FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 120

for use in the clinic. In addition to the in vivo studies, certain toxicity assessments
(e.g., mutagenicity) are carried out in vitro using cell culture systems.
The objectives of individual studies are rarely as simple as compiling data
that inherently supports/rejects development of the drug candidate. Although some
studies can be crafted with such a “go/no go” outcome defined, the goal is more often
to better characterize and understand observed toxicities in order to minimize their
impact, thus providing a way forward. For example, if a drug designed to treat anemia
in cancer patients receiving chemotherapy is oncogenic, that drug series will probably
be terminated. However, if an antihistamine causes stomach ulcers, there may be
a chance to move forward if this liability can be abrogated in following studies by
changing the dosing regimen, formulation, etc.
6.2 FLOW CYTOMETRY AND BIOMARKERS
Toxicology studies include clinical observation, clinical pathology/chemistry end
point measurements, and macroscopic/microscopic pathologic examination at and
postnecropsy (for terminal studies). Tissues are typically harvested at necropsy and
either examined immediately (e.g., blood) or preserved (i.e., chemically fixed and/or
frozen) for future examination. Various tissues collected provide many opportunities
to apply flow cytometry in preclinical toxicology/safety assessment. Obviously, body
fluids such as peripheral blood and urine, routinely collected for clinical pathologic
analyses, are amenable to flow cytometric analysis. Perhaps just as obvious, isolation
of hematopoietic cells frombonemarrow, spleen, and lymph nodes for flow cytometry
is simply achieved. In general, as long as a single-cell suspension can be achieved
by homogenization and/or digestion protocols, flow cytometric analysis can be
performed on any solid tissue as well. Some examples of solid tissues that can be
analyzed by flow cytometry include kidney, liver, eye, testis, and pancreas. Even fetal
brain and whole embryos can be homogenized and single-cell suspensions prepared
with appropriate protocols.
But capability does not equate with applicability; nor does it define benefit. For
instance, flow cytometric analysis is superfluous in an MTD study where cell-specific
information is not necessary to define dose-limiting toxicity (e.g., gastric ulceration).
On the other hand, flow cytometric end points could prove to be extremely useful as
correlative data in a dose range-finding study or provide the primary data in cases
where a defined toxicity affects hematopoiesis or is immunomodulatory. It is also
a useful technique to apply to mechanistic studies in order to identify affected cell
types in a heterogeneous tissue or to measure specific effects (e.g., protein expression
changes, ion flux, etc.) on select target cells. Given an appropriate analytical end
point, one could argue the benefits of including flow cytometric analysis in many
study designs. Preclinical toxicology may also be tasked with finding biomarkers of
toxicity. It may be possible to move the drug candidate forward if a robust biomarker
is available that predicts and/or correlates with pathology, regardless of the type of
toxicity observed. For terminal studies in animals, this could be provided by analysis
of virtually any tissue that can be obtained at necropsy. But it is highly desirable to