The Toxicologist - Society of Toxicology

concentrations in plasma or liver tissue. This two compartment model was compared
with a 10-compartment model composed of 5 sequential blood compartments
each connected to a liver tissue compartment. Gor some compounds the two
models gave very similar results, but differences were observed for compounds with
high active uptake. The multicompartment liver model is an approximation for the
dispersion or tube models. The majority of the analyses have been for published
human intravenous plasma profiles, but modeling for the rat has been completed
for a more limited set of compounds. In addition to modeling the in vivo data to
obtain estimates for the various processes, in vitro data from human liver microsomes
and sandwich cultured hepatocytes have been used. Comparisons of predictions
for plasma concentration profiles based upon in vitro results have been made
to estimate in vitro to in vivo extrapolation factors. The in vitro to in vivo extrapolation
factors vary for the different processes being scaled. The analyses demonstrate
the value of capturing mechanistic biological processes in pharmacokinetic modeling
to improve the ability to predict pharmacokinetic disposition.
939 A NOVEL PREDICTIVE PLATFORM FOR DILI: A
COMBINATION OF IN SILICO AND IN VITRO
METHODS TO PREDICT TOXICITY MECHANISMS
IN VIVO.
S. Das, R. Kumar, S. Raghavan and K. Subramanian. Strand Life Sciences,
Bangalore, India.
Prediction of hepatotoxicity is still a challenge in the drug development process.
Currently available methods are inadequate both for extrapolation of rodent information
to humans as well as in predicting the likelihood of idiosyncratic liver injury.
Species used in preclinical toxicity do not usually possess underlying aberrations
leading to idiosyncratic toxicity observed in humans in the clinic.
We have developed a dynamic systems model of the rat liver to predict DILI. The
metabolic network modeled integrates the biology behind different types of drug
induced toxicity. Important biological processes involved in fat metabolism (steatosis),
energy homeostasis (necrosis), oxidative stress (oxidative damage) are modeled
quantitatively. A set of differential equations predict changes in metabolite concentrations
The model was first validated by comparing model predictions under conditions
of normal liver homeostasis and DILI with observations published in the
literature. We then used the model to generate hypotheses for the mechanism behind
idiosyncratic toxicity caused by the anti-diabetic drug troglitazone.
Sensitivity analysis of the metabolic network identified a set of enzymes that are
predicted to be key players in causing different types of DILI. In vitro measurements
of these enzyme rates, using a primary hepatocyte system, in absence and
presence of well-known hepatotoxic drugs, are used as input to the model. Model
simulated outputs of toxicity for each drug matched well with the associated toxic
phenotype reported in the literature. The validation of this combinatorial (in silico
and in vitro) approach using known hepatotoxic drugs e.g. Diclofenac,
Cyclosporin, Tamoxifen, etc. yielded additional novel mechanistic insights.
The purpose of study is to develop a generalized liver toxicity prediction platform
integrating in vitro and in silico approaches. Such an approach allows us to predict
toxicity, generate mechanistic insights, and mechanism specific biomarkers.
940 APPLICATIONS OF A COMPUTATIONAL MODEL FOR
PREDICTING DRUG INDUCED LIVER INJURY (DILI).
B. A. Howell 1 , R. Kumar 3 , L. Wennerberg 3 , Y. Yang 2 , R. Ho 3 , A. Harrill 1 , C.
L. Kurtz 1 , M. E. Andersen 2 , H. J. Clewell 2 , S. Q. Siler 3 and P. B. Watkins 1 .
1 Drug Safety Sciences, The Hamner Institutes, Research Triangle Park, NC,
2 Chemical Safety Sciences, The Hamner Institutes, Research Triangle Park, NC and
3 Entelos, Inc., Foster City, CA.
Drug-induced liver injury (DILI) is the adverse drug event that most frequently
leads to termination of clinical development programs and regulatory actions on
drugs. We have developed a predictive model based on physiological processes involved
in DILI, focusing initially on acetaminophen toxicity. A physiologically
based pharmacokinetic (PBPK) model was developed to describe the disposition
and metabolism of APAP. Reactive metabolite production and subsequent covalent
protein binding are characterized. Glutathione (GSH) depletion and synthesis, oxidative
stress, mitochondrial dysfunction, and the cell life cycle, including regeneration
and death, are modeled. The model accurately reproduced APAP pharmacokinetic
measures and GSH depletion/recovery for rats, mice, and humans, and this
explained much of the variation in species specific susceptibility to APAP induced
DILI. In-depth comparisons were also made between rats and mice with regard to
their ability to re-synthesize GSH after GSH depletion. The use of N-acetyl-cysteine
(NAC) as a treatment for APAP overdose was incorporated into the model,
and treatment times were analyzed and compared to standard clinical practices to
predict optimal NAC use. Finally, the model was used to successfully predict the
species differences in hepatotoxicity of methapyrilene using in vitro to in vivo ex-
200 SOT 2011 ANNUAL MEETING
trapolation. The absorption, distribution, and metabolism of methapyrilene were
estimated from the physical characteristics of the compound and in vitro metabolism.
The model correctly predicted that rats would be less sensitive to acetaminophen
than mice or humans, but most sensitive to methapyrilene. A parametric
search for in silico rats, mice, and humans susceptible to methapyrilene DILI predicted
that methapyrilene would cause very little hepatotoxicity in humans clinically.
This finding agrees with clinical reports.
941 DEVELOPMENT OF A PBPK/PD MODEL FOR
ACETAMINOPHEN TOXICITY IN RATS.
Y. Yang, B. Howell, M. Yoon, M. Andersen and H. J. Clewell. The Hamner
Institutes for Health Sciences, Research Triangle Park, NC.
A whole-body physiologically based pharmacokinetic/pharmacodynamic
(PBPK/PD) model for acetaminophen (APAP) and its metabolites in the rat was
developed to quantitatively describe the processes that determine APAP hepatotoxicity.
The key features of the model include metabolic activation of APAP by cytochrome
P450s, detoxication of APAP via sulfate and glucuronide conjugation,
and binding of the reactive metabolite N-acethyl-p-benzoquinone imine (NAPQI)
to hepatic glutathione (GSH) and cellular macromolecules. Both sulfate and glucuronide
conjugation was described using Michaelis Menten equations. Sulfation
was modeled as a high affinity, low capacity pathway, while glucuronidation was described
as a low affinity, high capacity pathway. A description of the depletion of
the cofactor for sulfation (PAPS) and its precursor inorganic sulfate was required to
properly simulate APAP-sulfate concentrations in the rat blood. Enterohepatic recirculation
of the conjugates was also included. The net production of NAPQI is
determined by the balance between the bioactivation via cytochrome P450s and the
detoxification by conjugation with GSH. The model also describes the resulting depletion
and recovery of GSH by incorporating a feed-back regulation of GSH synthesis.
The model successfully simulated concentration-time profiles of APAP, sulfate
and glucuronide conjugates of APAP after oral, intraperitoneal, and
intravenous (iv) administrations ranging from 37.5 to 1000 mg/kg doses. The dosedependent
changes in hepatic GSH concentrations after IV dose were also well described.
This model will contribute to better understanding of the mechanism of
APAP hepatotoxicity by providing a tool for predicting the time-course of reactive
metabolite formation and glutathione depletion in studies measuring hepatic damage
and repair.
942 USING ZEBRAFISH TO STUDY BENZO(A)PYRENE-
MEDIATED CHANGES IN DNA METHYLATION
STATUS DURING DEVELOPMENT.
X. Fang 1 , C. Thornton 1 , B. E. Scheffler 2 and K. L. Willett 1 . 1 Department of
Pharmacology and Toxicology, the University of Mississippi, University, MS and
2 Genomics and Bioinformatics Research Unit, USDA Mid South Area Genomics
Laboratory, Stoneville, MS.
DNA methylation is one of the epigenetic mechanisms to control gene expression
and is vulnerable to in utero or early-life environmental toxicant exposures.
Constitutively, we confirmed that zygote demethylation is conserved in zebrafish
and characterized dynamic CpG methylation patterns in 5 genes (vasa, Ras-association
domain family member 1 (RASSF1), telomerase reverse transcriptase
(TERT), c-Myc and c-Jun). Direct waterborne benzo(a)pyrene (BaP) treatment at
100 μg/L from ~2 to 96 hours post fertilization (hpf) to zebrafish embryos significantly
decreased global cytosine 5’ methylation by 55% and gene-specifically reduced
promoter CpG methylation in vasa by 17.2%. Future study will measure the
vasa mRNA expression to determine correlated altered expression. However, BaP
did not change CpG island methylation in RASSF1, TERT, c-Myc and c-Jun at 96
hpf. To study the mechanisms of demethylaion, mRNA expression and enzyme activity
of 2 methylation related enzymes, DNA methyltransferase 1 (DNMT1) and
glycine N-methyltransferase (GNMT), were measured constitutively and after
treatment. BaP exposure at 1, 10 or 100 μg/L did not change DNMT1 and
GNMT mRNA expression at 48, 60 or 96 hpf or nuclear total DNMT and cytosolic
GNMT enzyme activity at 96 hpf. In summary, BaP is a potential epigenetic
modifier for global and gene specific DNA methylation status in zebrafish embryos
and the alteration is possibly not mediated by affecting DNMT1 and GNMT expression.
(Support: NIEHS R01ES012710).