Download A System That Helps Scientists See Drugs in Tissue Samples

Reprinted from American Biotechnology Laboratory July 2005
A System That Helps Scientists
See Drugs in Tissue Samples
by Mark Springer
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 LCMS-MS system (Applied Biosystems/MDS
SCIEX), a QqTOF system equipped with an
Figure 1
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.
QSTAR system and MALDI plate.
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 MALDITOF (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
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.
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.
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
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 MSMS 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 system 3 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 re-
sulting 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.: 650570-6667; fax: 650-638-6239; e-mail: SpringMN@
appliedbiosystems.com.