Seeing Drugs in Tissue Samples

Scientists from Schering-Plough are now employing mass spectrometry systems to apply a technique known as MS tissue imaging to obtain detailed information about the spatial distribution of drug candidate compounds hroughout tissue samples.

, den 12. Oktober 2005

Being able to see where a drug candidate is distributed in a targeted tissue helps drug developers to better assess the potential value of that compound as a pharmaceutical product. For these purposes, scientists at Schering-Plough are now using the Applied Biosystems/MDS SCIEX API QStar Pulsar i hybrid LC/MS/MS system, a QqTOF system equipped with an optional matrix-assisted laser desorption/ionization (Maldi) source.

Dr. Korfmacher, Director of Exploratory Drug Metabolism at Schering-Plough, and his team have visualized the different regions within rat and mouse tissues where candidate drug compounds were located. He oversees 13 scientists in two labs who work together with drug discovery teams to identify new compounds that the scientists then recommend for development into pharmaceutical products (figure 1).
“Typically, somewhere between five and ten percent of the compounds that go into development actually ever become a drug,” explains Dr. Korfmacher. “Even when you reach phase 1 (of clinical trials), only about 20 percent of compounds succeed and become a marketable drug. So we are continually trying to improve upon that record.”
Tracking the Whereabouts Early in the Discovery Phase
One tool that may improve the rate of success for candidate compounds becoming marketable drugs is a technique called MS tissue imaging, developed by Dr. Richard Caprioli, Director of the Mass Spectrometry Research Center, Vanderbilt University School of Medicine, and colleagues at Vanderbilt. This technique is a sophisticated application of Maldi-TOF MS/MS analysis and provides researchers with reliable localization information for small molecules.
By applying tissue imaging to drug discovery studies, researchers can track the whereabouts of a particular drug candidate within a target tissue early in the discovery phase before moving the compound to the costlier drug development phase. Besides helping researchers obtain detailed information about where a compound is distributed in a tissue sample, MS tissue imaging can provide answers to questions about the way compounds act in different tissue types. “One of the reasons that tissue imaging is enticing, and one of the reasons that we are looking at it, is for central nervous system projects,” notes Dr. Korfmacher.
At Schering-Plough, Dr. Korfmacher oversees two labs that are equipped with multiple mass spectrometry systems, including four Applied Biosystems/MDS SCIEX mass spectrometry systems: an API 3000 LC/MS/MS System, two API 4000 LC/MS/MS Systems, and an API QSTAR Pulsar i hybrid LC/MS/MS System that the exploratory drug metabolism labs use for the MS tissue imaging technique.
How the MS Tissue Imaging Technology Works
Dr. Caprioli, working as a consultant for Schering-Plough, first developed the MS tissue imaging technique for locating proteins in tissues samples. The technique was successful at locating proteins and peptides, which have relatively large molecular masses of between 2,000 and 50,000 daltons. Then, about three years ago, with urging from Dr. Korfmacher, Dr. Caprioli adapted the technique to detect small molecules in tissue samples such as drug candidates. The use of the API QStar Pulsar i system from Applied Biosystems/MDS SCIEX helped the researchers to overcome limits of detection, and clearly identify the signals generated by candidate drugs, compounds that generally have molecular masses of around 500 daltons (figure 2).
In MS tissue imaging, researchers place a tissue sample on a Maldi plate of a QStar system, and then view an image of the sample tissue on a computer screen. By using the Maldi system MS Imaging (MMI) software developed by Applied Biosystems/MDS SCIEX, they select the part of the tissue whose image they want. They then determine how closely they want to space successive laser shots that create the image of the sample. To increase the level of detail of a sampling, the researcher increases the number of laser shots and pixels generated per unit area. For example 4,000 pixels produce a much more detailed image of the sample than 200 pixels (s. figure 3).
To evaluate the results of a tissue analysis, the researcher reviews an image on a computer screen filled with colored spots at locations where a particular compound has been detected. The intensity of the color of a spot corresponds to the amount of signal that the laser detects at any one point or pixel (s. figure 4).
“We have shown that Maldi MS/MS imaging is semi-quantitative for MS tissue imaging,” states Dr. Korfmacher. “When the color is more intense in one part of the picture that means that there is more of the drug in that part of the tissue than in the other part. However, we cannot determine what the actual concentration of the compound is in different parts of the tissue.”
Selecting any pixel that is part of a colored spot on the image will display a mass spectrum or product-ion spectrum, representative of the compound present in that region of the tissue. “The product-ion spectrum can then be compared to the authentic standard. This gives you somewhat of a fingerprint match,” explains Dr. Korfmacher.
Alternative methods for identifying the presence of different candidate drug compounds in tissues include autoradiography, tissue homogenation followed by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI)-MS analysis. However, neither of these approaches can provide the kind of detailed localization information that can be obtained from the MS tissue imaging technique.
In a study undertaken in 2003, Dr. Korfmacher, Dr. Caprioli and a team of researchers were able to use MS tissue imaging to visualize the distribution of candidate drug compounds in different regions of the rat brain, and different areas of a mouse tumor tissue sample [2]. For this study, the researchers applied the Maldi imaging technique, using an API QStar Pulsar i hybrid LC/MS/MS system. Dr. Caprioli and his colleagues then applied the imaging capability of software now available with a QStar system to generate 2-D-images of mouse tumor tissue samples and rat brain tissue samples from animals previously dosed with candidate drug compounds.
Separating Small Molecules from Matrix Interference
Separating the background noise produced by the Maldi matrix from the signals generated by a candidate compound was the key to extending tissue imaging from experiments capable of locating larger-sized proteins in tissues to ones that can pinpoint the location of drug candidate compounds in tissue samples. “Dr. Caprioli had been doing everything on a Maldi-TOF system, but to do the drug imaging he had to use a QStar system, an MS/MS system that allowed him to do the imaging on a [small molecule] compound of interest,” explains Dr. Korfmacher.
What Caprioli’s lab discovered was that with a Maldi-TOF system the background ions in the matrix overload the small molecule signals. Therefore, they could only see the drug if there were extremely high levels of it in the sample. In order to detect the analyte – the drug candidate of interest – in a tissue, they would need the MS/MS capabilities of a tandem mass spectrometry system such as the API QStar Pulsar i hybrid LC/MS/MS system [3].
The QStar System Resolves a Matrix Interference Problem
In the product ion mass spectrum there is some signal from the matrix, and some from the analyte. With the QStar system it is possible to distinguish between the two signals. Besides allowing for clear discrimination between matrix and analyte, the higher mass accuracy and resolution of the QStar system compared to that of other MS/MS systems give the researchers added confidence when identifying compounds in a tissue image: “When you are looking at your data to see if indeed it is the analyte that you are trying to get your image of, the fact that you have the higher mass accuracy and resolution gives you increased confidence that (the compound) is indeed what you think it is,” explains Dr. Korfmacher.
A Future Routine Tool of Drug Discovery
While researchers in the exploratory drug metabolism labs at Schering-Plough are currently applying the MS tissue imaging technique to drug discovery projects, Dr. Korfmacher believes that the technique has even greater potential in the future as a tool for both drug discovery and drug development applications: “It is still aresearch tool, but the potential for it is very high. Hence, it will probably become a routine tool somewhere in the next two to five years.”