Issue 229 | December 2014

Biomarker Identification and Assay Development

Biomarker assays are now an integral part of the drug needs, and are offered as a stand-alone service or a fully
discovery and development process, acting as indicators of integrated drug discovery project. Below are just a few of the
drug efficacy, toxicity and disease progression, as well as applications for Charles Rivers biomarker identification and
assisting in patient selection and design of clinical trials1. assay development services.
In early stage drug discovery, biomarkers are used to validate
in vitro target modulation and routine cellular screening. Biomarker Identification
Typically, these assays are initiated in human cell lines,
The following illustrations demonstrate the breadth of
primary blood cells or isolated tissue cells. At this early stage,
Charles Rivers biomarker identification and validation
translational biomarkers are also investigated to identify
capabilities as applied in an oncology project. Using a
markers of drug sensitivity that ultimately may be used
validated tool compound, we were able to show significant
in selection of patient populations. As projects progress,
changes to primary and secondary pathway phosphoproteins
biomarker assays are developed for pharmacokinetic/
(Figure 1). This study was extended to look at modulation of
pharmacodynamic (PK/PD) models, profiling molecules prior
specific genes known to be dependent on the phosphotarget
to testing in longer term disease models as initial proof of
(Figure 2) and validated in the same experiment by
concept. PK/PD models can also assist in dose-to-man scaling
determining the gene signature elicited by short hairpin
predictions for use in clinical trials. The biomarker assays
RNAs. The biomarker was successfully translated into mouse
developed during the in vitro discovery phases are frequently
xenograft models with dose- and time-dependent inhibition
used as efficacy or toxicity endpoints in the clinic. Clinical trials,
of phosphoprotein observed in homogenized tumor material
particularly in oncology, are frequently designed around these
(Figure 3). Finally, the mechanism of action was confirmed
biomarkers.
by immunohistochemistry, with strong induction of apoptosis
observed in tumor sections from animals who had received
Charles River Early Discovery has broad expertise in
compound treatment for a set number of days (Figure 4).
biomarker identification and assay development across many
therapeutic areas, including, oncology, CNS, and metabolic
and respiratory diseases. We develop quantitative assays in
primary or immortalized cells or disease tissue that can be
used as pharmacodynamic or disease models. Ultimately,
these models will act as efficacy and translational markers
from the in vivo phase to the clinic. A broad range of endpoints
can be employed to support biomarker selection, including
genomic, proteomic, phosphoprotein and epigenetic markers.
Assay technologies include Luminex and Meso Scale for
multiplexed endpoints, although antibody-driven FACS analysis
and high-throughput mass spectrometry endpoints can also be
applied to ensure that the most relevant and sensitive assays
are employed.
Figure 1. Western blotting of compound-treated cells showing
Charles River has considerable experience in the development dose-dependent decrease in phosphoprotein levels (A), increased
of biomarker assays that have ultimately been used in the secondary protein marker (B+C), induction of apoptosis marker (D),
clinic. Our biomarker capabilities are catered to suit your and protein loading control (E).

To download previous Researcher issues on The SourceSM, please visit www.criver.com/thesource.

 Biomarker Assay Development
Charles River Early Discovery has extensive experience in
developing both efficacy and translational biomarker assays in
cell lines, primary blood cells and tissues. Formats range from
tissue mRNA levels, protein marker and signaling pathways to
cellular phenotypic changes. Assays are commonly developed
in immortalized cell lines initially, providing on-target cellular
readouts to support medicinal chemistry optimization projects.

These cellular assays can then be adapted for use in primary

Figure 2. Inhibition of gene expression in cell lines induced by tool
compounds or shRNA
blood and tissue cells, including human cells obtained from
an in-house donor panel, and from relevant species (typically
rodents) for PD and disease models. The efficacy biomarker
assays are used in PK/PD models to determine dosing
regimens, and in disease models to correlate target coverage
with disease-modifying effects. A range of assays for
pharmacodynamic models have been developed from

Apoptosis
receptor
occupancy in blood and tissue to functional readouts
125 of target function.
showing inhibition

100
Nuclear Receptor Antagonist Biomarker Assays

% Inhibition
As part of125
a glucocorticoid
75 receptor antagonist project, which
required compounds with good CNS penetration, ex vivo
100
binding assays
Vehicle
50 were developed Compound
in brain and other tissues to
determine the extent and duration of antagonist binding.2
% Inhibition
7525
The time course of antagonist binding in brain tissue,
50 0
together with compound levels, was monitored to allow
PK/PD modelling (Figure 5). Data from this model were then
25
-25compounds for evaluation in a longer term
used to select
disease model, 1 10 100 1000
0 and for functional effects in different regions of
the brain.2 [Compound] (nM)
-25
1 10 100 1000
[Compound] (nM)
Figure 3. Pharmacodynamic inhibition of phosphoprotein by four
tool compounds in mouse xenograft tumors. Protein levels were
compared to vehicle-treated control to determine percentage
inhibition of phosphoproteins.
Apoptosis

Figure 5. Antagonist nuclear receptor binding in rat brain at 2, 4 and 6

hours following a single oral dose of test compound.

Vehicle Compound

Figure 4. Changes in apoptotic protein levels (stained brown) in

mouse xenograft tumors after several days of treatment with tool
compounds. 125

100
bition

75
GPCR Antagonism Assay Tissue Biomarkers
FACS analysis is one of the most versatile formats for A lung phosphomarker was established to support an inhaled
biomarkers assays (Researcher issue 224, June 2014). kinase inhibitor program to determine the inhibitor duration

on
A FACS whole blood leukocyte shape change assay was of action (Figure 8). The inhibitors showed slow dissociation

C
established as a biomarker assay to support a GPCR kinetics from the target kinase, which gave prolonged
antagonist project. Antagonist effects were monitored as pharmacodynamic properties despite rapid clearance of the
blockade of agonist-induced shape change (Figure 6). compound from the lung.
The whole blood assay was used in the PK/PD model to
optimize the molecules, and ultimately in dose-to-man
predictions prior to clinical trials. The PD assay was used in a
Phase I trial to demonstrate inhibition of shape change at the
predicted human dose. The Phase I shape change data was
subsequently used to determine the dose range for Phase II
proof of concept studies, and also to assist in patient selection
for the
trials.

125

100
% Inhibition

75
Figure 8. Effect of a kinase inhibitor on lung phosphomarker levels in
50 mice at various times following a single intratracheal dose.

25
The key goal for the program in the following example was to
0 maximize the therapeutic index by topical compound delivery
-25 to the target tissue with the aim of restricting peripheral
1 10 100 1000 pharmacological activity (and potential side-effect liability).
[Compound] (nM) A tissue-based biomarker assay was established that enabled
parallel measurement of compound target engagement in both
Figure 6. Inhibition of leukocyte shape change by a GPCR antagonist the target and peripheral tissue post topical compound dosing
in human whole blood. in mice. Using a functional pathway endpoint measurement,
it was successfully established that topical compound dosing
Blood and Tissue Phosphomarker Assays to the target tissue achieved selective pharmacological
pathway modulation in the absence of peripheral
Blood Cell Biomarkers
pharmacological activity (Figure 9).
A phosphobiomarker assay was developed in isolated human
peripheral blood mononuclear cells (PBMCs) and in whole

blood to determine the impact of plasma protein binding on Peripheral Tissue
Pharmacology
the activity of target kinase inhibitors (Figure 6). Initially, these

assays were used to optimize the protein binding properties of 3000
14%
inhibitors. Subsequently, the whole blood assay was used in ns

pharmacodynamic models as an efficacy marker and in clinical Target Tissue
studies as a measure of target engagement and efficacy. 2000 Pharmacology

32%
p<0.01Tissue
Peripheral
1000 Pharmacology
3000
14%
ns

0 Tissue
Target
2000 Pharmacology
nd

nd
ity

ity
ity

ity

iv
iv

u
iv

iv
po

po
ct
ct
ct

ct

A
A

32%
A

A
om

om
d
d
e

p<0.01
ce
ce

C
tiv

tiv

+
+

du
du
itu

itu

1000
ity
ity

In
In
st

st
iv

iv
on

on
ct

ct
C

C
A

A
ed

ed
c

c
du

du

In

In

0
nd

nd
ity

ity
ity

ity

Figure 9. Tissue-based biomarker measurement demonstrates

iv
iv

u
iv

iv
po

po
ct
ct
ct

ct

A
A
A

om
om

Figure 7. Effect of a kinase inhibitor on phosphomarker levels in separation of target and peripheral tissue pharmacology is achieved
d
d
e

ce
ce

C
C
tiv

tiv

+
+

du
du

isolated human PBMCs and whole blood from the same donor.
itu
itu

by topical compound dosing.

ity
ity

In
In

st
st

iv

iv
on

on
ct

ct
C

C
A

A
ed

edc
c

du
du

In
In
Summary
Charles River Early Discovery has broad expertise in biomarker identification that encompasses genomic, proteomic, epigenetic,
phosphoprotein and phenotypic biomarkers across a range of cellular and tissue types. These assays have been successfully
employed to demonstrate pharmacological target engagement, establish translational PK/PD relationships to support clinical
dose projections and enable early clinical characterization of compound pharmacology for optimal dose setting for Phase II proof
of concept studies.

References
1. Colburn, W.A. Biomarkers in drug discovery and development: from target identification through drug marketing.
J Clin Pharmacol. 43 (4), 329-41 (2003).

2. Zalachoras, I., Houtman, R., Atucha, E., Devos, R., Tijssen, A.M., Hu, P., Lockey, P.M., Datson, N.A.,
Belanoff, J.K., Lucassen, P.J., Jols, M., de Kloet, E.R., Roozendaal, B., Hunt, H., Meijer, O.C.
Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator.
Proc Natl Acad Sci U S A., 110 (19), 7910-5 (2013).

For additional information, please visit The SourceSM, a secure portal that provides registered users with direct access to the
technical, scientific and educational resources available from Charles River. To register, visit www.criver.com/thesource.

askcharlesriver@crl.com
www.criver.com