Biomedical Engineering Reference
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including CD13, CD14, and CD19. This problem can be overcome if needed by
adding the appropriate immunophenotypic antibody to the whole blood sample prior
to the fixation step, although the possibility that antigen binding might activate cell
signaling, as it occurs with the T cell receptor, needs to be considered.
7.2.4 Cell Signaling: Use in Pharmacodynamic Monitoring
7.2.4.1 Surrogate Marker Assays
Particularly during the early phase of clinical
testing, where the goal is to establish safe treatment schedules for subsequent efficacy
testing, pharmacodynamic monitoring of drug effects on normal tissues can be
informative. In phase I clinical trials of anticancer agents, this is often done using
buccal mucosa or skin biopsies as a source of normal epithelium. However,
quantitative analysis of this material can be problematic, and successful detection
of pharmacodynamic effects depends on the relevant pathway being constitutively
active, which is not always the case. The use of flow cytometry to measure effects in
normal peripheral blood cells avoids these problems due to the inherently quantitative
nature of flow cytometry and the ability to acquire large numbers of events for
statistical analysis. Although most signal transduction pathways are quiescent in
normal peripheral blood cells, many can be acutely activated ex vivo, and the extent of
activation relative to an unstimulated control sample can be used as a measure of
pathway inhibition.
Using phorbol ester to activate the ERK pathway in blood samples, we found that
although target inhibition occurred at sorafenib concentrations in the tens of
micromole range when isolated cells were resuspended in tissue culture medium,
drug concentrations of several hundred micromoles were needed to produce an
equivalent effect in whole blood [18]. This is explained by the high levels of protein
binding that occur with this agent, and suggests the potential to compare signaling
inhibition in serum-freemediumversus whole blood as a rapid screen for the plasma
or red cell binding of a novel compound. In our original phase I trial of sorafenib in
solid tumor patients, we were unable to detect a pharmacodynamic effect at the
maximum tolerated dose, and this result now appears to be most likely explained by
the inability to achieve sufficient free drug to inhibit Raf kinase in peripheral blood
due to protein binding rather than failure of the assay. In addition to its potential to
monitor pharmacodynamic effects of ERK pathway inhibitors, phorbol ester
stimulationinwholebloodhasalsobeenadapted to monitor effects of the protein
kinase C inhibitor enzastaurin, using an antibody that recognizes PKC phosphor-
ylation sites in general [19].
The ERK pathway can also be acutely activated in normal peripheral blood
monocytes by bacterial lipopolysaccharide (LPS). Although this acts through
Toll-like receptors rather than growth factor receptors, downstream signaling
proceeds via MEK and is more physiological than phorbol ester. The other MAP
kinase pathways SAP/JNK and p38 are also acutely activated by LPS, suggesting the
potential to use this as an assay for monitoring anti-inflammatory agents that target
these pathways [20, 21]. Other signaling pathways that can be acutely activated in
whole blood include JAK/STAT, which responds to GM-CSF or G-CSF (Figure 7.1a).
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