an analytical instrument technology like DESI can have many useful applications, Kissinger said.

"DESI was invented about five years ago and received attention for its potential to detect explosive residues and help homeland security," he said. "Next it was shown to be effective for testing fruit for pesticide residues or harmful bacteria. Now we are beginning to fulfill the technology's potential as a health-care tool."

Cooks' team is associated with several research centers at Purdue including the Center for Analytical Instrumentation Development, Bindley Bioscience Center in Purdue's Discovery Park and the Center for Sensing Science and Technology.

Research funding was provided by the Office of Naval Research and through the Indiana 21st Century Research and Technology Fund.

Writer: Elizabeth K. Gardner, (765) 494-2081, ekgardner@purdue.edu

Sources: R. Graham Cooks, (765) 494-5262, cooks@purdue.edu

Peter T. Kissinger, (765) 497-5801, kissingp@purdue.edu

Justin M. Wiseman, (317) 278-6110, wiseman@prosolia.com

Purdue News Service: (765) 494-2096; purduenews@purdue.edu

Ambient ionization methods for MS enable direct, high-throughputmeasurements of samples in the open air. Here, we report onone such method, desorption electrospray ionization (DESI), which is coupled to a linear ion trap mass spectrometer and used to recordthe spatial intensity distribution of a drug directly from histologicalsections of brain, lung, kidney, and testis without prior chemical treatment. DESI imaging provided identification and distributionof clozapine after an oral dose of 50 mg/kg by: i) measuring the abundance of the intact ion at m/z 327.1, and ii) monitoring the dissociation of the protonated drug compound at m/z 327.1 to its dominant product ion at m/z 270.1. In lung tissues, DESI imaging was performed in the full-scan mode over an m/z range of 200- 1100, providing an opportunity for relative quantitation by using an endogenous lipid to normalize the signal response of clozapine. The presence of clozapine was detected in all tissue types, whereas the presence of the N-desmethyl metabolite was detected only in the lung sections. Quantitation of clozapine from the brain, lung, kidney, and testis, by using LC-MS/MS, revealed concentrations ranging from 0.05 _g/g (brain) to a high of 10.6 _g/g (lung). Comparisons of the results recorded by DESI with those by LCMS/ MS show good agreement and are favorable for the use of DESI imaging in drug and metabolite detection directly from biological tissues.

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