A System That Helps Scientists See Drugs in Tissue Samples

For drug developers, being able to see where a drug candidate is distributed in a targeted tissue helps them better assess the potential value of that compound as a pharmaceutical product. To that end, one scientist, Dr. Walter Korfmacher, and researchers from Schering-Plough (Kenilworth, NJ) are employing mass spectrometry systems from Applied Biosystems/MDS SCIEX (Foster City, CA) to apply a technique known as mass spectrometry tissue imaging to obtain detailed information about the spatial distribution of drug candidate compounds throughout tissue samples.

Using the API QSTAR® Pulsar i hybrid LC-MS-MS system (Applied Biosystems/MDS SCIEX), a QqTOF system equipped with an optional matrix-assisted laser desorption ionization (MALDI) source, Dr. Korfmacher and a team of researchers have visualized the different regions within rat and mouse tissues in which candidate drug compounds were located.

Exploring applications of MS tissue imaging

As Director of Exploratory Drug Metabolism at Schering-Plough, Dr. Korfmacher oversees 13 scientists in two laboratories who work with drug discovery teams to identify new compounds that the scientists then recommend for development into pharmaceutical products.1 Typically, less than 10% of compounds that go into development ever become drugs. Only about 20% of compounds that reach Phase 1 clinical trials succeed and become marketable drugs. According to Dr. Korfmacher, an increase from 20 to 40% would be a big improvement.

One tool that may improve the rate of success for candidate compounds becoming marketable drugs is MS tissue imaging, which was developed by Dr. Richard Caprioli, Director of the Mass Spectrometry Research Center, Vanderbilt University School of Medicine (Nashville, TN), and colleagues. The technique, a sophisticated application of MALDI-TOF (time-of-flight) MS-MS analysis, provides researchers with reliable localization information for small molecules. By applying tissue imaging to drug discovery studies, researchers can track the location of a particular drug candidate within a target tissue early in the discovery phase before moving the compound to the costlier drug development phase.

In addition to 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. For example, one question that often confronts drug researchers is: Why are some compounds that are intended to treat brain disorders able to get into the brain, but are not active once they get there? MS tissue imaging can answer that question by showing if a compound is present in the diseased area of the brain.

Moving more compounds from discovery to development

At Schering-Plough, Dr. Korfmacher oversees two laboratories 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 used by the exploratory drug metabolism laboratories for the MS tissue imaging technique.

How MS tissue imaging works

Dr. Caprioli, who also works as a consultant for Schering-Plough, first developed MS tissue imaging for locating proteins in tissue samples. The technique was successful at locating proteins and peptides, which have relatively large molecular masses of 2000–50,000 D. Then, about three years ago, with urging from Dr. Korfmacher, Dr. Caprioli adapted the technique to detect small molecules, such as drug candidates, in tissue samples. Use of the API QSTAR Pulsar i system helped the researchers overcome limits of detection and clearly identify the signals generated by candidate drugs, compounds that generally have molecular masses of around 500 D.

Figure 1 - QSTAR system and MALDI plate.

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 (see Figure 1). By using the MALDI system MS imaging (MMI) software (Applied Biosystems/MDS SCIEX), they select the part of the tissue for which they want an image. 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, 4000 pixels produce a much more detailed image of the sample than 200 pixels.

To evaluate the results of a tissue analysis, the researcher reviews an image on a computer screen filled with colored spots at locations at which a particular compound has been detected. The color intensity of a spot corresponds to the amount of signal detected by the laser at any one point or pixel.

MS tissue imaging is semiquantitative. Color that is more intense in one part of the picture indicates there is more of the drug in that part of the tissue than in the other part. However, it is not possible to determine the actual concentration of the compound 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 known standards, as is done when making a fingerprint match.

Alternative methods for identifying the presence of different candidate drug compounds in tissues include autoradiography, or tissue homogenization followed by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI)-MS analysis. However, neither approach can provide the kind of detailed localization information that can be obtained from MS tissue imaging.

Visualizing candidate drug compounds

Figure 2 - Example of MS tissue imaging technique. The letters S and P were “written” on a rat brain tissue slice using a compound that was then detected by the MALDI-MS-MS technique. The imaging software then produced the images shown in this figure.

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 colleagues then applied the imaging capability of software now available with the 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 (see Figure 2).

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 applications 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 using a MALDI-TOF system, but, to perform the drug imaging, he had to use a QSTAR system, an MS-MS system that allowed the imaging to be done on a small-molecule compound of interest.

The laboratory discovered that, with a MALDI-TOF system, the background ions in the matrix overload the small-molecule signals. Therefore, the drug could be seen 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, the MS-MS capabilities of a tandem mass spectrometry system such as the API QSTAR Pulsar i hybrid LC-MS-MS system3 would have to be used.

Resolution of a matrix interference problem

The product ion mass spectrum generated by the QSTAR system shows some signal from the matrix and some from the analyte, which allows users of the system to distinguish between the two signals. In contrast, with the single time-of-flight system, there is no distinction between the signal generated from the analyte and the matrix. The two signals show up as one peak. In addition to allowing for clear discrimination between the matrix and analyte, the higher mass accuracy and resolution of the QSTAR system compared to that of other MS-MS systems gives users added confidence when identifying compounds in a tissue image.

Not only do drug developers obtain higher mass accuracy and resolution, the QSTAR system uses an orthogonal reflectron time-of-flight analyzer in place of a third scanning quadrupole. The use of a TOF analyzer in the system instead of a third quadrupole provides drug developers with additional information in the resulting product ion mass spectrum that helps them confirm the results of a tissue image analysis.

The system generates a spectrum of everything that comes out of a collision cell, whereas a third quadrupole is typically used to select a single ion of interest. The TOF analyzer provides the necessary information without losing sensitivity; in contrast, on a triple quadrupole, to obtain the same information would result in an extreme loss in sensitivity.

Conclusion

While researchers in the exploratory drug metabolism laboratories at Schering-Plough are currently applying MS tissue imaging to drug discovery projects, the technique will have even greater potential in the future as a tool for drug discovery and drug development applications. Today, it is a research tool, and researchers are still refining the technique. However, in the next two to five years, MS tissue imaging will likely become a routine tool for drug discovery and development.

References

  1. Korfmacher WA. Bioanalytical assays in a drug discovery environment. In: Korfmacher W, ed. Using mass spectrometry for drug metabolism studies. Boca Raton, FL: CRC Press, 2002:305–28.
  2. Reyzer ML, Hsieh Y, Ng K, Korfmacher WA, Caprioli RM. Direct analysis of drug candidates in tissue by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spec 2003; 38:1081–92.
  3. Reyzer ML, Caprioli RM. MS imaging: new technology provides new opportunities. In: Korfmacher W, ed. Using mass spectrometry for drug metabolism studies. Boca Raton, FL: CRC Press, 2004:305–28.

Mr. Springer is a Senior Science Writer, Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, U.S.A.; tel.: 650-570-6667; fax: 650-638-6239; e-mail: [email protected].

Comments