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Biomarkers in Drug Discovery and Development: From Target Identification through Drug Marketing
Wayne A. Colburn
J. Clin. Pharmacol. 2003; 43; 329
DOI: 10.1177/0091270003252480

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ARTICLE
BIOMARKERS
COLBURN
10.1177/0091270003252480
DRUG DEVELOPMENT
IN DRUG DISCOVERY AND DEVELOPMENT
Biomarkers in Drug Discovery
and Development: From Target
Identification through Drug Marketing
Wayne A. Colburn, PhD, FCP
Biomarkers of disease play an important role in medicine and
have begun to assume a greater role in drug discovery and de-
velopment. The challenge for biomarkers is to allow earlier,
more robust drug safety and efficacy measurements. Their
role in drug development will continue to grow for the fore-
seeable future. For biomarkers to assume their rightful role,
greater understanding of the mechanism of disease progres-
sion and therapeutic intervention is needed. In addition,
greater understanding of the requirements for biomarker se-
lection and validation, biomarker assay method validation
and application, and clinical endpoint validation and appli-
cation is needed. Biomarkers need to be taken into account
while the therapeutic target is still being identified and the
concept is being formulated. Biomarkers need to be incorpo-
rated into a continuous cycle that takes what is learned from
the discovery and development of one series of biomarkers
and translates it into the next series of biomarkers. Optimum
biomarker development and application will require a team
approach because of the multifaceted nature of biomarker se-
lection, validation, and application, using such techniques as
pharmacoepidemiology, pharmacogenetics, pharma-
cogenomics, and functional proteomics; bioanalytical
T he current medical revolution began just prior to
World War II with the advent of antibiotics. New
approaches to treating infection brought about a move
to culture infections to improve the cure rate. This
could have been the beginning of the use of biomarkers.
This medical revolution continues today. Biomarkers
of disease play an important role in medicine and have
begun to assume a greater role in drug discovery and
development. The challenge for biomarkers is to allow
earlier, more robust measures of drug safety and
efficacy.
From MDS Pharma Services, Phoenix, AZ. Submitted for publication No-
vember 10, 2002. Revised version accepted January 24, 2003. Address
for reprints: Deborah L. Keefe, MD, MPH, FCP, P.O. Box 500, New Ro-
chelle, NY 10804-0500.
DOI: 10.1177/0091270003252480
J Clin Pharmacol 2003;43:329-341
DRUG DEVELOPMENT
method development and validation; disease process and
therapeutic intervention assessments; and pharmacokinetic/
pharmacodynamic modeling and simulation to improve and
refine drug development. The potential for biomarkers in
medicine and drug development will be limited by the least
effective component of the processes. The team approach
will minimize the potential for the least effective component
to be fatal to the rest of the process. As scientific/regulatory
foundations for biomarkers in medicine and drug develop-
ment begin to be established, successes and applications
will need to be effectively communicated with all of the
stakeholders, including not only internal and external
drug developers and regulators but also the medical com-
munity, to ensure that biomarkers are totally integrated into
drug discovery and development as well as the practice of
medicine.
Keywords: Biomarkers; drug discovery and development;
pharmacokinetic/pharmacodynamic modeling;
surrogate and clinical endpoints
Journal of Clinical Pharmacology, 2003;43:329-341
©2003 the American College of Clinical Pharmacology
Biochemical and molecular markers have been used
in medicine for disease characterization and diagnosis
for centuries. Together, biomarkers from the past and
present provide greater possibilities to get safer and
more effective drugs to market faster. Biomarkers are
factors that are objectively measured and evaluated as
indicators of normal biological processes or pathogenic
processes and/or as indicators of pharmacological re-
sponses to therapeutic intervention. Clinical endpoints
are variables that can be used to measure how patients
feel, function, or survive. Surrogate endpoints are
biomarkers that are intended to substitute for a clinical
endpoint. In a few select cases, biomarkers have
evolved to become partial surrogate endpoints.1 No sin-
gle biomarker has predicted an adequate portion of the
observed clinical safety and efficacy outcome to war-
329
 COLBURN
Table I Biomarkers, Surrogate Endpoints, and Clinical Endpoints/Outcomes
Biomarkers
Leukotrienes, cytokines, and chemokines
Pulmonary function tests
Glucose, fructosamine, glycosylated albumin, and HbA1c
Retinal evaluations, nephropathy measures, and peripheral
neuropathy assessments
Cytokines
Angiotensin-I, angiotensin-II, plasma renin, aldosterone,
and angiotensin-converting enzyme (ACE) activity
Blood pressure and heart rate measures
Surrogate endpoints
HbA1c
HIV/CD4
Blood pressure
Tumor size
Cholesterol (HDL, LDL, VLDL)
rant ideal surrogate endpoint status,2 so biomarkers
that have successfully predicted partial clinical out-
comes have been called surrogate endpoints. As more
than 100,000 proteins are in the human body and most,
if not all, act on a variety of biological processes, why
should a single protein biomarker provide that kind of
insight? At best, a single biomarker will predict a por-
tion of the overall effect. For example, CD4 cell counts
account for about 30% of the increased survival time,
whereas CD4 plus HIV viral load accounts for approxi-
mately 70%a (Table I). It will take more than one
biomarker to describe disease and treatment, while it
will take more than one additional biomarker to de-
scribe treatment-related adverse events. Although sev-
eral biomarkers may be needed to create an ideal surro-
gate endpoint cluster to truly characterize clinical
endpoints, individual biochemical/molecular markers
can be used as biomarkers to evaluate disease progres-
sion and to evaluate the effects of therapeutic interven-
tion early in development. Mechanism-based
biomarkers should be used in drug discovery and de-
velopment to support early and correct decisions. They
will also be useful in later development to optimize
dose selection, stratify patients to better understand
drug effects/select treatments, and ensure prognosis.
Throughout the drug discovery and development
pipeline, biomarkers are identified that create the foun-
a. Personal communications with FDA biostatisticians involved
in the decision to use CD4 cell counts plus HIV viral burdens as sur-
rogates for AIDS outcomes.
330 • J Clin Pharmacol 2003;43:329-341
Clinical Endpoint/Outcome
Asthma, chronic obstructive pulmonary disease, and
rheumatoid arthritis
Asthma and chronic obstructive pulmonary disease
Type 1 and 2 diabetes mellitus
Type 1 and 2 diabetes mellitus
Type 1 diabetes mellitus
Hypertension
Hypertension
Retinopathy, cardiomyopathy, neuropathy, and nephropathy
Survival time
Myocardial infarct/stroke—survival time
Survival time
Myocardial infarct/stroke—survival time
dation for a continually evolving biomarker team (Fig-
ure 1). A continuous cycle, as well as improvements in
drug development and medicine, can occur by apply-
ing biomarkers. Because of the complexities with and
the multifaceted nature of biomarker selection and val-
idation, a multifunctional team is needed to integrate
biomarker concepts across discovery and develop-
ment. These teams should include the areas of
pharmacogenetics, pharmacogenomics, and functional
proteomics; bioanalytical method development and
validation; disease process and therapeutic interven-
tion assessments; and use of biomarkers for
pharmacokinetic/pharmacodynamic (PK/PD) model-
ing and simulation. The potential for biomarkers in
medicine and drug development is limited by the least
effective component of these activities. The team mini-
mizes the potential for the least effective component to
be fatal to the rest of the process. Once the scientific and
regulatory basis for biomarkers in medicine and drug
development has been established, successes and ap-
plications need to be effectively communicated with
all of the stakeholders, including, but not limited to, us-
ers of the data, corporate management, regulators, in-
ternal and external experts, and the medical commu-
nity, to ensure that biomarkers are totally integrated
into medicine and drug development.
This article is divided into five sections, including
(1) mechanism-based biomarkers, (2) PK/PD modeling
concepts, (3) biomarker assay methods designed to
meet drug development objectives, (4) clinical end-
point issues, and (5) issues and opportunities. Each of
 BIOMARKERS IN DRUG DISCOVERY AND DEVELOPMENT

Other Providers
For biomarkers to be useful in drug development,
Stakeholders they should reflect biochemical/molecular changes
Clinical Laboratories
Hospitals Concept
Diagnostics that occur due to the disease process and are altered by
HMOs/PPOs
therapeutic intervention prior to a downstream impact
Insurers Marketing Discovery on the clinical endpoint.3-5 The changes are generally
Physicians Chemistry
Phase IIIb > Phase IV/V HTS
mediated through receptor or enzyme induction or in-
Pharmacists
hibition. In some cases, the effects are mediated
Patients
through direct interaction with the deleterious bio-
Etc. Preclinical chemical agent, such as when antibodies or receptors
Regulators Development are used to bind cytokines. Ultimately, some of these
Pharmacology
Toxicology biomarkers may become surrogate endpoint clusters
PK/DM that predict observed outcomes. For example, develop-
Clinical ing the first angiotensin-II (A-II) antagonist with the be-
Development lief that it would reduce blood pressure and result in re-
Phase I > Phase Ib > Phase II a >
duced incidence of strokes and myocardial infarctions
Phase II b > Phase III
represented a sequential process. First, it was impor-
tant to show that the drug bound to the A-II receptor
Figure 1. The team takes center stage. Stakeholders and other func-
and altered the renin-angiotensin system, as was pre-
tions that can produce and receive information and knowledge from
the team occupy the surrounding area. The schematic emphasizes dicted from its intended mechanism of action. Changes
the critical team concept as well as the critical nature of continuous, in biomarkers such as angiotensin-I, angiotensin-II,
effective, and efficient communication. plasma renin, and angiotensin-converting enzyme
(ACE) inhibition were used for this purpose.6 This,
however, did not ensure that blood pressure would be
reduced or that this mechanism of blood pressure re-
duction would result in reduced stroke and heart at-
these topics contributes to the impact that biomarkers tacks. Each sequential step that contributed to linking
are having and can have on drug discovery and devel- the biomarker to the clinical outcome was important to
opment. In the final analysis, the goal is to identify a se- the total understanding of the process and to establish-
ries of biomarkers that can characterize the disease pro- ing surrogate endpoints to predict clinical outcomes.
cess, be measured robustly, and assist in early and Biomarkers can provide great value in early drug devel-
correct decisions for compound selection and efficient opment if they reflect mechanism-based intervention,
drug discovery and development. even if they do not ultimately become part of surrogate
endpoint clusters.7,8 An increase in our understanding
MECHANISM-BASED of disease and drug mechanisms of action at the bio-
BIOMARKERS chemical and molecular level is producing greater num-
bers of biomarkers (see Table I for a few examples).6
Clearly, all biomarkers should represent mechanism- Useful biomarkers provide value because they occur
based processes. Early in discovery and development, earlier than clinical endpoints/outcomes and/or are
the biomarker should at least reflect activity that is me- measured by more robust methods.6 Biomarkers that
diated through the theoretical disease mechanism of are proximal to the clinical endpoint but occur earlier
action. Later in development, the biomarker should in time make drug development and approval more ef-
represent mechanism-based processes that are critical ficient. Proximal, rather than distal, biomarkers mini-
to disease progression and that are appropriately al- mize the potential influence of disparity between the
tered by effective therapeutic interventions. However, biomarker and effect that can result from other inter-
some biomarkers are simply associated with the dis- vening variables, but the biomarkers still must occur
ease, do not drive the disease process, or are not altered earlier in time and/or be measured by a more robust
by therapeutic intervention that acts on the disease method than the clinical endpoint. As earlier drug ap-
mechanism. False-positive results occur when there is provals occur using biomarkers of efficacy, biomarkers
an assumption that the biomarker is a critical part of the of drug safety will also be needed to evaluate safety and
disease process when in fact it is only loosely associ- efficacy in parallel. For example, observing seizures
ated with the disease process or a random event that that result from drug-induced epileptiform activity in
has inadvertently coincided with disease diagnosis an EEG will no longer suffice. A biochemical/molecular
and progression. marker that predicts the EEG changes and resulting sei-

DRUG DEVELOPMENT 331

 COLBURN
zure early enough to anticipate and avoid the seizure is
needed.
Biomarkers have been used for diagnosis and, in
some cases, for prognosis for centuries, for example,
when physicians tasted urine to determine whether a
person had diabetes. Biomarkers are used when look-
ing for hypersecretion of glucocorticoids to help in the
diagnosis of Cushing’s syndrome. When elevated HER2
concentrations are used to indicate the appropriate
women for Herceptin breast cancer treatment, a
biomarker is being used. Biomarkers, such as CD4 cell
counts, and viral loads have been instrumental to fast-
track drug development and approval, as well as pre-
dict the need for treatment changes to optimize ther-
apy. In contrast, there have been examples of
biomarkers such as those that reduce premature ven-
tricular contractions, which were expected to reduce
the incidence of sudden death but actually increased
sudden death.9
Differences that exist between biochemical/molecular
biomarker concentrations or clinical biomarker mea-
sures in disease and health may or may not be related to
the disease mechanism. Differences in biomarker val-
ues may occur because of the disease process if the
biomarkers are mechanism based or may occur because
of circadian rhythms or other physiological, pathologi-
cal, or pharmacological processes that are not based in
the disease of interest. In addition, apparent differ-
ences can occur because of lack of reproducibility in
the analytical method. Or, differences between disease
and health may not be robust enough to guide future
drug development or prognosis (Figure 2).6-8,10-12 For
biomarkers and PK/PD modeling to be efficient and ef-
fective, there must be more than just an association be-
tween drug concentrations and response—there must
be a correlation.
Identifying proteins and their functions using
genomic information—proteomics—allows for more
rapid identification of new biomarkers.13-17 Comparing
a homogeneous subpopulation along with familial
tracking and comparing genes within a heterogeneous
population database that encompasses the target dis-
ease are two ways to identify disease-related genes. In
both approaches, bioinformatics software is used to de-
tect differences in gene expression from healthy as well
as mild and severely diseased tissues. Gene and protein
sequencing and synthesis can be used to find, evaluate,
and confirm the appropriate genes and biomarker gene
products for target diseases.
Differing protein profiles from diseased and normal
tissues can also lead to biomarker discovery and identi-
fication. Knowledge of the protein will lead to gene
identification. Protein targets such as enzymes and re-
332 • J Clin Pharmacol 2003;43:329-341
Disease Process
Biomarker 1a Biomarker 1 Biomarker 2 Biomarker 3
Indirect Direct Direct Not Linked
Reliable Unreliable Reliable Reliable
Surrogate Surrogate
Clinical Endpoint/Outcome Clinical Endpoint/Outcome
Figure 2. Biomarkers can contribute to or detract from drug devel-
opment. Biomarkers 1a and 2 can contribute positively to develop-
ment, whereas biomarkers 1 and 3 will detract from the development
process. Biomarker 1 is a potentially useful marker but cannot be as-
sayed effectively and therefore will detract from team progress.
Biomarker 3 appears to be, but is not, part of the therapeutic interven-
tion process.
ceptors that are linked to downstream protein
biomarkers can then be used as part of high-throughput
screening for discovery. These screening tools are then
used to set the course for biomarker selection during
preclinical and clinical drug development. Animal
models that incorporate relevant biomarkers can be es-
tablished to reduce the number of drug candidates that
will be considered as potential lead compounds by
looking at preclinical drug effects on biomarkers of hu-
man disease.12,18
Endogenous ligands compete with drugs for recep-
tor occupancy and can thereby influence therapeutic
drug intervention.19 This competition is consistent
with certain protein biomarker changes observed be-
tween health and disease. The observation is also con-
sistent with dosing of receptor agonists/antagonists
causing a transient increase in the circulating concen-
tration of a naturally occurring agonist/antagonist. In
addition, this observation is consistent with dosing of
an enzyme inducing or inhibiting agent-altering meta-
bolic processes that result in increased or decreased
concentrations of a substrate. For example, it has been
shown that antibodies or biotechnology-derived recep-
tors (etanercept) that bind endogenous ligands such as
TNF-α reduce ligand concentrations that may be found
in excess in certain disease states and thereby cause the
disease to revert to a more healthy state.19 These exam-
ples support using changes in biochemical/molecular
marker concentrations as one way to monitor the ef-
fects of disease treatment (Figure 3).20
 BIOMARKERS IN DRUG DISCOVERY AND DEVELOPMENT
Figure 3. Enzyme/receptor binding of endogenous ligands in
health and disease and the influence of binding to an exogenous
antibody/receptor on the unbound amount of endogenous ligand
during therapy of the disease. In segment a, endogenous receptors
are half-filled in health. In segment b, endogenous receptors are to-
tally saturated in disease. In segment c, administration of exogenous
antibody or receptor reestablishes the healthy half-filled balance.
Adapted from Colburn.19
DRUG DEVELOPMENT
PK/PD MODELING
Biomarkers create value throughout the drug devel-
opment process by providing early decision-making
information and input for PK/PD modeling.20-24
Pharmacokinetics is concerned with what the body
does to the drug. Drug concentration-time profiles are
generated. Pharmacodynamics is concerned with what
the drug does to the body. Response-time profiles are
generated. PK/PD modeling is a mathematical treat-
ment of the PK and PD data in an effort to gain greater
insight into how drug concentrations and effects relate
to each other, as well as how the models can be used to
predict study outputs under a variety of different con-
ditions. More modeling of disease processes without
therapeutic intervention is needed so the disease com-
ponent of the PK/PD model can be better understood
and applied. Several recent review articles discuss the
potential for PK/PD modeling in drug discovery and
development.18,25-32 This section focuses on important
issues and concepts that apply to PK/PD modeling and
biomarkers.
A series of questions need to be answered when
identifying PK inputs for PK/PD modeling and
simulation.
Question 1: What needs to be measured? A series of ques-
tions that relate to providing sufficient input for infor-
mative PK/PD modeling need to be asked and an-
swered at this time, such as the following:
Will drug or drug plus active metabolites be measured?
If active metabolites are present, do they contribute to
the activity profile following single and/or repeated
doses of the drug?
Will unbound drug concentrations or total (unbound
plus bound) drug concentrations be assayed? Is bind-
ing linear or nonlinear? If binding is nonlinear, un-
bound concentrations need to be measured to provide
appropriate input for PK/PD modeling.
Does the drug compete with endogenous ligands for re-
ceptor binding? The answer is most likely yes. Can this
competition be built into the PK/PD models?
Question 2: Do biomarker responses parallel drug and/or
active metabolite concentrations? Generally, they do
not because there is a temporal delay between concen-
trations and effects. PK/PD models are used to better
understand this relationship and how it might change
as a function of drug input and other variables.
Any decision on which approach to take is based on lo-
gistics as well as experience and receptivity of the
study sites and investigators to each approach.
Having only one active moiety rather than several or
one that dominates the beneficial and toxic effects of
therapeutic intervention makes PK/PD studies simpler.
In addition, determining whether to measure unbound
333
 COLBURN

or total (unbound plus bound) drug concentrations can 100

be another variable. If drug-plasma binding is linear in

Unbound/Total Plasma Dry Concentration

90
the therapeutic and toxic range, unbound and total 80
concentrations are simple ratios. For example, if bind- 70
ing is 75% and linear, unbound concentrations are 60
25% of the total drug concentration across the entire 50
concentration range: 2.5 unbound at 10 total, 25 un- 40
bound at 100 total, and 250 unbound at 1000 total. In 30
contrast, nonlinear plasma binding requires unbound 20
drug concentrations to be measured because unbound 10
concentrations increase more than proportionately 0
with increasing total drug concentrations, as shown in

0
10
20
30
40
50
60
70
80
90
0
0
0
0
0
0
0
0
0
0
0
10
11
12
13
14
15
16
17
18
19
20
Figure 4. Concentration
Competitive endogenous ligands complicate the PK
Figure 4. Impact of linear and nonlinear binding. This graph shows
and PD inputs33 for PK/PD models. Environmental, di- the percentage of unbound/total drug concentration as a function of
etary, and endogenous substances compete with drugs total plasma drug concentrations under linear and nonlinear (satu-
for receptor occupancy in vivo.19 Should drug develop- rable) binding conditions. In a linear situation, percent unbound re-
mains constant as plasma drug concentrations increase. In contrast,
ers identify these competitive substances and use them during saturable binding, the unbound/total drug concentration ra-
as biomarkers to track the effects of their drug develop- tio increases as total drug concentrations increase. In this example,
ment programs? Or, are these competing substances almost no drug is unbound at concentrations less then 10 units, but
just obstacles that complicate assay development and about 90% is unbound at concentrations in excess of 100 units.
When plasma drug binding is saturable, drug assays should measure
validation as well as modeling efforts? As these sub- unbound concentrations because they vary as a function of total drug
stances compete with the drug for receptor binding, concentrations, and only unbound drug elicits drug effects.
they must be acting to stimulate or inhibit physiologi-
cal activity that could be a predecessor to pharmacolog-
ical activity at greater concentrations.34-36 It can be an-
ticipated that changes in endogenous ligand
concentrations following drug dosing may be similar to
changes observed in angiotensin-I, angiotensin-II, and
plasma renin following doses of an angiotensin-II an-
tagonist, as shown in Figure 5.6 It is likely that baseline
activity is associated with endogenous competing sub-
stances, even in the absence of drug. For comprehen-
sive PK/PD modeling, it is important to determine con-
centrations of the competing ligand(s) after both active
and placebo treatment so that competition can be built
into the models.33
PK/PD models should never be more complicated
than required to fit existing data and predict future
data. Models should only include rate-limiting,
mechanism-based components of the system that are
required to describe the data.25,37,38 For example, if the
rate-limiting PD step is receptor mediated, then the
model should reflect receptor binding. In contrast, if
Figure 5. Biomarker changes as a function of angiotensin-II inhibi-
the rate-limiting step is hormone secretion or protein
tor dosing. Angiotensin-I (A-I), angiotensin-II (A-II), and plasma
production, then the model should reflect the secretion renin activity increase from placebo/baseline in response to the ad-
process. If PK/PD models are not simple and unique, ministration of an A-II receptor antagonist. There is a direct effect of
model-predicted endpoints may provide inappropriate the antagonist on A-II when the antagonist inhibits the binding of A-II
outputs during simulation under different conditions. to the receptor. The resulting increase in A-II concentrations (䊏) then
works through a feedback loop in the renin-angiotensin system to
Sensitivity analysis has been used to identify unique
also increase A-I (starts) and plasma renin activity (䊉). This process
model solutions when transitioning from fitting a represents the types of changes that occur when a therapeutic inter-
model to data to using the model to simulate several po- vention affects biomarkers of disease to elicit a clinical endpoint.
tential outputs.39 Adapted from McShane et al.41

334 • J Clin Pharmacol 2003;43:329-341

 BIOMARKERS IN DRUG DISCOVERY AND DEVELOPMENT
BIOMARKER ASSAY METHODS
Whether chemical assay methods or binding assays are
used, biomarker assay development and method vali-
dation tend to be more complicated than those for most
drug assays.27,40,41 Several factors that can contribute to
this complexity will be discussed below. For example,
biomarkers have diverse molecular structures ranging
from simple electrolytes and amino acids to large pro-
teins to complete microorganisms such as viruses and
bacteria. As a result, it is generally not possible to vali-
date biomarker analytical methods according to strict
good laboratory practices (GLPs). Recent efforts have
looked at providing GLP-like validation by evaluating
all aspects of the method, including precision,
reproducibility, and reliability according to GLP crite-
ria, but without attempting to determine accuracy.42-45
GLP-like validation provides scientifically valid infor-
mation about the biomarker that can be used to deter-
mine changes in biomarkers associated with disease
progression as well as therapeutic intervention provid-
ing reliable data for critical decision making.11,28,43,45,46
Choice of Matrix
The first challenge is to identify and select an accessi-
ble, meaningful sample matrix. Readily accessible ma-
trices such as whole blood, plasma, serum, urine, sa-
liva, or sweat are preferred over more difficult to obtain
sample matrices such as biopsy or bronchoalveolar la-
vage samples. For example, plasma or urine drug con-
centrations are often used as surrogates for drug con-
centrations in the target tissue during pharmacokinetic
studies. It is convenient to measure biomarkers in these
same matrices. However, the site of biomarker produc-
tion as well as the physiology and distribution of the
biomarkers must be taken into account before an ac-
ceptable matrix can be selected. Biomarker concentra-
tions are generally lower in the systemic circulation
than they are at the site of production. For example,
cytokines can be assayed in bronchoalveolar lavage
fluid, sputum, plasma, peripheral mononuclear cells,
or whole blood from asthma patients.47-51 The first three
matrices can be assayed directly for cytokines, whereas
the latter two matrices must be stimulated to evaluate
individualized cytokine production. If the assay
method has sufficient sensitivity, plasma or sputum is
preferred for ease of collection and analysis. If analyti-
cal sensitivity is not sufficient to measure the
biomarker in these matrices, bronchoalveolar lavage
can become the preferred matrix because of greater
concentrations, but variability and complexity of sam-
ple collection and preparation are also greater.
DRUG DEVELOPMENT
Reference Standards
Another challenge for biomarker assays is obtaining
biomarker/analyte-free matrices to prepare calibrator
standards. Several approaches have been used such as
nonspecific or affinity-based removal of desired
biomarkers, use of matrix from a different species, or
use of a similar protein-buffer solution. However, ma-
trices produced by any of these methods are not the
same as an authentic sample matrix. Therefore, assay
performance must be tested during prestudy validation
to show that assay performance is similar between au-
thentic and nonauthentic matrices.46 In some cases,
pure reference standards are not available.
Heterogeneity and Quantification
Macromolecular biomarkers exist in heterogeneous
forms. For example, vascular endothelial growth factors
(VEGF) are important biomarkers for carcinogenesis.52
VEGF is expressed in at least four forms with 121, 165,
189, and 206 amino acid residues. These forms are dis-
tributed differently into vascular space and
extracellular fluids.53-55 It might be possible to obtain
one pure form and use it as a reference standard to
quantify VEGF samples as standard equivalents with-
out specifying exact composition. This is analogous to
using nonspecific radioactivity measurements of drug
plus breakdown products to evaluate mass balance fol-
lowing a dose of radiolabeled drug.
Biomarkers can also exist in various bound states,
as is the case for prostate-specific antigen (PSA),
which occurs in serum both free and bound to α1-
antichymotrypsin, α1-antitrypsin, protein C, and α1-
macroglobulin. The World Health Organization is
attempting to standardize reference PSA material as
a characterized mixture.56,57 Similarly, standardiza-
tion of biomarker calibrators is an ongoing effort for
clinical chemists and bioanalytical chemists work-
ing to improve biomarker assay methodology for
drug development.25,26
In contrast to the term definitive quantification,
which is used to describe assay results for well-
characterized small drug molecules that can be accu-
rately measured with a single standard calibrator, rela-
tive quantification is a term that is used when endoge-
nous macromolecular biomarkers are measured and
accuracy cannot be determined.45 An example is when
VEGF-165 is used as the standard calibrator to quantify
all VEGF isoforms. For relative quantification,
postdose sample values are generally compared to
predose sample values from the same subject to deter-
mine the relative effect of drug intervention. This ap-
335
 COLBURN
proach minimizes the need for accuracy but still de-
pends on precision, reproducibility, and an otherwise
robust assay method. Data are normalized and ex-
pressed as percent change from predose baseline
biomarker concentration. More definitive data can be
obtained by comparing biomarker concentrations over
the same sampling schedule following placebo and ac-
tive treatment.
Semiquantitative assays are used for antidrug anti-
body, enzymatic, or receptor or cell-based assay meth-
ods when well-characterized reference standards are
not available.58-60 Categorical or classification results
are often used when assay methods do not exhibit con-
tinuous concentration-response relationships. Dilu-
tion assays are those in which concentrations are based
on all or none activity at dilutions of 1:2, 1:4, 1:8, 1:16,
1:32, and so forth. Biomarker assays based on biologi-
cal systems can exhibit discontinuous categorical
changes such as none, low, medium, and high or di-
chotomous changes such as yes/no or 0/1. The term
classification assay has been used to describe categori-
cal assays that are used to provide descriptive order
such as +/– or 0, 1, 2, 3, 4, 5 to biological reactions.61-63
Categorical quantification is more akin to clinical end-
point measures than to typical bioanalytical results.64-66
Therefore, to provide added value, less robust and less
quantitative categorical biomarker assays should be re-
served for intermediate biomarkers that occur much
earlier than the clinical endpoint.
Immunoassay methods are generally more compli-
cated than chemical methods for biomarker measures.
For example, tight-binding soluble receptors such as Il-
2R may bind biomarkers differently between healthy
and diseased subjects, within patient groups, or in
the same patient at different times. 45,46 Since
immunoassays are based on binding, each form of a
heterogeneous group of biomarkers can react differ-
ently and thereby affect assay results in an antibody-
based assay system. Biological activity of various forms
may or may not parallel antibody-binding response
curves. Although functional biological assays such as
enzyme, cell-based, or tissue perfusion assays have
greater variability and are not as sensitive as immuno-
chemical or chemical methods, they may complement
quantitative analyses.67-69 In general, functional assays
are labor intensive and time-consuming, so they would
not be practical for use as a primary measurement tool
for large clinical trials.
Multiple Biomarkers
Several biomarkers from a multitude of discovery/
preclinical markers can be selected to test during ex-
336 • J Clin Pharmacol 2003;43:329-341
ploratory clinical research and development. The prac-
ticality of multiple biomarker assays, sample volume
limitations, requirements for adequate sample collec-
tion and handling, and multiple assay methods must be
considered. The number of biomarkers may be nar-
rowed to just a few that are able to show differential,
mechanism-based effects during early drug develop-
ment. A final cluster of biomarkers can be selected for
larger Phase III proof-of-safety and efficacy trials. Out-
put data must be reliable and robust to pass Food and
Drug Administration scrutiny during the end of Phase
II meetings and, ultimately, new drug application re-
view for market approval. Using information and
knowledge gained during early development, method-
ology and assay dynamic ranges should be optimized,
and proper method validation should be carried out be-
fore designing later phase clinical protocols.70-72
What Is Reasonable?
It is not practical or necessary to perform full validation
of biomarkers during early exploratory phases of drug
development, as long as the methods provide reliable
data, information, and knowledge.28 As drug develop-
ment progresses, validation should keep pace with the
required precision and reliability needed to achieve
program objectives.6,43 Prestudy validation should be
completed before clinical studies are begun and should
set the foundation for establishing method acceptance
criteria. Prestudy validation should include a pilot
study to provide insight into the differences that exist
between disease and health as well as differences in
biomarker variability between health and disease.
Bioanalytical results will be used to assess whether the
method can provide the sensitivity, precision, and ro-
bustness to distinguish the differences between disease
and health as well as to quantitatively evaluate the pro-
gression from one state to the other.73,74 Assay perfor-
mance acceptance criteria should be established a pri-
ori based on the objectives of the study and the known
assay variability.74
In-study sample analyses and validation must use
quality control samples (QCs) to document analytical
performance during clinical studies.42 In addition, QCs
are used for the acceptance or rejection of an analytical
run during sample analysis. Similar to drug assay vali-
dation, QCs are generally prepared to evaluate the
lower, middle, and upper limits of standard curve
ranges. QCs are made in the same matrix as the study
samples, so assay performance determined from the
QCs reflects that of authentic samples. Therefore, even
if assay standards are prepared in a nonauthentic ma-
trix, QCs must be prepared in an authentic matrix. Au-
 BIOMARKERS IN DRUG DISCOVERY AND DEVELOPMENT
thentic matrix samples are screened to determine basal
biomarker concentrations. Low basal concentrations
are selected, and then various middle and upper QC
concentrations can be prepared by adding additional
known biomarker amounts. For semiquantitative
methods, QCs can be prepared by combining samples
with high, middle, and low observed concentrations.74
Validation using these approaches provides precise
and stable data but does not provide accurate data.
CLINICAL ENDPOINT ISSUES
Intuitively, it can be assumed that clinical endpoints
that measure how a patient feels, functions, or survives
are the best measures of disease progression and effec-
tiveness of therapeutic intervention. This is true for the
practice of medicine but not necessarily in drug devel-
opment and regulatory review. Except for survival or
cure, clinical endpoints have questionable value for
determining whether a biomarker can predict reason-
able clinical endpoints in the current clinical develop-
ment environment. From a drug development perspec-
tive, clinical endpoints and clinical endpoint measures
are inextricably linked. If the clinical endpoint cannot
be measured or cannot be measured reliably across sev-
eral patients, several sites, or several studies, it has no
value within the context of drug development and the
regulatory approval process. Clinical endpoint ratio-
nalizations are often found deep in the history of dis-
ease, diagnosis, and prognosis. In many cases, results
from one clinical investigative site cannot be repro-
duced at another site because measurement methods
are somewhat subjective and dependent on operator-
specific performance. Generalizing a clinical endpoint
to another patient population because it was useful in
one patient population can be misleading. It is likely
that false-positive and false-negative clinical endpoint
measures have contributed to incorrect understanding
and inappropriate application of clinical endpoints.
Biomarker assay results tend to be more robust than
clinical endpoint measures, but they must be based on
both disease and therapeutic mechanisms.23,24
From a drug discovery and development perspec-
tive, clinical endpoint measurements such as visual
analogue scales, pulmonary function tests, and cogni-
tive function tests that assess how a patient feels or
functions should meet the same GLP-like acceptance
criteria that are required for biochemical/molecular
biomarker assay methods. Clinical endpoints must be
based on known mechanisms of action or the disease
process that is being treated. As stated earlier, preci-
sion, reproducibility, and reliability are more impor-
tant than accuracy for clinical endpoint measurement
DRUG DEVELOPMENT
methods as well as for biochemical marker methods
since relative outputs are used for decision making
during drug development. Accuracy becomes impor-
tant to ensure continuity throughout the development
process when results from one method need to be com-
pared to another or one method replaces another dur-
ing drug development. Clinical endpoint measure-
ments need to generate outputs that are as useful and
informative as drug assays and biochemical/molecular
marker assays.6,20 Clinical endpoint validation criteria
are outlined in Table III.
It should be easier to obtain reproducible GLP-like
clinical endpoint and biomarker output from a single
site than from several independent clinical trial sites.
Therefore, a single site or a small number of common
Table II Evolution of Biomarkers
during Drug Discovery and Development
Theoretical foundations for disease processes and drug
mechanisms
Experimental foundations for disease processes and drug
mechanisms
Preclinical In vitro binding to enzyme or receptor
In vivo in animal models (transgenic?)
Clinical Healthy human subjects in Phase I
Healthy human disease models in Phase Ib
Human patients in Phase IIa
Human patients in Phase IIb/III
Epidemiological evidence
Previous clinical experience with therapeutic class
Previous clinical experience with biomarker
Simulated biological systems
Table III Clinical Endpoint
Method Validation Requirements
Create uniform instrument specifications
Document maintenance and calibration
Require study participants to use same instrument
Create consistent/controlled environment for testing
Implement uniform staff and study participant training
procedures
Document training procedures (standard operating
procedures)
Document that training and proficiency testing is complete
Implement quality control procedures
Use replicate (n ≥2) measurements if possible
Use baseline and placebo-controlled measurements for
reference
337
 COLBURN
Table IV Types of Validity to Evaluate Biomarkers
Content validity—measuring the right thing
Construct validity—the empirical relationship is consistent with the theoretical relationship
Criterion-based validity such as (1) concurrent—consistent measures, (2) predictive—later evidence supports,
and (3) prescriptive—used to select treatment
standard operating procedure sites are preferred during
early clinical development to optimize data that can
provide insight into the disease process and the influ-
ences of therapeutic intervention.75-77 At the same time,
it will be important to be able to generalize the proce-
dures that have been used and the results that have
been obtained during the early phases to later
multicenter trials if biomarkers are to continue to pro-
vide insight during later clinical phases of develop-
ment.6 Following specific validation guidelines can
minimize site-to-site variability (Table IV). It is critical
to ensure that biomarkers and clinical endpoints meet
both content and construct validity requirements. If
biomarkers meet predictive validity criteria, they be-
come surrogate endpoints, and it is now becoming
more common to use prescriptive biomarkers to select
appropriate treatment. Appropriate selection of both
predictive and prescriptive biomarkers is dependent
on clinical endpoints that, themselves, are appropriate
and measured reliably. For example, it would be appro-
priate to select a treatment that reduces IL-4 or TNF-α
concentrations if IL-4 or TNF-α concentrations are ele-
vated in the patient and are mechanistically based in
the disease process. In contrast, it is pointless to take
this approach if IL-4 or TNF-α concentrations are in the
normal range or are not involved in the disease process
(Figure 3).
ISSUES AND OPPORTUNITIES
Comprehensive use of biomarkers or endpoints for PK/
PD modeling has typically been initiated during clini-
cal development. Starting to think about modeling dur-
ing clinical development is too late. Plans to incorpo-
rate biomarkers, clinical endpoints, and PK/PD
modeling in drug discovery and development need to
occur when a new therapeutic target is being identified
and the discovery process is starting. High-throughput
screening targets can serve as initial sources of poten-
tial biomarkers. For example, if the discovery process is
going to target a specific pathogen, enzyme, or receptor,
these targets should serve as potential biomarkers or as
producers of downstream biomarkers such as amino
338 • J Clin Pharmacol 2003;43:329-341
acids, peptides, proteins, or electrolytes that can be
used for preclinical and early clinical assessments.
Although preclinical models will probably not be
directly applicable to the clinical setting, they establish
a preliminary foundation for future clinical models.78
For example, transgenic animals or other animal mod-
els or in vitro tests that closely reflect human disease
should be used to start the creation of PK and/or PD
models that will ultimately be tested in patients to
build clinical information and knowledge. This foun-
dation can then be expanded and modified to incorpo-
rate PK/PD model components that reflect human
pharmacokinetics, biology, physiology, and pharma-
cology as development moves from Phase I through
Phase III and finally into postmarketing. These PK/PD
modeling tools are also being built into regulatory re-
view and approval processes.79-81
Biomarkers and PK/PD models will guide com-
pound selection and retention. Even if the selected
biomarkers are never linked to clinical endpoints,
mechanism-based biomarkers will improve early drug
discovery and development by simplifying decision-
making processes. Only compounds with a high proba-
bility of medical and commercial success will enter
Phase III. The cost of drug development will decrease
in response to this more efficient and effective process.
Although our current PK/PD models are not suffi-
ciently mechanism based for effective prospective pre-
dictions,82-84 biomarkers, together with PK/PD model-
ing and simulation, will provide a continuous process
to link what has been learned from today’s drug devel-
opment cycle to the next generation of biomarkers, as-
says, and models. Biomarkers, together with PK/PD
modeling and simulation, will provide a foundation to
bridge effective, multidirectional communication.
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