The Toxicologist - Society of Toxicology

and the percentage of responders per group was determined. The percentage of responders
ranged from 80-100 and 0-20 for the positive (ofloxacin, amoxicillin, sulfamethoxazole)
and negative (metformin, acetaminophen, phenobarbital) control
drugs, respectively, indicating that the model is able to differentiate between positive
and negative controls. These data suggest that this model may be a useful tool
for identifying compounds with the potential to produce hypersensitivity responses.
1498 HLA-SPECIFIC T-CELL ACTIVATION AS AN
UNDERLYING CAUSE OF DRUG-INDUCED
HYPERSENSITIVITY.
J. M. Goodwin 1 , T. Kawabata 2 and M. T. Pletcher 1 . 1 Compound Safety Prediction,
Pfizer, Groton, CT and 2 Immunotoxicology CoE, Pfizer, Groton, CT.
Drug hypersensitivity is a major safety issue in drug discovery. Though it occurs in
only a small fraction of the patient population, the results are often severe.
Genome-wide association studies have been employed to better understand the underlying
mechanisms and have shown an association between alleles of the major
histocompatibility complex (MHC) region and an individual’s hypersensitivity to
certain drugs. One hypothesis that has been put forth to explain the genetic relationship
between adverse events and the MHC is the Pharmacological Interactions
(PI) concept. It states that certain drugs can bind to T-cell receptors, mimicking a
ligand, resulting in T-cell activation in an MHC-dependent fashion. We sought to
further investigate the validity of this hypothesis by determining a transcriptional
response indicating T-cell activation in PBMCs with hypersensitive haplotypes.
PBMCs with known reactive and non-reactive MHC haplotypes were dosed with
their corresponding reactive drugs (HLA-B*5701 with Abacavir and HLA-B*1502
with Carbamazepine) and RNA was profiled on microarrays covering 172 genes associated
with T and B cell activation. We identified 27 candidate genes, including
IL-12beta, CD80, IL-7, IL-13, CXCR3, and CSF2, whose expression was altered
only in the hypersensitive haplotype treated with reactive drug. We then examined
the expression changes of these selected genes on a larger panel of PBMCs from patients
with various non-reactive and reactive haplotypes to confirm the transcriptional
fingerprint. In conclusion, we have identified a set of genes that are regulated
in an MHC-dependent fashion in PBMCs treated with compounds known to induce
hypersensitivity reactions in patients carrying these specific alleles. This identification
of the genetic and transcriptional factors involved in the development of
immune-mediated idiosyncratic tissue injury provides the insight necessary to develop
diagnostic tools to identify patient populations at increased risk and new
treatment strategies.
1499 EARLY CHANGES IN AROMATIC AMINE-INDUCED
IDIOSYNCRATIC DRUG REACTIONS.
W. Ng and J. Uetrecht. Pharmacy, University of Toronto, Toronto, ON, Canada.
Virtually all drugs that contain a primary aromatic amine group are associated with
a high incidence of idiosyncratic drug reactions (IDRs) and this is considered a
structural alert for drug development. This suggests that this functional group has
biological effects that might be used as a biomarker to predict IDR risk as well as
provide clues to the mechanism of IDRs. A previous microarray study to determine
the early changes (12 h) in hepatic gene expression in Brown Norway rats treated
with the aromatic amine drugs sulfamethoxazole (SMX, 150 mg/kg), dapsone
(DDS, 20 mg/kg), and aminoglutethimide (AMG, 80 mg/kg) provided the basis
for our present studies. The pattern of mRNA changes was significantly different
for the 3 drugs. Three distinct changes from the microarray data were pursued in
terms of their downstream effects: early insulin-induced gene (Eiih), chemokine
(C-X-C) 1 (Cxcl1), and genes involved with the Nrf2-ARE pathway. PCR analysis
of Eiih confirmed that it was up-regulated at 12 h in the liver with all drugs tested,
although the change was greater for AMG and DDS than SMX. The function of
Eiih is presently unknown but these data suggest that it could be a potential biomarker
of aromatic amine-induced IDRs. Despite moderate changes in hepatic
Cxcl1 and an up-regulation of Cxcl1 protein levels in the blood of AMG-treated
rats this was not the case for SMX- or DDS-treated animals. This increase in Cxcl1
coincided with a transient increase in peripheral blood neutrophils 24 h after AMG
treatment. This finding may be related to the agranulocytosis that sometimes occurs
in AMG-treated patients. Up-regulation of the Nrf2-ARE pathway was also
observed: increased glutathione-S-transferase and thioredoxin reductase in the liver
48 h after AMG treatment. This suggests that these aromatic amines induce oxidative
stress. These results demonstrate the diverse effects of aromatic amine drugs
and illustrate the difficulties in finding a common biomarker that predicts IDR
risk. This research was funded by grants from the Canadian Institutes for Health
Research.
322 SOT 2011 ANNUAL MEETING
1500 THE USE OF MAST CELLS DERIVED FROM HUMAN
CD34+ HEMATOPOIETIC STEM CELLS AS A TOOL
FOR PREDICTING PSEUDOALLERGIC DRUG
REACTIONS.
J. Whritenour 1 , C. Homiski 1 , K. Haskell 2 , M. Thiede 2 and T. T. Kawabata 1 .
1 Immunotoxicology CoE, Drug Safety Research and Development, Pfizer, Inc., Groton,
CT and 2 Genetically Modified Mice CoE, Pfizer, Inc., Groton, CT.
Pseudoallergic (anaphylactoid) reactions are the result of direct mast cell activation
and degranulation via an IgE-independent mechanism, resulting in the release of
specific mediators such as histamine, tryptase, and cytokines. Assays to predict the
potential of drugs to produce pseudoallergic reactions in humans are currently unavailable
but would be a useful tool during drug development. In this study, we explored
the utility of CD34+ hematopoietic stem cell (HSC)-derived mast cells for
predicting the pseudoallergic potential of various drugs using histamine release as
an indicator of mast cell degranulation. CD34+ HSC from human bone marrow
donors were cultured over a period of ~68 days with various cytokines and differentiated
into cells expressing a mast cell phenotype (FcεR1+, CD45+, CD117+).
Mast cells were plated (5x10 4 cells/well) and incubated for 30 min, 60 min, or 24
hr with either vehicle (PBS), or compounds known (tubocurarine, morphine) or
not (metformin, acetaminophen) to produce pseudoallergic reactions in humans.
Following incubation, contents from each well were centrifuged to remove the cells
and the supernatants were collected and analyzed for histamine content using a
commercially available ELISA kit. While the overall magnitude of the histamine response
appears to be donor-dependent, CD34+ HSC-derived mast cells consistently
responded to the known pseudoallergens. Histamine levels were similar between
cells treated with the negative controls and the vehicle. No significant
differences were measured in the quantity of histamine released when mast cells
were incubated with compounds for 30 min, 60 min, or 24 hr. These data indicate
that human CD34+ HSC-derived mast cells may be used to predict for pseudoallergic
potential of drug development candidates.
1501 EFFECT OF RETINOIC ACID ON NEVIRAPINE-
INDUCED SKIN RASH AND THE INVOLVEMENT OF
TH17 CELLS.
X. Chen and J. P. Uetrecht. Faculty of Pharmacy, University of Toronto, Toronto,
ON, Canada.
Introduction: Nevirapine (NVP) is used to treat AIDS but is associated with a high
incidence of skin rash and liver toxicity. We found that it also causes a similar skin
rash in the Brown Norway (BN) rats. The NVP-induced skin rash in BN rats is
clearly immune-mediated and we think the mechanism is similar to that in humans.
For example, a low CD4 + T cell count decreases the risk of rash in both rats
and humans. Furthermore, CD4 + T cells can transfer the sensitivity from sensitized
animals to naïve animals. We found that IL-17 was produced by lymphocytes from
NVP-sensitized animals suggesting that IL-17 and/or Th17 cells might be involved
in the pathogenesis. Retinoic acid (RA), a vitamin A metabolite, is reported to inhibit
Th17 cells. In this preliminary study we examined the effect of RA on NVPinduced
skin rash and the involvement of Th17 cells. Methods and Results:
Female BN rats co-treated with NVP and RA for 21 days had no symptoms of
NVP-induced idiosyncratic reactions, i.e. red ears and/or skin rash. However,
slightly lower plasma levels of NVP and/or its metabolites were observed. In the peripheral
blood, NVP treatment significantly decreased the percentage of cells that
express CD4 + /CD161 low , which in humans is the precursor of Th17 cells. We also
observed a big spike of CD4 - CD161 low cells on day 4 following NVP treatment but
they deceased to baseline levels in 1 week. These might be CD8 + or NK cells and
they need to be further characterized. Conclusions: The protective role of RA on
NVP-induced skin rash might be a result of decreased drug plasma levels. However,
this needs to be confirmed by using a higher dose of NVP or a P450 inhibitor with
co-administration of RA. The involvement of Th17 cells (CD4 + IL17 + ) will also be
investigated using a more direct method, i.e. intracellular staining. Completion of
these studies will provide a better understanding of the mechanism of NVP-induced
skin rash and, in turn, this may provide possible ways to prevent and/or treat
idiosyncratic drug reactions. This work was supported by grants from CIHR.