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
- 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.
- 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.
- 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].