Biomarkers
of Toxicity and Surrogate Endpoints for Safety
Objectives
·
Review
the current status of toxicology biomarkers in drug development and
clinical safety assessments
-
Do we
need more information on biomarkers, and are we using our current
information wisely?
·
Demonstrate
that biomarkers can serve as early predictors of insidious adverse effects
related to chronic drug exposures
-
What
is needed to establish the utility of a biomarker as an indicator of
clinically significant toxicology?
•
Discuss the development of genetically modified rodent models as
better predictors of drug effects in humans
-
Can or
should transgenic humanized models be developed in other species besides
the mouse?
•
Discuss the clinical application of pharmacogenetics to predict and
prevent adverse drug effects
-
Do the
benefits outweigh the costs/risks associated with clinical pharmacogenetic
profiling?
•
Identify technological approaches that will provide opportunities
for developing more and better toxicology biomarkers
-
What
are the roles of industry, academia, and the government in the development
of toxicology biomarkers?
-
What
are the current barriers to the development of toxicology biomarkers?
Agenda
Moderators:
Frank Sistare, Ph.D., Center for Drug Evaluation and Research, U.S.
Food and Drug Administration
Introduction
Kenneth
Olden, Ph.D., Director, National Institute of Environmental Health
Sciences
Presentation
I
The Status of Toxicology Biomarkers and Safety Evaluation Approaches
James
T. MacGregor, Ph.D., Center for Drug Evaluation and Research, U.S. Food
and Drug Administration
Presentation
II
Cardiac Troponin T as a Biomarker for Monitoring Chronic Doxorubicin
Cardiomyopathy
Eugene
H. Herman, Ph.D., Center for Drug Evaluation and Research, U.S. Food and
Drug Administration
Steven
Lipshultz, M.D., University of Rochester Medical Center
Presentation
III
Preclinical Toxicology Model: Comparing
Different Human Variant Alleles in a Mouse Model
Daniel
W. Nebert, M.D., University of Cincinnati Medical Center
Pharmacogenetics as Applied to Human Drug Safety Testing
Richard
M. Weinshilboum, M.D., Mayo Clinic and Foundation
The Role of Mass Spectrometry in the Development of Biomarkers
Ian
A. Blair, Ph.D., University of Pennsylvania
Toxicogenomics in Safety Assessment
Ronald
D. Tyler, D.V.M., Ph.D., GlaxoWellcome Company, United Kingdom
Presentation
IV
Gene Expression Analysis for Toxicology: Moving Beyond Phenomenology
Spencer
B. Farr, Ph.D., Phase I Toxicology
Open
Discussion
Summary
of Session Recommendations
Jason
D. Morrow, M.D.
Frank
Sistare, Ph.D.
ABSTRACTS
The Status of Toxicity Biomarkers and Safety
Evaluation Approaches
James
T. MacGregor, Ph.D.
Safety
evaluation has traditionally relied on the measurement of clinical
chemistry and hematology parameters that serve as biomarkers of cellular
integrity (e.g., serum transaminases) and homeostasis of the organism
(e.g., blood urea nitrogen, creatinine, electrolytes) or as indicator cell
populations (e.g., white blood cell, host‑defense cell counts).
Recently, advances in biological understanding have created the
opportunity for new surrogate markers of toxicity, including (1) inducible
damage‑specific responses; (2) transgenic animals with humanized
characteristics, accelerated tumor development, and other favorable
traits; (3) in vitro human cell models that retain in vivo
characteristics; (4) chemokine and/or cytokine signals as surrogates for
tissue damage; (5) more specific and sensitive molecular markers of organ
damage; and (6) the opportunity to use hybridization array, proteomic, and
imaging technologies to improve conventional biomarker assays.
These new technologies can be expected to improve the clinical and
nonclinical interface and improve the approaches to both nonclinical and
clinical safety studies.
Key
References
MacGregor
JT. Transgenic animal models for mutagenesis studies: Role in mutagenesis
research and regulatory testing. Environ Mol Mutagen
1998;38:106‑109.
MacGregor
JT, Farr S, Tucker JD, Heddle JA, Tice RR, Turteltaub KW. New molecular
endpoints and methods for routine toxicity testing. Fund Appl Toxicol
1995;26:156‑173.
MacGregor
JT, Tucker JD, Eastmond DA, Wyrobek AJ. Integration of cytogenetic assays
with toxicology studies. Environ Mol Mutagen 1995;25:328‑337.
Cardiac
Troponin T as a Biomarker for Monitoring Chronic
Doxorubicin
Cardiomyopathy
Eugene
H. Herman, Ph.D.; Jun Ahang, M.D.; Steven Lipshultz, M.D.; Nader Rifai,
Ph.D.; Kazuyo Takeda, M.D.; Zu‑Xi Yu, M.D.; Douglas P. Chadwick,
B.S.; and Victor Ferrans, M.D., Ph.D.
The
present study sought to determine whether cardiac troponin T (cTnT), a
cardiac protein used to diagnose ischemic myocardial injury, also may be
useful in detecting doxorubicin (DXR) cardiotoxicity.
Spontaneously hypertensive rats (SHRs) were given 1 mg/kg DXR
weekly. Myocardial tissues
and serum samples were collected and analyzed after 2,4,6,8,10, or 12 weeks
of treatment. DXR lesion
scores were assessed semiquantitatively by light microscopy, and serum
levels of cTnT were quantified by an ELISA method (Enzyum).
Increases of cTnT (0.03‑0.05 ng/mL) in serum and lesion
scores of 1 or 1.5 were noted in 1/5 and 2/5 SHRs given 2 or 4 mg/kg DXR,
respectively. All SHRs given
6 mg/kg or more DXR had elevations of cTnT in serum and myocardial
lesions. The average cTnT
concentrations and the average lesion scores increased with the cumulative
DXR dose (0.13 versus 0.40 ng/ml and 1.4 versus 3.0) in SHRs given 6 and
12 mg/kg DXR, respectively. It
is concluded that cTnT is released from DXR‑damaged myocytes, and
measurements of serum levels of this protein can provide a sensitive means
for assessing the early and continuing cardiotoxic effects of DXR.
Key
References
Herman
E, Lipshultz S, Rifai N, et al. Use of cardiac troponin T levels as an
indicator of doxorubicin‑induced cardiotoxicity. Cancer Res
1998;58:195‑197.
Lipshultz
SE, Rifai N, Sallan SE, Lipsitz SR, Dalton V, Sacks DB, Ottlinger ME.
Predictive value of serum cardiac troponin T (cTnT) in pediatric patients
at risk for myocardial injury. J Am Coll Cardiol 1997;96:2641-2648.
Preclinical
Toxicology Model: Comparing
Different Human Variant Alleles in
a Mouse Model
Daniel
W. Nebert, M.D.; Michael J. Carvan III, Ph.D.; Gary W. Stuart, Ph.D.; and
Tim
Dalton, Ph.D.
Striking
interindividual differences in environmental toxicity and cancer
susceptibility among human populations often reflect polymorphisms in
drug‑metabolizing enzymes (DMEs) and receptors that control DME
levels. Compared with interspecies (human‑mouse) differences of
twofold to perhaps twentyfold, human interindividual variability in DME
activities or DME receptor affinity has often been shown to vary fourfold
to more than 10,000‑fold. How
can these different human alleles be studied most effectively and
efficiently in an experimental animal model?
Transgenic knock‑in technology can be used to insert unique
human alleles in place of the orthologous mouse genes.
The knock‑in of each gene is a separate targeting event,
however, requiring (1) construction of the targeting vector and
transfection into embryonic stem cells, (2) generation of a targeted
mouse, and (3) backcross breeding of the knock‑in mouse (at least
six times) to produce a suitable genetically homogeneous (>99 percent)
background (for decreasing interindividual variability).
These experiments require years to complete—making this very
powerful technology inefficient for routine applications. If, on the other hand, the initial knock‑in targeting
vector includes sequences that would allow the knocked‑in gene to be
exchanged (possibly, even repeatedly) for yet another new allele, then
testing a battery of human polymorphic alleles in transgenic mice could be
accomplished in several months instead of several years.
This gene swapping can be done by zygotic injection of the human
allele cassette into the fertilized ovum of the parental knock‑in
mouse strain or by cloning mice from fibroblasts containing the nucleus
wherein each human allele has already been swapped.
In mouse cells, we have succeeded in gene swapping by exchanging
one gene (including its regulatory regions) flanked by heterotypic lox
sites with a second gene (including its regulatory regions) flanked by
heterotypic lox sites. This
research is supported in part by National Institutes of Health grants R01
ES07058, R01 ES08147, R01 AG09235, R01 ES06321, and P30 ES06096.
Pharmacogenetics
as Applied to Human Drug Safety Testing
Richard
M. Weinshilboum, M.D.
A
large number of functionally significant, common, genetic polymorphisms
for enzymes that participate in drug and xenobiotic biotransformation have
been described over the past three decades.
We now appreciate that genetic polymorphisms for both phase I and
phase II drug‑ and xenobiotic‑metabolizing enzymes can be
important factors for variation in toxicity after exposure to these
agents. Prototypic examples of genetically polymorphic
drug‑metabolizing enzymes will be described in the context of the
rapid advances that are occurring in human genomics as well as molecular
pharmacology and toxicology.
The
Role of Mass Spectrometry in the Development of Biomarkers
Ian
A. Blair, Ph.D.
The
quantitation and identification of DNA adducts in biological samples as
molecular dosimeters of both exogenous and endogenous carcinogens require
sophisticated analytical methodology because adducts are normally present
in such low concentrations. Mass
spectrometric techniques have high sensitivity and are highly specific
because only ions derived from the analyte of interest are monitored.
The development of liquid chromatography/mass spectrometry
methodology based on the use of atmospheric pressure
ionization‑based techniques of electrospray ionization, ionspray,
and atmospheric pressure chemical ionization has had a profound effect on
our ability to identify analytes of interest when only trace amounts of
material are available. These
techniques are extremely robust and are amenable to the quantitation of
DNA adducts in biological samples. High
sensitivity can be attained by the use of selected reaction monitoring of
a specific product ion after collision‑induced dissociation has been
performed on the protonated molecular ion derived from the analyte of
interest. The ability to use
this methodology as a dosimeter for environmental chemicals was
exemplified in recent studies we have conducted on butadiene exposure.
Liquid chromatography/mass spectrometry, in combination with stable
isotope methodology, provided a robust, sensitive, and accurate means to
quantify the butadiene derived N7‑guanine adducts.
Using this methodology, it was possible to demonstrate the presence
of two diastereomeric forms of trihydroxybutylguanine in liver DNA and to
assess the excretion of these DNA adducts in the urine.
Studies on endogenous carcinogens have relied more heavily on the
use of gas chromatography/electron capture negative ion chemical
ionization mass spectrometry because of the high sensitivity of this
technique. However, liquid
chromatography/mass spectrometry has been very useful for identifying
novel targets for molecular dosimeters.
Using this methodology, we have recently identified a new reactive
electrophilic aldehyde from the breakdown of lipid peroxides that form
covalent adducts with DNA bases. The
characterization of these DNA adducts has provided two novel biomarkers
that can be employed to assess exposure to oxidative stress.
This research is supported by National Institutes of Health grant
CA63878.
Key
References
Chaudhary
AK, Nokubo M, Marnett LJ, Blair IA. Analysis of the malondialdehyde‑2'‑
deoxyguanosine adduct in rat liver DNA by gas chromatography/electron
capture negative chemical ionization mass spectrometry. Biol Mass Spectrom
1994;23:457‑464.
Chaudhary
AK, Nokubo M, Oglesby TD, Marnett LJ, Blair IA. Characterization of
endogenous DNA adducts by liquid chromatography/electrospray ionization
tandem mass spectrometry. J Mass Spectrom 1995;30:1157‑1166.
Chaudhary
AK, Nokubo M, Reddy GR, Yeola SN, Morrow J, Blair IA, Marnett LJ.
Detection of endogenous malondialdehyde‑deoxyguanosine adducts in
human liver. Science 1994;265:1580‑1582.
Chaudhary
AK, Reddy GR, Blair IA, Marnett LJ. Characterization of an N6‑oxopropenyl‑2'‑deoxyadenosine
adduct in malondialdehyde‑modified DNA using liquid chromatography/electrospray
ionization tandem mass spectrometry. Carcinogenesis
1996;17:1167‑1170.
Kambouris
SJ, Chaudhary AK, Blair IA. Liquid chromatography/electrospray ionization
tandem mass spectroscopy (LC/ESI MS/MS) analysis of 1,2‑epoxybutene
adducts of purine deoxynucleosides. Toxicology 1996;113:331‑335.
McCoull
KD, Rindgen D, Blair IA, Penning TM. Synthesis and characterization of
polycyclic aromatic hydrocarbon ortho‑quinone depurinating
N7‑guanine adducts. Chem Res Toxicol 1999;In press.
Muller
M, Belas FJ, Blair IA, Guengerich FP. Analysis of 1,N2‑ethenoguanine
and 5,6,7,9‑tetrahydro‑7‑hydroxy‑9‑oxoimidazo[1,2‑a]purine
in DNA treated with 2‑chlorooxirane by high performance liquid
chromotography/electrospray mass spectrometry and comparison of amounts to
other DNA adducts. Chem Res Toxicol 1997;10:242‑247.
Oe
T, Kambouris SJ, Walker VE, Meng Q, Recio L, Wherli S, Chaudhary AK, Blair
IA. Persistence of N7‑(1,2:3,4‑trihydroxy)guanine adducts in
the livers of mice and rats exposed to 1,3‑butadiene. Chem Res
Toxicol 1999;12:247‑257.
Rouzer
CA, Chaudhary AK, Nokubo M, Ferguson DM, Reddy GR, Blair IA, Marnett LJ.
Analysis of the malondialdehyde‑2'‑deoxyguanosine adduct
pyrimidopurinone in human leukocyte DNA by gas chromatography/electron
capture/negative chemical ionization/mass spectrometry. Chem Res Toxicol
1997;10:181‑188.
Toxicogenomics
in Safety Assessment
Ronald
D. Tyler, D.V.M., Ph.D.
Most
toxicities that are not lethal within minutes are likely to have some
impact on gene expression, be a result of gene expression, or both.
Some of the gene expression changes (GECs) that occur as toxicity
develops are expected to be unique to the mechanism of toxicity (e.g.,
free radical production, inhibition of cellular respiration).
Some GECs are expected to be unique to the type of toxicity (e.g.,
apoptosis, nongenotoxic carcinogenicity) but common among mechanisms that
cause the same type of toxicity. Other
GECs are expected to be adaptive in response to changes in such things as
blood pressure, nutrition, and so forth.
Determination of GECs that reliably indicate the above would allow
development of gene expression‑based biomarkers of toxicity.
The most valuable GECs will probably be critical core genes that
have been conserved during evolution.
These GECs will be shared among species, greatly improving
interspecies comparisons and extrapolations.
If gene expression analysis in discovery and toxicology studies
identify GECs indicative of toxicity and in vitro studies with human and
animal cell lines establish that the human gene homologs behave as the
animal genes do, GECs can be used as biomarkers of toxicity in clinical
trials and patients. These
biomarkers will be especially valuable when they occur in easily
assessable tissues (e.g., lymphocytes) and for early detection of chronic
toxicity. Although functional
or morphologic evidence of chronic toxicities may take weeks to years to
develop, characteristic GECs likely occur in hours or days.
Identification of these GECs would enable early detection of the
chronic toxicity, markedly shortening necessary animal studies and
protecting clinical trial subjects and patients.
To fully harvest the benefit of gene expression analysis, reliable
broad gene coverage technologies, computer systems capable of manipulating
large data sets, and scientists that understand pathophysiology at the
molecular and transcriptional level are necessary.
Development of these technologies, systems, and scientists should
be a major focus of future resource commitment.
Gene
Expression Analysis for Toxicology: Moving
Beyond Phenomenology
Spencer
B. Farr, Ph.D.
The
science of toxicology is changing radically.
There are two fundamental forces compelling this change:
(1) high‑throughput chemical library synthesis and screening
and (2) the human genome project and attendant technology for rapid
polymorphism and expression analysis.
Toxicologists are faced with the challenge of developing
high‑throughput toxicity screens to keep pace with the increased
number of “hits” derived from upstream screening activities.
In addition, toxicologists will need to better understand the
interaction between a potential drug and the individual genetic makeup of
the patient exposed to that compound.
To make meaningful progress in either area, toxicologists will have
to effectively assimilate and wisely use an avalanche of information.
Without appropriate bioinformatics developed specifically for these
challenges, the effort will be stunted.
The purpose of this presentation is to present an overview of the
interplay among screening, molecular toxicologic analysis, and associated
informatics.
Additional
Thoughts on Toxicity Biomarkers: The
Isoprostanes, Indices of Oxidative Stress In Vivo
Jason
D. Morrow, M.D.
Over
the past decade, the role that oxidant stress plays in human
pathophysiology has received considerable attention.
A number of biomarkers have been developed to measure oxidative
injury although most have serious shortcomings when applied to the
assessment of oxidant stress in vivo.
We have previously described a series of prostaglandin
F2‑like compounds, termed F2‑isoprostanes (F2‑IsoPs),
produced independently of the cyclooxygenase enzyme by the
free‑radical‑catalyzed peroxidation of arachidonic acid in
vivo in humans. A large body of evidence suggests that quantification of
these compounds is an accurate index of lipid peroxidation in vitro and in
vivo. F2‑IsoPs can be
quantified by mass spectrometry or immunologically.
A particular interest of mine is the role of oxidative injury in
atherosclerosis and neurodegenerative disorders such as Alzheimer's
disease. We have found that
risk factors for atherosclerosis are associated with markedly increased
circulating levels of F2‑IsoPs and that antioxidants such as vitamin
E, which decrease the incidence of atherosclerosis, suppress plasma
F2‑IsoP levels. Furthermore, F2‑IsoPs are selectively increased in
cerebrospinal fluid from patients with Alzheimer's disease, and levels
correlate with disease severity. Therefore,
these studies suggest that quantification of F2‑IsoPs may provide a
useful biomarker of oxidant stress in humans.
Key
References
Montine
TJ, Morrow JD. Increased CSF F2‑isoprostane levels in probable
Alzheimer's disease. Neurology 1999;52:562‑565.
Morrow
JD, et al. The isoprostanes‑unique bioactive markers of lipid
peroxidation. Prog Lipid Res 1997;36:1‑21.
Roberts LJ, Morrow JD. Isoprostanes as markers of lipid peroxidation in atherosclerosis. In: Molecular and Cellular Basis of Inflammation. Serhan CN (ed.) Totowa NJ: Humana Press, 1998;141‑163