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Issue 229 | December 2014
Biomarker Identification and Assay Development
Biomarker assays are now an integral part of the drug
discovery and development process, acting as indicators of
drug efficacy, toxicity and disease progression, as well as
assisting in patient selection and design of clinical trials1.
In early stage drug discovery, biomarkers are used to validate
in vitro target modulation and routine cellular screening.
Typically, these assays are initiated in human cell lines,
primary blood cells or isolated tissue cells. At this early stage,
translational biomarkers are also investigated to identify
markers of drug sensitivity that ultimately may be used
in selection of patient populations. As projects progress,
biomarker assays are developed for pharmacokinetic/
pharmacodynamic (PK/PD) models, profiling molecules prior
to testing in longer term disease models as initial proof of
concept. PK/PD models can also assist in dose-to-man scaling
predictions for use in clinical trials. The biomarker assays
developed during the in vitro discovery phases are frequently
used as efficacy or toxicity endpoints in the clinic. Clinical trials,
particularly in oncology, are frequently designed around these
biomarkers.
Charles River Early Discovery has broad expertise in
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.
Charles River has considerable experience in the development
of biomarker assays that have ultimately been used in the
clinic. Our biomarker capabilities are catered to suit your
needs, and are offered as a stand-alone service or a fully
integrated drug discovery project. Below are just a few of the
applications for Charles River’s biomarker identification and
assay development services.
Biomarker Identification
The following illustrations demonstrate the breadth of
Charles River’s biomarker identification and validation
capabilities as applied in an oncology project. Using a
validated tool compound, we were able to show significant
changes to primary and secondary pathway phosphoproteins
(Figure 1). This study was extended to look at modulation of
specific genes known to be dependent on the phosphotarget
(Figure 2) and validated in the same experiment by
determining the gene signature elicited by short hairpin
RNAs. The biomarker was successfully translated into mouse
xenograft models with dose- and time-dependent inhibition
of phosphoprotein observed in homogenized tumor material
(Figure 3). Finally, the mechanism of action was confirmed
by immunohistochemistry, with strong induction of apoptosis
observed in tumor sections from animals who had received
compound treatment for a set number of days (Figure 4).
Figure 1. Western blotting of compound-treated cells showing
dose-dependent decrease in phosphoprotein levels (A), increased
secondary protein marker (B+C), induction of apoptosis marker (D),
and protein loading control (E).
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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.
Apoptosis
% Inhibition
% Inhibition
Figure 2. Inhibition of gene expression in cell lines induced by tool
compounds or shRNA
These cellular assays can then be adapted for use in primary
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
receptor
occupancy in blood and tissue to functional readouts
125 of target function.
showing inhibition
100
Nuclear Receptor Antagonist Biomarker Assays
As part of125
a glucocorticoid
receptor antagonist project, which
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required compounds with good CNS penetration, ex vivo
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binding assays
in brain and other tissues to
determine the
extent
and
duration
of antagonist binding.2
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The time course of antagonist binding in brain tissue,
together with
compound levels, was monitored to allow
50 0
PK/PD modelling (Figure 5). Data from this model were then
25
-25compounds for evaluation in a longer term
used to select
1
10
100
1000
disease model,
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[Compound] (nM)
the brain.2
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1
10
100
1000
[Compound] (nM)
Apoptosis
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.
Figure 5. Antagonist nuclear receptor binding in rat brain at 2, 4 and 6
hours following a single oral dose of test compound.
Compound
Figure 4. Changes in apoptotic protein levels (stained brown) in
mouse xenograft tumors after several days of treatment with tool
compounds.
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bition
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Tissue Biomarkers
A lung phosphomarker was established to support an inhaled
kinase inhibitor program to determine the inhibitor duration
of action (Figure 8). The inhibitors showed slow dissociation
kinetics from the target kinase, which gave prolonged
pharmacodynamic properties despite rapid clearance of the
compound from the lung.
C
on
GPCR Antagonism Assay
FACS analysis is one of the most versatile formats for
biomarkers assays (Researcher issue 224, June 2014).
A FACS whole blood leukocyte shape change assay was
established as a biomarker assay to support a GPCR
antagonist project. Antagonist effects were monitored as
blockade of agonist-induced shape change (Figure 6).
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
75
Figure 8. Effect of a kinase inhibitor on lung phosphomarker levels in
mice at various times following a single intratracheal dose.
Peripheral Tissue
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Target Tissue
Pharmacology
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Figure 9. Tissue-based biomarker measurement demonstrates
separation of target and peripheral tissue pharmacology is achieved
by topical compound dosing.
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Figure 7. Effect of a kinase inhibitor on phosphomarker levels in
isolated human PBMCs and whole blood from the same donor.
Pharmacology
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Peripheral
p<0.01Tissue
1000
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Blood Cell Biomarkers
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
the activity of target kinase inhibitors (Figure 6). Initially, these
assays were used to optimize the protein binding properties of
inhibitors. Subsequently, the whole blood assay was used in
pharmacodynamic models as an efficacy marker and in clinical
studies as a measure of target engagement and efficacy.
om
Blood and Tissue Phosphomarker Assays
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Figure 6. Inhibition of leukocyte shape change by a GPCR antagonist
in human whole blood.
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[Compound] (nM)
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ce
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The key goal for the program in the following example was to
maximize the therapeutic index by topical compound delivery
to the target tissue with the aim of restricting peripheral
pharmacological activity (and potential side-effect liability).
A tissue-based biomarker assay was established that enabled
parallel measurement of compound target engagement in both
the target and peripheral tissue post topical compound dosing
in mice. Using a functional pathway endpoint measurement,
it was successfully established that topical compound dosing
to the target tissue achieved selective pharmacological
pathway modulation in the absence of peripheral
pharmacological activity (Figure 9).
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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., Joëls, 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).
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