National Capital Area SOT, Spring
2004 Symposium
“Novel Methods for Detecting Hepatotoxic Agents”
Speaker Abstracts and Slide Presentations
Detecting Liver Injury: Drug-Induced
or Not?
John R. Senior, M.D., FDA, Center for Drug Evaluation and
Research
Correct
attribution of causality as to whether a case of acute liver injury is
drug-induced or not is among the more difficult problems in medicine. Drugs or
other substances, or their metabolites produced by the liver, may cause liver
injuries that mimic all known liver diseases. There is no pathognomonic test or
finding, including biopsy, that proves the injury is drug-induced. The
diagnosis therefore must be made by ruling out all other possible causes. This
is often not done well or thoroughly, so that the data gathered too frequently
allow concluding only that the liver injury was “possibly” or “probably” drug-induced.
We chose to address one aspect
of this problem: whether we could be reasonably certain of detecting and
diagnosing serious liver disease that was NOT drug-induced. The work was done
as an innovative research collaboration between the Food and Drug
Administration and the Merck Research Laboratories, where the clinical
representative served not as a regulator but as a cooperative research partner
with the pharmaceutical industry statistician. We examined a large database of
3301 study participants randomized to placebo and observed repeatedly (20
times) over more than 5 years.
We found that serum
transaminase elevations were very poorly predictive of serious liver disease,
were often transient and unexplained. Only when the transaminase elevations (3-fold)
were persistent or progressive and either accompanied or followed by rises in
serum bilirubin (2-fold) was the liver disease clinically significant or
serious. The combination of a test for hepatocellular injury and a measure of
overall liver function was as sensitive as the transaminase alone but far more
specific in detecting 6 of 6 cases of serious liver disease incident among 3248
ambulatory participants on placebo for 5 years.
Thus, we could be reasonably
sure we could detect serious non-drug-induced liver injury or disease by this
combined injury-function test as a biomarker, the same combination that the
late Hyman Zimmerman reported for drug-induced liver injury. The methods for
exclusion of non-drug causes, and correct and quantitative attribution of
causality, will require further work.
Biomarkers of Liver Toxicity and
Disease
Yvonne Dragan, Ph.D., FDA, National Center for
Toxicological Research
Hepatotoxicity is the number
one reason for drug recall and hence it is of concern to the FDA and to the
American public. Drug induced toxicities account for nearly half of the cases
of acute liver failure in adults over age 50 in the US. There is a need for
better predictive tests for agents that have the potential to cause liver
toxicity in humans. In addition, early biomarkers of adverse liver
effects from exposure to agents are needed and should be developed.
Factors that trigger hepatic cell death, permit hepatocyte survival, and
trigger hepatocyte proliferation are potential biomarkers. Techniques
such as Seldi based proteomics and NMR-based metabonomics provide tools for the
development of biomarkers of toxicity and disease. Specific, sensitive, and
predictive biomarkers for the induction of liver toxicity and the potential for
progression to fulminant liver failure need to be identified and are the long
term goal of this proposal.. Animal toxicity data can be used as a surrogate to
predict potential human liver toxicity.
Predictive Hepatotoxicity: Correlations in a
Structural Database
James H. Kelly, Ph.D., Amphioxus Cell Technologies
We have employed a human
liver cell line and a series of high throughput in vitro toxicology assays to examine the structural basis of
hepatotoxicity in several families of compounds. The data show that such a system can provide
fine discrimination among structural variants.
We have examined thiazolidinediones, nonsteroidal anti-inflammatories,
fluoroquinolones, and antihistamines with known hepatotoxic properties to show
that there is a clear structural basis for the problems encountered with these
compounds. With this idea in mind, we
are screening a large number of compounds in an attempt to build a predictive
toxicology database which, when coupled to the experimental system, can be used
to eliminate hepatotoxic liabilities early in the drug development
process. The data are gathered directly
into software for experiment management, MDL Assay Explorer®. The results are then collated to the
associated structure and functional information for each compound using
ChemBioAE®. Using cyp1A induction as an
example, we are able to show that compounds with similar mechanisms of action,
such as ERK inhibition, are common inducers.
Likewise, unrelated compounds with similar structural features, such as
aristolochic acid and apomorphine, also induce.
In Vitro Assessment of Necrosis and
Apoptosis Using Human Hepatocytes
Neal Jensen, Ph.D., In Vitro Technologies
Hepatotoxicity is the most
common side effect of new chemical entities (NCE). While hepatotoxicity is generally described
as a necrotic event, apoptosis may also play an important role in chemically-induced
hepatotoxicity. A number of different
models have been used for evaluating the hepatotoxicity of NCEs, including in
vivo animal studies and various cell-based assays. Isolated human hepatocytes have been used to
study the metabolism and toxicity of drug candidates. Human hepatocytes offer several advantages as
an in vitro model. They provide
human-specific results, testing can be carried out with small amounts of
compounds, and animal use is minimized.
Human hepatocytes can be obtained in different formats for use in the
evaluation of hepatotoxicity by necrotic or apoptotic pathways. Freshly isolated hepatocytes can be used in
suspension or plated as monolayers for short- or long-term toxicity studies. In addition, hepatocytes can be cryopreserved,
thawed as needed, and used in suspension for evaluation of drug toxicity. Recently, cryopreserved hepatocytes have
become available which, when thawed, can form monolayers to be used in place of
fresh hepatocyte monolayers for metabolism or toxicity studies. Different methods for measuring necrosis
(MTT, ATP, LDH) and apoptosis (Caspase, DNA fragmentation) using hepatocytes in
various formats will be discussed.
Susceptibility
Factors in Idiosyncratic Drug-Induced Liver Injury
Steven Yee, Ph.D., NIH, National Heart, Lung and
Blood Institute
Drug-induced liver disease
(DILD) is a common cause of life-threatening, acute liver failure and is a
major reason drugs are removed from clinical development and widespread
use. Because of its idiosyncratic
nature, accurate prediction of which new drugs will cause DILD and who will be
at risk for the development of this disease is difficult. Likewise, the pathogenesis of DILD is complex
and appears to involve the formation of reactive drug metabolites that affect
critical biochemical functions or elicit an immune response. Recent studies, however, have demonstrated
that an alteration in the balance of protoxicants and protectants in the liver
can also influence susceptibility to DILD.
Environmental influences, such as the inflammagen bacterial
lipopolysaccharide, and/or genetic polymorphisms may affect this balance. Indeed, concurrent inflammation can enhance
susceptibility to a variety of hepatotoxicants, including drugs, and result in
the generation of numerous injurious, protoxicant inflammatory mediators. Moreover, the deficiency of key protective
factors in the liver – such as anti-inflammatory cytokines and others – can
lead to increased tissue susceptibility, often through unchecked protoxicant activities. Hence, the balance between these
susceptibility factors may play a critical role in the overall pathogenesis,
with increased levels of protoxicants or decreased levels of protectants
leading to this disease. Identification
of such factors through the use of toxicogenomics, proteomics and metabonomics
will be ideal in better understanding the mechanism and facilitating the
prediction of DILD, thereby preventing the removal of otherwise beneficial
drugs from the market.
Multi-endpoint Profiling of Suspect Hepatotoxicants
in Cultured Hepatocytes
Thomas Flynn, Ph.D., FDA, Center for Food Safety and
Applied Nutrition
Hepatotoxicity is the
leading cause of post-market withdrawal of drugs and of warning notices for
dietary supplements. The present study evaluated the effects of model
compounds, some with known hepatotoxicity, on human (HepG2/C3A, WRL-68) and rat (Clone-9) hepatocyte cell lines. Cells
were exposed to multiple concentration levels of test agent (up to the limit of
aqueous solubility but not more than 1 mg/mL) for up to 48 hr. Specific endpoint assays, which
could all be conducted in a plate reading fluorometer or luminometer and which
model known mechanisms of hepatotoxicity, included: generation of reactive
oxygen species (dichlorofluorescin); depolarization of mitochondria (rhodamine
123); steatosis (nile red); induction or inhibition of cytochrome P450
activities (EROD, BOROD); cell viability (liver enzyme release, Alamar blue,
total ATP); and apoptosis (caspase 3). All parameters were normalized for total
double stranded DNA content (H33258) per well. Each model compound generated a
unique response profile based on which endpoint showed a concentration-related
increase, decrease, or no change. Only 3 of 12 compounds tested showed
significant cytotoxicity. For some model compounds that are human drugs and
have known hepatotoxicity (e.g., valproic acid), some endpoints responded at concentrations
comparable to therapeutic blood levels and in ways consistent with the
compound’s known mechanism of toxicity. These findings suggest that this test
system may serve as a screening assay for hepatotoxicity of food- or dietary
supplement-related compounds.
The Use of Microarray Technology to
Predict Hepatotoxicity in Humans
William Mattes, Ph.D., Genelogic, Inc.
Because numerous industrial
and pharmaceutical chemicals have been found to cause damage to the liver,
testing for hepatotoxicity remains a key component in the safety assessment of
a new chemical entity. While standard
animal models generally predict human hepatotoxicity, there are a number of
examples of compounds whose human liver injury was not forseen in rodent
models. Gene Logic has taken the premise that gene expression data from livers
of compound-treated rats may be contain signals predictive of human
hepatotoxicity. To this end studies were conducted with a series of known human
and rodent hepatotoxins, as well compounds known to be safe. A database of gene
expression results for various treatment times and doses was constructed, and
then used to build predictive models composed of hundreds of gene responses. The models are of three types: a model
predicting general hepatoxicity, models predicting various pathologies or
mechanisms, and models identifying similarity of an unknown compound to one in
the reference database. Examples of the results of these models for two
paradigm compounds, tacrine and felbamate, will be presented.