C H A P T E R

17
Biomarkers in Nonclinical Drug Development
A.D. Aulbach, C.J. Amuzie

O U T L I N E

Biomarkers in Nonclinical Safety Assessment 448 Clusterin457

Cystatin C 457
Biomarker Validation, Qualification, and Application
Enzymuria458
in Nonclinical Studies 449
Glomerular Injury Biomarkers 458
Biomarker Qualification 449
Total Protein, Albumin, and Microglobulins 458
Biomarker Assay Kits 449
Future Directions 458
Fit-for-Purpose (Analytical) Validation 450
Biomarker Application in Nonclinical Studies 451 Cardiac and Skeletal Muscle Injury Biomarkers 458
Lab-to-Lab and Method-to-Method Diversity 451 Biomarkers of Cardiac Structural Injury and Necrosis 458
Biomarkers of Cardiac Functional Alterations 460
Biomarkers of Liver Injury 451
Biomarkers of Skeletal Muscle Injury 460
Traditional Biomarkers 452
Biomarkers of Hepatocellular Injury 452 Vascular Injury Biomarkers 461
Alanine Transaminase 452
Biomarkers of the Immune System 461
Aspartate Transaminase 453
Indicators of Immunosuppression 462
Glutamate Dehydrogenase 453
Hematology and Clinical Chemistry 462
Sorbitol Dehydrogenase 453
Increased Incidence of Tumors 462
Total Bile Acids 454
Increased Incidence of Infections 462
Biomarkers of Hepatobiliary Injury 454 Histopathology and Lymphoid Organ Weight 463
Alkaline Phosphatase 454 Immune Function Studies 463
Total Bilirubin 454 Primary Antibody Assays 463
γ-Glutamyl Transferase and Other Hepatobiliary Natural Killer Cell Activity 464
Markers454 Macrophage Function 464
Emerging Biomarkers 454 Indicators of Adverse Immunostimulation 464
Glutamate Dehydrogenase 455 Acute-Phase Proteins 464
Malate Dehydrogenase 455 Cytokine Evaluation 464
Paraoxonase-1455 Indicators of Hypersensitivity 465
Purine Nucleoside Phosphorylase 455 Indicators of Immunogenicity 466
Other Emerging Biomarkers of Liver Injury 455 Indicators of Autoimmunity 466
Biomarkers of Toxicity in the Central Nervous System 466
Renal Injury Biomarkers 456
Biomarkers of Toxicity in the Reproductive System 467
Tubular Injury Biomarkers 456
Kidney Injury Molecule-1 Conclusion467
(Kim-1/TIM-1/HAVCR-1)456
References468
Neutrophil Gelatinase-Associated Lipocalin or
Lipocalin-2457

A Comprehensive Guide to Toxicology in Nonclinical Drug Development, Second Edition 447

http://dx.doi.org/10.1016/B978-0-12-803620-4.00017-7 © 2017 Elsevier Inc. All rights reserved.
448 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
BIOMARKERS IN NONCLINICAL SAFETY
ASSESSMENT
The use of biomarkers in nonclinical drug develop-
ment covers a broad range of disciplines, modalities,
and development stages that extend from the identi-
fication and assessment of drug targets to issue reso-
lution during the clinical trial of investigative drugs.
Biomarkers became more applicable in drug devel-
opment after the complete sequencing of the human
genome and the expansion of omics technologies that
followed. The National Institutes of Health (USA) has
defined biomarkers as a characteristic that is objec-
tively measured as an indicator of biological processes
or pharmacological responses [1]. When adapted to
the context of nonclinical drug development, a bio-
marker may be defined as any specific parameter that
is objectively measured and evaluated as an indicator
of normal biological processes, pathologic processes, or
pharmacologic responses to a drug or test article, which
helps to diagnose or monitor a disease and/or the out-
come of therapeutic intervention [2,3].
In safety assessment, biomarkers are most commonly
used for monitoring early signs of toxicity; as such they
are often called “safety” biomarkers in nonclinical and
clinical environments. However, scientists in drug dis-
covery are aware that biomarkers are used in a broader
context than “safety” and can sometimes be selected as
a molecular index to evaluate the efficacy of new ther-
apeutic entities. For example, the human epidermal
growth factor receptor (HER2) is a protein in 20% of
breast cancers and its presence is associated with very
aggressive tumor behavior, yet it is a target for several
effective drugs targeting breast cancer [4]. This example
illustrates that a discussion on the utility of biomarkers
should be broad to include their physiology, expose the
range of possible applications, and also keep the appli-
cable details to their most common uses.
There are three broad categories of biomarkers in
nonclinical drug development based on the drug expo-
sure–effect continuum. They are biomarkers of expo-
sure, susceptibility, and effect. Most of the discussion
in this chapter will be focused on biomarkers of effect,
but the other categories will be discussed briefly. A bio-
marker of exposure is the administered test article or a
metabolite that is measured in a specific tissue or body
fluid as evidence that the animal or human biological
system was exposed to the test article. For example,
acetaminophen is an oral analgesic that is metabolized
to a reactive intermediate N-acetyl-p-benzoquinone
imine (NAPQI). Both acetaminophen and NAPQI can
be measured as biomarkers of exposure to help investi-
gators understand acetaminophen exposure and toxic-
ity. Note that in acetaminophen overdose, NAPQI levels
are used more like a biomarker of effect to monitor the
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
magnitude of acetaminophen toxicity or the effective-
ness of acetaminophen antidote, N-acetyl cysteine. This
NAPQI example illustrates that a single biomarker can
provide a picture of both exposure and effect of the test
article, highlighting that biomarker categories can over-
lap and should be contextually understood in order to
be meaningfully applied.
Biomarkers of susceptibility are specific genotypes in
animals or people that make them more prone to a partic-
ular disease or more likely to be affected by a particular
therapy. Warfarin (Coumadin) is an anticoagulant used
to manage thrombotic disorders and acts by inhibiting
vitamin K-dependent clotting factors. Specific alleles of
the gene that encode the metabolizing cytochrome P-450
enzyme (CYP2C9*2 and CYP2C9*3) are associated with
reduced metabolism of Warfarin, a higher circulating
drug concentration, and an increased risk for bleeding
disorders in patients that are taking Warfarin [5]. The
genotyping of patients provides information from these
alleles, from which patients are ranked according to their
susceptibility to bleeding disorders while taking Warfa-
rin. Therefore patients with thrombotic disorders who
also have these alleles are given lower doses of Warfarin
to mitigate this risk. The understanding of biomarkers of
susceptibility and their application to a particular drug
or target is useful in selecting the correct animal model
of disease and/or safety in drug discovery and develop-
ment, understanding toxicokinetics in the appropriate
haplotype, and selecting the right patient population for
early clinical trials.
Pharmacodynamic (efficacy) or toxicity (safety) bio-
markers are essentially biomarkers of effect. They indi-
cate that a specific alteration or injury has occurred in a
receptor, tissue compartment, or body fluid after a test
article has been administered. The ideal biomarker of
effect is a characteristic that can be objectively measured
as an index of test article-related alterations in a mini-
mally invasive way. Therefore biomarkers that can be
obtained in body fluids (saliva, urine, blood, and plasma)
or imaging are generally preferred to those that can be
obtained by biopsies or other invasive techniques. The
identification of biomarkers of effect requires a thorough
understanding of the effect pathway in a specific tissue
or the signaling pathway and their interaction(s) with
other systems within the whole organism. For example,
growth hormone is released from the anterior pituitary
and binds to growth hormone receptors in the liver to
produce an elevation of insulin-like growth factor-1
(IGF-1) and its acid-labile binding subunit (IGFALS) in
the plasma. Therefore molecules that alter growth or
growth hormone signaling, such as the toxin deoxyniva-
lenol, are also expected to change the levels of IGF-1 or
IGFALS in plasma, and these circulating proteins can be
used as biomarkers to determine the effect of a xenobi-
otic on growth hormone signaling [6]. In general, a good
 Biomarker Validation, Qualification, and Application in Nonclinical Studies
understanding of the circulating kinetics of the test arti-
cle and the biomarker is absolutely necessary to properly
understand and interpret any biomarker of effect data.
BIOMARKER VALIDATION,
QUALIFICATION, AND APPLICATION IN
NONCLINICAL STUDIES
It is clear that biomarkers are playing an increasingly
important role in pharmaceutical development and are
receiving increased attention from both regulators and
industry. Although there are many biomarkers that
would be considered “traditional,” in the sense that they
have been in use for many years and utilize standardized
methodologies, most of the newly emerging biomark-
ers discussed in this section are quantified by relatively
novel immunoassay methodologies, often using propri-
etary commercially available assay kits. Many of these
kits and their associated reagents are not consistently
regulated, monitored, or standardized across indus-
try. These factors result in inconsistent and sometimes
improper use and application of biomarkers across dif-
ferent organizations/laboratories and emphasize the
importance of stringent validation and assay character-
ization practices. In this section some of the challenges in
biomarker validation and their application in nonclinical
toxicology studies are reviewed.
Biomarker Qualification
The need for high-quality biomarkers that are robust,
predictive, and cost effective has been thoroughly dis-
cussed by a variety of industry and regulatory groups for
a number of years [7–11]. Regulatory organizations like
the Food and Drug Administration (FDA), the Organ-
isation for Economic Co-operation and Development
(OECD), and the European Medicines Agency (EMEA)
have engaged in a dialogue with the pharmaceutical
industry on what they deem necessary to develop a
qualified biomarker. The term “qualification” should not
be confused with the term “validation,” since the latter
represents the performance characterization of a method
or assay. In a regulatory context, qualification represents
the endorsement of a particular biomarker by a regula-
tory body under a very specific set of conditions (eg,
“…may be used for the characterization of renal effects
in rats…”). The FDA has established a Biomarker Quali-
fication Review Team (BRQT) that provides evaluations
of qualification data for biomarkers submitted as part of
IND (investigational new drug)/NDA (new drug appli-
cation) applications to the FDA [12]. The BRQT aims to
ensure that biomarkers are integrated in the review pro-
cess and to encourage development of biomarkers that
add value to the development program of a given drug
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
449
candidate. Submission of qualification data is largely
driven by international consortiums such as the Predic-
tive Safety Testing Consortium (PSTC), the International
Life Sciences Institute—Health and Environmental Sci-
ences Institute (ILSI-HESI), whose work ultimately leads
to the discovery, critical evaluation, and application of
promising new biomarker candidates. Once qualified,
results from specific biomarkers are then officially “con-
sidered” by the endorsing regulatory body as part of
nonclinical data submission packages under a very spe-
cific set of circumstances as outlined by the agency. That
said, biomarkers that have not been officially qualified by
the FDA, but have demonstrated repeated utility in non-
clinical settings, are still routinely applied to nonclinical
safety data packages to provide additional perspective.
Biomarker Assay Kits
Unlike traditional clinical pathology, clinical chem-
istry, and other well-established testing methods, com-
mercially available first-to-market biomarker kits often
use ligand-based (immunoassay) testing methods and
reagents that vary greatly in their quality, performance,
and overall value. Despite the availability of a multitude
of commercial kits from numerous vendors, the extent of
assay characterization and critical reagent performance
of these assays is often insufficient to support their use.
Because these products are distributed as “research use
only,” there is essentially no oversight over the manufac-
ture, distribution, or marketing of such products. These
kits are often marketed for the broadest possible use and
are often not designed for specific applications in which
they are used (ie, cross-species, multiple matrices, insuf-
ficient stability). Additionally, the specific techniques
and performance assessment procedures used by the
end-users prior to undertaking sample testing varies
greatly. These factors can negatively impact the quality
of biomarker data used in making key decisions during
drug development. Hence when using novel biomarkers
to generate nonclinical data, it is critical to obtain assay
kits from reputable vendors, execute a robust site-spe-
cific fit-for-purpose performance characterization, and
maintain consistency of the materials and methods used
throughout a nonclinical program.
Two main categories of commercial biomarker kits
are used in drug development and research purposes:
in vitro diagnostic assays (IVD) and research use
only (RUO) assays. An understanding of these cate-
gories and their inherent differences is important to
gain insight into whether or not a given assay may
be suitable for the intended use. IVD assays make up
a large majority of the traditional clinical pathology
assays as well as a number of the newer biomarker
kits. These assays have been approved by the FDA
(defined in 21 CFR 809.33) for intended use in human
450 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
clinical diagnosis or patient care and undergo intense
screening/monitoring, and regulation as to how these
materials are manufactured, distributed, and used in
laboratories [13]. For these reasons, traditional clinical
pathology assays and IVD biomarker tests have been
better characterized, standardized from lab-to-lab, and
generally have better performance characteristics than
novel first-to-market biomarker assays. Conversely,
the large majority of commercially available novel bio-
marker kits used in nonclinical laboratories to which
most of this chapter is devoted fall under the RUO cat-
egory. Assays that are labeled RUO are exempt from
the regulatory requirements and approvals needed
for clinical diagnosis or patient management. They
are intended for use only in research and discovery
work and since there are no rigorous requirements or
guidelines for the RUO label, the extent of manufac-
turing quality compliance, assay characterization, and
documentation varies considerably across different
vendors and from assay to assay. Hence most novel
biomarker assays are not standardized between labo-
ratories and need to have a robust performance char-
acterization/validation prior to use.
Fit-for-Purpose (Analytical) Validation
The term validation is used in a multitude of dif-
ferent contexts, sometimes inappropriately, and often
interchangeably with the term qualification. Gener-
ally the term validation represents the actual ana-
lytical performance characterization of a method or
assay by executing various experiments (eg, spike
and recovery, dilutional linearity, stability, etc.) to
gain an understanding of the ability of the assay to
accurately quantitate the analyte of interest. For many
years industry investigators went without a consistent
approach to this process, which led to disharmony and
confusion between laboratories, particularly when
attempting to compare results between different labs.
Eventually leading ligand-binding scientists began to
harmonize this process by promoting the fit-for-pur-
pose validation model.
The fit-for-purpose paradigm has been in use for over
a decade now and was initially described in 2006 with
an intention to provide a framework for the validation
and performance characterization of ligand-binding
(immunoassay) biomarker kits [8]. The core principle
of the fit-for-purpose approach surrounds the notion
of executing a performance characterization/valida-
tion equal in robustness to the intended use of the data
being generated. For example, the level of robustness
of a validation for a renal biomarker assay being used
during an early non-GLP discovery phase would not be
as great as the validation of a cardiac biomarker being
used during a 13-week GLP study for a compound
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
in which there was a known cardiac liability. Briefly,
the key elements included in a fit-for-purpose valida-
tion include preparation of a validation plan, testing
of dynamic range, intra- and interassay precision and
accuracy, dilutional linearity, parallelism, establish-
ment of quality control (QC) samples in species-specific
matrix, normal baseline ranges from species of interest
in matrix, and short/long-term stability in matrix. In
accordance with the fit-for-purpose approach, the over-
all level of rigor (ie, number of runs, length of stability
testing, standard curve points, lot-to-lot variability test-
ing, etc.) is determined by the level of confidence one
puts into the data being generated. For a more detailed
and comprehensive discussion of an analytical fit-for-
purpose validation readers are directed to the refer-
ences discussed in this section.
Recommendations for fit-for-purpose biomarker
assay development and validation have been described
for immunoassays developed de novo at pharmaceuti-
cal companies and contract research laboratories [7,8].
However, specific regulatory guidance has been sparse
and incomplete, which does not help the pharmaceutical
end user determine the appropriate practices to select,
adapt, validate, and ensure long-term supplies of reliable
commercial biomarker assay kits for drug development
purposes. The most comprehensive of the nonbinding
recommendations on the subject are found in the 2013
Bioanalytical Method Validation, which that states the
following points regarding commercial biomarker assay
validation [14]:
• T he performance of diagnostic kits should be
assessed in the facility conducting (site-specific) the
sample analysis.
• Diagnostic kit validation data provided by the
manufacturer may not ensure reliability of the kit for
drug development purposes.
• Specificity, accuracy, precision, and stability should
be demonstrated under actual conditions of use.
• Quality control (QC) samples with known
concentrations should be prepared and used,
independent of the kit-supplied materials.
• Standards and QCs should be prepared in the same
matrix as the subject samples.
• If multiple kit lots are used within a study, lot-to-lot
variability and comparability should be addressed
for critical reagents.
  
Although these guidelines remain somewhat vague,
they are consistent with most published fit-for-purpose
documents as well as those inherent to bioanalytical
method validation practices, which share many features
of immunoassay biomarker methodologies. Currently,
most laboratories conducting biomarker analysis have
adopted some form of the fit-for-purpose validation
model when establishing new methods.
 Biomarkers of Liver Injury
Biomarker Application in Nonclinical Studies
Inherent to the use of biomarker assays in toxicology
studies is an understanding of the identification and
interpretation of a positive biomarker signal. We have
discussed the relative diversity of assays, levels of perfor-
mance, and lack of standardized methods for biomarker
assays. All of these factors contribute to the difficulty in
identifying a positive signal, which must be distinguished
not only from background biologic variability, but also
from the analytical “noise” that is generally much higher
than traditional clinical pathology tests. Thus it remains
crucial for the interpreting scientist to understand the
inherent variation of the specific assay/reagent set based
on the analytical validation data, as well as the biologic
variation, which can be determined by establishing his-
torical control data sets for the intended reference pop-
ulation. Once a positive signal has been identified, a
thorough understanding of what a positive signal repre-
sents is fundamental to accurate drug hazard identifica-
tion. For example, for cardiac biomarkers, some markers
are indicators of direct cardiac myocyte injury (troponins)
while others are indicators of a functional defect (natri-
uretic peptides). The renal biomarker NGAL (neutrophil
gelatinase-associated lipocalin) is an indicator of renal
tubular injury, but can also be seen in cases of systemic
and urinary tract inflammation [15].
A key feature to interpreting data from novel biomark-
ers is to have an appreciation for the heterogeneity of pos-
itive biomarker response signals in timing, magnitude,
and by species. Every individual biomarker will have its
own unique signature in response to a specific compound
and pathophysiologic mechanism. For example, in the
dog, compound A may induce a 10-fold increase in car-
diac troponin I (cTnI) within 24 h, but with compound B
the response is twofold and isn’t seen until 72 h postdose.
If this same example is used, replacing cTnI with another
commonly used cardiac biomarker like proBNP, the mag-
nitudes and timing would be completely different that
those observed with cTnI. These differences are related to
the specific pathology induced by the compounds both
in timing and severity, and what each biomarker actually
represents pathologically. In addition, some species may
show different magnitudes or even nonexistent responses
to injury when using certain biomarkers. The best exam-
ples of this phenomenon are seen with the acute-phase
proteins (eg, CRP, α2-macroglobulin, serum amyloid A,
etc.). These biomarkers show a large degree of species-
specific diversity among their individual responses to
the same inflammatory stimuli [16]. All of these factors
emphasize the importance of correlating biomarker data
with study data generated from more traditional end-
points (eg, pathology, clinical pathology).
Another important concept is to have an understand-
ing of the specific timing of individual biomarker signals.
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
451
For example, in the case of inflammation, IL-6 (6–24 h),
CRP (1–2 days), and fibrinogen (2–3 days) are all posi-
tive acute phase reactants that will increase in response
to the same inflammatory stimulus. However, each will
have a different window in which you can capture their
increases (listed in parentheses). Attempting to draw
blood at 3 days postdose for a cytokine like IL-6 will gen-
erally result in no positive signal, which can lead to mis-
characterization of the safety profile for the compound.
Lab-to-Lab and Method-to-Method Diversity
One final important feature in the application and
interpretation of biomarkers in nonclinical studies is
related to the lack of standardization between laborato-
ries and methods. For any given biomarker, the methods
and reagents used for quantification will vary by labora-
tory and by kit or platform. This results in a number of
different methods being used by different laboratories
to measure the same biomarker. Objective comparisons
between methods/kits from different laboratories have
been made for a number of routinely used biomarker
assays, and have generally demonstrated the lack of con-
cordance between them. A recent study comparing renal
biomarkers across different platforms revealed differ-
ences in individual biomarkers that ranged up to 15-fold
and showed that these differences were probably due
to the use of different antibodies with varying degrees
of affinity and specificity [17]. This phenomenon is not
uncommon and emphasizes the importance of establish-
ing laboratory/method-specific reference ranges.
BIOMARKERS OF LIVER INJURY
The liver is a critical organ because of its broad role
in nutrient homeostasis, as well as metabolism/detoxi-
fication and excretion of xenobiotics or endogenous
compounds. Examples of liver function are synthesis of
albumin and blood clotting factors, synthesis and storage
of glucose, use and recycling of bilirubin and cholesterol,
metabolism of hormones, and detoxification of various
drugs. In order to accomplish these functions the liver
is organized into lobular units that comprise cords of
hepatocytes that are separated by sinusoidal spaces. The
sinusoids contain spaced-out (fenestrated) endothelial
cells and resident macrophages (Kupffer cells) as well as
stellate cells that reside in small spaces (Space of Disse)
between the hepatocyte cords and sinusoidal basement
membrane. Within each lobular cord, individual hepa-
tocytes are separated by bile canaliculi, which are useful
for bile transport and recycling. Each liver lobule has a
portal (periportal) blood input that supplies the peripor-
tal hepatocytes with the most oxygenated blood within
the lobule, hence the periportal area has been referred to
452 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
as Zone 1—the highest oxygen tension within a hepatic
lobule. A central (centrilobular) vein with least oxygen-
ated blood (Zone 3) is at the opposite end of the peripor-
tal blood supply within a liver lobule, while the midzone
(Zone 2) is an intermediate zone of oxygenation that
resides between Zones 1 and 3. The gradient of metabo-
lizing enzymes such as cytochrome p450 is opposite to
that of oxygenation, so hepatocytes around the central
vein (Zone 3) have the most metabolizing enzymes, while
those around the periportal area usually have the least
metabolizing enzymes. This simplified understanding of
the liver architecture, oxygen, and metabolizing enzyme
gradients is frequently used to understand and interpret
patterns of liver injury in toxicology. For example, the
liver injury related to acetaminophen overdose is often
driven by the reactive intermediate N-acetyl-p-quinone
imine and is predominately centrilobular because of the
high concentration of metabolizing enzymes that create
the reactive intermediate around the central vein. On the
other hand, injury related to iron toxicity/overdose is
predominately periportal because iron toxicity involves
redox cycling of ferrous and ferric iron, which occurs
more in the zone with higher oxygen tension. The zoning
of injury within the liver is a useful and relatively com-
mon practice in safety assessment because it can help
identify potential mechanism(s) of injury and is useful
for investigative toxicologists who design experiments
to further understand toxicity or characterize the injury.
Drug-induced liver injury (DILI) is among the leading
causes of pharmaceutical withdrawals from the market
in the last three decades [18,19], and these withdrawals
are impactful for the patients who need/rely on the drug
and the drug maker who often has large economic loses
and liabilities. Efforts to use biomarkers for improved
prediction of DILI in nonclinical safety assessment are
increasing in scope and complexity. In order to appreci-
ate the scope of biomarkers of liver injury, we need to
remember that xenobiotic-related liver injury may take
several forms and involve different cell types depending
on the chemical stimuli, dose, and/or duration of expo-
sure. Examples of key biological events in different types
of toxic liver injury are cell death, alteration of canalicu-
lar patency and function, disruption of cytoskeleton,
increased accumulation of lipid molecules, alteration of
sinusoidal patency and function, activation of Kupffer
cells and inflammation, hapten-mediated liver injury,
and increased fibrosis/collagen deposition.
Several traditional markers indicate liver injury in
both clinical and nonclinical species. Some of these
markers may be occasionally and often incorrectly
termed “liver function tests,” or LFTs. The concepts of
liver injury and liver function need to be carefully delin-
eated in order to understand and communicate toxic
responses of the liver to injury. The details of such delin-
eation during nonclinical safety data acquisition and
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
interpretation is outside the scope of this chapter and
has been addressed elsewhere [19–21]. It is important to
remember that there may be a biologically and/or sta-
tistically significant alteration in markers of liver injury
without a corresponding effect on the indices of liver
function, and vice-versa.
There are increasing numbers of in vitro systems that
are useful for prioritizing chemical and biological entities
in development. These in vitro systems use alterations
in several molecular markers to predict toxicity. They
are predominately used in non-GLP discovery settings
and will not be discussed specifically in this chapter. The
discussion of biomarkers in this section will be largely
focused on liver injury that occurs in vivo in integrated
animal systems. Currently there are two broad classes
of liver injury biomarkers, called traditional and novel/
emerging biomarkers. The traditional markers are most
often routine clinical chemistry markers, most of which
are part of a standard drug development program and
will be described in detail below. Most of the emerging
biomarkers and their associated methods have not been
standardized or fully qualified but are currently being
used on a case-by-case basis to understand or further
characterize xenobiotic-related liver injury.
Traditional Biomarkers
The current best practice recommendation for non-
clinical safety assessment is that a minimum of four
serum parameters should be used to assess hepatocellu-
lar (minimum of two markers) and hepatobiliary (mini-
mum of two markers) injury [19]. Any two markers from
alanine transaminase (ALT) activity, aspartate trans-
aminase (AST) activity, sorbitol Dehydrogenase (SDH)
activity, and glutamate dehydrogenase (GLDH) will
assess hepatocellular injury, while any two from alkaline
phosphatase (ALP) activity, gamma-glutamyltransferase
(GGT) activity, total bilirubin (TBILI), total bile acids
(TBA), and 5′-nucleotidase will assess hepatobiliary
injury. Among these serum parameters, ALT, AST, ALP,
and TBILI are also used clinically to identify liver injury
in humans, so they tend to be used relatively more fre-
quently. It is important to note that these markers vary
in their utility among nonclinical species. Therefore the
proper selection of liver injury markers for each program
is a thoughtful deliberation that often involves consulta-
tion with a qualified and experienced pathologist.
Biomarkers of Hepatocellular Injury
Alanine Transaminase
Alanine transaminase (ALT), which may be referred
to in other literature as alanine aminotransferase (ALAT)
or serum glutamate-pyruvate transaminase (SGPT),
is found in blood and many tissues. ALT catalyzes the
 Biomarkers of Liver Injury
transfer of an amino group from alanine to alpha-keto-
glutarate to yield glutamate and pyruvate as a part of
amino acid metabolism and gluconeogenesis. Dam-
aged hepatocytes leak their ALT into the extracellular
space and ultimately plasma, so that ALT activity and/
or amount will be increased in animals with damaged
hepatocytes when compared to those with normal
hepatocytes. Among the traditional markers of hepato-
cellular injury, ALT is considered to be a sensitive and
translatable indicator of hepatocellular in the common
preclinical species. Serum ALT activity is the most fre-
quently relied upon indicator of hepatotoxic effects of
drugs [22], although it does not always correlate well
with histopathology data [23]. Increases in ALT activ-
ity may be observed with enzyme induction in rats and
dogs [24,25] or muscle injury [19]. Furthermore, there
may be instances of hepatic injury where ALT activity
is not elevated due to inhibitory factors such as Vitamin
B12 deficiency [26], or interference by the presence of
pyridoxal-5′-phosphate inhibitors such as isoniazid or
lead [23]. Due to the nonspecificity of ALT to hepatocel-
lular injury, ancillary clinical chemistry and/or histopa-
thology tests are often used to help interpret ALT values.
In human clinical trials, it is an acceptable and recom-
mended practice to interpret a greater than 3× elevation
of ALT above the upper limit of normal (ULN) combined
with TBILI elevation of greater than 2× above ULN as
indicative of severe injury with/without any other evi-
dence. This practice in human clinical medicine is called
Hy’s Law. A related recommendation for ALT interpre-
tation in the absence of correlative histopathology in a
nonclinical setting is the biomarker of two rule, which
considers an ALT elevation of 3× ULN combined with
a significant alteration in another liver injury biomarker
as a sign of liver injury [27]. In general, caution should
be exercised when applying human clinical recommen-
dations to preclinical species because enzymes such
as TBILI that reflect biliary function in humans do not
accurately reflect liver function in all preclinical species
[26]. ALT may also have reduced sensitivity and specific-
ity to liver injury in some species such as swine, guinea
pigs, and in some strains of rats [19]. The interpretation
of ALT response in short-term nonclinical studies, espe-
cially in the absence of other indices of hepatic injury,
needs to consider the so-called “adapter” phenomenon
where transient, nonclinically relevant ALT elevations
occur after the introduction of new drugs, but return to
normal following continuous exposure [28]. Due to the
challenges associated with using ALT, some have sug-
gested an inclusion of immunoassay for ALT1, an ALT
isozyme that is more specific for the liver, as a measure
of hepatocellular injury, although these tests have not
yet reached widespread acceptance on routine nonclini-
cal studies [23]. Since ALT has not always been predic-
tive of DILI in the clinical setting another marker(s) is
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
453
necessary for nonclinical predictive testing, hence the
need for emerging biomarkers, discussed below.
Aspartate Transaminase
Aspartate transaminase (AST), which may also be
referred to as aspartate aminotransferase or serum
glutamic-oxaloacetic transaminase (SGOT) in other lit-
erature, catalyzes the reversible transfer of amino group
between aspartate and glutamate. Like ALT, AST is
found in the cytoplasm of hepatocytes and other tissues,
including skeletal muscle. Injury to hepatocytes causes
leakage of AST into the extracellular compartment with
subsequent elevation in serum AST activity. AST activity
may also be elevated in times of skeletal muscle injury
in preclinical species. The magnitude of ALT elevation
is usually greater than AST when both are elevated due
to hepatocellular injury because of the longer half-life
of ALT and the greater fraction of AST that is bound to
the mitochondria [29]. The practice of considering a high
ratio of AST/ALT to be more indicative of skeletal mus-
cle injury [23] has been suggested and may aid in dis-
tinguishing muscle versus liver injury. Animal restraint
procedures leading to mild muscle trauma may also ele-
vate AST activity in nonclinical studies [30]. Given the
nuances associated with interpreting leakage enzymes in
the liver, careful integration of enzyme kinetics, enzyme
half-lives, and all other data by well-qualified personnel
is very important in the practice of safety assessment.
Glutamate Dehydrogenase
Glutamate dehydrogenase (GLDH) is a mitochondrial
enzyme that reversibly converts glutamate to alpha-
ketoglutarate as part of the urea cycle. GLDH activity
increases with liver injury and the magnitude of eleva-
tion may be higher and longer lasting when compared
to ALT [31]. Unlike ALT and AST, compounds that inter-
fere with pyridoxal-5′-phosphate are less likely to alter
the GLDH activity assay [23]. Unlike ALT, serum GLDH
activity is neither affected by skeletal muscle damage
nor induced by corticosteroids. GLDH is not currently
used routinely in nonclinical settings often due to the
limited availability of reliable reagents, but there may be
instances where GLDH is an appropriate adjunct to help
delineate liver injury in preclinical species. Interest in
GLDH as a more specific nonclinical biomarker of hepa-
tocellular injury in some species when compared to ALT
or AST is increasing and will be discussed further in the
section “Emerging Biomarkers.”
Sorbitol Dehydrogenase
Sorbitol dehydrogenase (SDH) is present in several tis-
sues where it catalyzes the reversible oxidation reduction
454 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
of sorbitol, fructose, and NADPH (nicotinamide adenine
dinucleotide phosphate). SDH is a sensitive and specific
indicator of acute hepatocellular injury in rodents, dogs,
and nonhuman primates and has been reported as valu-
able in humans [23]. However, SDH has a short half-
life (4 h in canine) and has limited stability compared
to most other clinical chemistry analytes requiring it to
be analyzed as soon as possible after necropsy [27]. To
our knowledge, no large study has determined whether
SDH adds any additional value to ALT quantitation.
Total Bile Acids
Total bile acids are important in the hepatic metabo-
lism of cholesterol and absorption of fat and fat-soluble
vitamins. The levels of circulating bile acids are influ-
enced by diet and fasting but their elevation may also be
a sign of hepatocellular injury and/or functional change
in the liver [32]. TBA is a useful adjunct to assess the
extent and consequence of liver injury because it is an
index of hepatocellular function in preclinical species.
BIOMARKERS OF HEPATOBILIARY
INJURY
Alkaline Phosphatase
Alkaline phosphatase (ALP) is a hydrolase that
removes the phosphate group from various proteins and
nucleotides. ALP is a leading biomarker of hepatobiliary
injury in common preclinical species. Serum ALP levels
increase when the patency of the bile duct is reduced, so
ALP is widely used in nonclinical and human clinical set-
tings as a marker of cholestatic liver injury. Like ALT, ALP
is not specific for hepatobiliary injury as increased activity
of ALP may be seen in conditions of bone growth and dis-
ease, glucocorticoid treatment, and microsomal enzyme
induction [25,33]. Fluctuations of ALP need to be inter-
preted cautiously in nonclinical settings because there is
an intestinal isoform of ALP that drives a decrease in ALP
activity, particularly in rodents, during fasting or anorexia
and a transient increase postprandially [19]. Specific ALP
isoforms may be assessed to separate liver from bone and
intestinal sources, but isoform measurement does not
offer any additional diagnostic information in preclinical
species compared to total ALP [19].
Total Bilirubin
Total bilirubin is a yellowish-green breakdown prod-
uct of hemoglobin from aging red blood cells. TBILI is
made of hepatic/conjugated and extrahepatic/uncon-
jugated sources. Elevations in TBILI are usually indica-
tive of cholestasis, but may be a reflection of increased
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
bilirubin production from erythrocyte destruction,
altered bilirubin metabolism from drug-related inhibi-
tion of metabolizing enzymes such as uridine diphos-
phate glucuronosyltransferase, or faltering liver function
[34]. A careful and integrated assessment of all study
data associated with hyperbilirubinemia is often needed
to identify a specific cause of TBILI elevations.
γ-Glutamyl Transferase and Other Hepatobiliary
Markers
γ-glutamyl transferase (GGT) is a liver canalicular
enzyme responsible for the transfer of glutamyl moi-
ety to other acceptors as part of gluthathione recycling,
and is present in other tissues. GGT is elevated through
induction in conditions of cholestasis and may be used
to confirm ALP activity elevations that are associated
with cholestasis. In dogs and cats GGT is a less sensitive
marker of cholestasis than ALP, but is more specific [19].
Two other hepatobiliary markers, 5′-nucleotidase
(5′NT) and total bile acids (TBA), may be used to assess
biliary function in nonclinical studies. However, they do
not offer additional information when compared to ALP
and TBILI and will not be discussed further.
Emerging Biomarkers
Due to the specificity issues with existing liver injury
biomarkers such as ALT efforts are being made to iden-
tify additional biomarkers that are more specific and
those that may provide additional information about
the zone of injury and/or affected cell types. Within the
last decade, proteomic approaches were used by inves-
tigators to identify a group of 19 putative liver injury
biomarkers from a mechanistically diverse group of hep-
atototoxins based on early onset of biomarker alteration
relative to time of liver [35]. The Hepatototoxcity Work-
ing Group of the Predictive Safety Testing Consortium
(PSTC) has listed some promising classic and newly
identified markers of injury for further qualification.
The first set of biomarkers for qualification includes glu-
tamate dehydrogenase (GLDH), malate dehydrogenase
(MDH), paraoxonase-1 (PON1), and purine nucleoside
phosphorylase (PNP). PSTC also listed a second set of
biomarkers sorbitol dehydrogenase (SDH), arginase 1
(ARG-1), and gluthathione-s-transferase alpha (GSTα)
for qualification while a circulating microRNA marker
(miR-122) is being considered for qualification under the
third set. This decision is based, in part, on published
evidence in specific liver injury situations indicating that
these biomarkers are likely to provide additional value
in predicting liver injury. A robust qualification process
is currently ongoing and some of these markers may
become more routine in nonclinical testing panels after
the qualification process.
 Biomarkers of Hepatobiliary Injury
Glutamate Dehydrogenase
Glutamate dehydrogenase (GLDH) is a mitochon-
drial enzyme that is predominantly located in the
centrilobular (Zone 3) region of the liver lobule [36].
GLDH is a classic serum enzyme like ALT and AST.
Unlike ALT, serum GLDH activity is neither affected
by skeletal muscle damage nor induced by cortico-
steroids, and is not sensitive to interference by pyri-
doxal phosphate inhibitors. GLDH activity increases
with liver injury and the magnitude of elevation may
be higher and longer lasting when compared to ALT
[31]. However, in the past there has been limited avail-
ability of reliable GLDH reagents in the United States.
Recently, GLDH was reported as a specific biomarker
of liver injury in a cohort of human clinical cases of
drug-induced liver injury [37]. Furthermore, a human
reference interval has been established for GLDH from
a relatively large cohort. GLDH is emerging from a less
commonly used classic biomarker to one that may be
used more routinely to detect acute liver injury based
on possible increases in sensitivity compared to ALT,
and transnational use in humans.
Malate Dehydrogenase
Malate dehydrogenase (MDH) is a predominately
periportal enzyme that is expressed highly in the extra-
mitochondrial cytoplasm of the liver, although 10% of
MDH has been reported in the mitochondria [23]. It is an
enzyme in the citric acid cycle that catalyzes the revers-
ible conversion of malate into oxaloacetate. Serum MDH
activity is correlated with liver injury, although cardiac
injury can cause an elevation in activity. Like GLDH,
MDH levels are stable in healthy patients across gender
and age groups, but are elevated with liver injury [37].
Due to the predominantly periportal location of MDH
and possible translation to human clinical setting, MDH
is among the first set of biomarkers undergoing qualifi-
cation through the PSTC.
Paraoxonase-1
Paraoxonase-1 (PON1) is an esterase that protects
low-density lipoproteins from oxidation and detoxifies
organophosphates. PON1 is predominantly produced
in the liver, although enzyme activity has been detected
in the kidney and brain. Unlike MDH and GDH, PON1
decreases during liver injury, probably due to decreased
synthesis and/or secretion. Initial baseline data suggests
that PON1 varies among healthy people based on their
ethnicity [37]; therefore it is not clear how PON1 will
translate from nonclinical to clinical settings. However,
since all other liver injury biomarkers are elevated in
times of injury, PON1 is an attractive biomarker candidate
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
455
because it decreases with injury. PON1 is currently under-
going qualification as a liver injury biomarker.
Purine Nucleoside Phosphorylase
Purine nucleoside phosphorylase (PNP) is an enzyme
that catalyzes phosphorolysis of nucleosides in the
purine salvage pathway. It is located in the cytoplasm
of hepatocytes, Kupffer cells, and endothelial cells and
is released into the sinusoids in times of hepatocellular
necrosis [23]. PNP has also been shown to be relevant
in clinical settings. PNP is a good biomarker candidate
because it also has the possibility to detect nonparen-
chymal hepatic injury [26]. PNP is among the first set
of markers that are currently being qualified for liver
injury.
Other Emerging Biomarkers of Liver Injury
Arginase 1 (Arg-1) is a liver-specific hydrolase that
catalyzes the breakdown of arginine to urea and orni-
thine. In a limited number of investigative studies
ARG-1 has been shown to be an early onset marker of
liver injury [23]. Arginase is not commonly measured in
routine nonclinical safety assessment.
Gluthathione-s-transferase (GST) is a phase II detoxi-
fying enzyme with four isozymes (alpha, pi, mu, and
theta) expressed in mammals. The alpha isozyme, GSTα,
is found in high concentrations in centrilobular hepato-
cytes; as such, it is a good biomarker candidate to iden-
tify injuries that occur in the metabolic zone of the liver.
The occurrence of GSTα within the metabolic zone of the
liver makes it a good candidate for qualification.
miR-122 is a single-stranded noncoding (micro)
RNA with about 22 nucleotides that is involved in post-
transcriptional control of protein synthesis within the
eukaryotic genome. miR-122 is predominately expressed
in the liver and has been shown to be elevated in circula-
tion during conditions of drug overdose [26]. Although,
the evidence for miR-122 association with DILI is lim-
ited, qualification of a microRNA biomarker is poten-
tially useful because of its stability and the availability
of more sensitive PCR-based assays compared to colori-
metric and ligand-based assays for current biomarkers.
In conclusion, there are several markers currently
available to assess liver injury, and these markers offer
a lot of useful information about liver injury when inter-
preted properly. However, the occurrence of DILI after
drug approval indicates that more predictive biomarkers
are necessary in nonclinical safety assessment. PSTC has
taken the lead in qualifying new markers. As the new
markers become qualified and adapted, further under-
standing of their comparative relevance in different pre-
clinical species should be an integral part of improving
nonclinical safety assessment.
456 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
RENAL INJURY BIOMARKERS
Nephrotoxicity and acute kidney injury (AKI) is a
common occurrence in nonclinical safety studies and
continues to rank among the top reasons for drug attri-
tion of promising new drug candidates. The kidneys are
uniquely susceptible to a wide array of xenobiotics due
to their receiving a large proportion of circulating blood
volume (up to 25% of cardiac output) resulting in high
exposure to compounds and/or their metabolites in cir-
culation. The nephrons are also involved in the urine
formation process, which further serves to concentrate
toxicants in renal tubular fluid. Some of the most com-
mon nephrotoxins include heavy metals (eg, chromium,
lead, mercury), nonsteroidal antiinflammatories thera-
peutics (eg, acetaminophen), aminoglycoside antibiotics
(eg, gentamicin), and immunosuppressive and chemo-
therapeutic agents (eg, cyclosporine, cisplatin) [38].
The use of traditional clinical pathology endpoints
such as blood urea nitrogen (BUN), serum creatinine
(sCr), phosphorus, and urine-specific gravity as indi-
cators of renal function have long been established;
however, these parameters are relatively insensitive
indicators of renal injury and will not demonstrate dis-
cernible alterations until a functional deficit of up to
65–75% has occurred [39]. Clearly, the performance of
these markers is not sufficient for monitoring kidney
injury in many nonclinical settings. The need for renal
biomarkers with improved sensitivity has led to more
recent efforts, largely driven by collaborative groups
like the Predictive Safety Testing Consortium (PSTC)
and the International Life Sciences Institute—Health
and Environmental Sciences Institute (ILSI–HESI),
resulting in the discovery of a number of promising
next-generation renal injury biomarkers that provide
earlier and more specific detection of renal injury for
use in drug development.
Initially, a set of seven urinary biomarkers was quali-
fied by the FDA for use in the rat for monitoring drug-
induced kidney injury in conjunction with BUN and SCr
[40]. These seven biomarkers include kidney injury mol-
ecule-1 (Kim-1), albumin, total protein, β2-microglobulin
(B2M), cystatin C, clusterin, and trefoil factor-3. Addi-
tional data for four more biomarkers were later submit-
ted to the FDA and EMA for nonclinical qualification
and two of them, clusterin and renal papillary antigen-1
(RPA-1), were accepted for use in detecting acute drug-
induced renal tubule alterations in rat. In addition to this
list, a fair number of other renal biomarkers have been
described (eg, osteopontin, NGAL, GST-α, calbindin)
and are being used in drug development settings [17,41].
It is worth noting that qualification of a biomarker by a
regulatory body represents their endorsement of its use
under very specific circumstances, in a specific species,
and does not discount the utility or prevent the inclusion
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
of data from other biomarkers for informational pur-
poses, or to provide additional perspective.
When measuring urinary biomarkers it is important
to utilize metabolism cages to collect urine and insti-
tute efforts to minimize urine sample contamination by
environmental materials such as food, bedding, feces,
and other debris, which can introduce undesired pre-
analytical variation resulting in poor datasets. When
quantifying any urinary-based analyte it is also critical
to normalize analyte concentration to urine concentra-
tion or volume by performing either a creatinine ratio
or an analyte over timed urine volume ratio (eg, ana-
lyte/16 h) to correct for fluctuations in urine output.
Although the release kinetics of each biomarker into the
urine will be compound-dependent and vary slightly,
collecting urine for 12–16 h at least 2–3 days following
anticipated renal injury is a standard approach in non-
clinical toxicology studies. Some biomarkers have been
shown to be released into the urine within hours fol-
lowing an acute insult [42].
Renal biomarkers generally are classified into sev-
eral groups indicating their association with injury to a
specific part of the nephron (eg, proximal tubule, glom-
erulus, collecting duct). However, recent work suggests
that there is a fair amount of overlap and redundancy
among individual biomarkers in regards to their specific
nephron segment, hence classifying biomarker effects
to either the glomerulus or the tubules appears to be
the most appropriate approach in most cases [41]. The
magnitude, timing, and sensitivity of positive signals
among specific biomarkers will differ between test com-
pounds, and between assay methodologies and labora-
tories, hence the selection of specific biomarkers should
be made with consideration of assay availability, spe-
cies, anticipated pathophysiology, and analyte stability
[17,43]. Lastly, it is important to have an understanding
of the relationship between positive biomarker signals
and microscopic pathology as histologic effects on the
kidney as assessed by light microscopy continues to be
the reference standard used to assess the overall perfor-
mance and sensitivity of the emerging renal biomarkers.
Tubular Injury Biomarkers
Kidney Injury Molecule-1 (Kim-1/TIM-1/
HAVCR-1)
Kidney injury molecule-1 (Kim-1/TIM-1/HAVCR-1)
is probably the most popular and commonly utilized of
next-generation or novel renal biomarkers for detecting
acute kidney injury (see Table 17.1). It is an 85-kDa type
I cell membrane glycoprotein conserved across many
species including, rodents, dogs, primates, and humans
and is classically considered a proximal tubular marker
[44]. Kim-1 is shed from tubular epithelial cells into the
urine in response to injury (eg, toxic, ischemic, septic,
 Renal Injury Biomarkers
TABLE 17.1  Summary of Renal Injury Biomarkers
Biomarker Sources
Kidney injury molecule 1 (KIM-1, TIM-1, Proximal tubules
HAVCR-1)
Neutrophil gelatinase-associated lipocalin Proximal and distal tubules, neutrophils,
(NGAL, lipocalin-2) bone marrow
Clusterin Proximal and distal tubules
Cystatin C All nucleated cells, renal tubules
Alkaline phosphatase (ALP) Renal tubules
N-acetyl-β-glucosaminidase (NAG) Renal tubules
Gamma glutamyltransferase (GGT) Renal tubules
Total protein Plasma protein
Albumin (micro albumin) Plasma protein
α1/β2-microglobulin Plasma protein
GFR, glomerular filtration rate.
and transplant), and has been shown to be a sensitive
and early diagnostic indicator of renal injury in a variety
of animal models and human disease states [42,45–47].
In humans, increased levels have also been shown to
be predictive of adverse disease outcomes [48]. Kim-1
is routinely used in rat and nonhuman primate studies;
however, anecdotal evidence suggests Kim-1 may not be
a useful indicator of renal effects in dog. Kim-1 assays
are widely available and are often included in species-
specific renal injury panels on multiplex platforms [43].
Neutrophil Gelatinase-Associated Lipocalin or
Lipocalin-2
Neutrophil gelatinase-associated lipocalin (NGAL)
(lipocalin-2) is a 25-kDa protein originally observed
within specific granules of the neutrophil produced
during granulocyte maturation in bone marrow [49].
However, more recently it has been shown to be unregu-
lated in the proximal and distal tubules in response to
a variety of renal injury conditions including ischemia
and chemical toxicity [41,50,51]. Although it has not
specifically been qualified for use by regulatory agen-
cies, NGAL has generated much interest as a biomarker
for the early detection of acute proximal tubular injury
and is routinely used in nonclinical studies. In healthy
individuals NGAL is expressed at low levels in various
tissues, filtered at the glomerulus, and reabsorbed by
the proximal tubule [52]. In addition to increasing renal
production of NGAL following injury, damage to the
nephron may enhance NGAL presence in the urine by
impairing proximal tubular reabsorption.
Given the involvement of NGAL in neutrophil
function, false-positive increases in NGAL can be
induced in neutrophils by various stimuli including
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
457
Interpretation
Toxic, ischemic, septic renal tubular injury
Renal tubular injury (urinary), inflammatory
(blood)
Toxic, ischemic, septic renal tubular injury
Plasma levels inversely related to GFR; renal
tubular injury (urine)
Renal tubular injury
Renal tubular injury
Renal tubular injury
Glomerular injury
Glomerular injury
Glomerular injury
inflammation, infections (eg, urinary tract), and neo-
plasia, which should be considered when positive
NGAL signals are seen [53,54].
Clusterin
Clusterin is a 76–80 kDa protein expressed on the
dedifferentiated tubular cells after injury and is another
promising marker for the detection of acute tubular
injury. In the context of kidney injury, clusterin has
been suggested to play an antiapoptotic role and to be
involved in cell protection, lipid recycling, cell aggrega-
tion, and cell attachment [55]. Increased urinary clus-
terin levels have been seen in a variety of conditions of
both proximal and distal tubular injury, but not in those
associated with glomerular injury [45,56–58].
Cystatin C
Cystatin C is a low molecular weight (13.3 kDa) basic
protein (cysteine protease inhibitor) produced by all
nucleated cells at a constant rate and is freely filtered
by the kidneys. It is a good candidate as both an index
of function as related to ​GFR as well as a tubular injury
indicator. It is not secreted in normal urine and is com-
pletely reabsorbed by proximal tubule epithelia [59].
Plasma/serum levels of cystatin C are inversely related
to GFR, so that reduced GFR is reflected in increased
cystatin C concentrations. Serum concentrations appear
to be independent of sex, age, and muscle mass. Cys-
tatin C does not appear in the urine of normal animals
but will be increased in the urine in conditions of renal
tubular dysfunction [60,61]. The PSTC recently reported
that urinary cystatin C can outperform BUN and sCr in
detecting early impairment of tubular reabsorption due
to glomerular alterations/damage in rat studies [55].
458 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
Enzymuria
Enzymes released from damaged renal tubular cells
of the proximal and distal tubule have been used as
markers of kidney injury in animals for several decades;
however, their use in human studies has not been widely
accepted. This group of renal injury biomarkers is not
generally considered part of next generation or novel
renal biomarkers, partly because of the traditional meth-
odology in which most of them are measured (nonim-
munoassay-based methods). A variety of enzymes (see
Table 17.1) have been utilized for this purpose includ-
ing alkaline phosphatase (ALP), the lysosomal enzymes
N-acetyl-β-glucosaminidase (NAG) and gamma glutam-
yltransferase (GGT), and the glutathione S-transferases
(GSTs) [62]. The appearance of these enzymes in the urine
is specific to proximal and distal tubule damage, and
they have been shown to be fairly sensitive indicators
of nephrotoxicity and injury even preceding increases in
sCr [62–64]. These assays are available on most modern
clinical chemistry analyzers.
Glomerular Injury Biomarkers
Total Protein, Albumin, and Microglobulins
The glomerular capillaries create a barrier for the
passage of proteins any larger than those about the size
of albumin (36 Å) into the urine. Depending on charge,
smaller proteins (eg, α and β microglobulins) will also
pass through the filtration barrier into the tubular fluid
ultimately being reabsorbed or broken down by the
proximal tubules. For those reasons, the detection of
increased concentrations of such proteins in the urine is
an indicator of glomerular (albumin or larger proteins)
and/or proximal tubular injury (smaller proteins like
the microglobulins). In the case of glomerular damage,
injury results in large proteins leaking into the urine,
which overwhelms the modest ability of the proximal
tubule to remove them, whereas injury to the proximal
tubule will impair the ability to remove smaller proteins
such as α1 or β2-microglobulin. They have also been
shown to have good sensitivity and specificity in detect-
ing renal injury due to a variety of causes [17,43].
Future Directions
Given the complexity of renal structure and function,
the variety of traditional and emerging renal biomarkers
in use, and the wide array of new test compounds and
associated pathophysiologic observations, it is unlikely
that a single biomarker will be the lone answer for the
detection of renal injury in toxicology studies in the
future. It is more likely that we will continue to use pan-
els of serum and/or urine biomarkers for general assess-
ment, patient monitoring, and investigative nonclinical
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
and clinical applications for the early detection of renal
injury. We anticipate the continued emergence of new
biomarkers and those selected for routine use should be
ones with a sufficient body of evidence to support their
intended use.
CARDIAC AND SKELETAL MUSCLE
INJURY BIOMARKERS
Cardiotoxicity can be caused by a variety of xenobiot-
ics and chemical therapeutics including antiarrhythmic
agents (eg, β-adrenergic blockers), positive inotropic
compounds (eg, digoxin, isoproterenol), anesthetics (eg,
isoflurane, propofol), narcotics (eg, cocaine), and anti-
neoplastic agents (eg, doxorubicin, cyclophosphamide).
Cardiotoxicity, and the biomarkers used to identify such
effects, should be distinguished from cardiovascular, or
drug-induced vascular injury (DIVI) biomarkers, which
are generally aimed at identifying injury to blood vessels
and the vascular system, discussed later in this chapter.
Biomarkers used for the identification and characteriza-
tion of cardiac injury effects can be broadly categorized
as those that identify structural or direct cardiac myo-
cyte injury (eg, troponins, fatty acid binding protein)
and necrosis, and those that indicate alterations to car-
diac function (eg, natriuretic peptides). Although cardiac
injury may be detected through a variety of modalities,
including electrocardiogram, imaging, and other func-
tional assessments (eg, blood pressure, cardiac output),
this discussion will focus on the measurement of bio-
markers in blood and other body fluids (serum/plasma),
which are more widely used in nonclinical research and
toxicology studies.
Biomarkers of Cardiac Structural Injury and
Necrosis
The current gold standard for the detection of acute
cardiac myocyte injury/necrosis includes the measure-
ment of cardiac troponin I (cTnI) and/or troponin T
(cTnT) in serum/plasma (see Table 17.2). Both of these
molecules are involved in regulating the excitation and
coupling process of the contractile apparatus within the
cardiac myocyte, and are released into circulation fol-
lowing cell injury and necrosis. These biomarkers have
repeatedly demonstrated their high sensitivity and spec-
ificity in detecting acute myocardial injury in a variety
of disease and experimental conditions in both humans
and animals [65–67]. Elevations in these biomarkers
will often precede the first histologic evidence of car-
diac injury and correlate well with effects on echocar-
diography and overall histologic injury scores [68,69].
In healthy animals, cardiac troponins circulate in low,
nearly undetectable levels and can increase within hours
 Cardiac and Skeletal Muscle Injury Biomarkers
TABLE 17.2  Summary of Cardiac and Skeletal Muscle Biomarkers
Leakage Markers
Cardiac troponin I (cTnI)
Cardiac troponin T (cTnT)
Heart-type fatty acid binding protein (H-FABP or FABP3)
Myoglobin
Myosin light chains (Mlc)
Creatine kinase (CK)
CK-MM
CK-MB
Lactate dehydrogenase (LD)
Aspartate aminotransferase (AST)
Skeletal muscle troponin I (sTnI)
FUNCTIONAL MARKERS
Atrial natriuretic peptide (ANP)
Brain natriuretic peptide (BNP, proBNP, NT-proBNP)
following a single cardiac insult [68]. The positive sig-
nal is short-lived as clearance of cTn molecules is rapid
and occurs within 24–48 following a single insult. Given
the sensitivity of many commercial troponin assays, it is
generally considered necessary to observe a greater than
twofold increase in cTnI values before considering the
signal to be biologically/toxicologically significant [70].
As with many of the newer biomarker assays, the lack
of standardized methods across industry makes com-
parison of absolute values between different methods
and laboratories problematic. Careful consideration of
method and laboratory-specific historical ranges should
be made before interpretation of positive signals is
determined. Similar troponin molecules also are present
in skeletal muscle (skeletal troponin; sTnI) but are anti-
genically distinct from their cardiac counterparts adding
to the specificity of both groups of biomarkers for the
detection of cardiac and skeletal muscle injuries, respec-
tively [71]. Although cTnI and cTnT have similar inter-
pretive utility and kinetic characteristics, cTnI assays
have been better characterized, are more widely avail-
able, and have demonstrated good cross-reactivity and
translatability across multiple species. Cardiac troponin
I is the most consistently utilized biomarker for detect-
ing myocardial injury in drug safety assessments [66,67].
Heart-type fatty acid-binding proteins (H-FABP
or FABP3) have recently been investigated as an early
marker for cardiac myocyte injury and associated cell
membrane disruption. FABPs are a family of low molec-
ular weight proteins found in multiple tissues including
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
459
Sources Interpretation
Cardiac muscle Cell injury/necrosis
Cardiac muscle Cell injury/necrosis
Cardiac and skeletal muscle Cell injury/necrosis
Cardiac and skeletal muscle Cell injury/necrosis
Cardiac and skeletal muscle Cell injury/necrosis
Cardiac and skeletal muscle, brain, GI tract Cell injury/necrosis
Cardiac and skeletal muscle Cell injury/necrosis
Cardiac and skeletal muscle Cell injury/necrosis
All muscle types, liver, RBCs Cell injury/necrosis
All muscle types, liver, RBCs Cell injury/necrosis
Skeletal muscle Cell injury/necrosis
Primarily cardiac atria Atrial wall stretch
Primarily cardiac ventricles Ventricular wall stretch
cardiac and skeletal muscle, brain, liver, and small intes-
tine [72]. FABP3 contained in heart muscle is rapidly
released into circulation within hours following myo-
cardial injury [73,74]. It has good sensitivity for cardiac
injury and has very similar release (within hours) and
clearance (≤48 h) kinetics compared to the cTnI; however,
it may lack the specificity of the cardiac troponins as sig-
nificant amounts of FABP3 have been demonstrated in
skeletal muscle in rat [75]. Similarly, myosin light chains
(Mlc) are a component of the myosin molecule found in
cardiac and slow twitch skeletal muscle cells that have
been used as indicators of myocardial injury [76]. They
have been incorporated into multiplex panels and used
in conjunction with other markers to aid in the character-
ization of myocardial damage.
Myoglobin is found in the cytoplasm of both skeletal
and cardiac muscle and is primarily used for the early
detection of acute myocardial infarction in humans.
However, it lacks specificity for cardiac muscle and
occurs in higher concentrations in skeletal muscle pre-
cluding its use as an independent marker of cardiac
injury [77]. Myoglobin has a short half-life (as short as
20 min in humans) and is used for postcardiac injury
monitoring in clinical settings [78].
Historically, a variety of other enzyme biomarkers
have been utilized for the detection of cardiac myocyte
injury in humans and animals. These include aspartate
aminotransferase (AST), lactate dehydrogenase (LD),
creatine kinase (CK), and creatine kinase MB isoenzyme
(CK-MB), which are no longer recommended for use as
460 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
cardiac biomarkers due to their low cardiac specificity,
inconsistent performance across species, wide tissue dis-
tribution, and lack of clinical relevance [77]. However,
many of these markers are well suited as biomarkers of
skeletal muscle injury, discussed later in this chapter.
Additionally, a number of proinflammatory biomarkers
(eg, high-sensitivity CRP, TNF-α, and myeloperoxidase)
have been used for cardiac injury detection, and occa-
sionally included in cardiac injury biomarker panels;
however, these types of endpoints only serve to identify
general inflammatory signals and should always be used
in conjunction with other more cardiac-specific markers.
Biomarkers of Cardiac Functional Alterations
The natriuretic peptides (see Table 17.2) are a family
of related hormones produced in the myocardium that
have potent vasoactive and diuretic properties used in
the regulation and homeostasis of blood pressure, renal
function, and maintenance of cardiac output [79]. Sev-
eral variants of these peptides exist including ANP (atrial
natriuretic peptide), BNP (brain natriuretic peptide),
and their N-terminal prohormones (N-terminal proatrial
natriuretic peptide (NT-proANP) and N-terminal pro-
brain natriuretic peptide (NT-proBNP)), all of which are
rapidly released into circulation in response to myocar-
dial wall stretch [80]. BNP is secreted from the ventricles
but may also be secreted from the atria in conditions of
heart failure [81]. Of this class of peptides, NT-proBNP
is the most commonly used biomarker in nonclinical
toxicology studies. NT-proBNP has been shown to be a
sensitive and specific biomarker in the diagnosis of heart
failure in humans and animals and has a positive correla-
tion to ventricular systolic dysfunction [80,82]. BNPs are
also elevated in conditions of dilated and hypertrophic
cardiomyopathy, diastolic dysfunction, and systemic
hypertension, and have been shown to be a good gen-
eral prognostic indicator of cardiovascular disease states
[83–85]. However, natriuretic peptides are also greatly
increased in a number of noncardiac conditions related
to increased vascular fluid volume and blood pressure
including pulmonary embolism, liver cirrhosis, renal
disease, and hyperthyroidism [79]. Natriuretic peptides
are particularly useful in drug safety assessments when
used in conjunction with more specific biomarkers of
cardiac structural injury/necrosis, such as cTnI, to pro-
vide an overall characterization of both structural and
functional effects on the heart.
Biomarkers of Skeletal Muscle Injury
Skeletal muscle toxicity and/or injury is a common
finding in nonclinical safety studies following expo-
sure to various chemical compounds (eg, cholinester-
ase inhibitors, carbofuran) or surgical manipulation in
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
medical device studies. Unlike the extensive research
devoted to the development of diagnostic assays for
myocardial disease, much less effort has been expended
on the development of biomarkers for skeletal muscle
injury detection. A number of enzymes (eg, CK, AST, LD)
and protein biomarkers (eg, myoglobin) have tradition-
ally been used for this purpose; however, they generally
lack the sensitivity and specificity needed to reliably dis-
tinguish subtle effects on skeletal muscle from effects on
other tissues.
Creatine kinase is a cytosolic enzyme found in a vari-
ety of tissues including skeletal and cardiac muscle,
brain with lesser amounts in the gastrointestinal tract,
uterus, bladder, and kidney [86]. CK is released into cir-
culation following myocyte injury and is rapidly cleared
with a half-life of only 2 h in dog [39]. It is the most
widely used biomarker to detect general muscle injury
and has three distinct isoenzymes (CK-MM, CK-BB, and
CK-MB), which can be determined through electropho-
retic separation. Traditionally CK-MM and CK-MB have
been used as markers of skeletal and cardiac muscle
injury, respectively; however, the majority of CK activ-
ity in both cardiac and skeletal muscle is attributed to
CK-MM [77]. There is also a high degree of variability in
CK content and isoenzyme proportions between specific
muscle locations, muscle types (fast twitch/slow twitch),
and between species, particularly in regards to myocar-
dial CK-MB activity [87–89]. CK is a good biomarker for
skeletal muscle injury but the CK-MB isoenzyme lacks
the specificity and consistency across species to be used
alone as a cardiac-specific biomarker.
Lactate dehydrogenase (LD) is an enzyme found in
most nucleated and nonnucleated cell types including
skeletal and cardiac muscle, liver, and erythrocytes, with
highly variable distribution among different tissue types
and between species [39,90]. Given its wide tissue distri-
bution, increases in LD activity are not specific to a single
organ. LD activity is generally higher in skeletal muscle
and liver compared to heart muscle, which supports its
use as a marker of skeletal muscle and liver injury [86].
Attempts to use isoenzymes of LD (eg, LD1, LD2, etc.)
to increase cardiac specificity have been previously per-
formed; however, much like the use of CK isoenzymes,
there is considerable variability between specific muscle
groups, species, and concentrations within the heart pre-
cluding it as a useful cardiac injury biomarker [77].
Aspartate aminotransferase (AST) and alanine amino-
transferase (ALT) are both enzymes that have multior-
gan tissue distributions that include skeletal muscle and
liver. Increased AST activities are generally associated
with effects on liver and skeletal muscle, but are also
seen with hemolytic conditions [39]. ALT elevations are
classically associated with hepatocellular injury; how-
ever, cases of significant muscle injury have also been
associated with increases in ALT activity [91].
 Biomarkers of the Immune System
More recently, next-generation biomarkers with
improved specificity for skeletal muscle have been dis-
covered and are gaining wider acceptance. The most
promising of these markers is skeletal troponin I (sTnI),
which has been shown to be a sensitive and specific bio-
marker for skeletal muscle injury [92]. Skeletal muscle
troponin is similar to, but antigenically distinct from its
cardiac-specific counterpart cTnI, and has been shown to
have the power to distinguish between skeletal and car-
diac injury in rats using a variety of cardiac toxins and
myotoxins [93]. This biomarker has also been included
in several multiplex muscle injury panels aimed at dif-
ferentiating between cardiac and skeletal muscle injury.
VASCULAR INJURY BIOMARKERS
Drug-induced vascular injury (DIVI) refers to any of
a variety of pathologic processes leading to or result-
ing in injury to blood vessels. It is a significant cause of
drug attrition as drug companies seldom proceed into
the clinics following identification of a DIVI liability in
nonclinical studies. The diagnosis of DIVI in nonclinical
animal species generally relies on biomarkers related to
the release of endothelial cell (EC) adhesion molecules,
EC activation markers, and/or nonspecific acute-phase
inflammatory proteins [94]. Although a long list of
potential biomarkers can be gathered from a literature
search, more recent evidence suggests that the vast
majority of markers will not apply equally to all animal
species and/or types of injury. This list becomes even
shorter when one considers the lack of species-specific
assays for all DIVI biomarkers.
The most significant DIVI work in a nonclinical spe-
cies (rat) to date was performed by the Vascular Injury
Working Group (VIWG) of the PSTC. The VIWG aimed
to identify candidate biomarkers that would be useful
in translating into humans, but also were independent
of pathophysiologic mechanism. The latter point being
critical as the mechanisms of vascular disease as seen in
humans (ie, immune-mediated) appear to be distinctly
different than those involved in DIVI in most nonclini-
cal animal species (ie, altered hemodynamics, direct EC
toxicity) [95,96]. These differences highlight the chal-
lenge in identifying biomarkers that have translatability
into man. The VIWG investigated dozens of protein and
molecular biomarkers and ultimately identified several
candidates that largely fell into one of two general cate-
gories: biomarkers of EC activation/injury and biomark-
ers of inflammation.
Biomarkers of EC injury make up a significant por-
tion of DIVI biomarkers covered in the literature and
include the vascular cell adhesion molecules (VCAMs),
intracellular adhesion molecules (ICAMs), integrins/
selectins, as well as signaling molecules involved in
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
461
vascular regeneration and repair. Though these fami-
lies of molecules have many members, only a select few
were deemed as useful for characterization of DIVI in
the rat by the VIWG. Candidate biomarkers from this
group included angiopoietin-2, endothelin-1, E-selec-
tin, thrombospondin-1, and VEGF-α [96]. Following
administration of a known vascular toxicant (PDE3i—a
phosphodiesterase inhibitor), positive signals in these
circulating EC biomarkers were modest (2–5-fold) and
tended to be most pronounced at 24–72 h postinjury.
Notably, most of these biomarkers exhibited increases
following injury, while E-selectin showed a decrease
from baseline following injury.
The second main category of biomarkers that was
found to be helpful in characterizing DIVI by the VIWG
were those involved in the inflammatory component.
Although these markers are extremely sensitive indica-
tors of inflammation, they lack the specificity to localize
injury to the vasculature and require the use of EC-
specific markers to be useful in DIVI identification. The
markers found to be useful included Timp-1, lipocalin-2,
KC/GRO (Cxcl1), α-1 acid glycoprotein 1, and total
nitric oxide. These markers tended to give more robust
positive signals following PDE3i administration (up to
200-fold) and were observed earlier (as soon as 1–4 h)
relative to biomarkers of EC activation/injury.
BIOMARKERS OF THE IMMUNE SYSTEM
The immune system is a complex regulatory net-
work of barriers, specialized effector cells, and mol-
ecules organized into a defense system that is capable
of distinguishing “self” from “nonself.” The cells of the
immune system include lymphocytes [bone marrow-
derived (B-cells), thymus-derived (T-cells), natural
killer (NK cells), macrophages/monocytes, neutrophils,
eosinophils, basophils, and mast cells. Functionally, the
immune system is broadly divided into innate and adap-
tive immunity based on whether or not the specific pro-
tection requires immunologic memory. Innate immunity
does not require immunologic memory and includes
defensive barriers such as anatomic (skin and mucous
membranes), physiologic (temperature and pH), and cel-
lular (phagocytic cells and pathogen-associated molecu-
lar patterns)]. The innate immunity mediates its function
through an array of soluble (cytokines, chemokines) and
cellular (phagocytic and cytolytic) mediators. Acquired
immunity requires previous exposure of lymphocytes
to the specific antigen, in the context of antigen present-
ing cells and major histocompatibility complex, which
creates immunologic memory for the next encounter of
that specific antigen. Any subsequent encounter with the
same antigen may elicit humoral (antibody-mediated)
or cell-mediated immune response. The details of basic
462 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
immunology are outside the scope of this chapter. Per-
haps the most important point to recognize about safety
assessment of the immune system is the complexity
and redundancy of the system, which is further compli-
cated by large species differences. Successful nonclinical
assessment of immune safety requires a thorough appre-
ciation of these complexities within and across species.
For example, recent observation of systemic organ fail-
ure during a Phase I clinical trial of anti-CD28 mono-
clonal antibody, which was not predicted by nonclinical
safety assessment, is a reminder of both complexity and
species difference within the immune system [97]. Addi-
tional information on the details of immunobiology and
immunotoxicity can be found in Chapter 22 of this book
and elsewhere [98–100].
Xenobiotic-related injury or alteration of the immune
system can be organized into five broad categories:
immunosuppression, hypersensitivity, immunogenicity,
autoimmunity, and adverse immunostimulation [101].
These categories are similar for chemical and biological
entities, although molecular weight is a large determi-
nant of immunogenicity. For example, entities with a
molecular weight of 1000 Da are not likely to be immu-
nogenic unless their metabolites are attached to endog-
enous molecules in the form of a haptens.
Biomarker testing of the immune system involves
a wide range of strategies including use of cell-based
(eg, hematology, immunophenotyping), tissue-based
(eg, histopathology, IHC), and protein-based (eg,
acute-phase proteins, cytokines, complement) assays.
The foundation of immunotoxicity testing involves
a tiered approach beginning with the evaluation of
standard toxicology endpoints including hematology
and clinical chemistry panels, with evaluation of lym-
phoid tissues for effects on organ weights, and gross
and microscopic morphology. As further investiga-
tions are warranted, more focused assays such as host
resistance, delayed-type hypersensitivity, autoimmu-
nity, and/or macrophage/natural killer cell function
are incorporated as needed.
Indicators of Immunosuppression
FDA guidance for immunotoxicity assessment states
that all investigational new drugs should be evalu-
ated for the potential to produce immunosuppression.
This is usually accomplished by a 28-day daily dos-
ing of the test article through the applicable route(s)
of administration in humans. Due to the complexity
of the immune system, the markers of immunosup-
pression are often not one molecule as is common in
other systems. Clear suppression of T cell-dependent
antibody response (TDAR) may be considered as one
marker of immunosuppression but a conclusion of
immunosuppression in nonclinical safety assessment
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
often requires several concordant lines of evidence
and uses a weight-of-evidence approach.
Hematology and Clinical Chemistry
Evaluation of the complete blood count (CBC) with
differential continues to be a highly effective biomarker
in identifying alterations to the immune system for both
signals of immunosuppression as well as proinflamma-
tory immune conditions. Lymphoid and/or bone-mar-
row toxicities are routinely associated with decreases
in lymphocyte, neutrophil, and total leukocyte counts
ultimately resulting in immunosuppression. Concur-
rent decreases in platelets and reticulocytes may also be
seen. These findings often correlate to decreased organ
weights and microscopic cellularity of lymphoid and
bone-marrow tissues. The first appearance of mild cyto-
penias on the hematology panel often precedes decreases
in bone-marrow cellularity as seen microscopically on
tissue sections.
Stress is common in toxicology studies and causes
mild-to-moderate reductions in lymphocytes and
eosinophils, often with concurrent increases in neutro-
phils and/or monocytes. These findings must be dis-
tinguished from primary effects on the immune system
by careful consideration of other related endpoints (eg,
histopathology, organ weights, acute-phase proteins) as
specifically suggested in the FDA S8 guidance [102].
Increased Incidence of Tumors
Test article-related increase in the incidence of tumors
in 2-year rodent bioassays or other toxicity studies that
are not due to genotoxic, hormonal, or other well-under-
stood carcinogenic mechanism need to be further inves-
tigated for the likely role of immunosuppression [101].
In cases where further investigation is necessary tumor-
host resistance assays with B16F10 melanoma cells or
other xenograft models may be the appropriate assay
to further evaluate the role of immunosuppression in
tumorigenesis.
Increased Incidence of Infections
Treatment-related increase in infections, especially
those that involve weakly pathogenic organisms such as
Candida albicans, are suggestive of immunosuppression.
The increased incidence of infection may not be related
to alterations in the function and/or number of lympho-
cytes but the barrier systems of innate immunity. For
example, ozone exposure before an aerosol challenge of
infectious agents results in treatment-related infections
[103]; however, this increase in infection was related to
alterations in the innate immune barriers and macro-
phage phagocytic system. Investigators need to remem-
ber that some cases of increased infection may show no
effect on TDAR because the test article altered the innate
component of the immune system.
 Biomarkers of the Immune System
Histopathology and Lymphoid Organ Weight
Decreases in spleen and thymic weights in conjunc-
tion with correlative histopathology findings may
indicate immunosuppression [104]. The associated
histopathology findings are usually decreases in lym-
phocytes within specific compartment(s). Lymphoid
organ weight is highly variable within and across
species and this variability should be carefully con-
sidered while interpreting organ weight data. The
variability of median organ weight within thymus of
mature Sprague Dawley rats may be up to 70%, while
the variability in Beagle dogs may be up to 170% [98].
The weighing of lymph nodes and Peyer’s patches is
even more variable and the quality of data it gener-
ates rarely justifies the effort it requires. The weight of
the spleen is also variable across species. For example,
the defense spleen has a large amount of lymphocytes
and is present in rats, mice, nonhuman primates, and
rabbits, while the storage spleen, which may store up
to one-third of the circulating blood volume, is seen
in dogs [98]. The swine spleen is an intermediate
between defense and storage spleen. The methods of
euthanasia and exsanguination may affect the spleen
weight in several species, but this influence tends
to be greater with storage spleen than with defense
spleen. In general, spleen and thymus weights are
often useful in rodents and should always be collected
but their utility in nonrodents is limited and collection
of lymphoid organ weights in nonrodents should be
addressed case-by-case [105].
To enhance the detection of immunosuppressive
effects, the Society of Toxicologic Pathology and the
majority of the toxicologic pathology community rec-
ommend a descriptive semiquantitative approach to
immune system evaluation [104,106,107]. There are
nuances to the approaches reported by these authors,
but they all emphasize a semiquantitative description of
changes in compartments of the lymphoid organs, which
derives from the recognition that (1) separate compart-
ments within each lymphoid organ support-specific
functions, (2) each compartment should be evaluated for
change(s), and (3) descriptive is better than interpretive
terminology for characterizing changes that occur in the
lymphoid system.
Immune cell phenotyping of lymphoid tissues and/
or whole blood may be used to further characterize the
nature of identified changes in immune system and their
effect(s) on circulating subpopulations of immune cells.
Flow cytometry analysis of surface markers of T cells
(CD4+ and CD8+), B cells, and NK cells are often utilized
as tier I assays in immunotoxicity assessments as recom-
mended by the FDA [102]. Immunophenotyping by flow
cytometry is generally not considered to be an adequate
standalone test of drug effects on immune function and
should be used in context with other immune function
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
463
assessments [108]. Immunohistochemistry of the same
markers within lymphoid tissues, when feasible, is a nec-
essary adjunct to further integrate changes in cell popu-
lation with the histopathology findings and hematology
observations. Overall an indication of immunosuppres-
sion by some of the above indices necessitates immune
function studies to further characterize/understand the
specific signal.
Immune Function Studies
These studies are designed to investigate functions
of components of the immune system when there is
reason to be concerned about immunosuppression or
occasionally immunostimulation. The principles from
FDA guidance indicate that these studies are relevant
when (1) the drug is being designed for HIV patients
or other immune-compromised population, (2) mol-
ecule is structurally similar to known immunosup-
pressive compounds, (3) pharmacokinetics and/or
toxicokinetics indicate that the compounds occurs in
immune tissues or cells at relatively high concentra-
tions, and (4) signs suggestive of immunosuppression
are seen in clinical trials [101]. In immune function
studies, the dose, duration, and route of administra-
tion should be consistent with the study in which the
original immune effect was observed. The functional
studies are divided into tier I and tier II studies based
on the ease of incorporating them into standard toxic-
ity studies (STS). Tier I studies are generally easier to
incorporate into STS than tier II, which often require
separate studies.
Primary Antibody Assays
T-cell dependent antibody response (TDAR) is a
tier I assay. It is the most acceptable, general-purpose
functional assay because it depends on the appropriate
function of antigen-presenting cells, helper T cells, sol-
uble cytokines, and B cells. The TDAR assay evaluates
an individual’s ability to mount a humoral immune
response to a standardized antigenic challenge (eg,
KLH, tetanus toxoid, SRBC). Sheep red blood cells
(SRBC) were previously used in a plaque assay, but
this is increasingly being replaced by Keyhole Lim-
pet Hemocyanin (KLH) and ELISA (enzyme-linked
immunosorbent assay) measurement of antigen-spe-
cific immunoglobulins. Evaluation of the immune sys-
tem using a TDAR assay should be performed when
assessing compounds with known or potential sup-
pressive or modulatory effects on the immune system
[101]. Decreases in antigen-specific antibody levels
are indicative of decreased immune function, which
is often associated with effects on related endpoints
including decreased lymphoid organ weights and
microscopic cellularity, decreases in cell counts on the
hemogram, and occasional clinical infections.
464 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
Natural Killer Cell Activity
Natural killer (NK) cell activity assay is a tier I assay.
This assay measures the cytolytic function of NK cells
and has been traditionally performed by quantifying test
article-related radioactivity in 51Cr-radiolabeled YAC-1
tumor cells. Ideally this assay should also include appro-
priate immunosuppression and immunostimulation con-
trols. Due to radioactivity concerns, a more-sensitive flow
cytometry-based NK cell assay has been developed and is
increasingly being used in drug development [109].
Macrophage Function
This is a tier II assay that evaluates the ability of mac-
rophages to phagocytose-labeled antigens. The assay
can be performed in vitro with primary peritoneal mac-
rophages isolated from animals that received the test
article or in vivo, which involves an antigen challenge
to test article-exposed animals and subsequent examina-
tion of marker antigen within macrophages in tissues.
Apoptosis and cytokine profiling are tier II assays that
help in identifying mechanisms of immunotoxicity or
immunosuppression. The measurement and interpreta-
tion of circulating cytokines need to consider that xeno-
biotics induce different cytokines in a variable temporal
pattern within and across tissues [6,110]. Therefore the
temporal pattern of cytokine induction is just as important
for understanding the mechanism as the induced cyto-
kine. Depending on the nature of observed effects (acute
vs. chronic) the time points should be chosen to help inter-
pret pathogenesis of the observed findings. At least three
time points of cytokine measure will help in elucidating
the mechanism in a repeat-dose study. Interpretation of
cytokines needs to consider that cytokine quantification
does not provide any information about the biological
activity/significance of the protein being measured [99].
Host resistance is a tier II assay that may be used to
detect the influence of xenobiotics on the host response
to bacterial (Listeria monocytogenes and Streptococcus
pneumonia), parasitic (Plasmodium yoelli), viral (influenza
A2), and tumor (B16F10 melanoma) pathogens. Host-
resistance assays are typically standalone studies and
can be more challenging to perform as the results are
often highly variable, thus a greater number (compared
to tier I) of animals may be necessary to achieve statisti-
cal significance [99].
Indicators of Adverse Immunostimulation
Some compounds are capable of overstimulating the
immune system to produce a range of unintended findings
that may include cytokine release, leucocyte infiltration
in multiple tissues, complement activation, and chronic
inflammation. Vaccines, adjuvants, medical devices,
and monoclonal antibody therapeutics may also initiate
responses typical of inflammation/immune stimulation
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
including increases in neutrophils, lymphocytes, acute-
phase protein (eg, fibrinogen, CRP), cytokines, and other
inflammatory markers. The mechanism of adverse immu-
nostimulation should be investigated/understood in other
ways to contextualize potential human risk.
Standard hematology panels can be used to detect
increases in leukocytes, neutrophils, and/or lymphocytes
that are typical of immune/inflammatory stimulation,
either as a primary immunotoxic effect, or secondary to
infection or other morbidity. These increases are often
associated with bone-marrow hypercellularity, lym-
phoid organ hyperplasia, extramedullary hematopoiesis
in the spleen, and/or other inflammatory lesions. Stan-
dard clinical chemistry panels include albumin (negative
acute-phase protein) and globulin (positive acute-phase
protein), which are both fairly sensitive in the detection of
inflammatory and immune signals [39]. There are also a
variety of other acute-phase protein assays (eg, C-reactive
protein, fibrinogen, serum amyloid A) available on most
standard clinical chemistry or coagulation analyzers that
are helpful in identifying proinflammatory signals.
Acute-Phase Proteins
Acute-phase proteins (APPs) are circulating blood
proteins primarily synthesized in the liver in response to
upstream inflammatory signals. APPs serve as excellent
biomarkers of immune system perturbation and are used
in context with other markers of inflammation to identify
subtle inflammatory/immune signals. Acute-phase pro-
teins can either be positive APPs, which increase with
inflammation (eg, fibrinogen, C-reactive protein (CRP),
serum amyloid A), or negative APP (eg, albumin), which
decrease with inflammation. APPs have strong species-
specific behaviors and can be classified as either a major
or minor APP in any given species (Table 17.3). It is criti-
cal to understand which APP is most appropriate for the
species in use to avoid mischaracterization of compound
liabilities. For example, in the nonhuman primate CRP
is major APP, eliciting strong increases in response to
inflammatory stimuli while in rodents, the CRP response
will be comparatively minor or nonexistent [16]. Most
APPs will increase in 24–72 h following an inflammatory
stimulus, which should be considered when choosing
blood-draw intervals and during data interpretation.
Results from these biomarkers should always be inter-
preted in context with other inflammatory biomarkers
(eg, hematology, cytokines, etc.). APP assays are widely
available on both automated clinical chemistry analyz-
ers and as single or multiplex ELISA platforms.
Cytokine Evaluation
Cytokines are a heterogeneous collection of peptides
that serve as signaling molecules between cells and elicit
biological responses, including cell activation, prolifera-
tion, growth, differentiation, migration, and apoptosis.
 Biomarkers of the Immune System 465
TABLE 17.3  Summary of Major and Minor Positive Acute-Phase Proteins by Species
Species Major Minor

Human C-reactive protein, serum amyloid A α-1 acid glycoprotein, haptoglobin

Nonhuman primate C-reactive protein α2-macroglobulin, serum amyloid A

Rat α-1 acid glycoprotein, α2-macroglobulin C-reactive protein, fibrinogen

Mouse Haptoglobin, serum amyloid A C-reactive protein, fibrinogen

Dog C-reactive protein, serum amyloid A α-1 acid glycoprotein, haptoglobin

Rabbit Haptoglobin, serum amyloid A C-reactive protein, fibrinogen

Pig Haptoglobin, serum amyloid A α-1 acid glycoprotein

The evaluation of cytokines in immunotoxicity charac-
terization, both in vitro and ex or in vivo, have been in
use for some time now for the purposes of hazard iden-
tification, mechanistic characterization, as markers of
pharmacodynamic activity and/or efficacy, as well as
to monitor for adverse drug reactions [111–113]. Blood
cytokine measurements are an attractive option due
to the ability to perform serial monitoring using read-
ily accessible samples on a multitude of available assay
platforms. However, the use of cytokines as biomarkers
poses several challenges related to their short half-life
and release dynamics, lack of organ specificity, undetect-
able baseline levels, and lack of standardized method-
ologies. The overall complexity of the immune system
and related cytokine interactions emphasizes the need
for focused and deliberate strategies when evaluating
these endpoints.
Measurement of serum cytokines may have the most
utility in programs or studies that evaluate intended
or unintended inflammation and immunomodulation
produced by therapeutics. When applying cytokine
measurements to nonclinical studies in animals, it is
important to take samples at intervals that are physi-
ologically relevant to the specific immunomodulation
or immunopathology being induced. Cytokines are
released quickly and equally rapid in their clearance,
hence serial blood sampling time points should occur at
several intervals within the first 24 h of dose administra-
tion [111]. A standard approach is to sample 2–3 times
within 24 h of dose administration (eg, 2–6, 12, and 24 h
postdose) in order to capture the positive cytokine sig-
nals, although this will need to be customized to the
particular project based on the goals of the study and
the known pharmacology of the compound. Ongoing
immune/inflammatory stimulation may allow positive
signals to be detected at later intervals, although this is
more the exception than the rule. Increases in cytokines
(2–24 h) will usually precede increases in APPs (24–72 h).
In general, larger multiplex panels including multiple
cytokines are used for these purposes. Some of the most
common markers evaluated include IL-1, IL-6, TNF-α,
and INF-γ with more advanced panels including MCP-1,
VEGF, IL-2, IL-4, IL-8/KC/GRO, IL-10, IL-12, and IL-13.
Although the evaluation of cytokines in animals contin-
ues to be common in nonclinical studies, recent examples
have demonstrated that in vivo measures of cytokines in
animal species are not always predictive of adverse reac-
tions in humans [114]. This sometimes disastrous lack of
concordance between nonclinical and human cytokine
responses has led to more widespread use of ex and in vivo
cytokine release assays (CRAs) in an attempt to bridge the
gap between nonclinical species and humans. Although
various approaches to CRAs have been published, most
often, peripheral blood leukocytes or mononuclear cells
(PBMCs) are isolated from blood and incubated with test
compounds either in an aqueous solution or solid phase/
dry-coat (immobilized on plate) format [112]. Small sam-
ples of cell-suspension media are then measured for cyto-
kines at various intervals (eg, 6–72 h) following incubation.
The notion of performing CRAs in solution or in solid
phase or in whole blood versus PBMCs is still under debate
with 62% of respondents performing aqueous phase CRAs
according to a recent industry survey [112]. It has been
argued that whole blood more closely mimics in vivo con-
ditions by involving soluble factors, platelets, and other
cells types when compared to PBMCs. In addition, whole-
blood CRAs require less technical effort to execute. Soluble
or aqueous phase CRAs may use whole-blood or PBMCs
and are attractive due to their simplicity but have been crit-
icized for their lack of predictivity, particularly when using
TGN1412, when compared to solid-phase CRAs [113].
Indicators of Hypersensitivity
Type I hypersensitivity reactions are usually IgE-
mediated, in humans and many nonclinical species,
although IgG can also mediate systemic anaphylaxis in
guinea pigs, rabbits, rats and mice [98]. Type I hyper-
sensitivity could be systemic (anaphylaxis or urticarial)
or respiratory (asthma). The systemic reaction involves
the release of one or more vasoactive amines from hista-
mine, kinins, and serotonin. FDA requires that all drugs

III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS

466 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
developed for dosing by inhalation route be evaluated
for ability to induce Type I hypersensitivity [101]. Thera-
peutic proteins may also elicit Type I reactions in mon-
keys and other nonclinical species [115], but the relevance
of such reaction to human risk assessment needs to con-
sider the species source of the protein. The demonstra-
tion of drug-specific IgE should be considered a hazard
with or without the elevation of Th2 cytokines such IL-4,
IL-5, IL-10, and IL-13 [99,101]. The murine local lymph
node assay (LLNA) has undergone extensive validation
since the first report [116] and has almost replaced other
sensitization assays in guinea pigs. It measures lympho-
cyte proliferation after topical exposure to xenobiotics.
A chemical is considered a sensitizer if, at any dose, it
induces threefold or greater cell proliferation in local
lymph node when compared to concurrent control. For
human risk assessment, the comparative thickness of
human versus murine skin, which is thinner and more
permeable, needs to be considered [117].
Types II and III hypersensitivities are antibody-­
mediated and tend to cooccur in xenobiotic exposed ani-
mals, although Type III is more common in animals dosed
with therapeutic proteins [101,115]. They result from IgG
and/or IgM mediated antibody-dependent/complement-
mediated cytotoxicity or lysis of somatic cells (Type II) or
immune complex-related complement-mediated destruc-
tion of tissue (Type III). Pathology findings may include
one or more of vasculitis, glomerulonephritis, anemia, and
thrombocytopenia. Type III hypersensitivity is increas-
ingly being observed in nonclinical safety assessment as
the testing of larger molecule biopharmaceuticals increase.
Complement-related pathology is thought to arise from
failure of the clearance system for soluble immune com-
plexes. In some cases (subclinical) increases in circulat-
ing immune complexes and circulating IgM have been
reported prior to or concurrent with the drug reaction
[115]. The kinetics of IgG/IgM and circulating complement
on days preceding the drug reaction may be informative
when investigating drug reactions. The investigation of
immune complex-mediated pathology requires a weight
of evidence approach using some of the following informa-
tion (1) plasma and/or serum drug and antidrug antibody
(ADA) levels; (2) histopathology findings from animals
with drug reactions; (3) hematological indices such as
erythrocyte numbers and morphology, and platelet num-
bers; (4) circulating complement component levels, which
may not be available due to the manner of death, and is
rapid in some cases; and (5) immunohistochemical dem-
onstration of granular deposits of drug, endogenous IgG,
IgM, and other complement components [115].
Type IV hypersensitivity is a T-cell mediated delayed
type hypersensitivity. Drugs intended for topical use need
to be evaluated for their ability to sensitize [101], and the
LLNA is increasingly being accepted for the sensitization
aspect of Type IV reaction, in addition to Type I testing.
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
Indicators of Immunogenicity
Molecules above 1000 Da may be immunogenic, thus
raising a twofold concern. The first is alteration of phar-
macodynamics and loss of efficacy due to binding of
the test article to antidrug antibodies (ADA) in circula-
tion, while the second is allergenicity. In general ADA
are used to determine the immunogenicity while mod-
els of allergenicity testing have not undergone robust
validation.
Indicators of Autoimmunity
Xenobiotic-related autoimmunity has been associ-
ated with several drugs from different classes. They are
related to the formation of hapten for molecules less
than 1000 Da and often include a genetic predisposi-
tion such as the HLA-DR3 allele in humans [110]. Due
to the complex nature of autoimmune diseases there are
no generally recognized nonclinical testing approaches.
Drugs that cause autoimmune diseases tend to cause
hematological disorders such as neutropenia, thrombo-
cytopenia, and anemia. Biotherapeutics are now known
to cause unexpected hematological findings and some
of these are immune-mediated and not predicted from
nonclinical studies. However, nonclinical studies have
predicted clinical hematotoxicity for hemophagocytosis
(occurs with recombinant cytokines and growth factors)
and thrombocytopenia, which is observed with anti-
CD40L monoclonal antibodies [118].
In conclusion safety assessment of the immune sys-
tem requires a weight-of-evidence approach using a
variety of modalities to evaluate lymphoid organs and
bone marrow with careful understanding of the test
article and the immune system of the species in which
the test is being conducted. A tiered approach starting
with standard toxicology endpoints (eg, hematology,
lymphoid organ, and bone-marrow pathology) with
the addition of standalone immunotoxicity studies as
needed is the current paradigm for characterization of
the immune system.
Biomarkers of Toxicity in the Central Nervous
System
The central nervous system (CNS) contains a vast
array of neuronal cell bodies (nuclei) that have very
specific functions. These cell bodies are protected from
most xenobiotics by the blood–brain barrier. However,
some molecules are designed to cross the blood–brain
barrier or do so by nature of their physicochemical
properties. There are no routinely used molecular/
chemical validated biomarkers to assess xenobiotic-
related injury to the nervous system. Functional obser-
vation battery (FOB) or other analogous test is required
 Conclusion
for assessing the effects of test articles on indices of
neurobehavioral function such as coordination, motor
activity, behavioral changes, sensory/motor reflex
responses, and body temperature [119]. The brain and
spinal cord are also evaluated histologically as part of
most standard nonclinical safety assessment programs.
The current neurotoxicity testing paradigm relies heav-
ily on morphological examinations, but the future will
probably include additional methods for understand-
ing receptor dynamics as well as cellular and biochemi-
cal alterations in the brain [120]. Two methods that
might increase the sensitivity of detection and help elu-
cidate mechanisms of CNS toxicity are cerebrospinal
fluid (CSF) analysis and molecular imaging.
Cerebrospinal fluid analysis may reveal test article-
related changes in CNS alteration through changes in
the color and quantity of CSF, number and composi-
tion of mononuclear cells in the CSF, and biochemical
alterations of CNS [121]. Neurotoxic effects can result
in the alteration of neurotransmitters and their metab-
olites such as dopamine, glutamate, γ-aminobutyric
acid, epinephrine, serotonin, homovanillic acid,
5-hydroxyindolacetic acid. For example, homovanillic
acid can be measured in the CSF of Collie dogs that
develop neurologic signs after Ivermectin exposure
[121]. The concentrations of total protein, albumin,
C-reactive protein, myelin basic protein, and S-100 can
also be indications of toxicant-related alterations in
blood–brain barrier and/or CNS. With the continued
refinement of metabolomics and spectrometry tech-
niques, CSF analysis might become a better-utilized
adjunct for CNS safety assessment.
Recently imaging techniques such as magnetic
resonance imaging (MRI), positron-emission tomog-
raphy (PET), and single photon-emission computed
tomography (SPECT) have been used in drug dis-
covery and development. These imaging techniques
have been used to (1) detect activated microglia in
neuroinflammation by using a radiotracer to target
a translocator protein (TP-18), (2) access the quan-
tity and integrity of adhesion molecules (CD62E) and
P-glycoptoteins(CD243) in the blood–brain barrier,
and (3) CNS glucose metabolism by using [18F]fluo-
rodeoxyglucose uptake [122]. As these imaging tech-
nology capabilities improve and reduce in cost, they
are likely to provide more comprehensive and specific
approaches to assess CNS safety of xenobiotics.
Biomarkers of Toxicity in the Reproductive
System
The reproductive system has a complex hormonal
regulatory network that ranges from gonadal sex steroid
hormones to centrally released luteinizing hormone (LH)
and follicle stimulating hormone (FSH). In nonclinical
III.  CLINICAL PATHOLOGY, HISTOPATHOLOGY, AND BIOMARKERS
467
safety assessment there are elaborate toxicity studies
designed to assess the effects of xenobiotics on reproduc-
tion. However, these studies are expensive, longer term,
and tend to occur later in the drug development process.
Ideally, noninvasive biomarkers of reproductive toxic-
ity that can be measured in a 28-day repeat-dose toxicity
study will be good predictors to provide early warning
about reproductive toxicity and help contextualize the
morphological findings that occur in the reproductive
system during shorter-term studies.
Currently, hormones such as estrogen, progesterone,
prolactin, testosterone, LH, and FSH are sometimes used
to provide additional context when there is a signal of
reproductive effect. However, hormone endpoints can be
difficult to assess in large toxicology studies due to the
difficulty in controlling variations due to stress, diurnal
variation, and menstrual/estrus cycling [123]. For female
animals, estrus cycle evaluation provides another index
of reproductive health, although the variable length of
estrus cycle in different species makes it more useful
in those with short cycles (eg, rat) and very difficult in
species with longer cycles (eg, dog). For testicular tox-
icity, histopathology and organ weight of accessory sex
organs are currently the most sensitive and reliable ways
of detecting test article-related effects [124]. However,
there is industry-wide interest in validating the utility
of inhibin B or other potential toxicogenomic mark-
ers that will translate to human clinical trial scenario
[125]. Androgens stimulate sertoli cells to secrete inhibin
B, which regulates spermatogenesis in seminiferous
tubules. Clinically, serum inhibin B tends to be higher in
fertile men than in infertile men [126]. Therefore inhibin
B appears to be a good biomarker for testicular toxicity
because it can be measured in a nonterminal longitudi-
nal fashion and is translatable.
CONCLUSION
Several routinely and seldom used biomarkers were
presented for different tissues and organ systems. A
key feature of interpreting data from these biomarkers
is to have an appreciation for the heterogeneity of posi-
tive biomarker response signals in timing, magnitude,
and by species, and to have a good understanding of
the analytical performance each individual assay. Such
appreciation frequently involves professionals who
are trained and experienced in the molecular and com-
parative physiological foundations of these biomarkers
and their clinical relevance. A careful case-by-case and
integrative interpretation of data from these biomark-
ers often provides a better perspective on the potential
risk(s) of xenobiotic-related safety issues and informs
on the appropriate risk mitigation strategies in drug
development.
468 17.  BIOMARKERS IN NONCLINICAL DRUG DEVELOPMENT
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