This study investigated the immune response of mice to the drug phenytoin to determine if it could predict the risk of idiosyncratic drug reactions in humans. The researchers found that phenytoin treatment in mice led to an early increase in macrophages between 2-7 days. This early response appeared to influence later changes in T cell and NK cell populations. Phenytoin treatment also resulted in an immune response that resolved within 3 weeks, supporting the hypothesis that most patients mount an immune response to phenytoin that results in immune tolerance rather than an adverse reaction. Future studies will examine macrophage and T cell subtypes and extend this research to human patients taking phenytoin.

1. INTRODUCTION
THE IMMUNE RESPONSE OF MICE TO PHENYTOIN:
DOES THIS RESPOSE PREDICT THE RISK OF IDIOSYNCRATIC DRUG REACTIONS?
UNIVERSITY OF TORONTO | LESLIE DAN FACULTY OF PHARMACY
Alexandra Barany | Jack Uetrecht
METHODS AND MATERIALS DISCUSSION
CONCLUSION
ACKNOWLEDGEMENTS
CELLULAR ISOLATION
AND STAINING
FLOW CYTOMETRY AND
STATISTICAL ANALYSIS
• Mice were female C57BL/6 between 6 and 8
weeks of age
• Dosed orally with 50 mg/kg/day of phenytoin in
oil suspension or oil vehicle first in a short term
experiment and then in a longer term (3 week)
study
ANIMALS AND
DRUG TREATMENT
RESULTS
1 2 3Idiosyncratic drug reactions (IDRs) are unpredictable, sometimes life-
threatening reactions that cannot be explained based on the drug’s
pharmacology. Phenytoin is an example of a drug that is associated
with a variety of IDRs including a generalized hypersensitivity reaction.
It is also associated with lymphadenopathy and pseudolymphomas.
The data in Figure 2 suggest that there is an increase in macrophages
early in the treatment, e.g. between two and seven days. This early
response appears to lead to later changes in T cell and NK cell
populations. We did not observe changes in other leukocytes such as
cytotoxic T cells, B cells, T regulatory cells, and myeloid derived
suppressor cells.
• Lymphocytes and monocytes were isolated from the
spleen and cervical lymph nodes after 7, 14, and 21
days of treatment
• Cells were stained for helper, cytotoxic and regulatory
T-cells, B-cells, natural killers cells, macrophages, and
myeloid derived suppressor cells
• Cells were counted on a x20 (4 laser)
cytometer and a minimum of 50 000 single live
cells per sample was acquired
• Mean and standard deviations were calculated
for each group, and data were analyzed using
a two-way analysis of variance
References: 1.Uetrecht,J.2007.Idiosyncraticdrugreactions:currentunderstanding.AnnuRevPharmacolToxicol.47:513-39. 2.Uetrecht,J.&Naisbitt,D.2013.Idiosyncraticadversedrugreactions:currentconcepts.PharmacolRev.65:779-808.
F4/80(B695/40)
CD11b(R660/20)
Figure 2: Flow cytometry staining of monocytes and leukocytes from the spleen and cervical lymph nodes.
PhenytoinTreatmentControl
D
ay
7
D
ay
14
D
ay
21
0
2
4
6
8
Spleen CD3- NK+
%Frequency
Control
Treated
**
**
D
ay
7
D
ay
14
D
ay
21
0
10
20
30
40
50
Lymph Th Cells (CD3+ CD4+)
%Frequency
Control
Treated
D
ay
2
D
ay
8
0
2
4
6
Spleen Macrophages (CD11b+ F/40+)%Frequency
Control
Treated
* *
Clinical data and circumstantial evidence suggest that such reactions
are caused by reactive metabolites and are immune mediated (Figure
1). There is now evidence that the dominant response to drugs that can
cause IDRs is immune tolerance (1); therefore, it is possible that most
patients treated with such drugs have an immune response, but it does
not result in an IDR.
CD4(B525/50)
CD3(V450/50)
PhenytoinTreatmentControl
NK1.1(YG780/60)
CD3(V450/50)
PhenytoinTreatmentControl
Figure 1: The hapten hypothesis (1). A chemically reactive drug (or
more likely a reactive metabolite) covalently binds to proteins, and this
adduct results in an immune response.
Hypothesis: Most patients have an immune response to phenytoin, but
the usual immune response results in immune tolerance rather than an
idiosyncratic drug reaction.
Phenytoin treatment does appear to lead to an immune response that
resolves by 3 weeks. This is consistent with the working hypothesis.
Future Studies:
These studies will be followed by studies to determine the activation
status and subtype of the macrophages (M1 vs M2), helper T cells
(Th1 vs Th2), and NK cells. When studies in mice are complete the
studies will be extended to patients who are being started on phenytoin
to determine if a similar immune response is observed in humans.
Such studies may help us understand the initial steps in the phenytoin-
induced immune response that can lead to a serious IDR, but only in a
few patients.
Figure 3: CD4+ T cell subsets involved in immune regulation and
pathology (2).
Spleen Macrophages (CD11b+ F4/80+)