Biomedical Engineering Reference
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in natural killer (NK) cells to provide a surrogate biomarker, since it is easier to obtain
sequential samples from normal blood cells rather than tumor tissue. In this case, it is
reasonable to expect that the levels of target inhibition in NK cells reflect those in the
cancer. They used rhodamine 123 as the Pgp substrate rather than a fluorescent
anticancer drug, since this has a much greater quantum efficiency and is therefore
detectable using low concentrations (50 nM). This limits the rate of bulk flow into the
cells, so that its efflux via Pgp is more evident. Because LY335979 tightly binds to
Pgp, target inhibition is maintained during mononuclear cell isolation, whereas a
weakly binding inhibitor is expected to dissociate. To standardize the assay, they
compared rhodamine uptake in the absence or presence of excess LY335979 added
ex vivo to completely inhibit Pgp and expressed the results for NK cells (identified by
dual staining with anti-CD56) using a formula based on these values. This assay was
then applied to a large number of clinical trial patients, and it clearly identified a wide
range of drug target inhibition during treatment, with almost 100% inhibition in some
patients. Importantly, Pgp inhibitionmeasured by the flow cytometry assay was found
to correlate closely with the plasma drug concentration in the same blood samples
(Figure 7.4) [32]. This paper therefore provides an excellent example of the rational
development and standardization of a flow cytometry assay to monitor a complex
process in a clinical trial and the correlation between the pharmacodynamics and
pharmacokinetics of a novel agent.
7.3.2 Cell Cycle Regulation
Flow cytometry is well suited to the study of normal and abnormal cell cycle
regulation. This is fundamental to understanding the action of cancer chemotherapy
FIGURE 7.4
Results from Rubin et al. [32], showing the close correlation between a flow
cytometry assay for P-glycoprotein functional activity in peripheral blood NK cells, and the
corresponding plasma concentrations of the P-glycoprotein inhibitor zosuquidar obtained in a
phase I clinical trial.
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