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Investigating the Mechanisms of Idiosyncratic Drug Reactions: Lessons from Nevirapine-induced Skin Rash in
Female Brown Norway Rats
by
Xin Chen
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Pharmaceutical Sciences University of Toronto
© Copyright by Xin Chen 2014
ii
Investigating the Mechanisms of Idiosyncratic Drug Reactions:
Lessons from Nevirapine-induced Skin Rash in Female Brown
Norway Rats
Xin Chen
Doctor of Philosophy
Graduate Department of Pharmaceutical Sciences
University of Toronto
2014
ABSTRACT
Idiosyncratic drug reactions (IDRs) cause significant morbidity and mortality. At present it is
impossible to predict who will have such reactions because the mechanisms involved are not
understood. This work used nevirapine-induced skin rash in female Brown Norway rats as a
model to investigate the mechanisms of IDRs. Specifically, we hypothesized that CD4+ T cells
are mediating the rash, and the reactive sulfate metabolite of nevirapine induced the immune
response by directly activating antigen presenting cells (APCs).
Independent of which chemical species induced the rash (treatment with nevirapine or 12-OH-
nevirapine), CD4+ T cells isolated from the lymph nodes of animals responded vigorously to the
parent drug, but not to 12-OH-nevirapine even though oxidation of nevirapine to 12-OH-
nevirapine pathway is required to induce the rash. This falsifies the basis for the PI hypothesis,
which assumes that what the lymphocytes respond to is what induced the IDR.
In the APC activation study it was found that nevirapine, 12-OH-nevirapine, and 12-OH-
nevirapine sulfate all appeared to activate APCs to some degree. For instance, CD40 was
upregulated in RAW264.7 cells and bone marrow-derived dendritic cells, while CD86 was
iii
upregulated in THP-1 cells. However, the effects were small and not limited to the reactive
sulfate.
Several interventions were also used to modulate the rash caused by nevirapine to learn more
about possible risk factors; however, no results showed that any of them had a significant effect
on the rash. This included depleting B cells and treatment with buthionine sulfoximine, retinoic
acid, 1-methyl-tryptophan, lipopolysaccharide, imiquimod, or vitamin D.
This animal model of an IDR allowed us to test mechanistic hypotheses that would be impossible
to test by any other method. It provided insights into the mechanism of this IDR, and by
extension, into the mechanisms of other IDRs.
iv
ACKNOWLEDGMENTS
I will never forget that sunny day in February 2007 when I visited Toronto for the first time and
met eight supervisors in this top University to decide where I want to spend my next five years of
time. Jack was the last one I interviewed with. He greeted me warmly with a father’s big smile
and asked a question no one else ever cared: “Where are you gonna be in ten years from now?” I
didn’t get it the first time. He repeated and elaborated: “Describe where you will be and what
you will be doing.” I clearly remembered my answer: “Working in a Pharma, and I don’t mind
going to other countries.” He then started to talk about his education, his career path, and a lot of
other things that are not very relevant to sciences, for at least 30 minutes or so. I went back to
the hotel and said to myself: “This is an interesting and challenging person. Most importantly,
he looks like the Santa Claus!”
It has been 6 and a half years now since I joined Jack’s lab and time proved that my decision was
right. Throughout my Ph.D, Jack has provided tremendous help and guided me to become an
independent scientific investigator and a critical thinker, which have made me stand out from
most other people starting my day one in industry. He is probably thanked a million times for his
achievement in science and academic training, but I would like to say, it is the non-scientific part
that I learned from him will benefit me the rest of my life. He is like a father to us, strict but with
love, fair and reasonable to everyone. I truly appreciate what Jack has taught me and thank him
for being such a great mentor and role model.
Along with great science and great supervisor, there is always a great team or you could call it a
family. I sincerely thank all my lab mates for the great moments and conversations during my
Ph.D - we shared our happiness and frustration throughout the graduate life. Special thanks to
my big “brothers” Robert, Ervin, Ping and Feng, for their consistent support and inspiration for
both better science and personal life. Great thanks to Connie for the amazing food and receipts,
and Max for the fun time - he is way too adorable.
I also want to thank my friends outside the lab who supported me during my “heartbroken” and
“backbroken” times: Hui Wang, Carl Song, Yanan Chu, Jing Jing, Yanrong Shi. They made me
stronger.
v
Lastly, thanks to my uncle’s family, my parents, and my fiancé Kenny Liao for their generous
love. I love them.
This research was conducted in the spirit of the following (quotes from Jack):
“Good research always leads to more questions.”
“It is better to get embarrassed early than late.”
“The only reason I know more than you do is because I made more mistakes.”
“It is very important to publish negative results so others know what is not working!”
“You have to be open-minded!”
The same applies to life.
vi
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................... ii
ACKNOWLEDGMENTS ........................................................................................................... iv
TABLE OF CONTENTS ............................................................................................................ vi
LIST OF PUBLICATIONS ........................................................................................................ ix
LIST OF ABBREVIATIONS ...................................................................................................... x
LIST OF TABLES ...................................................................................................................... xii
LIST OF FIGURES ................................................................................................................... xiii
LIST OF APPENDICES .......................................................................................................... xvii
CHAPTER 1 .................................................................................................................................. 1
INTRODUCTION ......................................................................................................................... 1
1.1. Overview of Adverse Drug Reactions ................................................................................ 2
1.2. Idiosyncratic Drug Reactions .............................................................................................. 5
1.2.1. Clinical Manifestations .................................................................................................... 5
1.2.2. Risk Factors ...................................................................................................................... 6
1.3. Hypotheses of Mechanisms of IDRs ................................................................................... 6
1.3.1. Hapten Hypothesis ........................................................................................................... 7
1.3.2. Danger Hypothesis ......................................................................................................... 10
1.3.3. Pharmacological Interaction (PI) Hypothesis ................................................................ 13
1.4. Animal Models .................................................................................................................. 15
1.4.1. NVP-induced Skin Rash in Female BN Rats ................................................................. 15
1.4.2. D-penicillamine-Induced Autoimmune Diseases .......................................................... 20
1.5. Investigating the Mechanisms of IDRs ............................................................................. 22
vii
1.5.1. Detection of Reactive Intermediate Formation and Covalent Binding .......................... 22
1.5.2. T Cell Based Assays....................................................................................................... 23
1.5.3. Co-administration Studies .............................................................................................. 25
1.5.4. Pharmacogenetics in Clinical Research ......................................................................... 25
1.6. Rationale of the Present Studies ....................................................................................... 26
CHAPTER 2 ................................................................................................................................ 27
A STUDY OF THE SPECIFICITY OF LYMPHOCYTES IN NEVIRAPINE-
INDUCED SKIN RASH ........................................................................................................ 27
2.1. ABSTRACT ...................................................................................................................... 28
2.2. INTRODUCTION ............................................................................................................ 29
2.3. METHODS ....................................................................................................................... 31
2.4. RESULTS ......................................................................................................................... 35
2.5. DISCUSSION ................................................................................................................... 44
CHAPTER 3 ................................................................................................................................ 46
INDUCING SKIN RASH IN FEMALE BN RATS BY TOPICAL TREATMENT OF
NVP AND/OR 12-OH-NVP ................................................................................................... 46
3.1. INTRODUCTION ............................................................................................................ 47
3.1.1. Topical Treatment of NVP on Sensitized Animals ........................................................ 47
3.1.2. Attempts to Induce a Skin Rash in Naïve Rats by Topical Treatment with 12-OH-
NVP ................................................................................................................................... 48
3.2. MATERIALS AND METHODS ...................................................................................... 50
3.3. RESULTS ......................................................................................................................... 51
3.4. DISCUSSION ................................................................................................................... 55
CHAPTER 4 ................................................................................................................................ 57
FACTORS THAT MAY INFLUENCE THE INCIDENCE AND SEVERITY OF NVP-
INDUCED SKIN RASH IN FEMALE BN RATS .............................................................. 57
4.1. INTRODUCTION ............................................................................................................ 58
4.2. MATERIALS AND METHODS ...................................................................................... 61
viii
4.3. RESULTS ......................................................................................................................... 69
4.4. DISCUSSION ................................................................................................................... 85
CHAPTER 5 ................................................................................................................................ 89
POTENTIAL ACTIVATION OF ANTIGEN PRESENTING CELLS BY NVP
AND/OR ITS METABOLITES ............................................................................................ 89
5.1. INTRODUCTION ............................................................................................................ 90
5.2. MATERIALS AND METHODS ...................................................................................... 91
5.3. RESULTS ......................................................................................................................... 97
5.4. DISCUSSION ................................................................................................................. 119
5.5. CONCLUSION ............................................................................................................... 122
CHAPTER 6 .............................................................................................................................. 123
CONCLUSIONS AND FUTURE DIRECTIONS .................................................................. 123
6.1. Summary ......................................................................................................................... 124
6.2. Implications and Future Directions ................................................................................. 126
REFERENCES .......................................................................................................................... 129
APPENDIX ................................................................................................................................ 140
ix
LIST OF PUBLICATIONS
1. Metushi, I. G., X. Zhu, et al. (2014). "Mild Isoniazid-Induced Liver Injury in Humans Is
Associated with an Increase in Th17 Cells and T Cells Producing IL-10." Chem Res Toxicol.
27(4): 683-689.
2. Ng, W., A. R. Lobach, et al. (2012). "Animal models of idiosyncratic drug reactions." Adv
Pharmacol 63: 81-135.
3. Zhang, X., F. Liu, et al. (2011). "Involvement of the immune system in idiosyncratic drug
reactions." Drug Metab Pharmacokinet 26(1): 47-59.
4. Chen, X., T. Tharmanathan, et al. (2009). "A study of the specificity of lymphocytes in
nevirapine-induced skin rash." J Pharmacol Exp Ther 331(3): 836-841.
5. Dugoua, J. J., M. Machado, et al. (2009). "Probiotic safety in pregnancy: a systematic review
and meta-analysis of randomized controlled trials of Lactobacillus, Bifidobacterium, and
Saccharomyces spp." J Obstet Gynaecol Can 31(6): 542-552.
6. Chen, X and J. Uetrecht. Factors that may influence the incidence and severity of
nevirapine-induced skin rash in female Brown Norway rats. In preparation.
7. Chen, X and J. Uetrecht. Potential activation of antigen presenting cells by nevirapine and/or
its metabolites. In preparation.
x
LIST OF ABBREVIATIONS
2-ME β-mercaptoethanol
ADR Adverse Drug Reaction
ALN Auricular Lymph Nodes
APC Antigen Presenting cell
AUC Area Under the Curve
BCA Bicinchoninic Acid
BMDC Bone Marrow-Derived Dendritic Cell
BN Brown Norway
BSO Buthionine Sulfoximine
CD Cluster of Differentiation
CFSE Carboxyfluorescein Succinimidyl Ester
CO2 Carbon Dioxide
CYP Cytochrome P450
DAPK1 Death-associated Protein Kinase 1
DC Dendritic Cell
DMEM Dulbecco's Modification of Eagle's Medium
DMSO Dimethyl Sulfoxide
ECL Enhanced Chemiluminescence
EDTA Ethylenediaminetetraacetic Acid
ELISA Enzyme-linked Immunosorbent Assay
ELISPOT Enzyme-linked Immunosorbent Spot
FBS Fetal Bovine Serum
FcγII Fc gamma receptor II
FITC Fluorescein Isothiocyanate
FOXP3 Forkhead Box P3
G-CSF Granulocyte Colony-stimulating Factor
GM-CSF Granulocyte Macrophage Colony-stimulating Factor
GRO-KC Growth-related oncogene
H & E Hematoxylin and Eosin
HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic Acid
HIV Human Immunodeficiency Virus
HLA Human Leukocyte Antigen
HMGB1 High Mobility Group Box 1
HPLC High-performance Liquid Chromatography
HSPs Heat Shock Proteins
IgG Immunoglobulin G
LC/MS Liquid chromatography–mass spectrometry
IDO Indoleamine Dioxygenase
IDR Idiosyncratic Drug Reaction
xi
IFN Interferon
IgE Immunoglobulin E
IL Interleukin
IP-10 Interferon gamma-induced Protein 10
IU International Unit
LPS Lipopolysaccharide
LTT Lymphocyte Transformation Test
MFI Mean Fluorescence Intensity
MCP-1 Monocyte Chemoattractant Protein 1
MHC Major Histocompatibility Complex
MIP-1 Macrophage Inflammatory Protein 1
NNRTI Nonnucleoside Reverse Transcriptase Inhibitor
NOD Nucleotide-binding Oligomerization Domain
NLRs Nucleotide-binding Oligomerization Domain Receptors (NOD-like receptors)
NVP Nevirapine
OD Optical Density
PBMC Peripheral Blood Mononuclear Cell
PBS Phosphate-buffered Saline
PE Phycoerythrin
PE-Cy7 R-Phycoerythrin-Cyanine Dye 7
PerCP Peridinin Chlorophyll Protein Complex
PI Pharmacological Interaction
PMA Phorbol Myristate Acetate
Poly I:C Polyinosinic: polycytidylic acid
RA Retinoic Acid
RANTES Regulated on Activation, Normal T cell Expressed and Secreted
RPMI Roswell Park Memorial Institute
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
SMX Sulfamethoxazole
TBST Tris-buffered Saline Tween-20
TCR T Cell Receptor
Th T Helper (cells)
TLR Toll Like Receptor
TNF Tumor Necrosis Factor
Treg Regulatory T (cells)
TRIM63 Tripartite Motif Containing 63
VEGF Vascular Endothelial Growth Factor
xii
LIST OF TABLES
Table 1. Classification of adverse drug reactions. ......................................................................... 4
Table 2. Similar characteristics of NVP-induced skin rash in humans and female BN rats. ....... 17
Table 3. Percentage of cell types before and after the depletion of lymphocyte subsets with
magnetic beads. ............................................................................................................................. 43
Table 4. Design of NVP and anti-mouse CD20 antibody†
cotreatment experiments in naïve BN
rats. ................................................................................................................................................ 66
Table 5. Design of NVP and anti-mouse CD20 antibody cotreatment experiments in
splenectomized female BN rats. ................................................................................................... 67
Table 6. Design of BSO and NVP cotreatment experiments in female BN rats. ........................ 68
xiii
LIST OF FIGURES
Figure 1. Hapten hypothesis. .......................................................................................................... 9
Figure 2. Danger hypothesis. ....................................................................................................... 12
Figure 3. PI hypothesis. ............................................................................................................... 14
Figure 4. Structures of NVP, 12-OH-NVP, 4-Cl-NVP and 12-Cl-NVP. .................................... 30
Figure 5. IFN-γ secretion by lymphocytes in response to NVP, 12-OH-NVP, 4-Cl-NVP and 12-
Cl-NVP. ........................................................................................................................................ 37
Figure 6. IL-10 secretion by lymphocytes in response to NVP, 12-OH-NVP, 4-Cl-NVP and 12-
Cl-NVP. ........................................................................................................................................ 38
Figure 7. Frequencies of lymphocytes responding with the production of IFN-γ when stimulated
with NVP or its analogs/metabolites from NVP-rechallenged rats using an ELISPOT assay. .... 39
Figure 8. Cell proliferation in response to NVP, 12-OH-NVP and 4-Cl-NVP as determined by
the reduction of alamar blue. ........................................................................................................ 40
Figure 9. Concentration of cytokines/chemokines produced by lymphocytes from control (n=4),
primary treated (n=6), and rechallenged (n=7) animals in response to NVP as determined by a
Luminex assay. ............................................................................................................................. 41
Figure 10. Production of IFN-γ in response to NVP by lymphocytes from rechallenged rats
before and after depletion of CD4+ and/or CD8
+ T cells. ............................................................. 42
Figure 11. Proposed chemical mechanisms of NVP-induced skin rash by formation of 12-OH-
NVP sulfate in the skin. ................................................................................................................ 49
Figure 12. H&E staining of skin samples from rats rechallenged with topical NVP (2.5 mg/mL
in acetone/olive oil, 1:1/v:v) or vehicle only. ............................................................................... 52
Figure 13. H&E staining of skin samples from naïve rats treated with topical 12-OH-NVP (15
mg/kg/day in DMSO) or vehicle only. ......................................................................................... 54
xiv
Figure 14. Percent of cells stained with CD45RA/B in peripheral blood following anti-mouse
CD20 antibody injections. ............................................................................................................ 71
Figure 15. Percent of cells stained with CD45RA/B in peripheral blood following anti-mouse
CD20 antibody injections and NVP treatment. ............................................................................. 72
Figure 16. B cell levels in the spleen, ALN, and peripheral blood on D22 of NVP treatment. .. 73
Figure 17. Spleen weight for selected groups on Day 22 of NVP treatment. .............................. 74
Figure 18. The effect of NVP treatment and anti-mouse CD20 antibody on plasma IgE levels. 75
Figure 19. Plasma levels of NVP and its metabolites in BSO-NVP co-treated or NVP-treated
female BN rats. ............................................................................................................................. 77
Figure 20. 24 hours urinary excretion of NVP and its metabolite in BSO-NVP co-treated and
NVP-treated female BN rats. ........................................................................................................ 78
Figure 21. Liver weights and glutathione levels in the liver of BSO-NVP co-treated or NVP-
treated female BN rats. ................................................................................................................. 79
Figure 22. Effect of RA on the incidence of NVP-induced skin rash. ......................................... 81
Figure 23. Effect of RA on plasma levels of NVP and its metabolites. ...................................... 82
Figure 24. NVP and 12-OH-NVP plasma concentrations in animals that received RA (20
mg/k/day in oil, gavage) and an escalating dose of NVP (started from 15 mg/kg/day and
escalated to 175 mg/kg/day). ........................................................................................................ 83
Figure 25. Plasma concentrations of cytokines/chemokines during a 21-days treatment course
with NVP or RA-NVP determined by a Luminex assay. ............................................................. 84
Figure 26. Expression of CD40 on RAW 264.7 cells after a 24 hours stimulation with 12-OH-
NVP sulfate. .................................................................................................................................. 98
Figure 27. Expression of CD40 on RAW264.7 cells in response to various concentrations of
NVP, 12-OH-NVP, or its sulfate metabolite expressed in two different ways. ............................ 99
xv
Figure 28. Precent CD40 positive cells in “aging” RAW264.7 cells. ....................................... 100
Figure 29. Representative dot plots and histograms of surface marker staining of THP-1 cells
stimulated by various substances. ............................................................................................... 102
Figure 30. Change in cell surface marker expression on THP-1 cells in response to NVP or its
metabolites. ................................................................................................................................. 103
Figure 31. Phenotype of bone marrow-derived cells. ................................................................ 105
Figure 32. Representative dot plots and histograms of surface marker staining on BMDCs
stimulated by various substances. ............................................................................................... 106
Figure 33. Change in cell surface marker expression on BDMCs in response to NVP or its
metabolites. ................................................................................................................................. 107
Figure 34. Cytokine production by drug-stimulated BMDCs at the end of a 24 hour incubation.
..................................................................................................................................................... 108
Figure 35. Covalent binding of DMSO, NVP, 12-OH-NVP, and its sulfate to BMDCs after a 24
or 72 hour incubation. ................................................................................................................. 109
Figure 36. Change in cell surface marker expression on BMDCs in response to D-penicillamine
or isoniazid. ................................................................................................................................. 111
Figure 37. Change in cell surface marker expression on RAW264.7 cells in response to D-
penicillamine or isoniazid. .......................................................................................................... 112
Figure 38. Phenotype of CD4+CD25
- cells isolated from the spleen and ALNs of naïve female
BN rats using magnetic beads and column. ................................................................................ 114
Figure 39. Proliferation of αβ-TCR+ cells measured by CFSE staining. ................................... 115
Figure 40. Chromatographs of (A) 12-OH-NVP sulfate and (B) 12-OH-NVP after a 24 hour
incubation with immature BMDCs generated from naïve female BN rats. ................................ 117
xvi
Figure 41. Quantification of 12-OH-NVP sulfate in cell culture medium in the presence or
absence of BDMCs over time. .................................................................................................... 118
xvii
LIST OF APPENDICES
Appendix 1. Supplemental Data of APC activation by 12-OH-NVP sulfate. ........................... 140
1
CHAPTER 1
INTRODUCTION
2
1.1. Overview of Adverse Drug Reactions
Primum non nocere
(‘First, do no harm’)
Hippocrates (460-370 BC)
It is a Latin phrase that means “first, do no harm”, a long-held principle in medicine, which
unfortunately has never been achieved. As old as Medicine itself, adverse drug reactions
(ADRs) unfortunately have been one of the many ways that a patient can come to harm through
the practice of medicine. The World Health Organization defines ADRs as “harmful, unintended
reactions to medicines that occur at doses normally used for treatment” [1].
Although many ADRs are mild, some can be very severe and sometimes even life-threatening.
They are among the leading cause of mortality and morbidity ahead of pulmonary disease,
diabetes, and AIDS etc.; they account for about 7% of hospital admissions [2, 3]. It was
estimated that in 1994 overall 2,216,000 (1,721,000-2,711,000) hospitalized patients had serious
ADRs, and 106,000 (76,000-137,000) had a fatal ADR, making these reactions between the
fourth and sixth leading cause of death in UK and US [4, 5].
ADRs also represent a huge social and economic problem. Drug-related deaths from ADRs cost
more than $136 billion a year, and 10% of drugs have been withdrawn from the market or
received a Black Box warning in the past 25 years due to unacceptable safety profiles [2, 6, 7].
They represent a great cost to society because of the longer and advanced care needed for the
affected patients. Even when occurring in a small number of people, ADRs can cause a drug to
receive a black box warning or even be withdrawn from the market. This has been a big concern
to the pharmaceutical industry given the limited numbers of new products in their pipelines.
The clinical manifestations of ADRs are complex because they can be present in different organs
and mimic other disease processes. Some ADRs are caused by inappropriate use of medications;
however, ADRs will probably never be totally eliminated because different people respond to
drugs differently [8, 9]. Although randomized controlled trials are the gold standard in testing
the efficacy and safety of new drugs, they are not very effective in detecting ADRs, mainly
because of the limited sample size and duration of the study. Compared to the highly controlled
3
environment in a trial, the real clinical settings are much more dynamic and complex. The
inappropriate use of a drug may be caused by inappropriate dosage or duration of treatment,
drug-drug interactions, or interactions with the over-the-counter products and off-label use, etc.
Table 1. shows the 6-type classification of ADRs adapted from Edwards et al. and Pourpack et
al. to showcase the variety and features of different types of ADRs: A (augmented, namely an
enhanced pharmacological effect), B (bizarre or idiosyncratic, with unknown mechanisms but
most likely involving the immune system), C (chronic or time-related), D (delayed effects), E
(end-of–treatment effects), and F (failure of therapy) [10].
It is obvious that many types of ADRs are preventable and ideally should not occur. To conquer
this clinical and economic problem, many efforts have been applied jointly by regulatory
agencies, academia, and the pharmaceutical industry to study the mechanisms of ADRs and
develop post-marketing pharmacovigilance programs. However, it is also true to say that many
ADRs are uncommon and remain unpredictable, which makes it impossible to set up an effective
surveillance system or to prevent such reactions from happening. Most of the unpredictable
ADRs fall into the second category, the Type B (Bizarre) reactions, which will be the focus of
the present work.
4
Table 1. Classification of adverse drug reactions.
Adapted from [10, 11].
Category Clinical Characteristics Example Solutions
Augmented
Pharmacological
Effect
Predictable
Pharmacology related
Digoxin toxicity Reduce dose
Low mortality
Bizarre Unpredictable
Uncommon
Not related to any known therapeutic effect of the drug
Penicillin hypersensitivity Discontinue
treatment
Chronic Related to the cumulative
dose Corticosteroids
Reduce dose
Delayed Occurs or becomes apparent
some time after the use of the drug
Carcinogenesis
Reproduction defects
Withdrawal Occurs soon after withdrawal
of the drug β-blocker withdrawal
Reintroduce the
drug
Failure therapy Usually caused by drug-drug
interactions Inadequate dosage of an
oral contraceptive Increase dosage
5
1.2. Idiosyncratic Drug Reactions
The definitions of idiosyncratic drug reactions (IDRs) are inconsistent; here we refer to IDRs as
those ADRs that do not occur in most patients at any dose used clinically and do not involve the
known therapeutic effect of the drug [12]. IDRs accounts for 6 -10% of all ADRs and have a
typical incidence from 1/100 to 1/100000 [4, 13]. Although less common than other ADRs,
IDRs are unpredictable in nature and often life threatening. The low incidence makes IDRs
difficult to detect in clinical trials, and the unpredictable nature makes mechanistic studies on
human subjects virtually impossible, and most studies are retrospective. There are also very few
animal models for IDRs because IDRs are also idiosyncratic in animals, and only the ones that
share similar characteristics with the reactions in humans would be of value. Therefore IDRs
represent a big challenge to the pharmaceutical industry, and it is unlikely that much progress
will be made in preventing these reactions until their mechanisms are well understood.
1.2.1. Clinical Manifestations
From a broader view, IDRs can affect almost any organ with skin, liver, and blood cells being
the most common targets. Some drugs only causes one type of IDR, but many others can affect
multiple organs of the same person simultaneously, or affect different organs in different
individuals. Some drugs may cause IDRs with similar patterns; there are also some common
characteristics shared by many IDRs. However each of the reactions has its own unique
characteristics.
The most common feature of IDRs is that there is a delay between starting treatment with the
drug and the start of the symptoms, while the time to onset on rechallenge is usually shortened.
These characteristics provide evidence of an immune-mediated mechanism [14]. Depending on
the drug, the delay in onset of symptoms can vary from 1 week to a couple of years. Another
common characteristic is that the incidence of IDRs does not appear to increase with dose, and
they are often referred as “dose independent”. However, this is misleading, and no IDR is dose
independent. It is true that most patients do not experience IDRs at any dose, but this is due to
the relative narrow therapeutic windows used clinically. Given that the mechanism of the IDR
does not involve the therapeutic effects of the drug, there is no reason that the dose response
curve for the therapeutic effect and the dose response curve for the IDR should be similar.
Depending on where the dose-response curve of the IDR falls, the patients could experience an
6
adverse reaction well below the therapeutic dose of the drug (IDR dose-response curve falls
below the therapeutic dose), or never experience any IDRs if the IDR dose-response curve starts
well above the therapeutic window [14].
1.2.2. Risk Factors
Many factors have been identified as risk factors for IDRs such as age, gender, viral infection,
and genetic predisposition, etc. Some of the risk factors are weak and are not true for all IDRs.
For example, women are found to be more susceptible to halothane-induced hepatitis and
clozapine-induced agranulocytosis, but not other IDRs [15, 16]. The risk of liver toxicity
induced by isoniazid is found to be higher in elderly; however, the incidence is higher in infants
when induced by valproic acid [17]. Some viral infections such as human immunodeficiency
virus (HIV) infections and herpes virus seem to be associated with an increase incidence of
IDRs, but most patients develop IDRs without being infected [18, 19]. A strong genetic
component was found for some IDRs. For example, Mallal et al in 2002 reported that 50%
human leukocyte antigen (HLA)-B*5701 patients treated with abacavir will develop an IDR. A
cost-benefit analysis involving 19 countries has shown that prospective screening of this allele
can reduce the incidence of abacavir-induced hypersensitivity. However, the cost-effectiveness
may depend on patient populations, health care settings as well as the availability of appropriate
laboratory assays in those regions [20, 21]. The association between IDRs and HLA genes, i.e.,
major histocompatibility complex, either MHC I or MHC II, also provides strong evidence that
IDRs are immune mediated.
1.3. Hypotheses of Mechanisms of IDRs
Although the mechanisms of IDRs are unclear, it is a general consensus that most are immune
mediated. There is also overwhelming circumstantial evidence for the involvement of reactive
metabolites, although some exceptions appear to exist. Despite all the efforts made to
understand the mechanism of IDRs, it is also obvious that one single mechanism does not
explain the characteristics of all IDRs. All of the hypotheses discussed below center on an
immunological mechanism, and they are not mutually exclusive. One or more might be useful in
explaining a specific reaction because IDRs are likely complex cascades of metabolic and
immunological events.
7
1.3.1. Hapten Hypothesis
The concept of haptens emerged from experiments reported by Landsteiner in 1935. He showed
that small chemicals were unable to elicit an immune response unless they are chemically
reactive and bound to proteins [22]. With more advanced knowledge, the hapten hypothesis has
evolved to the following: a hapten refers to a chemically reactive compound or reactive
metabolite, which can irreversibly bind to proteins and form drug-modified protein adducts.
These adducts can be taken up by antigen presenting cells (APCs) and presented in the major
groove of the MHC molecule to T cells. The recognition of the drug-protein adduct by T cell
receptors (TCRs) is referred to as signal 1.
One example that is consistent with the hapten hypothesis is penicillin-induced allergy [23]. The
β-lactam ring of penicillin can irreversibly react with free amino and sulfhydryl groups on
proteins and form drug-protein adducts. In some patients, this will induce the production of
immunoglobulin E (IgE) antibodies against penicillin-modified proteins, which can stimulate
degranulation of mast cells and release histamine, leukotrienes, and other inflammation-
associated molecules [24]. These IgE antibodies are pathogenic and can mediate very severe
allergic reactions including anaphylaxis.
Unlike penicillin and other β-lactam class drugs, most drugs are not chemically reactive, and
therefore cannot covalently bind to proteins to form adducts. However, many drugs such as
carbamazepine and acetaminophen do form reactive metabolites, which can also act as haptens.
Both of these drugs can undergo bioactivation to form quinone metabolites, which are usually
detoxified by glutathione formation [25, 26]. When there is an imbalance between the reactive
metabolite formation and the detoxification process, the toxic metabolites will bind to other
proteins to form haptens [27, 28].
Regardless of whether the parent drug or the reactive metabolite is acting as the hapten, the
relationship between covalent binding of a drug to a protein (and the degree of binding) and the
risk for developing an IDR is vague. Recently, the β-lactam-albumin conjugates in patient
plasma samples were identified, and the profile of drug-protein adducts at specific lysine
residues with respect to dose and incubation time was determined [28]. The minimum levels of
modification associated with the stimulation of a clinically relevant drug-specific T-cell response
8
were characterized using piperacillin-induced immune reactions in patients [29, 30]. This is the
first direct evidence of how protein haptenation induced T cell responses at the cellular level.
The hapten hypothesis is the mostly widely accepted theory of IDRs, and it has been well
documented for contact hypersensitivity and respiratory allergens [31]. However, not all drugs
that form reactive metabolites are associated with a significant incidence of IDRs, and it is not
clear what determines which drugs will cause IDRs. Perhaps a better understanding of the
implications of covalent binding and characterization of the proteins that are typically affected is
needed to further support this hypothesis.
9
Figure 1. Hapten hypothesis.
Reactive chemicals or reactive metabolites covalently bind to a protein and form a drug-protein
adduct, which is then picked up by APCs and presented to T cells. The recognition of drug-
modified proteins by TCR that leads to an immune response is referred as signal 1.
10
1.3.2. Danger Hypothesis
Before introducing the danger hypothesis, we need to briefly review the history of
immunological models in order to understand the impact this hypothesis has had on the field.
The traditional model to address the specificity of the immune system is known as the self-
nonself model that was proposed by Burnet in 1961 [32]. The key principle is that lymphocytes
surface receptors can differentiate between the self and nonself factors and only respond to the
foreigners. This theory was widely accepted for decades, although it was modified to fit with the
results of later experiments. In 1989 Janeway proposed that induction of immune response
requires a second signal, and he referred to adjuvants as the immunologist’s “dirty little secret”
[33]. The first signal is the recognition of the antigen or antigen-protein complex by TCRs (as
described in Hapten Hypothesis); and the second signal is the interaction between costimulatory
molecules on APCs and T cells, e.g., interaction between B7 molecules and cluster of
differentiation (CD) 28. Although Janeway’s theory emphasized that the induction of immune
response depends on the recognition of pathogen by APCs not T cells, both the original self-
nonself model and Janeway’s theory are based on the recognition of foreignness.
For almost a half decade, the traditional self-nonself model dominated immunology, although it
does not explain many common phenomena. For example, gut bacteria are nonself substances,
but we all tolerate them without a problem. A pregnant woman’s immune system also does not
attack its fetus. If the nonself is the major determinant of an immune response, humans would
never be able to reproduce because many of the antigens are not produced until puberty, and
therefore would not have induced tolerance in the prenatal period. The self-nonself model also
does not explain the mechanisms of autoimmune diseases where no foreign substances are
introduced.
In 1994, Polly Matzinger presented the Danger model and proposed that it is cell damage rather
than nonself that determines whether an immune response will occur [34]. Using this
framework, the tissue that is injured or under stress releases danger signals, which then activate
APCs leading to upregulation of costimulatory molecules and providing the second signal [35].
The danger hypothesis has significantly changed our perspective on what is involved in the
induction of an immune response because it can explain why a wide variety of nonself exposures
11
do not trigger an immune response in the absence of significant cell damage as well as how
endogenous molecules can induce immune reactions.
The danger hypothesis also has had a big impact on our perspective of the mechanisms of IDRs.
It could also explain why not all drugs that form reactive metabolites are associated with a high
incidence of IDRs: the induction of immune-mediated IDRs may be determined by the ability of
reactive metabolites to induce danger signals rather than their ability to form haptenated proteins
[36, 37]. The danger hypothesis may also help with animal model development. If it is true, in
theory we could introduce some “danger signals” to overcome the immune tolerance, which has
been the greatest challenge to the development of animal models [38]. It is also possible that
other factors leading to cell damage, such as viral infection and surgery, may be risk factors for
IDRs [39].
As a follow up on the original hypothesis, many efforts have been made to answer two questions:
1) what is the identity of danger signals; 2) what is the link between production of danger signals
and the risk of inducing an immune response such as IDRs. Researchers have suggested that
hydrophobic biological molecules and stress-induced molecules from damaged cells such as high
mobility group box 1 (HMGB1), heat shock proteins (HSPs), and S100 proteins are good
candidates to be danger signals [40, 41]. Based on Matzinger’s finding, endogenous molecules
that are potential danger signals can bind to the same receptors that foreign antigens bind to, i.e.,
toll like receptors (TLRs) on APCs [35]. Although the danger hypothesis is very attractive, it is
so very hard to rigorously test. The range of danger signals is unknown to date, and more
evidence is needed to demonstrate to what extent this hypothesis is relevant to the mechanism of
immune responses.
12
Figure 2. Danger hypothesis.
Cells under stress can produce danger signals that can stimulate the costimulatory molecules on
APCs and provide signal 2 for T cell priming. The lack of signal 2 leads to immune tolerance.
This model does not address signal 1.
13
1.3.3. Pharmacological Interaction (PI) Hypothesis
The pharmacological interaction of drug with antigen-specific immune receptors, or the PI
hypothesis, proposed that chemically inert drugs can bind directly and non-covalently to the
MHC-peptide complex. The non-covalent binding between drug and MHC-peptide complex
may not be consequential per se, but a T cell with the appropriate TCR may bind to this drug-
MHC complex with a much higher affinity and lead to an immune response [42]. This
hypothesis was proposed by Werner J. Pichler based on the observation that T cell clones
generated from patients with history of sulfamethoxazole (SMX) proliferated in response to the
parent drug in the absence of metabolism [43]. He also found that when exposed to the parent
drug and the metabolites (nitroso-SMX and SMX-hydroxylamine), the generated T cell clones
responded much better to the parent drug than to the metabolites [44].
The PI hypothesis is relatively new and therefore less tested compared to the other hypotheses.
However, the use of T cell clones may lead to artifacts, and the response of these cells may not
represent the mechanism by which a drug induced an immune response. In fact, more recent
studies on SMX from Dean Naisbitt’s group showed the opposite results: lymphocytes from
patients with a history of SMX-mediated allergic reactions proliferated strongly with the nitroso
metabolite but very weakly to the parent drug [45]. Consistent with this observation, the same
group also showed that SMX can form metabolism-derived antigenic protein adducts in dendritic
cells, which then stimulate dendritic cell signaling and lead to a T cell-mediated immune
response [46, 47].
There is an implicit assumption on which the PI hypothesis is based - what the lymphocytes
respond to is what initiated the immune response. We were skeptical that this assumption is
correct, and we were able to test it with our animal model of nevirapine (NVP)-induced skin
rash. The details of this research will be discussed in Chapter 2 [48].
14
Figure 3. PI hypothesis.
The chemically unreactive parent drug can bind directly and reversibly to the MHC-peptide
complex on an APC and be recognized by T cells with a TCR that fits the drug-MHC complex.
This model does not address signal 2.
15
1.4. Animal Models
Given the low incidence of IDRs and their unpredictable nature, mechanistic studies of human
subjects are virtually impossible. Since IDRs are often not diagnosed until late, most human
studies are retrospective and cannot study the events that led up to the IDR. Therefore, it is very
difficult to test mechanistic hypotheses.
The most effective tool to study mechanisms of IDRs is to use a valid animal model. However,
drug reactions that are idiosyncratic in humans are also idiosyncratic in animals, and the model is
useful only if the mechanisms and clinical manifestations mimic what occurs in humans.
Therefore, it has been a great challenge to develop animal models, and as a result, very few good
models exist to date. Sulfonamide-induced hypersensitivity in dogs mimic clinical
manifestations in humans, and it appears to be a good model, but the incidence is about 1%,
mostly in large breed dogs, and it is not very practical to study large numbers of large dogs [49].
Overdoses of acetaminophen can lead to acute liver failure, which is characterized by
centrilobular hepatic necrosis both in humans and animals [25]. Although it has been
extensively studied, acetaminophen-induced idiosyncratic liver injuries in mice is a model of
direct hepatotoxicity rather than a model of an IDR [50]. Amodiaquine induces both
agranulocytosis and hepatotoxicity in humans. When rats are treated with amodiaquine, it
induces a similar immune response including slightly elevated liver transaminase levels and
leucopenia, but without histological evidence in liver damage or agranulocytosis [51, 52]. Our
group has shown that there is an immune adaptation component in the rodent model, and
overcoming immune tolerance appears to be the biggest challenge to develop an animal model
that mimics an IDR that occurs in humans [50].
Despite all of the challenges, we are still fortunate to have a few models that are good
representatives of mechanisms of human IDRs. Two of these models will be discussed in the
following sections: the NVP-induced skin rash in female Brown Norway (BN) rats, and the D-
penicillamine-induced autoimmune syndrome in BN rats.
1.4.1. NVP-induced Skin Rash in Female BN Rats
NVP (Viramune®
) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) used to treat
human immunodeficiency virus-1 infections. Soon after being marketed, it was found to cause
16
skin rash and liver toxicity including Stevens-Johnson syndrome and toxic epidermal necrolysis
[53]. In early studies, the incidence of skin rash was 16%, and that of clinically evident
hepatotoxicity was 1% [53]. However, the incidence is lower at present because patients are
started at a lower dose (200 mg once daily) for two weeks followed by the full dose (200 mg
twice daily). Our lab found that NVP also causes a skin rash in female BN rats with similar
characteristics to the rash that it causes in humans, and the detailed characteristics are listed
below in Table 2.
17
Table 2. Similar characteristics of NVP-induced skin rash in humans and female BN rats.
Characteristic Humans Rats
Time to onset Less than 6 weeks, mostly 1~3
weeks [53]
Develop red ears in 7 days and
skin lesions in 2~3 weeks [54]
Dose-response Incidence increases with dose
[55]
Incidence increases with dose
[54]
Female sex Increased susceptibility [56, 57] Increased susceptibility [54]
NVP plasma levels ~ 5 µg/mL [58] ~20-40 µg/mL [59]
Rechallenge Earlier onset and more severe
[55]
Earlier with red ears in <24
hours and skin rash in a week
[54]
Lead-in dose treatment A 2-week low dose (200
mg/day) followed by the full
dose (200 mg twice daily)
decreased the incidence [53]
A 2-week low dose (40
mg/kg/day) followed by the
full dose (mg/kg/day)
prevented the rash [54]
CD4 T cell count Low CD4+ T cells count is
protective [60]
Partial depletion of CD4+ T
cells delayed the rash [61]
Response of
lymphocytes from
patients/rats with a rash
T cells produce interferon
(IFN)-γ when stimulated with
NVP [62]
T cells proliferate and produce
IFN-γ when stimulated with
NVP [48]
18
Female patients are more susceptible to NVP-induced skin rash than males [56, 57]. Similarly,
NVP-induced skin rash in rats is strain- and sex-dependent [54]. When female BN rats are fed a
diet containing NVP at a dose of 150 mg/kg/day, they develop red ears in about 7 days and skin
rash in 14-21 days with an incidence of 100% [54]. The incidence in female Sprague Dawley
rats was 20%, whereas none of the male rats of either strain developed a rash. However, the
blood level of NVP and its 12-hydroxy-metabolite (12-OH-NVP) are also lower than in female
BN rats, and if these animals are cotreated with aminobenzotriazole to inhibit cytochrome P450,
the incidence of skin rash increases [54]. In addition to the fact that females have a higher
incidence of skin rash in both humans and in our model, the characteristics of the skin rash are
similar as well. For instance, the onset of rash occurs 2-3 weeks after starting NVP treatment,
and the syndrome resolves when the treatment is discontinued. Upon rechallenge with NVP, the
onset of rash is accelerated. A 2-week low dose (40 mg/kg/day) followed by the full dose (150
mg/kg/day) of NVP also prevented the rash in female BN rats [54]. A low CD4+ T cell count
decreases the risk of rash in humans. Similarly, partial depletion of CD4+ T cells delayed the rash
in female BN rats [61]. The fact that the characteristics of NVP-induced skin rash in female BN
rats are similar to that in humans suggests that the mechanisms are similar; therefore, we have
used this as an animal model to study IDRs. However, it should be noted that the skin rash in
humans can vary from a mild rash that resolves despite continued treatment to life-threatening
toxic epidermal necrolysis; the rash in rats is more like the milder form of rash in humans.
In patients, in addition to the faster onset on rechallenge and the presence of drug-specific T
cells, which are clear evidence of immune reactions, NVP-induced skin rash is also reported to
be associated with specific HLA genotypes including the MHC II allele HLA-DRB1*01 [63] and
HLA-DRB1*0101 [64], as well as the MHC I allele HLA-Cw8 [65]. These associations suggest
involvement of the adaptive immune system. A toxicogenomics study also suggested that the
genetic predisposition to NVP-induced IDRs varies between different ethnic groups [66].
Likewise, our animal studies also provided overwhelming evidence of an immune-mediated
mechanism in rats. First, histology of skin sections revealed the existence of an inflammatory
cell infiltration, primarily T cells and macrophages [54, 67]. Second, when rats with a skin rash
were removed from NVP until the rash resolved and then rechallenged with NVP, there was a
more rapid onset with red ears in less than 24 hours and skin lesions in about 1 week [54]. This
suggests an amnestic immune response. Moreover, this sensitivity can be transferred to naïve
19
animals with splenocytes or just splenic CD4+ T cells from rechallenged animals, and the onset
of the reaction for these naïve recipients followed the same time course as NVP-rechallenged
animals [54]. Pretreatment with immunosuppressants, i.e. cyclosporine and tacrolimus,
prevented NVP-induced skin rash, and it also led to resolution of the rash during NVP treatment,
further supporting an immune mechanism of the rash [61].
One fundamental question is whether an IDR is caused by the parent drug or a reactive
metabolite. Jie Chen et al. in 2009 showed that hydroxylation at the 12 position of NVP to form
12-OH-NVP is required to induce a skin rash [59]. Specifically, substitution of the methyl
hydrogen atoms at the 12 position of NVP with deuterium decreased the rate of 12-hydroxylation
and led to a decreased incidence of skin rash; treatment with the 12-OH-NVP metabolite also
causes a rash at a lower dose [59]. This demonstrates that the 12-OH-NVP pathway is
responsible for inducing the rash. 12-OH-NVP is not chemically reactive, and it is the same
oxidation state as the quinone methide so the rash cannot be caused by oxidation of 12-OH-NVP,
but it can be further metabolized to a benzylic sulfate. The most recent data showed that this
sulfate metabolite can covalently bind to proteins in the epidermis of rats, where the
sulfotransferases are located. Such binding was also present when the 12-OH-NVP metabolite
was incubated with homogenized human skin, but not with murine skin [68]. This may explain
why mice do not develop a rash following NVP treatment: they lack the sulfotransferase required
to form the reactive sulfate metabolite in the skin. Moreover, topical treatment with 2-
phenylhexanol, a sulfotransferase inhibitor, prevented the covalent binding as well as the rash,
but only where it was applied [69]. These results provide definitive evidence that 12-OH-NVP
sulfate formed in the skin is responsible for the rash. There is also a report that 12-mesyloxy-
NVP, a synthetic 12-sulfate-NVP surrogate, can form conjugates with glutathione, amino acids,
DNA, and haemoglobin [70-72]. This finding is irrelevant because the mesylate is far more
reactive than the sulfate, and it is not formed in biological systems.
Another question is how this IDR is initiated, which is virtually impossible to study in humans.
There are three major hypothesis explaining the initial steps of IDRs: the hapten hypothesis, the
danger hypothesis, and the PI hypothesis. The finding that the sulfate binds to skin proteins is
consistent with the hapten hypothesis. We also found that treatment of animals led to early (6
hours) changes in gene expression in the skin that are consistent with the danger hypothesis, and
there were many more changes after treatment with 12-OH-NVP than with NVP, which is
20
consistent with the fact that 12-OH-NVP is the obligate intermediate in the formation of the
sulfate reactive metabolite in the skin [73]. Our group also found that T cells from a sensitized
animal responded to incubation with NVP by producing cytokines such as IFN-γ and interleukin
(IL)-10, which implies that the rash was induced by NVP rather than a metabolite, and this is
consistent with the PI hypothesis. However, we already knew that the induction of rash depends
on 12-hydroxylation. This assumption was the basis for the PI hypothesis, and it is only with an
animal model that this could be tested.
NVP-induced skin rash in rats, as one of the very few animal models with characteristics similar
to the IDR that occurs in humans, has allowed testing of several hypotheses that could not be
tested in humans. It is the first study to use a valid animal model to demonstrate that a reactive
metabolite is responsible for an IDR, in this case a reactive metabolite formed in the skin. It
appears that both hapten formation and the induction of danger signals are involved, but there are
probably many factors that make this drug so effective in inducing an immune response and
many details remain to be studied. This thesis will further explore the sequence of events by
which a reactive metabolite leads to an IDR using this animal model.
1.4.2. D-penicillamine-Induced Autoimmune Diseases
Penicillamine is used in the treatment of Wilson’s disease and rheumatoid arthritis but associated
with a relatively high incidence of various autoimmune syndromes, including a lupus-like
syndrome, and myasthenia gravis [74]. It also causes an autoimmune reaction in BN rats, with
features of lupus in humans such as antinuclear antibodies and immune complex deposition in
the kidneys [75, 76]. The reaction is idiosyncratic because it only occurs in BN rats and the
incidence is ~50% even though this is a highly inbred strain of rats; and other strains are
resistant. The dose-response curve of penicillamine-induced autoimmune disease in BN rats is
very unique: the incidence is 0% at a dose of 5 to 10 mg/day; the incidence is between 50% and
80% at a dose of 20 mg/day and not increased by increasing the dose to 50 mg/day. A low dose
treatment (10 mg/day) for 2 weeks induces immune tolerance to a dose of 20 mg/day, which can
be transferred to naïve animals with spleen cells from a tolerant animal [77]. In contrast, the
protection induced by a low dose treatment in NVP-induced skin rash in BN rats is metabolic
tolerance, because it could not be transferred with splenocytes, and co-treatment with
aminobenzotriazole (a P450 inhibitor) breaks the tolerance [61].
21
D-penicillamine is chemically reactive without being metabolized; it reacts with aldehydes on
APCs which are normally involved in signaling between T cells and APCs [78]. Rhodes et al.
reported that the amines on T cells and the aldehydes on APCs can form a reversible imine bond,
which is essential for antigen-specific T cell activation [79]. Similarly, penicillamine can react
with aldehydes to form a thioazolidine ring, which is not readily reversible, and lead to activation
of APCs and an autoimmune syndrome [78, 80]. Partial depletion of macrophages with
clodronate-filled liposomes can decrease the incidence of penicillamine-induced autoimmunity;
however, it also inhibits tolerance during low-dose penicillamine treatment [81]. The above
studies suggested that macrophages play an important role in both the pathogenic mechanism
and mediating the tolerance induced by low dose treatment.
Recent studies found that 24 hours following the first dose of penicillamine, a spike in IL-6 was
observed only in rats that developed autoimmune syndrome later in the treatment course [82].
The percentage of T helper 17 (Th17) cells was significantly increased, but only in sick animals.
IL-17, a characteristic cytokine produced by Th17 cells, was increased in sick animals at both the
messenger RNA and serum protein level. Macrophages can be activated by penicillamine and
produce IL-6; IL-6 is also the driving force of Th17 cell differentiation [80]. Taken together,
these findings suggest that macrophages are directly activated by penicillamine, which in turn
induce Th17 cell differentiation and lead to penicillamine-induced autoimmunity.
Many immune modulators have been used to manipulate the immune system to influence the
incidence of penicillamine-induced autoimmune syndrome in BN rats. For example, the
incidence and severity are increased by poly I:C, a synthetic double-stranded polyribonucleotide
that functions as a viral RNA analog and can stimulate APCs via TLR 3. Lipopolysaccharide
(LPS), which is found in the outer membrane of Gram-negative bacteria and stimulates
macrophages via TLR 4, also had similar effects to that induced by poly I:C, but less pronounced
[77, 83]. In contrast to poly I:C and LPS, misoprostol (a prostaglandin E analog) and
aminoguanidine (an inhibitor of inducible nitric oxide synthase) were both protective [83]. On
the other hand, tacrolimus, a T cell immunosuppressant, not only prevented the autoimmune
syndrome and induced tolerance to continued treatment, but it could also reverse ongoing disease
and prevented recurrence of autoimmunity upon re-exposure to penicillamine [81].
22
Similar to the NVP-induced IDR in BN rats, the penicillamine-induced autoimmune syndrome
has a delay in onset of symptoms of about 3 weeks. However, unlike the NVP model, the onset
of disease is not accelerated on rechallenge of penicillamine. Although the mechanism is
obviously immune-mediated, the reason why there is no immune memory is unknown. In
addition, given that it is an autoimmune syndrome and the antigens remain, it is not known why
it does not persist after the drug is stopped.
We believe that IDRs are due to a failure of immune tolerance mechanisms in the patients who
develop a reaction; likewise, immune tolerance is the major reason why some animals do not
develop IDRs while others do. The fact that penicillamine only causes an IDR in certain BN rats
makes this model a perfect tool for comparative studies between rats that do get sick and rats that
do not. We were able to show some markers that can predict which rats will develop such
reactions and which don’t, e.g., the early spike in IL-6. We will continue to use this animal to
study the difference between sensitive animals and tolerant animals and hopefully provide more
mechanistic clues on how to predict which drugs will cause IDRs in humans.
1.5. Investigating the Mechanisms of IDRs
Understanding IDRs has been a real challenge due to the lack of valid animal models. However,
our research has progressed rapidly with the development of many advanced assays and growing
knowledge of immunology and the human genome. In this section, several state-of-the-art
approaches used for mechanistic studies will be discussed to showcase how they have advanced
our knowledge of drug hypersensitivity.
1.5.1. Detection of Reactive Intermediate Formation and Covalent Binding
In general, formation of drug-modified proteins following bioactivation of the parent drug to
form reactive intermediates is believed to be a required step for most IDRs. Many efforts have
been devoted to screen drug candidates for the formation of reactive metabolites such as
screening for suicide inhibition of drug metabolizing enzymes or glutathione conjugates.
However, these approaches usually cannot detect all reactive metabolites because the pathways
are complex [84]. Covalent binding, as suggested by the hapten hypothesis, appears to be a
crucial step in the pathogenesis of most IDRs. However it is important to note that many drugs
23
that do not lead to IDRs are also known to form reactive metabolites, and the quantity of
covalent binding does not necessarily correlate with the incidence of IDRs [84]. It is suggested
that the pathogenesis of the binding is determined by the target of binding and the association
with cytotoxicity in vivo. However, we have not yet identified any common pattern of binding or
unique target proteins associated with most IDRs. A good example of covalent binding that
leads to an IDR is the NVP-induced skin rash in female BN rats as discussed previously [69].
This is also the first study to use an animal model to demonstrate that a reactive metabolite is
responsible for an IDR, in this case the 12-OH-NVP sulfate formed in the skin. Future
characterization of the covalent binding in this model will provide more evidence on how that
translated into toxicity and contributed to the initiation of an immune response.
As discussed, reactive metabolite formation and covalent binding are probably necessary but not
sufficient for most IDRs. Screening drug candidates to avoid chemical structures that are likely
to form a reactive intermediate or covalent binding would likely decrease the likelihood of IDRs,
however, it also results in potential loss of useful drugs.
1.5.2. T Cell Based Assays
T cell based assays are routinely used in diagnosis of drug hypersensitivity and research in IDRs
because they pose no threat to patient safety. The most well known is the proliferation based
lymphocyte transformation test (LTT). This test reproduces T cell responses to a drug in vitro,
from which one concludes a previous in vivo sensitization [85]. Basically, drug-specific T cells
are isolated from patients with a history of drug hypersensitivity and cloned in vitro to increase
the sensitivity of the assay. T cell response is then determined by proliferation or cytokine
production. A positive LTT demonstrates the specificity of the T cells to the drug tested. It is
reported that the LTT has a general sensitivity of 60-70% and a specificity of 93% in detecting β-
lactam hypersensitivity [85]. However, numbers are different for different drugs. A large
prospective study on isoniazid-induced hepatotoxicity by Warrington showed that the specificity
of LTT was 83-90% and the sensitivity was only 50% [86]. This well designed study also
addressed the fact that a greater sensitivity was obtained when drug-modified proteins (instead of
the drug or its metabolite) were used to stimulate T cells in vitro. Despite that, the LTT is widely
accepted as a diagnostic tool. However, the mechanistic relevance of detecting drug reactive T
cells is indirect, e.g. it does not explain how the immune response is initiated or if the T cell
24
response is a cause or an effect of the IDR. In fact, what T cells respond to may not necessarily
be what induced the immune response in the first place. Such assumptions can only be tested by
using an animal model.
While the LTT is used to measure the response of long lasting antigen-specific T cells, there are
other T cell-based assays developed to predict the potential of a drug to cause hypersensitivity.
These assays typically utilize isolated human APCs (for example, monocytes-derived dendritic
cells) to recognize chemically reactive sensitizers such as 2,4-dinitroclorobenzene [87].
Basically, APCs isolated from humans are exposed to an allergen for 1-2 weeks in the presence
of naïve T cells to allow initial antigen presentation. Afterwards, T cells are re-stimulated under
the same experimental conditions to allow detectable levels of proliferation or cytokine
production. The APCs used in most studies are derived from peripheral blood mononuclear cells
(PBMCs) isolated from healthy volunteers. Responder T cells are usually purified by flow
cytometry or magnetic beads to exclude natural CD4+CD25
+FOXP3
+ regulatory T (Treg) cells.
The elimination of Treg cells lowers the barrier of T cell response to weakly immunogenic
allergens and increases the sensitivity of the assay [88]. The critical point of this assay is the
antigen presentation process: can APCs successfully deliver a message to T cells. Therefore, the
success depends on the availability of the APC as well as their ability to recognize and present
the antigens [89]. Allergens can be added either in the form of a reactive metabolite to directly
activate APCs or in the form of a hapten-protein conjugate. The modification of
cellular/extracellular proteins of APCs by a reactive metabolite is impossible to control, but it is
a determinant of the antigen presentation and T cell activation. Therefore, only a positive T cell
response is informative, and a negative result cannot be interpreted [90]. One possible
explanation of a negative T cell priming assay is that the modification of the APCs did not lead
to generation of epitopes that can be recognized by T cells. This could be because the type of
APC used did not have the MHC required to present the required peptide, or the wrong antigen
was used (i.e. a specific drug-modified protein/reactive metabolite may be needed instead of the
parent drug). Another possible explanation for a negative T cell priming assay could be that the
TCR repertoire used in the system does not include a TCR that can recognize the chemical [90].
In general, using hapten-protein conjugates has some advantages except that danger signals (such
as cytokines) that are produced in vivo may be missing and may need to be added to the assay in
order to successfully prime T cells [90].
25
T cell based assays provide strong evidence that a specific drug is involved in inducing an
immune response and are complementary to most other single cell based in vitro assays. Due to
the obvious complexity, these assays are still in development and need to be optimized
individually for each compound.
1.5.3. Co-administration Studies
Co-administration of a drug that causes IDRs with other substances helps to identify the risk
factors and is useful in animal model development. These substances usually include, but are not
limited to, metabolic inhibitors, immunosuppressants, and modulators. Some of them are used to
determine the involvement of the immune system. For example, co-treatment with
immunosuppressants or immune modulators can provide evidence of the involvement of the
immune system and which cells are involved. These modulators can be small molecules such as
agonists or antagonists of a specific pathway, or biological products such as neutralizing
antibodies. A good example is that the depletion of CD4 cells by anti-CD4 antibodies led to a
decreased incidence of NVP-induced skin rash in female BN rats. This suggested that CD4 cells
mediate this IDR [61]. TLR agonists such as LPS and poly I:C are also often studied because
they facilitate breaking immune tolerance, which has been the biggest challenge in animal model
development. Metabolic inhibitors manipulate the production and turnover rate of certain
metabolites in the biological system and identify their roles in inducing the IDRs. For example,
inhibition of detoxification pathways may result in accumulation of a reactive metabolite that is
responsible for an IDR and lead to an increased incidence. Nevertheless, all of these
manipulation studies either require an animal model that represents a similar mechanism of IDR
as that in humans, or aim to develop such models.
1.5.4. Pharmacogenetics in Clinical Research
Genomic studies of patients has provided strong evidence that genetic predisposition is a risk
factor for some IDRs. Identifying these risk factors could significantly decrease the incidence of
IDRs and improve the process of drug development. There are mainly two categories of genetic
predispositions that are associated with IDRs: polymorphisms involving drug metabolizing
enzymes and drug transporter genes, or the immune system in the case of a drug-induced allergic
reactions [91]. One example of the first category is the link between deficiency in glutathione
synthetase and increased hepatotoxicity of certain drugs such as acetaminophen [92, 93].
26
Glutathione conjugation is a major detoxification pathway of many drugs. A deficiency in
synthesizing the molecule may cause the accumulation of reactive drug metabolites, which in
turn leads to liver toxicity. The most intensively investigated example for the second category is
the association between IDRs and the polymorphism of HLA molecules. For instance, a tight
association was reported between carbamazepine-induced Steven-Johnson syndrome and toxic
epidermal necrolysis and the HLA-B*1502 allele in Han Chinese but not in Caucasians [94, 95].
HLA molecules play a key role in antigen presentation and initiating an immune response. The
fact that the polymorphism leads to different incidences in different ethnic groups suggested that
immune systems are involved in these reactions. Pharmacogenetics is perhaps the most
informative tool to study the mechanisms of IDRs in clinical settings, and it is an emerging area
that may allow us to identify potential biomarkers and establish a standard process for drug
screening.
1.6. Rationale of the Present Studies
IDR is a complex topic and requires comprehensive understanding of drug metabolism,
immunology, genetics, and other fields. In the present studies, this question is tackled from
different angles by a variety of methods using the NVP-induced skin rash in female BN rats as a
model. We aim to test the PI hypothesis and investigate the role of CD4+
T cells, review risk
factors, and determine the initial steps of the immune response induced by the metabolite of
NVP. These objectives will be discussed in the following chapters.
27
CHAPTER 2
A STUDY OF THE SPECIFICITY OF LYMPHOCYTES IN
NEVIRAPINE-INDUCED SKIN RASH
Chen, X., et al., A study of the specificity of lymphocytes in nevirapine-induced skin rash.
J Pharmacol Exp Ther, 2009. 331(3): p. 836-41.
0022-3565/09/3313-836–841$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 3
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
157362/3532294
JPET 331:836–841, 2009
Printed in U.S.A.
Reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics. All rights reserved.
28
2.1. ABSTRACT
Background: NVP treatment can cause a skin rash. We developed an animal model of this rash
and determined that the 12 hydroxylation metabolic pathway is responsible for the rash, and
treatment of animals with 12-OH-NVP also leads to a rash.
Objective: To determine the specificity of lymphocytes in NVP-induced skin rash.
Materials and Methods: BN rats were treated with NVP or 12-OH-NVP to induce a rash.
Lymph nodes were removed and the response of lymphocytes to NVP and its
metabolites/analogs was determined by cytokine production (ELISA, ELISPOT, and Luminex)
and proliferation (alamar blue assay). Subsets of lymphocytes were depleted to determine which
cells were responsible for cytokine production.
Results: Lymphocytes from animals rechallenged with NVP proliferated to NVP but not to 12-
OH-NVP or 4-chloro-NVP. They also produced IFN-γ when exposed to NVP, significantly less
when exposed to 4-chloro-NVP, and very little when exposed to 12-OH-NVP even though
oxidation to 12-OH-NVP is required to induce the rash. Moreover, the specificity of
lymphocytes from 12-OH-NVP-treated rats was the same, i.e. responding to NVP more than to
12-OH-NVP even though these animals had never been exposed to NVP. A Luminex
immunoassay showed that a variety of other cytokines/chemokines were also produced by NVP-
stimulated lymphocytes. CD4+
cells were the major source of cytokines.
Conclusions: The specificity of lymphocytes in activation assays cannot be used to determine
what initiated an immune response. This has significant implications for understanding the
evolution of an immune response and the basis of the PI hypothesis.
29
2.2. INTRODUCTION
NVP is a NNRTI used in the treatment of human immunodeficiency virus infections. Although
effective, its use has been limited due to its propensity to cause skin rash and liver toxicity. In
patients, skin rashes vary from mild erythematous, maculopapular rashes to more severe Stevens-
Johnson syndrome or toxic epidermal necrolysis [96, 97]. Our group discovered a novel animal
model of NVP-induced skin rash in rats. The characteristics of NVP-induced skin rash in BN
rats are very similar to the milder rashes that occur in humans, which suggests that the
mechanisms are very similar. Specifically, in both humans and rats there is a 2-3 week delay
between starting the drug and the onset of rash, and on re-exposure, symptoms are more severe
and accelerated [54, 55]. Females are more susceptible to developing rash than males in both
BN rats and humans. Furthermore, the sensitivity to NVP-induced skin rash can be transferred
with CD4+ T cells from NVP-rechallenged rats to naïve recipients [61]. Also, partial depletion
of CD4+ T cells delayed and decreased the severity of rash while depletion of CD8
+ T cells did
not prevent the development of NVP-induced skin rash which fits with the observation that the
incidence of rash is lower in patients with a low CD4+ T cell count [61].
There is circumstantial evidence that many IDRs involve reactive metabolites of the drugs rather
than the parent drug, but there is rarely definitive evidence for the involvement of a reactive
metabolite. We recently demonstrated that the NVP-induced skin rash is not caused by NVP
itself but requires 12-hydroxylation of NVP, presumably because the 12-hydroxy metabolite is
further converted to a more reactive sulfate in the skin. This conclusion was based on
experiments in which the 12-methyl hydrogens were replaced by deuterium, which decreases 12-
hydroxylation and rash but does not affect other properties of the drug. Furthermore, treatment
with 12-OH-NVP also led to a rash [59].
In the present study we used modifications of the LTT to determine the specificity of
lymphocytes from animals with NVP-induced skin rash. The structures of the compounds used
in these experiments: NVP, 12-OH-NVP and 4-chloro-NVP (4-Cl-NVP) are shown in Figure 4.
A chlorine atom and a methyl group are of approximately the same size and so noncovalent
binding of 4-Cl-NVP should be similar to that of NVP but the chlorine blocks oxidation to the
12-OH-NVP and subsequent formation of the sulfate, which is the putative immunogen.
30
Another analog, 12-chloro-NVP (12-Cl-NVP), which is more reactive than the sulfate of 12-OH-
NVP, was eliminated from later studies because of its cytotoxicity.
Figure 4. Structures of NVP, 12-OH-NVP, 4-Cl-NVP and 12-Cl-NVP.
31
2.3. METHODS
Chemicals. NVP was kindly supplied by Boehringer-Ingelheim Pharmaceuticals Inc.
(Ridgefield, CT). 12-OH-NVP [98], 12-Cl-NVP [99] and 4-Cl-NVP [100, 101] were synthesized
as previously described. Phosphate-buffered saline (PBS, pH 7.4), fetal bovine serum (FBS),
1640 RPMI-HEPES modified, MEM non-essential amino acids solution and Penicillin-
Streptomycin liquid were purchased from Invitrogen Canada, Inc. (Burlington, ON). Dimethyl
sulfoxide (DMSO), indomethacin, phorbol myristate acetate (PMA) and inomycin were
purchased from Sigma Aldrich (Oakville, ON). ß-mercaptoethanol (2-ME) was purchased from
Bio-Rad Laboratories (Canada) Ltd. (Missisauga, ON). Rat CD4 and CD8 MicroBeads were
purchased from Miltenyi Biotec (Auburn, CA). Antibodies for flow cytometry studies including
anti-rat CD4 phycoerythrin (PE) (mouse immunoglobulin G1 (IgG1)), anti-rat CD8a fluorescein
isothiocyanate (FITC) (mouse IgG1), mouse IgG1 PE (isotype control) and mouse IgG1 FITC
(isotype control) were purchased from Cedarlane Laboratories (Burlington, ON). Rat
cytokine/chemokine Luminex bead immunoassay kit, LINCOplex, 24 Plex, was purchased from
Millipore (Billerica, MA). Alamar blue solution was purchased from AbD Serotec (Oxford,UK).
IFN-γ and IL-10 enzyme-linked immunosorbent assay (ELISA), IFN-γ enzyme-linked
immunosorbent spot (ELISPOT) immunoassay kits and anti-rat CD32 (Fc-gamma receptor II or
FcγII) antibody were purchased from BD biosciences (Mississauga, ON).
Animal care. Female BN rats (150-175 g) were obtained from Charles River (Montreal, QC)
and housed in pairs in standard cages with free access to water and Agribrands powdered lab
chow diet (Leis Pet Distributing Inc., Wellesley, ON). The animal room was maintained at 22oC
with a 12:12 hour light:dark cycle. After one week of acclimatization period, the rats were either
continued on the same diet (control) or switched to a diet mixed with NVP (treatment group).
Primary-treated animals refer to rats that were treated with NVP at a dose of 150 mg/kg/day for
21 days. Rechallenged animals refer to rats that have recovered (4 weeks off drug) from primary
treatment and then reexposed to the same dose of NVP for 5 days. The amount of NVP mixed
with the diet was calculated based on the body weight of the rats and their daily intake of food.
All animals were monitored for the development of red ears, skin rash, food intake, and body
weight. At the termination of the experiment, rats were killed by carbon dioxide (CO2)
asphyxiation. All of the animal studies were conducted in accordance with the guidelines of the
32
Canadian Council on Animal Care and approved by University of Toronto’s animal care
committee.
Preparation of single cell suspension from auricular lymph nodes (ALNs). ALNs were
excised and put into Petri dishes containing culture medium (50 mL of FBS, 5 mL of MEM non-
essential amino acids, 5 mL of antibiotics, 5 mL of diluted 2-ME (35 µL of 2-ME in 100 mL of
distilled water) and 435 mL of 1640 RPMI-HEPES-modified medium. ALN cells were teased
out of the nodal capsule using the butt end of a sterile 3 mL syringe plunger and filtered twice
through a 40 µm nylon mesh cell strainer (BD falcon). The cell viability was assessed in 0.4%
Trypan Blue.
Determination of cell proliferation using an alamar blue assay. Single cell suspensions
made from control, primary-treated, or rechallenged animals were resuspended at a density of
106 cells/mL with 1 µg/mL of indomethacin. They were then plated at 200 µL/well in a 96-well
plate. The cells were incubated with various concentrations of NVP, 12-OH-NVP, or 4-Cl-NVP
dissolved in DMSO for 72 hours at 37°C in a 5% CO2 atmosphere. Cells were incubated with
equal volume (10µL) of PMA (50 ng/mL in DMSO) and inomycin (500 ng/mL in DMSO)
served as a positive control. DMSO alone-treated wells served as a negative control. Alamar
blue reagent was added to each well in an amount equal to 10% of the volume in the well (20
µL) at 24 hours during the incubation. Optical density (OD) values at 570 nm and 600 nm were
read at 72 hours. Alamar blue reagent incorporates an oxidation-reduction indicator that changes
color in response to the chemical reduction of growth medium resulting from cell growth. The
difference in its reduction between treated and control wells were calculated following the
manufacture’s instructions.
Luminex, ELISPOT and ELISA assays. Single cell suspensions made from control, primary-
treated, and rechallenged animals were incubated in the same manner as in the alamar blue assay
except that the cells were plated at 2 mL/well in a 24-well plate for the ELISA and Luminex
assays, and the addition of alamar blue was omitted. After 72 hours of incubation, the cell
culture supernatant was collected and stored at -20oC. Quantitation of IFN-γ and IL-10 were
achieved by using an ELISA assay and a broad screening of cytokines was performed using a
Luminex immunoassay kit from Millipore. The cytokines/chemokines measured were: IL-1α,
IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-18, granulocyte macrophage colony-
33
stimulating factor (GM-CSF), growth-related oncogene (GRO/KC), IFN-γ, monocyte
chemoattractant protein 1 (MCP-1), tumor necrosis factor α (TNF-α), IL-9, IL-13, IL-17,
Eotaxin, granulocyte colony-stimulating factor (G-CSF), leptin, macrophage inflammatory
protein 1α (MIP-1α), interferon gamma-induced protein 10 (IP-10), regulated on activation,
normal t cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF).
In the case of the ELISPOT assay, cells were resuspended at 2 X 106 cells/mL and 200 µL was
added into each well of the 96 well ELISPOT plate. After 72 hours of incubation, the
frequencies of cells that produce IFN-γ were analyzed by an automated enzyme-linked
immunospot counter (Cellular Technology Ltd., OH). All the immunoassays were performed by
following the manufacture’s instructions.
Depletion of CD4+
and/or CD8+ lymphocytes. In order to identify which cells produced IFN-
γ, CD4+ and/or CD8
+ cells were depleted from the single cell suspensions by labeling CD4
+
and/or CD8+ cells with immunomagnetic microbeads coated with anti-CD4 or anti-CD8a
antibodies, followed by passage through a magnetic column. The CD4+ and/or CD8
+ depleted
cells were resuspended at 106 cells/mL and 2 mL of cells were incubated with 12.5 µg/mL of
NVP in DMSO in the same way as the other immunoassays described above. To examine the
effect of the depletion procedure itself on cytokine production, a control experiment was
performed in which some CD4+ and/or CD8
+ depleted cells were combined with CD4
+ and/or
CD8+ cells (labeled by the corresponding antibodies) in the same portion as they were before the
depletion and then incubated with NVP. A small portion of cells without any processing was
also cultured the same way with NVP as a positive control (before depletion). DMSO alone-
treated cells served as negative control. The amount of IFN-γ released into culture medium was
determined by ELISA.
Flow cytometry. Single cell suspensions before and after the depletion of CD4+ and/or CD8
+
cells were surface-labeled, and one- or two-color immunofluorescence analysis was conducted.
Briefly, cells were resuspended at a density of 2 X 107 cells/mL in PBS/3% FBS and 50 µL of
these cell suspensions were aliquoted to wells in a 96-well plate. These cells were first incubated
with anti-CD32 antibody for 10 minutes at room temperature to reduce nonspecific binding.
Then monoclonal antibodies or suitable isotype controls were aliquoted to the appropriate wells
and incubated at room temperature for 20 minutes. The cells were washed twice and finally
resuspended in 400 µL of the same buffer. Samples were analyzed immediately with a FACS-
34
Calibur (Becton Dickinson) using the CellQuest software (Becton Dickinson). FlowJo (Tree
Star, Inc.) was used to analyze the difference in CD4+ and/or CD8
+ cell populations between
depleted and undepleted cells.
35
2.4. RESULTS
ALNs Cell Specificity as Determined by IFN-γ Production. Cytokine production was used as
a measure of T cell activation. It was found that IFN-γ was a sensitive marker of cell activation,
and the addition of indomethacin to the culture medium made the assay more sensitive and
consistent, presumably because it decreased the inhibitory effects of prostaglandin E production.
Drug-specific IFN-γ secretion from lymphocytes gradually increased from 6 hours until reaching
maximal concentrations on day 3; no IFN-γ could be detected in cultures in the absence of NVP
or in cultures of ALN cells from animals after 21 days of primary NVP exposure in the presence
of NVP (data not shown). Analysis of cell culture supernatants after 3 days showed that ALN
cells from secondary treatment rats incubated in the presence of NVP or 4-Cl-NVP produced
IFN-γ with only a minimal response to 12-OH-NVP and no response to 12-Cl-NVP (Figure 5).
12-Cl-NVP was found to be cytotoxic causing morphologic changes in the cells; therefore, it was
excluded from the rest of the studies. Likewise, ALN cells from animals in which the rash was
induced by 12-OH-NVP and rechallenged with 12-OH-NVP also produced IFN-γ on exposure to
NVP with a smaller response to 4-Cl-NVP and even less to 12-OH-NVP (Figure 5). Again, no
IFN-γ production was detected in cells from animals treated for 21 days on primary exposure to
12-OH-NVP (data not shown). The production of IL-10 by these lymphocytes was also
quantified and showed similar results. Independent of whether the rash was induced by NVP or
12-OH-NVP, lymphocytes always produced IL-10 on exposure to NVP with a smaller response
to 4-Cl-NVP and even less to 12-OH-NVP (Figure 6).
The IFN-γ ELISPOT assay showed the same specificity as the ELISA assay. For both control
and primary-treated animals, no response was observed (data not shown). The frequency of cells
from NVP-rechallenged animals that respond to NVP was approximately 1:4,000 compared to
1:40,000 for the frequency of cells responding to 4-Cl-NVP. Virtually no cells responded to 12-
OH-NVP (Figure 7).
ALN Cell Specificity as Determined by Cell Proliferation. The alamar blue assay was used
to measure cell proliferation in response to NVP, 12-OH-NVP, and 4-Cl-NVP. No increase in
proliferation was detected in cells from animals after primary exposure to NVP (data not shown).
For rechallenged animals, increased proliferation was detected in NVP-stimulated cells, which
was maximal at a NVP concentration of 6.25 µg/mL (Figure 8). No proliferation was detected in
36
response to 12-OH-NVP or 4-Cl-NVP. Higher concentrations of all drugs appear to be toxic and
led to less reduction of the alamar blue reagent compared to control wells.
Screening Cytokines/Chemokines Production by Lymphocytes. The Luminex immunoassay
was used to screen for the production of 24 cytokines/chemokines by lymphocytes. In response
to NVP, lymphocytes from primary-treated animals produced increased levels of IL-6, IL-10, IL-
17, IL-18, GMCSF, GRO/KC, RANTES, and MIP-1α (Figure 9). In contrast, rechallenged
animals had increased production of IL-17, GMCSF, GRO/KC, MIP-1α, TNF-α, IL-10, IL-18,
RANTES, and IFN-γ. In general, cells from rechallenged animals produced higher levels of
cytokines/chemokines than cells from primary-treated animals; however, their cytokine profiles
were slightly different. Primary-treated animals produced more IL-18 and produced IL-6, which
was not observed in rechallenged animals. In contrast, rechallenged animals produced TNF-α as
well as a large amount of IFN-γ, which were absent in cells from primary-treated animals (Figure
9). Stimulation of cells from both primary and rechallenged animals with NVP actually
appeared to decrease the basal production of MCP-1.
Production of IFN-γ by CD4+ and/or CD8
+ Depleted Lymphocytes To identify which cells
were responsible for cytokine production, CD4+ and/or CD8
+ cells were depleted from ALN
cells and then cultured with NVP. Flow cytometry was used to determine the degree of
depletion (Table 3). When only CD4+ cells were depleted, the production of IFN-γ was
decreased to levels similar to the negative control (Figure 10). When both CD4+ and CD8
+ cells
were depleted simutaneaously, IFN-γ production was virtually eliminated. However, when ALN
cells were depleted of CD8+ cells the production level of IFN-γ was only slightly decreased.
There was no significant difference in the production level of IFN-γ between undepleted
lymphocytes and lymphocytes combined after depletion procedure.
37
Figure 5. IFN-γ secretion by lymphocytes in response to NVP, 12-OH-NVP, 4-Cl-NVP and
12-Cl-NVP.
(A) lymphocytes from NVP-treated animals (n=6), (B) lymphocytes from 12-OH-NVP-treated
animals (n=4). CON indicates control animals; for example, CON/NVP indicates that the
lymphocytes were isolated from untreated control animals and incubated with NVP. The data
are expressed as the mean STD.
38
Figure 6. IL-10 secretion by lymphocytes in response to NVP, 12-OH-NVP, 4-Cl-NVP and
12-Cl-NVP.
(A) lymphocytes from NVP-treated animals (n=6), (B) lymphocytes from 12-OH-NVP-treated
animals (n=4). CON indicates control animals; for example, CON/NVP indicates that the
lymphocytes were isolated from untreated control animals and incubated with NVP. The data
are expressed as the mean ± STD.
39
Figure 7. Frequencies of lymphocytes responding with the production of IFN-γ when
stimulated with NVP or its analogs/metabolites from NVP-rechallenged rats using an
ELISPOT assay.
Cells incubated with a PMA/inomycin mixture served as a positive control while DMSO alone-
treated well served as a negative control. The numbers at the left corner of each well represents
the number of cells responding to the drug out of a total of 0.4 million cells/well.
40
Figure 8. Cell proliferation in response to NVP, 12-OH-NVP and 4-Cl-NVP as determined
by the reduction of alamar blue.
(A) lymphocytes from untreated control animals, (B) lymphocytes from NVP rechallenged
animals. The reduction of alamar blue in control wells (DMSO alone-treated) was set as 1. The
percentage difference in reduction of alamar blue in NVP and its metabolites and/or analog-
treated wells as compared to the control wells was plotted against the concentration of these
drugs in log scale and a decreasing manner. The data represent the mean from 4 rats STD.
Statistical significance between treated samples and control samples was determined using the
Mann Whitney test; values of p≤0.05 were considered statistically significant.
41
Figure 9. Concentration of cytokines/chemokines produced by lymphocytes from control
(n=4), primary treated (n=6), and rechallenged (n=7) animals in response to NVP as
determined by a Luminex assay.
The data are separated into panels A and B based on their different range of concentrations. The
levels are expressed as the mean SEM. Statistical significance between treatment group and
control group was determined using the Mann Whitney test; values of p≤0.05 were considered
statistically significant.
42
Figure 10. Production of IFN-γ in response to NVP by lymphocytes from rechallenged rats
before and after depletion of CD4+ and/or CD8
+ T cells.
Negative control was DMSO alone-treated cells. Combined samples were obtained by combining
CD4+ and/or CD8
+ depleted cells with CD4
+ and/or CD8
+ cells (labeled by the corresponding
antibodies) in the same portion as they were before the depletion and then incubated with NVP.
The data represent the mean from 4 rats SE. Statistical significance between depletion groups
and negative control group was determined using the Mann Whitney test; values of p≤0.05 were
considered statistically significant.
43
Table 3. Percentage of cell types before and after the depletion of lymphocyte subsets with
magnetic beads.
Rat Cell type
investigated
Before
depletion
(%)
CD4+
depletion (%)
CD8+
depletion
(%)
CD4/CD8+
depletion
(%)
1 CD4+ 40.3 0.0 -- 0.5
CD8+ 10.1 -- 1.2 1.3
2 CD4+ 56.3 0.0 -- 0.2
CD8+ 5.0 -- 0.6 0.4
44
2.5. DISCUSSION
In the present studies we determined the molecular specificity of lymphocytes from NVP-
rechallenged animals. Detection of drug-specific IFN-γ secretion by lymphocytes proved to be a
sensitive method for the detection of lymphocyte activation. A recent study of a patient with a
history of NVP-induced liver toxicity found that the patient’s T cells proliferated in response to
NVP but not to 12-OH-NVP, 2-OH-NVP, or descyclopropyl-NVP; they did not test the 4-Cl
analog [102]. In another study, T cells from a patient with NVP-induced skin rash responded to
incubation with NVP by producing IFN-γ (Keane NM et al., abstract MOPEB007, 4th
International AIDS Society Meeting, Australia, 2007). These clinical findings provide further
evidence that the immune response in the animal model is similar to that in NVP-induced
idiosyncratic reactions in humans. The finding that CD4+ T cells were the source of IFN-γ is
also consistent with the observation that depletion of these cells in both the animal model and
humans is protective where, at least in the animal model, depletion of CD8+ T cells actually
seemed to make the rash worse. Other cytokines were also produced and the pattern was
different in cells from animals after primary exposure to NVP than those from rechallenged
animals. The rash in animals is mild on initial exposure to NVP, but the reaction is systemic
with weight loss and a widespread infiltration of lymphocytes in the skin on rechallenge so it is
not surprising that the response and number of responding cells was markedly greater in cells
from rechallenged animals [67].
A quite surprising finding was that there was a complete disconnect between what induced the
skin rash and the specificity of the T cells. Not only do we know from independent experiments
that oxidation of NVP to 12-OH-NVP is required to cause a skin rash, but even when the skin
rash was induced by treatment with 12-OH-NVP and the animals had not been exposed to NVP
(12-OH-NVP is not converted to NVP), their T cells responded much better to NVP than to 12-
OH-NVP. This has significant implications for understanding how an immune response evolves.
It is likely that induction of this immune response requires covalent binding of a reactive
metabolite NVP derived from 12-OH-NVP because this results in modified protein (Hapten
Hypothesis) and/or because it causes cell damage (Danger Hypothesis). However, once induced,
there is much more drug present than modified protein and it is possible that some T cells cross-
react with parent drug and proliferate, i.e. epitope spreading [12]. NVP and 4-Cl-NVP are more
45
lipophilic than 12-OH-NVP and this may lead to higher affinity binding. The basis for why there
is a disconnect between what induces the immune response and the specificity of the T cells is
speculation; however, what is clear is that the specificity of T cells cannot be used to determine
what induced the immune response.
These findings also have implications for the PI hypothesis. This hypothesis was originally
based on the LTT results from patients with a history of an idiosyncratic reaction to SMX whose
cloned T cells showed a response to the parent drug in the absence of metabolism [103]. The
unstated assumption is that what T cells respond to is what induced the immune response which
no longer can be considered a valid assumption. This does not mean that the p-i hypothesis is
wrong in all cases; there are IDRs such as ximelagatran-induced liver failure in which a reactive
metabolite does not appear to be involved. Ximelagatran is structurally similar to a small peptide
and may be able to initiate an immune response through a p-i type of interaction; in fact there is
evidence that it can bind directly but reversibly to MHC, specifically DRB1*07 and DQA1*02
[104]. However, our study does have significant implications for the interpretation of LTTs.
Acknowledgments. We thank Boehringer-Ingelheim for their supply of nevirapine. Dr. Jack P.
Uetrecht is the recipient of the Canada Research Chair in Adverse Drug Reactions.
46
CHAPTER 3
INDUCING SKIN RASH IN FEMALE BN RATS BY TOPICAL
TREATMENT OF NVP AND/OR 12-OH-NVP
47
3.1. INTRODUCTION
12-OH-NVP is a major metabolite of NVP, and the 12-OH-NVP pathway was proven to be
responsible for the skin rash [59]. Moreover, we recently found that it is 12-OH-NVP sulfate that
is responsible for covalent binding in the epidermis and the skin rash that occurs when rats are
treated with NVP. Specifically, topical treatment of 1-phenyl-1-hexanol, a sulfotransferase
inhibitor, prevented both covalent binding where it was applied and the rash [68, 69]. These
results indicate that 12-OH-NVP sulfate formed in the skin is responsible for the skin rash, and
the proposed chemical activation of NVP is shown in figure 11.
Although the culprit metabolite has been identified, the mechanism of how the immune response
was elicited is yet to be fully defined. In addition to other attractive hypotheses, such as the
danger hypothesis, the data suggest that the initiation of this IDR occurs in the skin. The fact
that the covalent binding of 12-OH-NVP sulfate formed in keratinocytes causes the rash
suggested that NVP may share a similar mechanism to that of allergic contact hypersensitivity.
In the case of contact hypersensitivity, the chemical allergens covalently bind to proteins derived
from resident cells in the skin (i.e. keratinocytes, Langerhans cells, and dendritic cells, etc.). The
Langerhans and/or dendritic cells then travel to the local draining lymph nodes and present the
antigens to T cells, which leads to an immune response. It was found that the reactivity of
chemicals and their ability to covalently bind to proteins (hapten formation) are linked to their
immunogenicity and sensitization, and this led to tests to identify potential sensitizers [105-108].
If this hypothesis is right, it should be possible to induce a skin rash by topical treatment with 12-
OH-NVP.
3.1.1. Topical Treatment of NVP on Sensitized Animals
We first started this experiment with sensitized animals because on rechallenge, the immune
response is much more vigorous than that of the first-time sensitization, and the onset is also
accelerated.
When NVP-sensitized female BN rats are rechallenged orally with NVP, the onset of the disease
is accelerated and more severe (the animals develop red ears in 1 day and skin rash in 1 week).
The dose (oral treatment) needed to induce an immune response is as low as 1/30 of that needed
48
for primary treatment. We have also done ear patch tests on pre-sensitized animals, and the
results are similar to the oral treatment studies [67]. The ear patch tests also showed a systemic
reaction: when one ear of an animal was painted with NVP or 12-OH-NVP (the major metabolite
which is responsible for the skin rash), both ears turned red within 24 hours. However, we did
not test if the rat would develop skin rash after prolonged treatment. One thing that should be
emphasized is that, although NVP needs to be biotransformed to 12-OH-NVP sulfate in the skin
to initiate the immune response, the parent drug can induce the allergic reaction on rechallenge
without metabolism. It was demonstrated in chapter 2 that the lymphocytes from sensitized
animals can recognize and respond to the parent drug in the absence of metabolism on
rechallenge. Therefore, the use of 12-OH-NVP sulfate (the culprit metabolite) or 12-OH-NVP,
which can be sulfated and form the culprit metabolite by skin resident cells, is not necessary for
topical treatment on rechallenge.
In our animal model, although the development of red ears is used as the earliest sign of NVP-
induced IDRs, we also demonstrated that the redness was a result of vascular dilation. Our goal
is to understand the mechanisms of the immune responses in the skin, and to eliminate possible
interference of the vascular effect; therefore, we repeated the patch tests and used histology to
evaluate the response.
3.1.2. Attempts to Induce a Skin Rash in Naïve Rats by Topical Treatment with 12-OH-NVP
With the success of inducing skin rashes in pre-sensitized animals, we wanted to determine if
this would work with naïve animals. The sequence of events is that NVP is first oxidized in the
liver to 12-OH-NVP, which goes to the skin where sulfotransferase in keratinocytes forms the
reactive sulfate. Therefore, topical administration of NVP would not be expected to cause a rash.
If our hypothesis — that the skin is where the immune response is initiated and the mechanism is
similar to that of contact hypersensitivity — is correct, a direct topical 12-OH-NVP treatment
should be able to induce the skin rash.
49
Figure 11. Proposed chemical mechanisms of NVP-induced skin rash by formation of 12-
OH-NVP sulfate in the skin.
50
3.2. MATERIALS AND METHODS
Chemicals. NVP was kindly supplied by Boehringer-Ingelheim Pharmaceuticals Inc.
(Ridgefield, CT). 12-OH-NVP [98] was synthesized as previously described. Acetone was
purchased from Sigma Aldrich (Oakville, ON).
Animal care. Female BN rats (150-175 g) were obtained from Charles River (Montreal, QC)
and housed in pairs in standard cages with free access to water and Agribrands powdered lab
chow diet (Leis Pet Distributing Inc., Wellesley, ON). The animal room was maintained at 22 oC
with a 12:12 hour light:dark cycle. After a one-week acclimatization period, the rats were either
continued on the same diet (control) or switched to a diet mixed with NVP (treatment group).
Primary-treated animals refer to rats that were treated with NVP at a dose of 150 mg/kg/day for
21 days. Rechallenged animals refer to rats that have recovered (4 weeks off drug) from the
primary treatment and then reexposed to the same dose of NVP for 5 days. The amount of NVP
mixed with the diet was calculated based on the body weight of the rats and their daily intake of
food. All animals were monitored for the development of red ears, skin rash, food intake, and
body weight. At the termination of the experiment, rats were killed by CO2 asphyxiation. All of
the animal studies were conducted in accordance with the guidelines of the Canadian Council on
Animal Care and approved by University of Toronto’s animal care committee.
Inducing skin rash in rechallenged animals by topical treatment with NVP. Six female BN
rats received NVP in food (150 mg/kg/day) for 21 days or until the skin rash developed, and then
the drug was removed for at least 1 month to allow the animals to recover. After that, part of the
skin on the back of three rats was shaved (approximately 3 cm X 3 cm) and painted with NVP
(2.5 mg/mL in acetone/olive oil, 1:1/v:v). The others were treated with vehicle solution on a
daily basis. As a control for the effects of shaving, an additional area of skin was shaved distant
from where the drug or vehicle solution was applied.
Attempts to induce skin rash in naïve animals by topical treatment with 12-OH-NVP. To
test this hypothesis, 8 female BN rats were shaved on the back (approximately 3 cm X 3 cm).
Two of them were painted with 100 µL of 12-OH-NVP suspension in acetone/olive oil (1:1/v:v)
at a dose of 1.5 mg/kg/day; 2 were painted with 25 µL of 12-OH-NVP in DMSO at a dose of 15
mg/kg/day. The other 4 control animals were treated with the vehicle used in the treatment
groups.
51
3.3. RESULTS
Topical treatment of NVP-induced skin rash in sensitized animals. The rats that received
NVP applied topically to the back developed red ears within 24 hours and skin lesions in the
painted area in less than 1 week. Due to the irritation and burn caused by the solvent on the
lesion area, the treatment was stopped at approximately day 5. The vehicle control areas in
treated animals did not have apparent lesions, but the skin surface was dry and exfoliated. The
animals that received vehicle alone had no discomfort or symptoms at all, and the skin looked
normal. The vehicle alone appears to cause some edema and infiltration of a few lymphocytes,
but the histology is not significantly different from other areas on the same control rats (Figure
12. A and B).
The control area of NVP-treated rats (Figure 12. C) showed more lymphocyte infiltration in the
dermis and epidermis. It also had a thickened epidermal layer but few dead keratinocytes. The
histology of the skin where the topical NVP was applied (Figure 12. D-F) showed massive
lymphocyte infiltration everywhere, i.e. epidermis, dermis, and subcutaneous tissue. There was
also an infiltration of eosinophils and neutrophils as well as dead keratinocytes.
52
A) B)
C) D)
E) F)
Figure 12. H&E staining of skin samples from rats rechallenged with topical NVP (2.5
mg/mL in acetone/olive oil, 1:1/v:v) or vehicle only.
A) control rat, control area, 20X magnification; B) control rat, vehicle-application area, 20X
magnification; C) treated rat, vehicle-application area. 20X magnification; D-F) treated rat,
NVP-application area, 20X, 10X and 100X magnification, respectively.
53
Topical treatment with 12-OH-NVP did not induce a skin rash in naïve animals. The naïve
animals that were treated for 6 weeks with topical 12-OH-NVP had no evidence of skin rash nor
did they appear to have any discomfort. For rats that were treated with 12-OH-NVP in
acetone/olive oil (1:1/v:v) at a dose of 1.5 mg/kg/day, the histology (hematoxylin and eosin or
H&E staining) of the painted skin showed no difference between control and treated animals for
either control or treated areas (results not shown). This may be due to the very limited solubility
of 12-OH-NVP in this vehicle solution and the drug seemed to precipitate out on the surface of
the skin once the acetone had evaporated. Therefore the amount of absorption may have been
minimal. On the other hand, DMSO penetrates skin very well, and it was also the best solvent
for 12-OH-NVP by far that we tried. It seemed that it delivered the drug well and no
precipitation was observed.
Figure 13 shows that DMSO alone caused some skin edema (thicker epidermis) as well as
limited infiltration of lymphocytes in the epidermis and dermis compared to the control area on
the control rats (A). In 12-OH-NVP-treated rats, there seems to be more lymphocytes in the
dermal and subcutaneous areas. An eosinophil infiltration was also observed (C-E). However,
overall the histological changes were minimal, and the difference between drug-treated animals
and control animals were not significant.
54
A) B)
C) D)
E)
Figure 13. H&E staining of skin samples from naïve rats treated with topical 12-OH-NVP
(15 mg/kg/day in DMSO) or vehicle only.
A) control rat, control area; B) control rat, DMSO-application area; C) treated rat, DMSO-
application area; D-E) treated rat, 12-OH-NVP-application area. A-D): 10X magnification; E)
20X magnification.
55
3.4. DISCUSSION
Even though there is not much P450 in the skin, and NVP must to be metabolized to 12-OH-
NVP in order to induce a skin rash in naïve animals, we were still able to induce a skin rash by
topical treatment of the parent drug in sensitized animals. The reason is likely due to the
“epitope spreading” effect discussed in Chapter 2. Once the immune response is initiated, the
lymphocytes will “cross-react” and respond to both the parent drug and 12-OH-NVP. Therefore,
the parent drug is enough to trigger the immune response on rechallenge.
The fact that we were able to induce a skin rash by topical treatment of NVP suggested that, at
least on rechallenge, the immune response is initiated in the skin. The question is then which
cells are responsible for this “reactivation” and what is the mechanism.
In contact hypersensitivity, dendritic cells (CD11c+) and Langerhans cells (CD207
+) are the most
common antigen presentation cells in the skin [109, 110]. They migrate to the draining lymph
nodes and maybe the spleen once being activated by antigens. The signals are then presented to
T cells in the secondary lymphoid organs and an immune response may be elicited. On the other
hand, T cells expressing skin homing receptors could also be attracted by skin residential cells
expressing the corresponding ligands, and migrate to the local inflammatory site, then induce an
immune response. [111, 112]. In addition to the cell response to the drug, the mechanism
involved in inducing the systemic reaction by local topical treatment of sensitized rats may also
contain an autoimmune component. Although it is possible that there is sufficient absorption
through the skin to produce a significant circulating concentration of NVP, if the amount applied
to the skin is decreased, eventually no rash is induced, but there is no dose at which there is only
a local response. We have recently found the presence of autoantibodies in the sera of
rechallenged animals, which further supports the autoimmune component (Amy Sharma,
unpublished observations). Further characterization of the autoantibodies such as the time
course and classification will be determined.
Although no skin rash was induced by topical treatment of 12-OH-NVP in naïve animals, the
fact that the absolute low dose of drugs/metabolites which will get absorbed by the skin is
limited and may not be enough to induce an IDR. The histology results from primary exposure
experiments also suggested the possibility that 12-OH-NVP may have induced some immune
response although the changes were not that significant. Therefore this study does not directly
56
prove our hypothesis wrong. However, it is also possible that the successful induction of an
immune response in the case of NVP requires more than just the local reaction in skin, and the
only way to fully understand the mechanism is to vigorously test each hypothesis.
57
CHAPTER 4
FACTORS THAT MAY INFLUENCE THE INCIDENCE AND
SEVERITY OF NVP-INDUCED SKIN RASH IN FEMALE BN
RATS
58
4.1. INTRODUCTION
NVP (Viramune) is a NNRTI used in combination therapy to treat human immunodeficiency
virus-1 (HIV-1) infections. Soon after being marketed, it was found to cause skin rash and liver
toxicity including Stevens-Johnson syndrome and toxic epidermal necrolysis [53]. Early studies
showed the incidence of skin rash was 16%, and that of clinically evident hepatotoxicity was 1%
[53]. However, the incidence is lower at present. Starting patients at a lower dose (200 mg once
daily) for two weeks followed by the full does (200 mg twice daily) reduces the incidence of
rash, while treatment with steroids increases the incidence of rash [113]. Also, female patients
are more susceptible to skin rash than males [57].
We found that NVP also causes a skin rash in female BN rats and developed a novel animal
model of drug-induced idiosyncratic skin rash [54]. Specifically, when female BN rats were fed a
mash diet containing NVP at the dose of 150 mg/kg/day, they developed red ears in about 7 days
and skin rash in 14-21 days with an incidence of 100% [54]. The incidence in female Sprague
Dawley rats was 20%, whereas none of the male rats of either strain developed a rash. Besides
the fact that females are associated with higher incidence in both human and our model, the
characteristics of the skin rash are similar as well. For instance, the onset of rash occurs 2-3
weeks after NVP treatment and the syndrome resolves when the treatment is discontinued. Upon
the rechallenge with NVP, the onset of rash is accelerated. A 2-week low dose (40 mg/kg/day)
followed by the full dose (150 mg/kg/day) NVP also prevented the rash in female BN rats [54].
A low CD4+ T cell count decreases the risk of rash in humans; similarly, partial depletion of
CD4+ T cells delayed the rash in female BN rats. The characteristics of NVP-induced skin rash
in female BN rats suggest an immune-mediated mechanism, and we believe it is similar to that in
humans.
In the past few years we have used a variety of modulators to manipulate the incidence/severity
of NVP-induced skin rash to better illustrate the mechanisms of this IDR. For instance, we have
shown that a partial depletion of CD4+ cells by an anti-CD4 antibody is protective suggesting
that CD4+ cells play an important role in this IDR model [54]. On the other hand, a partial
depletion of CD8+ cells seemed to make the rash worse. The immunosuppressants, cyclosporine
and tacrolimus, also prevented NVP-induced skin rash and the associated increase in serum IgE,
which suggests that NVP-induced skin rash is immune-mediated [54]. Agents that were
59
demonstrated to change the severity/incidence in another immune-mediated IDR model
(penicillamine-induced autoimmunity in the BN rats) including poly I:C, misoprostol, and
aminoguanidine, had no effect on NVP-induced skin rash [61, 83]. The first sign of NVP-
induced skin rash was red ears, which suggested the involvement of histamine and/or serotonin,
and this led us to test a few anti-allergic drugs including cromolyn, ketanserin, and astemizole.
However, none of these agents had any effect on this IDR [61]. In this paper, we continued to
explore agents that may be able to manipulate the incidence and/or severity of this IDR with the
aim to better understand the mechanism.
Previous studies have shown that the number of B cells increased much more than any other cells
(CD4+ T cells, CD8
+ T cells, and macrophages) in ALNs of NVP-treated animals [67]. The
number of B cells expressing MHC II also significantly increased. In addition, there was a spike
of IgE on day 7 following NVP treatment [67]. All this evidence suggested that B cells may play
an important role in our model, and the best way to test this hypothesis is to deplete B cells in
vivo to see if it affects the NVP-induced skin rash. Rituximab has been used clinically to deplete
B cells for the treatment of many autoimmune diseases including multiple sclerosis and
rheumatoid arthritis. Because of its murine/human chimeric nature, it probably will not recognize
rat CD20 molecules; however, the anti-mouse CD20 antibody from Genetech Inc. seemed to
work in rats and was used to deplete B cells in the present study.
Glutathione reacts with electrophilic reactive intermediates derived from exogenous chemicals
such as drugs, and glutathione conjugation is an important detoxification pathway. It has been
shown that NVP forms a glutathione adduct at the 12 position in human liver microsomes and
cytochrome P450 3A4 (CYP3A4) cultures; therefore, it may represent a protective pathway
[114]. In the present study buthionine sulfoximine (BSO) was used to deplete glutathione to
determine if this would lead to a more severe reaction.
Retinoic acid (RA) is a vitamin A metabolite used to treat acne. It is also reported to inhibit
Th17 differentiation while promoting regulatory T cell differentiation [115]. In the D-
penicillamine-induced autoimmune disease model, in which we think Th17 cells are involved,
RA significantly increased the incidence [82]. In contrast, a small (N=2) pilot study showed that
RA significantly delayed the onset of NVP-induced skin rash. These preliminary findings led to
the investigation of the effect of RA on NVP-induced skin rash.
60
Indoleamine dioxygenase (IDO), an intracellular hemoprotein enzyme which catalyses
degradation of the essential amino acid tryptophan, appears important to promote immune
tolerance induction [116]. The levo form of 1-methyl-tryptophan was reported to inhibit IDO
produced by human dendritic cells [117]. Our previous studies have shown a role of regulatory T
cells in NVP-induced skin rash (unpublished observation). Treating animals with 1-methyl-
tryptophan might break the tolerance induced by regulatory T cells and result in an earlier onset
or a more severe disease or even liver toxicity.
Imiquimod and LPS can induce immune responses through toll-like receptors in a non-specific
manner. LPS is a major component of the outer membrane of Gram-negative bacteria; it
enhances an immune response by stimulating APCs through toll-like receptor 4 and upregulating
costimulatory molecules [118]. In another animal model of IDRs, D-penicillamine induced
lupus-like syndrome in male BN rats, LPS was able to reverse tolerance in a small percentage of
animals [77]. It was interesting to see if LPS would affect the disease progression of NVP-
induced skin rash as well. Imiquimod stimulates APCs through toll-like receptor 7 and can
induce the synthesis of IFN- and other cytokines in a variety of cell types [119]. Treatment of
imiquimod may accelerate the onset of NVP-induced skin rash or make the disease worse.
Vitamin D deficiency is associated with higher risk of drug-induced hypersensitivity syndrome
and might be a risk factor in the NVP model [120]. It is much easier to treat rats with Vitamin D
than to deplete it in vivo, so animals were dosed with vitamin D to see if it has any effect.
61
4.2. MATERIALS AND METHODS
Chemicals. NVP was kindly supplied by Boehringer-Ingelheim GmbH. Anti-mouse CD20
antibody 5D2 (mouse IgG2a) at a concentration of 17.2 mg/mL was kindly supplied by
Genetech. PBS (pH 7.4), and FBS were purchased from Invitrogen Canada, Inc. (Burlington,
ON, Canada). Anti-rat CD45RA/B PE antibody (mouse IgG1) and anti-mouse IgG1 PE antibody
(isotype control) were purchased from Cedarlane Laboratories (Burlington, ON, Canada). Anti-
rat CD32 antibody was purchased from BD Pharmingen (Mississauga, ON, Canada). Rat IgE
ELISA kit was purchased from GenWay (Hayward, CA, USA). Microvette
ethylenediaminetetraacetic acid-coated (EDTA-coated) tubes for blood samples were purchased
from Sarstedt (Montreal, QC, Canada). Ammonium chloride (NH4Cl), potassium bicarbonate
(KHCO3), ethylenediaminetetraacetic acid (EDTA), dextran, RA, LPS, and 1-methyl-tryptophan
were purchased from Sigma-Aldrich (Oakville, ON, Canada). L-buthionine - (S, R) -
sulfoximine and imiquimod were purchased from Toronto Research Chemicals Inc. (North York,
ON, Canada). Vitamin D3 was purchased from BioShop Canada Inc. (Burlington, ON, Canada).
Rat cytokine/chemokine Luminex bead immunoassay kit, LINCOplex, 24 Plex, was purchased
from Millipore (Billerica, MA). Glutathione assay kit was purchased from BioVision (Nountrain
View, CA, USA).
Animal care. Female BN rats (150–175 g) were obtained from Charles River Canada (Montreal,
QC, Canada) and housed in pairs in standard cages with free access to water and Agribrands
powdered lab chow diet (Cargill, Inc., Minneapolis, MN). The animal room was maintained at
22 °C with a 12:12 h light/dark cycle. After one week of acclimatization, the rats were either
continued on the same diet (control) or switched to a diet mixed with NVP (treatment group).
The amount of NVP mixed with the diet was calculated based on the body weight of the rats and
their daily intake of food. The treatment period was 21 days with a daily dose of approximately
150 mg/kg body weight unless otherwise specified. For B cell depletion experiments, the anti-
mouse CD20 antibody was injected into the tail vein with various dosing schedules. Vehicle
control animals received PBS injections. All animals were monitored for the development of red
ears, skin rash, food intake, and body weight. At the termination of the experiment, rats were
killed by CO2 asphyxiation. All of the animal studies were conducted in accordance with the
Guidelines of the Canadian Council on Animal Care and approved by University of Toronto’s
animal care committee.
62
Cotreatment of NVP and anti-CD20 antibody. 1. Testing the efficacy of anti-mouse CD20
antibody in rats. First of all, the efficacy of this anti-mouse antibody in rats was tested. Three
female BN rats received 3 different doses of anti-mouse CD20 antibody by i.v. injection (1 mg, 2
mg or 3 mg per rat). To determine the B cell levels in various tissues, anti-rat CD45RA/B was
used for flow cytometry due to the lack of an anti-rat CD20 antibody.
2. Depleting B cells with various dosing regimens of anti-mouse CD20 antibodies. Five
different dosing regimens with various injection schedules were tested to see if any of them
would change the incidence or severity of NVP-induced skin rash. Twenty eight female BN rats
were divided into 7 groups, and their corresponding dosing schedules are shown in Table 4. All
groups except the control were treated with NVP from Day 0 to 22 at a dose of 150 mg/kg/day.
Each injection of anti-CD20 antibody was 1 mg/rat.
3. Effect of B cell depletion on NVP-induced skin rash in splenectomized rats. The antibody
did not deplete B cells in the spleen and so another group of animals was splenectomized to
remove this B cell refuge. The rats were anesthetised with isoflurane prior to surgery. The hair
was removed from the surgical site and the skin washed with iodine and alcohol. A small
incision (2-3 mm long) was made at the lower left abdominal, the spleen was then identified and
removed after ligating the vessels. The abdominal musculature was closed with 3-0 interrupted
Vicryl sutures and the skin closed with 3-0 Vicryl interrupted sutures. The surgery was
performed using sterile technique. After the surgery, animals were heated to keep them warm
and monitored for 30 minutes before moving them back to normal cages. In the splenectomy
experiments, all animals receiving surgery were closely monitored for their well being and
allowed to recover before they were started on another treatment.
To test the effect of B cell depletion on NVP-induced skin rash in splenectomized rats, three
female BN rats received a splenectomy and were allowed to recover for 2 weeks. They were then
treated with NVP mixed in mash diet at a dose of 150 mg/kg/day. Meanwhile, another 3 healthy
rats received the same NVP diet and served as a control. On day 5, the splenectomized rats
started to develop pink ears, which were not very obvious until day 6 or 7. The control group had
developed red ears by the end of day 7. It seems that splenectomy had little effect on the onset
(not significant) and no effect on severity of NVP-induced skin rash. Therefore, a controlled
study was performed to see if depleting B cells would make a difference. This study involved 5
63
groups of rats with 2 in each group. All splenectomised animals were allowed to recover for 1
month before receiving any treatments. The experimental design was outlined in Table 4.
Preparation of single-cell suspension from peripheral blood, ALNs, and spleen. Blood was
drawn from the tail vein and mixed with an equal volume of saline with 3% of dextran and left at
room temperature for 20 minutes. The top straw-colored layer was obtained and mixed with 1
mL of red cell lysis buffer (155 mM NH4Cl/10 mM KHCO3/0.1 mM EDTA) and left at room
temperature for 10 minutes. The cell suspensions were then centrifuged at 1800 rpm for 10
minutes at 4°C. The cell pellet was obtained and resuspended in staining buffer (PBS/3% of
FBS) and proceeded to flow cytometry analysis. ALNs were excised and put into staining
buffer. ALN cells were teased out of the nodal capsule by using of the butt end of a sterile 3-mL
syringe plunger and filtered twice through a 40 µm nylon mesh cell strainer (BD falcon, BD
Biosciences). The cell viability and concentration were assessed in 0.4% trypan blue. The
splenic cells were prepared in the same way as for the ALNs except that an additional step of red
cell lysis was required.
Flow cytometry. Single-cell suspensions were surface-labelled, and single immunofluorescence
analysis was conducted. In brief, cells were resuspended at a density of 2 x 107 cells/mL in
staining buffer, and 50 µL of these cell suspensions were aliquoted to wells in a 96-well plate.
These cells were first incubated with anti-rat CD32 antibody for 10 minutes at room temperature
to reduce nonspecific binding. Then, monoclonal antibodies or suitable isotype controls were
aliquoted to the appropriate wells and incubated at room temperature for 20 minutes. The cells
were washed twice and finally resuspended in 400 µL of the same buffer. Samples were
analyzed immediately with a FACS-Calibur (BD Biosciences) with use of CellQuest software
(BD Biosciences). FlowJo (Tree Star, Inc., Ashland, OR) was used to analyze the stained
population.
Plasma IgE levels. The blood was drawn from the tail vein and IgE levels were determined
using the protocol included in the ELISA kit.
Cotreatment of BSO and NVP in naïve animals. Sixteen female BN rats were divided into 4
groups with 4 animals in each group and treated with the experimental plan described in Table 6.
For metabolic studies, animals in the NVP and BSO-NVP groups were put in metabolic cages
one day a week for urine collection, i.e. on days 7, 14, and 21. To avoid the contamination of
64
urinary samples with food and ensure adequate uptake of the interventional substances, when
they were in metabolic cages, all rats received NVP by gavage and had free access to regular
food and tap water. The gavaging dose was pre-optimized based on food and water intake: NVP
and BSO were suspended in corn oil at a dose of 75 mg/kg/day and 250 mg/kg/day, respectively.
Both drugs were gavaged twice a day with a 6 hour interval. Control and BSO rats received the
same amount of corn oil as vehicle control on that day. Plasma samples were taken right before
the first dose, and urine samples were collected at the end of the 24 hours. All rats were
monitored for red ears and skin rash on a daily basis and euthanized on day 22. Concentrations
of NVP and its major metabolites (12-OH-NVP, 3-OH-NVP, 4-COOH-NVP, 2-OH-NVP) were
determined using liquid chromatography-mass spectrometry (LC/MS) using the previously
reported method by our group [59]. Total glutathione levels in the liver were determined
following instructions of the glutathione assay kit.
Cotreatment of BSO and NVP in sensitized animals. Eight female BN rats were treated with
NVP at a dose of 150 mg/kg/day in food for 21 days followed by 4 weeks of recovery period,
then rechallenged with NVP at the same dose for 7 days. Four of these rats received BSO at a
dose of 500 mg/kg/day (with 2% of glucose) starting 1 week prior to rechallenge until the end of
this study. All animals were monitored for red ears and skin rash. The levels of NVP and its
metabolites in the plasma and urine were quantified using LC/MS using the previously reported
method by our group [59].
Cotreatment of RA and constant dose of NVP. Female BN rats were co-treated with NVP
(150 mg/kg/day) in food and RA at two different doses (5 mg/kg/day or 20 mg/kg/day). RA was
suspended in corn oil and administered by oral gavage. Each of the three co-treatment groups
had 4 animals and the treatment lasted 21 days.
Cotreatment of RA and an escalating dose of NVP. Twelve female BN rats were divided into
3 groups: 1 control, 1 NVP and 1 cotreatment group. The NVP group received a constant dose of
NVP at 150 mg/kg/day while the cotreatment group received 20 mg/kg/day of RA and NVP with
a starting dose of 150 mg/kg/day in food. The plasma levels of NVP and its major metabolites
were determined by LC/MS, and the dose of NVP in the cotreatment group was escalated to
approximately 175 mg/kg/day over the 21-day treatment course. Serum cytokine levels were
65
determined by using a Luminex multiplex assay, which can simultaneously measure 24
cytokines/chemokines.
Cotreatment of NVP with 1-methyl-tryptophan, LPS, imiquimod, or vitamin D. For each of
the four test substances, one cotreatment group with 4 female BN rats and one NVP group with 4
animals (treated with 150 mg/kg/day NVP in food) were used. The dose and administration
routes are outlined below: 1-methyl-tryptophan at a dose of 500 mg/kg/day in water by oral
gavage; LPS in water at a dose of 5 mg/kg/week by i.p.; imiquimod in water at a dose of 30
mg/kg twice per week by oral gavage; vitamin D at a dose of 40 international unit(IU)/rat/day in
oil by oral gavage. All animals were monitored for red ears and skin rash.
66
Table 4. Design of NVP and anti-mouse CD20 antibody† cotreatment experiments in naïve
BN rats.
Group Day -7 Day 0‡ Day 4 Day 7
Control (N=4) -- -- -- --
NVP (N=4) -- -- -- --
Anti-CD20 D-7 (N=4) Injection 1 -- -- --
Anti-CD20 D-7 &0 (N=4) Injection 1 Injection 2 -- --
Anti-CD20 D-7 & 7 (N=4) Injection1 -- -- Injection 2
Anti-CD20 D4 (N=4) -- -- Injection 1 --
Anti-CD20 D4 & 7 (N=2) -- -- Injection 1 Injection 2
†Anti-mouse CD20 was injected i.v. at a dose of 1 mg/rat.
‡Starting on Day 0, all animals except
the control group were started on a NVP mash diet at a dose of 150 m/kg/day.
67
Table 5. Design of NVP and anti-mouse CD20 antibody cotreatment experiments in
splenectomized female BN rats.
Group (n=2) Splenectomy NVP Anti-CD20 injection
Control -- -- PBS on days 1&4
NVP -- 150 mg/kg/day PBS on days 1&4
Splen-NVP Yes 150 mg/kg/day PBS on days 1&4
Splen-CD20-NVP Yes 150 mg/kg/day 1 mg/rat on days 1&4
CD20-NVP -- 150 mg/kg/day 1 mg/rat on days 1&4
68
Table 6. Design of BSO and NVP cotreatment experiments in female BN rats.
Group BSO† NVP
Control Tap water with 2% glucose Regular food
BSO Tap water with 20 mM of BSO & 2% glucose Regular food
NVP Tap water with 2% glucose 150mg/kg/day in food
BSO-NVP Tap water with 20 mM of BSO & 2% glucose 150mg/kg/day in food
† Treatment with 20 mM of BSO in 2% glucose resulted in a dose of approximately 500
mg/kg(BW)/day
69
4.3. RESULTS
Anti-mouse CD20 antibody effectively depleted B cells in peripheral blood. As shown in
Figure 14, a single injection of anti-CD20 antibody dramatically decreased B cell blood levels to
less than 5% in 3 days. The B cell level started to gradually recover from day 7, and the time
required to fully recover appeared to depend on the dose. The above results suggest that: 1) in
peripheral blood, B cells were effectively depleted by this anti-mouse CD20 antibody; 2) higher
doses (2 mg/rat and 3 mg /rat) did not lead to a better depletion than the lowest dose did; in fact,
they appeared less effective, and the level of B cells returned to normal levels faster.
B cell depletion had no significant effect on NVP-induced skin rash in normal animals.
During the 22 days of NVP treatment, only group “Anti-CD20 D4 & 7” rats that received 2
injections of anti-CD20 antibody on days 4 and 7 following NVP treatment seemed to have an
altered onset of the disease: they started to develop red ears on day 10, which did not become
obvious until day 12, whereas other groups developed red ears on day 7. The skin rash of all rats
that received 2 antibody injections seemed less severe than the others.
Blood was taken weekly and CD45+ cell levels were determined using flow cytometry. Since
there was no change observed in most groups, data for group “Anti-CD20 D4 & 7” (which
seemed to have an effect) and group “Anti-CD20 D-7 & 0” were selected (both groups received
two injections at 1 week intervals) as a representative of this study (Figure 15). B cell levels in
group “Anti-CD20 D-7 & 0” that received the antibody injections on days -7 and 0 gradually
increased from 10% (day 0) to 20% (day 20) while the levels in group “Anti-CD20 D4 & 7”
remained less than 10% during the entire examined period.
B cell depletion in other tissues. By day 22, all rats treated with NVP developed a skin rash and
were euthanized. At the end of the treatment course, the % B cells was ~20% in the peripheral
blood for group “anti-CD20 D-7 & 0” and ~3% for group “Anti -CD20 D4 & 7”, both
significantly less than that of the NVP and control groups (Figure 16). No significant difference
in B cell blood levels was observed between the control and NVP groups. There are about 10%
more B cells in the ALNs of the NVP group (60%) compared to that of control group (50%), and
the levels were significantly less in “anti-CD20 D-7 & 0” group (40%). Although N=2, the
“Anti-CD20 D4 & 7” group seemed to have a reduced % B cells as well (29%). On the other
70
hand, there was no difference in splenic B cell levels between the NVP group and the antibody-
treated groups.
Effect of B cell depletion on spleen weights and plasma IgE production. The spleen sizes of
the antibody-treated animals were similar to that of the control animals. Animals in the NVP-
treated group had enlarged spleens compared to all other groups (Figure 17). A previous study
reported a spike in IgE observed in NVP-treated animals on Day 7, which returned back to
baseline by day 14 and remained the same throughout the rest of the NVP treatment [61]. The
same trend was observed in both the NVP-treated and “anti-CD20 D4 & 7” group (Figure 18).
Interestingly, the IgE level started to climb on day 4 following NVP treatment in the “anti-CD20
D-7 and 0” group and peaked on day 7 before returned to baseline.
Depleting B cells in splenectomized rats had no effect on the NVP-induced skin rash. The
results of this experiment were the same as the previous one with normal rats: There was a
significant decrease in B cell levels in the peripheral blood and ALNs (data not shown).
However, there was no change in the onset of symptoms, incidence or severity of this NVP-
induced IDR.
71
B cell percentage in peripheral blood
0 3 7 14 18 25 28 37
0
20
40
60
1mg/rat
2mg/rat
3mg/rat
Control N=4
Treatment time (days)
% B
cells
Figure 14. Percent of cells stained with CD45RA/B in peripheral blood following anti-
mouse CD20 antibody injections.
Three female BN rats each received a single dose of anti-mouse CD20 antibody on Day 0 (1 mg,
2 mg, and 3 mg, respectively). Four control rats received PBS injections. The control data
represent the average of 4 animals ± SE and each of the other three lines represents one animal.
72
B cell levels in peripheral blood
0 5 10 15 20 250
20
40
60
Control
NVP
anti-CD20 (D-7 & 0)
anti-CD20 (D4 & 7)
NVP Treatment time (Days)
% B
cells
Figure 15. Percent of cells stained with CD45RA/B in peripheral blood following anti-
mouse CD20 antibody injections and NVP treatment.
Control groups (N=4) received regular food while all other groups received a NVP diet at a dose
of 150 mg/kg/day starting on Day 0. Group “anti-CD20 D-7 & 0” (N=4) received anti-CD20
antibody injections on days -7 and 0; group “anti-CD20 D4 & 7” (N=2) received the injections
on days 4 and 7. Each injection was 1 mg/rat. Data represent the mean ± STD for group “Anti-
CD20 D4 & 7” and the mean ± SE for all other groups.
73
B cell level in various tissues on D22
0
20
40
60
80
Control
NVP
anti-CD20 (D-7 & 0)
anti-CD20 (D4 & 7)
Spleen AuricularLymph Nodes
Blood
*
*% B
cells
Figure 16. B cell levels in the spleen, ALN, and peripheral blood on D22 of NVP treatment.
The control group received regular food while all other groups received a NVP diet at a dose of
150 mg/kg/day starting at day 0. Group “anti-CD20 D-7 & 0” received anti-CD20 antibody
injections on days -7 and 0; group “anti-CD20 D4 & 7” received the same injections on days 4
and 7. Each injection was 1 mg/rat. The data represent the average of 2 animals ± STD for
group “anti-CD20 D4 & 7” and mean of 4 animals ± SE for all other groups. Statistical
significance between cotreatment groups and NVP alone group was determined using the Mann
Whitney test; values of p≤0.05 were considered statistically significant.
74
0.0
0.2
0.4
0.6
0.8
ControlN=4
NVPN=4
Anti-CD20(D-7 & 0)
N=4
Anti-CD20(D4 & 7)
N=2
Spleen Weight
Sple
en w
eig
ht (g
)
Figure 17. Spleen weight for selected groups on Day 22 of NVP treatment.
The control group received regular food while all other groups received a NVP diet at a dose of
150 mg/kg/day. Group “anti-CD20 D-7 & 0” received anti-CD20 antibody injections on days -7
and 0; group “anti-CD20 D4 & 7)” received the same injections on days 4 and 7. Each injection
was 1 mg/rat. The data represent the mean ± STD for group “anti-CD20 D4 & 7” and mean ±
SE for all other groups.
75
Plasma IgE levels
0
500
1000
1500
D-7
D0
D7
D14
D21
ControlN=4
NVPN=4
Anti-CD20(D-7 & 0)
N=4
Anti-CD20(D4 & 7)
N=2
**
* P
lasm
a I
gE
(ng/m
L)
Figure 18. The effect of NVP treatment and anti-mouse CD20 antibody on plasma IgE
levels.
The control group received regular food while all other groups received a NVP diet at a dose of
150 mg/kg/day. Group “anti-CD20 D-7 & 0” received anti-CD20 antibody injections on days -7
and 0; group “anti-CD20 D 4 & 7” received injections on days 4 and 7. Each injection was 1
mg/rat. The data represent the mean ± STD for group “anti-CD20 D4 & 7” and mean ± SE for
all other groups. Statistical significance in IgE levels between baseline and different time points
was determined using the Mann Whitney test; values of p≤0.05 were considered statistically
significant.
76
BSO decreased the incidence of NVP-induced skin rash in naïve animals but not in
sensitized animals. In the primary treatment experiments, 2 (out of 4) rats in the BSO-NVP
group developed red ears on day 18, and the other 2 had no symptoms until the end of study (day
21). All animals in the NVP group developed red ears on day 7 and skin rash by day 21. For
rechallenge studies, pre-treatment of BSO for 7 days did not change the time to onset or
incidence of NVP-induced skin rash. All animals rechallenged with NVP developed red ears on
day 1 and skin rash by day 7.
Effect of BSO on NVP metabolism on primary treatment. Figure 19 shows that the plasma
levels of both 12-OH-NVP and the parent drug were significantly lower in the BSO/NVP
cotreatment group compared to that of the NVP group. No significant difference was observed
for 2-OH-NVP and 4-COOH-NVP. 3-OH-NVP was not detectable in either group. It seems
that the protective role of BSO on NVP-induced skin rash was a result of decreased NVP and/or
12-OH-NVP plasma levels; however, how BSO affected NVP metabolism remains unknown.
The urinary excretion of NVP was also lower in the cotreatment group than that of the NVP
group, but there was no difference in any other major metabolites (Figure 20).
Effect of BSO on liver weight and glutathione levels on primary treatment. A decrease in
liver weight and glutathione level was also observed. The increase in liver weight in this study
can be explained by the fact that NVP is a P450 inducer. Compared to the NVP group, there is
less of an increase in liver weight in the BSO-NVP group, which might be a result of glutathione
depletion. The glutathione level in the cotreatment group is lower than that of the NVP and
control groups, but higher than that of the BSO group.
77
Figure 19. Plasma levels of NVP and its metabolites in BSO-NVP co-treated or NVP-
treated female BN rats.
NVP was given at 150 mg/kg/day in food and BSO was given at 20 mM/day in tap water with
2% glucose. A) NVP, B) 12-OH-NVP, C) 2-OH-NVP, D) 3-OH-NVP, E) 4-COOH-NVP.
The data represent the mean concentration SE (N=4 for each group).
NVP plasma level
0 7 14 21
0
20
40
60
80
NVP
BSO-NVP
Day
A
g
/mL
12-OH-NVP plasma level
0 7 14 21
0
20
40
60
NVP
BSO-NVP
B
Day
g
/mL
2-OH-NVP plasma level
0 7 14 210
1
2
3
4
5
NVP
BSO-NVP
C
Day
g
/mL
3-OH-NVP plasma level
0 7 14 210.0
0.2
0.4
0.6
0.8
1.0
NVP
BSO-NVP
D
Day
g
/mL
4-COOH-NVP plasma level
0 7 14 210
1
2
3
NVP
BSO-NVP
E
Day
g
/mL
78
Figure 20. 24 hours urinary excretion of NVP and its metabolite in BSO-NVP co-treated
and NVP-treated female BN rats.
NVP was given at 150 mg/kg/day in food and BSO was given at 20 mM/day in tap water with
2% glucose. A) NVP, B) 12-OH-NVP, C) 2-OH-NVP, D) 3-OH-NVP, E) 4-COOH-NVP. The
data represent the mean SE (N=4 for each group).
Urinary excretion of NVP in 24 hrs
0 7 14 21
0
500
1000
1500
NVP
BSO-NVP
A
Day
g
/24 h
rsUrinary excretion of 12-OH-NVP in 24hrs
0 7 14 21
0
500
1000
1500
2000
2500
NVP
BSO-NVP
B
Day
g
/24 h
rs
Urinary excretion of 2-OH-NVP in 24hrs
0 7 14 21
0
1000
2000
3000
4000
NVP
BSO-NVP
C
Day
g
/24 h
rs
Urinary excretion of 3-OH-NVP in 24hrs
0 7 14 21
0
500
1000
1500
2000
NVP
BSO-NVP
D
Day
g
/24 h
rs
Urinary excretion of 4-COOH-NVP in 24hrs
0 7 14 21
0
1000
2000
3000
4000
NVP
BSO-NVP
E
Day
g
/24 h
rs
79
Liver weight
0
5
10
15
BSO NVP BSO-NVPControl
*
liver
weig
ht (g
)
Total glutathione level
0.0
0.2
0.4
0.6
0.8
1.0
BSO BSO-NVPNVPControl
*
mg/g
wet liv
er
tissue
Figure 21. Liver weights and glutathione levels in the liver of BSO-NVP co-treated or
NVP-treated female BN rats.
NVP was given at 150 mg/kg/day in food and BSO was given at 20 mM/day in tap water with
2% glucose. Tissues were collected at the end of the 21 day treatment. The data represent the
mean SE (N=4 for each group). Statistical significance between the cotreatment group and
NVP group was determined using the Mann Whitney test; values of p≤0.05 were considered
statistically significant.
80
RA decreased the incidence of NVP-induced skin rash but also decreased the plasma
concentrations of NVP and 12-OH-NVP. The incidence of skin rash in RA-NVP co-treated
animals was lower than that of NVP-treated group in a dose dependent manner (Figure 22.). All
animals in NVP-treated group developed red ears in 7~9 days and skin rash in 2 weeks. Animals
in low dose RA-NVP group had no red ears, but 2 out of 4 rats had redness on some part of the
skin. High dose RA-treated animals experienced no symptoms at all. The protective effect is
dramatic; however, it seems to be a result of the significantly decreased plasma levels of both the
parent drug and the 12-OH metabolite (Figure 23.). There is literature evidence that RA can
induce CYP3A4 [121], which may also be responsible for metabolizing NVP and 12-OH-NVP.
The induction of P450 would accelerate the metabolism of both the parent drug and 12-OH-NVP
and lead to lower plasma levels of both substances.
However, even the compromised plasma levels seemed high enough to induce a skin rash and
therefore this protection may be partially immune-mediated. Two simple ways to test this
hypothesis would be by using a P450 inhibitor (for example, aminobenzotriazole) or
manipulating the drug plasma levels in the cotreatment group by using a higher dose of NVP.
The former involves the co-administration of three substances and can lead to complicated
results. Therefore, I took the second path.
RA is protective in animals with comparable drug plasma levels with that of the NVP-
treated animals. In this experiment, animals in the cotreatment group received an escalating
dose of NVP to compensate the reduction of drug plasma levels caused by the induction of P450
by RA. Even though the drug plasma levels were similar in both groups, the RA-NVP
cotreatment group had a lower incidence (75%) and the onset of the disease in RA-NVP group
was delayed by a minimum of 5 days. The severity of the skin rash and ear redness were also
much milder in all rats that received RA. The Luminex multiplex assay results showed that RA
prevented the increase in many of the pro-inflammatory cytokines, and this suggests that it may
have a regulatory role in NVP-induced skin rash.
Cotreatment with 1-methyl-tryptophan, LPS, imiquimod, and vitamin D. None of these four
substances had any effect on the incidence or severity of NVP-induced skin rash. There was also
no change in the onset of symptoms compared to that of NVP-treated group.
81
Figure 22. Effect of RA on the incidence of NVP-induced skin rash.
Female BN rats were co-treated with NVP (150 mg/kg/day in food) and RA at two different
doses with 4 animals in each group for 21 days. A low dose of RA refers to 5 mg/kg/day and a
high does refers to 20 mg/kg/day in oil by oral gavage. NVP group received the standard dose of
NVP and corn oil by oral gavage.
82
NVP plasma level
0 5 10 15 20 250
20
40
60
80NVP
RA(low)-NVP
RA(high)-NVP
*
*
**
*
Time of Treatment (Day)
l/
mL
12-OH plasma level
0 5 10 15 20 25
0
10
20
30
40NVP
RA(low)-NVP
RA(high)-NVP
*
* **
*
Time of Treatment (Day)
l/
mL
2-OH plasma level
0 5 10 15 20 25
0
1
2
3NVP
RA(low)-NVP
RA(high)-NVP
Time of Treatment (Day)
l/
mL
Figure 23. Effect of RA on plasma levels of NVP and its metabolites.
Female BN rats were co-treated with NVP (150 mg/kg/day in food) and RA at two different
doses with 4 animals in each group for 21 days. A low dose of RA refers to 5 mg/kg/day and a
high does refers to 20 mg/kg/day in oil by oral gavage. Drug plasma concentrations were
determined by LC-MS. The data represent the mean of 4 animals ± SE. Statistical significance
between cotreatment groups and NVP alone group was determined using the Mann Whitney test;
values of p≤0.05 were considered statistically significant.
83
NVP
0 5 10 15 20 25
0
10
20
30
40
50NVP
RA-NVP
Time of Treatment (Day)
l/
mL
12-OH-NVP
0 5 10 15 20 25
0
10
20
30
40NVP
RA-NVP
*
Time of Treatment (Day)
l/
mL
Figure 24. NVP and 12-OH-NVP plasma concentrations in animals that received RA (20
mg/k/day in oil, gavage) and an escalating dose of NVP (started from 15 mg/kg/day and
escalated to 175 mg/kg/day).
Drug plasma concentrations were determined by LC/MS. The data represent the mean of 4
animals ± SE. Statistical significance between RA-NVP group and NVP alone group was
determined using the Mann Whitney test; values of p≤0.05 were considered statistically
significant.
84
IFN-
0 5 10 15 20 250
50
100
150
200
NVP
RANVP
Treatment time (day)
pg/m
LIL-13
0 5 10 15 20 250
10
20
30
40
NVP
RANVP
Treatment time (day)
pg/m
L
IL-18
0 5 10 15 20 250
50
100
150
NVP
RA-NVP
Treatment time (day)
pg/m
L
Treatment time (day)
Leptin
0 5 10 15 20 250
500
1000
1500
2000
2500NVP
RANVP
Treatment time (day)
pg/m
L
IL-2
0 5 10 15 20 250
20
40
60
80
100NVP
RANVP
Treatment time (day)
pg/m
L
MCP-1
0 5 10 15 20 250
200
400
600
800
1000
NVP
RA-NVP
Treatment time (day)
pg/m
L
Treatment time (day)
RANTES
0 5 10 15 20 250
2000
4000
6000NVP
RA-NVP
Treatment time (day)
pg/m
L
Treatment time (day)
Figure 25. Plasma concentrations of cytokines/chemokines during a 21-days treatment
course with NVP or RA-NVP determined by a Luminex assay.
Female BN animals in the NVP group received a dose of NVP at 150 mg/kg/day; animals in the
cotreatment group received RA (20 mg/kg/day in oil by gavage) and an escalating dose of NVP
(started from 150 mg/kg/day and escalated to 175 mg/kg/day). The data represent the mean of 4
animals ± SE.
pg
/mL
p
g/m
L
pg
/mL
85
4.4. DISCUSSION
B cells have multiple functions in many autoimmune diseases. In addition to acting as precursors
of antibody-producing plasma cells, they also negatively regulate autoimmunity through B10 cell
(B cells that produce IL-10) function and serve as critical adjuvants for CD4+ T-cell activation
[122, 123]. The increase in both the total number of B cells and MHC-II-expressing B cells
suggested their potential involvement in presenting antigens to T helper cells; the presence of
IgE following NVP treatment also suggested a role in antibody production [67]. We employed
an anti-mouse CD20 antibody to deplete B cells in vivo to determine if it could prevent the rash.
It is also possible that B cells are negative regulators or involved in downstream events of this
immune response. If that is the case, the depletion would not prevent the animals from getting a
rash and could make it worse.
The present study showed that this anti-mouse CD20 antibody significantly decreased B cell
levels in the peripheral blood and in ALNs; however, it was not effective in the spleen. A delay
in the onset of red ears was observed in the group that received 2 antibody injections on days 4
and 7 following NVP treatment, and that also corresponds to a lower level of B cells in the blood
and ALNs compared to other groups. These results suggest that B cells may play a role in
initiating or mediating the rash. The finding that the spike of IgE on day 7 in the NVP group
declined to a baseline level by day 14 is the same as what was previously reported [67].
However, the IgE started to increase on day 0 in group “Anti-CD20 D-7 & 0” which received
antibody injections on days -7 and 0; this may be the result of the injection of anti-mouse
antibody because these animals had not been treated with NVP on day 0. The peak on day 7 for
group “Anti-CD20 D-7 & 0” and “Anti-CD20 D4 & 7” is hard to interpret due to the lack of an
isotype antibody serving as a control. We do not know how long and to what level the increase
in IgE caused by anti-CD20 injections are. However, the spike on day 7 may be a combination
of anti-CD20 injection and NVP treatments. In any case, the goal to test IgE levels was to
determine if functions of plasma cells, which are derived from B cells, were reduced by anti-
CD20 injections; however, no difference between NVP group and antibody-injected groups was
observed.
The spleen is the largest lymphoid organ in the body and contains many different immune cells.
The reason that splenic B cell levels were not effectively depleted may be due to the anti-mouse
86
nature of the antibody. The antibody may be cleared by phagocytic cells such as macrophages.
As a result, the formation of plasma cells, which occurs in the spleen and lymph nodes, was not
affected. This may explain why we did not observe a significant delay in the onset of red ears. If
we could somehow effectively deplete B cells in the spleen it might result in a longer delay of
onset or change in incidence. Therefore, we decided to perform a study in splenectomized
animals.
The removal of spleen can simply avoid the resistance of splenic B cells to antibody clearance,
but it may also lead to other effects on the immune response. The spleen clears dead red blood
cells and many other old cells. Splenectomy may lead to an accumulation of dead cells, which
could potentially release “danger signals” and in turn accelerate or increase the possibility of
developing IDRs on exposure to NVP. Therefore the effect of splenectomy alone on NVP-
induced IDRs was determined first. The pilot study showed that following NVP treatment, the
ears of splenectomized rats started to turn red on day 5, but it was not obvious until day 7. Such
an effect might be due to the short recovery time (2 weeks), but the severity and all other
symptoms were similar to that of the healthy rats that received NVP. To make sure the B cells
were depleted prior to the development of IDRs, antibody injections were administered on days 1
and 4, although the previous study showed it might have a better effect if given on days 4 and 7.
However, the results showed again that depleting B cells in splenectomized rats had no effect on
either the onset or severity of NVP-induced skin rash.
In summary, depleting B cells using this anti-mouse CD20 antibody did not change the incidence
of NVP-induced skin rash in female BN rats. The depletion was not complete, which might be
due to the fact that it is an anti-mouse antibody and could be cleared in the spleen. However,
even in splenectomized rats, it did not have a significant effect. In contrast, a partial depletion of
CD4+ cells can significantly delay or prevent NVP-induced skin rash [61]. Although the
evidence is not conclusive, it is likely that B cells are not essential for the induction of a skin rash
in our model.
The second modulator tested was BSO. It can irreversibly inhibit gamma-glutamylcysteine
synthetase, and it has been used to deplete glutathione in rats and mice [124]. However,
glutathione depletion inhibits the antigen presenting process and shifts a Th1 immune response
pattern to Th2 [125]; therefore, it might change the incidence or severity of skin rash induced by
87
NVP treatment in female BN rats. On the other hand, glutathione protects cells against oxidative
stress, and its depletion may lead to liver toxicity.
The cotreatment of BSO changed the incidence of NVP-induced IDRs in female BN rats;
however, this is likely due to decreased plasma levels of parent drug and metabolites. One study
suggested that BSO can simultaneously induce cytochrome P450 2E1 and alcohol
dehydrogenase in mouse livers [126]. In our case, it might interact with P450 and result in less
metabolism and/or more clearance of NVP which would lead to a decreased covalent binding of
NVP and, therefore, a delayed onset of the disease. There was no difference in urinary excretion
of NVP metabolites that were tested between the BSO-NVP and NVP groups, but the parent
drug excretion was significantly less in the latter group. It is not likely that BSO induced P450
in this case because we did not observe higher plasma levels or urinary excretion of the
metabolites. On the other hand, BSO may have accelerated the clearance of NVP given that the
metabolite excretion levels were similar but that of parent drug was significantly reduced. The
mechanisms by which BSO is protective in NVP-induced skin rash is complicated, and it would
be quite difficult to determine the exact mechanism of its effects.
RA came to our attention originally because of its potential to alter the balance between Th17
cells and Treg cells, and we speculated that it might have an effect on the NVP-induced IDR,
which is mediated by CD4 T cells. However, recent reports showed that RA may have both
positive and negative regulatory roles. With respect to Th17 cells, it may suppress them at
higher concentrations (in vitro) but induce Th17 cells expressing gut-homing receptors at
physiological levels (in vivo)[115, 127, 128]. In the present study, RA seems to inhibit the
production of many pro-inflammatory cytokines. However, at the same time, it might have also
induced P450, which led to reduced drug plasma levels. When the dose of NVP was increased
so that the blood levels of NVP were similar RA appeared to be protective. However, because of
the complex effects of RA on the immune response which would be very difficult to sort out, we
decided not to pursue this further.
The present study attempted to manipulate the NVP-induced skin rash from different angles such
as suppressing the immune system, breaking immune tolerance, and activating the innate
immune system, etc. While most of the modulators do not have any significant effect, the very
few that worked seemed to involve different mechanisms than we expected. Based on the BSO
88
and RA experiments in which both reagents seemed to have an impact on the metabolism of
NVP and resulted in lower drug levels in the plasma, one can never conclude the effect of any
modulator on drug-induced adverse reactions without testing its effect on metabolism. Although
it was difficult to find something that has a “clean” effect on the NVP-induced IDR, it is
important to continue to explore such risk factors. Only with an valid animal model will we be
able to vigorously test all these hypotheses and eventually determine what is going on in humans.
89
CHAPTER 5
POTENTIAL ACTIVATION OF ANTIGEN PRESENTING
CELLS BY NVP AND/OR ITS METABOLITES
Co-author: Amy Sharma (covalent binding study)
90
5.1. INTRODUCTION
One fundamental question in drug-induced IDRs is whether it is the parent drug or the reactive
metabolite that leads to the reaction. In the case of NVP-induced skin rash, we were able to
show that 12-OH-NVP pathway was involved in the induction of skin rash in female BN rats
[59]. The most recent data further confirmed that this was due to the formation of 12-OH-NVP
sulfate in the epidermis, which covalently binds to proteins and induces an immune response [68,
69].
Although we have demonstrated that 12-OH-NVP sulfate causes the rash, we have not studied
how this may activate the immune system. In order to elicit an immune response, the drug may
form a protein adduct by covalent binding, which is then picked up by an APC and presented to
T cells in the presence of co-stimulatory signals. Alternatively, the drug may also directly bind
and activate APC similar to what we saw with the D-penicillamine-induced autoimmune disease
[78, 80]. A recent paper by Kevin Park’s group showed that SMX and its protein-reactive
metabolite, nitroso SMX, can stimulate dendritic cell (DC) co-stimulatory signaling. In
particular, the response of DCs to the nitroso reactive metabolite is much stronger than that to the
parent drug [46]. A similar study was also reported by Reuter et al. where the co-stimulatory
molecule CD86 was found to be upregulated on PBMC-derived DCs in an in vitro model for
allergic contact dermatitis [129]. In our NVP model, the interaction between reactive metabolite
and the APCs has not been studied. We hypothesized that 12-OH-NVP sulfate can directly
activate APCs, and this may lead to the idiosyncratic skin rash caused by NVP in female BN
rats. To test this hypothesis, APCs from different sources were stimulated by 12-OH-NVP
sulfate, 12-OH-NVP and/or the parent drug using the expression of co-stimulatory molecules as
a measure of APC activation.
91
5.2. MATERIALS AND METHODS
Chemicals. NVP was kindly supplied by Boehringer-Ingelheim Pharmaceuticals Inc.
(Ridgefield, CT). The synthesis of 12-OH-NVP, 12-OH-NVP sulfate, and preparation of NVP
antiserum were described previously [59, 98, 130, 131]. D-penicillamine was purchased from
Richman Chemical Inc. (Lower Gwynedd, PA). RPMI 1640 medium, dulbecco's modification of
Eagle's medium (DMEM) high glucose medium, PBS (pH 7.4), FBS, penicillin and streptomycin
concentrated solution, 2-ME, and normal goat serum were purchased from Invitrogen Canada,
Inc. (Burlington, ON, Canada). Microvette EDTA-coated tubes for blood samples were
purchased from Sarstedt (Montreal, QC, Canada). Isoniazid, RIPA buffer, DMSO, indomethacin,
PMA, inomycin, ammonium chloride (NH4Cl), LPS, potassium bicarbonate (KHCO3), EDTA,
horseradish peroxidise-conjugated goat antirabbit IgG (H + L chains), and dextran were
purchased from Sigma-Aldrich (Oakville, ON, Canada). Anti-rat CD4 allophycocyanin, anti-rat
CD25 PE, anti-rat CD80 PE, anti-rat CD86 FITC, anti-rat MHC II PE, anti-rat CD161 FITC,
anti-rat pan-macrophage PE, anti-rat αβ-TCR peridinin chlorophyll protein complex (PerCP),
anti-rat rat recombinant IL-4, and GM-CSF were purchased from Cedarlane (Mississauga, ON,
Canada). FITC hamster anti-mouse CD40, anti-rat CD32, and anti-mouse CD16/CD32
antibodies were purchased from BD Pharmingen (Mississauga, ON, Canada). Anti-human Fc
binding receptor, anti-human CD40 FITC, anti-human CD80 PE, anti-human MHC II antibody
conjugated with R-Phycoerythrin-Cyanine dye 7 (PE-Cy7), anti-human CD86 conjugated with
allophycocyanin, anti-mouse CD80 PE, and anti-mouse CD86 allophycocyanin antibodies were
purchased from eBioscience (San Diego, CA, USA). SDS and Tween-20 were obtained from
BioShop (Burlington, ON). Stock acrylamide/bis solution, nonfat blotting grade milk powder,
and nitrocellulose membranes were purchased from BioRad (Hercules, CA). Amersham
enhanced chemiluminescence (ECL) Plus Western Blotting Detection System was obtained from
GE Healthcare (Oakville, ON). Protein concentrations were determined using a bicinchoninic
acid (BCA) protein assay kit (Novagen, EMD Biosciences Inc.).
Animal Care. Female BN rats (150-175 g) were purchased from Charles River (Montreal, QC)
and housed in pairs in standard cages with free access to water and Agribrands powered lab
chow diet (Leis Pet Distributing Inc, Wellesley, ON). The animal room was maintained at 22 o C
with a 12:12 hour light:dark cycle. Rats were killed by CO2 asphyxiation. All of the animal
92
studies were conducted in accordance with the guidelines of the Canadian Council on Animal
Care and approved by University of Toronto’s Animal Care Committee.
Studies of Potential Activation of Freshly Isolated Cells from Naïve Rats and PBMC
Monocyte-derived DCs by NVP and Its Metabolites. The first study was to test the response
of cells isolated fresh from PBMCs, ALNs, and the spleen of a naïve female BN animal to NVP
and the metabolites. However, even the positive control, LPS, a strong stimulant of APCs, did
not induce significant changes in the markers that were tested. A possible explanation is that
these tissues have diverse cell populations, and the APC abundance might not be high enough to
detect any changes. However, even when APCs from ALNs were concentrated using magnetic
beads (APCs MACS cell separation kit, Miltenyi Biotec), no upregulation of any markers was
observed, but a significant decrease in MHC II expression was observed (shown in Appendix 1).
The second attempt was to derive DCs from PBMCs isolated fresh from naïve animals using the
method described by Kevin Park’s group [46]. Although this seems to be the “standard”
approach for studies involving human cells, it was not practical for rat studies because the yield
of cells is too low.
General Procedures for Direct Activation of APCs by NVP and Its Metabolites. NVP, 12-
OH-NVP, and 12-OH-NVP sulfate were dissolved in 0.5% DMSO at various concentrations: 1.0,
2.0, 3.9, 7.8, 15.6, 31.3, 62.5, and 125 µg/mL. APCs from different sources were populated at a
concentration of 1 million/mL (unless specified) in complete medium and incubated with drugs
at 37 o C, 5% of CO2 for 24 hours. At the end of the culture period, expression of co-stimulatory
molecules CD40, CD80, CD86, and MHC II on these cells were tested by flow cytometry.
Direct Activation of RAW264.7 Cells by NVP and Its Metabolites: The RAW 264.7 cell line
was purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA) and
maintained according to the manufacturer’s instruction. The day before the APC activation
experiment, the cells were seeded at 0.5 million/mL in a 24-well plate overnight in DMEM high
glucose medium containing 10% FBS and antibiotics, allowing the cells to adhere to the culture
plate. The next morning, non-adherent cells were washed off and fresh culture medium
containing various concentrations of 12-OH-NVP sulfate, 12-OH-NVP, or NVP were added.
After 24 hours of stimulation, the cells were harvested and surface markers were tested by flow
cytometry.
93
Direct Activation of APCs by NVP and Its Metabolites: THP-1 Cells. The THP-1 cell line
were purchased from ATCC (Manassas, VA, USA) and maintained according to the
manufacturer’s instruction. The procedures for stimulating THP-1 cells were the same as
stimulating RAW264.7 cells except that RPMI1640 medium was used.
Direct Activation of APCs by NVP and Its Metabolites: Bone Marrow-Derived Dendritic
Cells (BMDCs). The complete culture medium used to generate BMDCs (referred as the
“complete medium” hereafter) was prepared as follows: RPMI 1640 medium was supplemented
with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-ME, 5
ng/mL recombinant rat GM-CSF, and 5 ng/mL IL-4.
Bone marrow was prepared from the femur bones of naïve female BN rats. The bones were
placed in a 100-mm dish and washed with 70% alcohol for 2 minutes. After removing the
remaining muscle tissue, they were transferred into a new dish with RPMI medium. Both ends
of the bones were cut and the bone marrow was flushed out with a syringe and a 25-gauge needle
using 10 mL of RPMI medium. The cells were passed through two 40 µm cell strainers and
centrifuged at 350 g. To derive DCs from the bone marrow cells, the cell pellet was resuspended
in 10 mL of the complete medium and plated on 100-mm petri dishes at a density of 5 X 105
cells/mL. On day 3, 10 mL of freshly prepared complete medium was added to the culture. On
day 6, the supernatant was removed, and 10 mL of freshly prepared complete medium was
added. On day 8 or 9, the cells were harvested by scraping the plate and resuspended at a
concentration of 5 X 105 cells/mL. The single cell suspension was then seeded at 1 mL per well
in a 24-well tissue culture plate and incubated with various concentrations of NVP, 12-OH-NVP,
or 12-OH-NVP sulfate. The cells incubated with LPS served as positive control whereas the
DMSO-treated cells were used as negative control.
Covalent Binding of NVP and Its Metabolites to BMDCs. To determine if 12-OH-NVP and/or
its sulfate covalently bind to the BMDCs, incubations of these compounds as well as NVP, or
DMSO was incubated with BMDCs. Immature BMDCs were harvested on day 8 and
resuspended at a concentration of 1x106 cells/mL and a total of 10 x10
6 cells were used for each
incubation. The concentration of NVP and its metabolites in the incubations was 62.5 µg/mL,
the incubation time was 24 or 72 hours at 37 o
C in 5% CO2. After incubation, the cells were
lysed using RIPA buffer, and the proteins were pelleted. The protein concentration was
94
determined by a BCA protein assay kit (Novagen, EMD Biosciences Inc., Mississauga, ON), and
the proteins were loaded on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) gel at a concentration of 40 µg/lane for 24 hours samples and 35 µg /lane for 72 hours
samples. SDS-PAGE and western blotting procedures were described previously by Amy
Sharma[69]. Specifically, SDS-PAGE gels were hand-cast (stacking gel, 5% bisacrylamide;
resolving gel, 8% bisacrylamide) and run at ~110 V. Electrophoresis running buffer (BioRad,
Mississauga, ON) consisted of 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3. Transfer to
nitrocellulose membrane (pore size 0.2 µM, BioRad) was performed at 0.13 mA for 90 minutes
at 4 ºC using Protean-3 minigel system (BioRad). Tris-glycine transfer buffer consisted of 25
mM Tris, 192 mM glycine, and 20% methanol at pH 8.5. Membranes were washed twice in
Tris-buffered saline Tween-20 (TBST) wash solution for 5 minutes. Membranes were then
blocked in 5% nonfat milk blocking solution in TBST for 90 minutes at room temperature and
washed with three changes of TBST for 5 minutes each. A 1:500 dilution of the primary anti-
NVP antiserum and 10% normal goat serum in TBST was added to the membranes and
incubated overnight at 4 ºC. After a 20 minute wash on the next day, the membranes were
incubated with a secondary goat anti-rabbit horseradish peroxidase antiserum (1:2000) for 90
minutes. Blots were washed 3 times with TBST and incubated with enhanced
chemiluminescence stain for 5 minutes and imaged for 3 minutes using a FluroChem CCD
imager.
Activation of APCs by D-penicillamine and Isoniazid. BMDCs generated by the procedures
described above or RAW264.7 cells were stimulated with D-penicillamine or isoniazid at various
concentrations for 24 hours: 0.1, 0.4, 1.2, 3.6, 11, 33, 100, and 300 µg/mL, and the expression of
surface markers was determined by flow cytometry. D-penicillamine and isoniazid were shown
to activate RAW264.7 cells by binding the aldehyde groups on RAW264.7 cells, and therefore it
should be possible to use them as positive controls in this study [132, 133].
T Cell Activation Assay.
1. Naïve T Cell Isolation. Autologous T cells were isolated from the spleen and ALNs of female
BN rats. Naïve T cells were isolated by negative selection using a CD4+ T cell isolation kit
followed by a positive selection of CD25+ cells (Miltenyi Biotec, Anburn, CA, USA). The bead
95
isolation procedures were carried out according to the manufacturer’s instructions. The
phenotype of the isolated cells was determined by flow cytometry using anti-rat CD4 APC and
anti-rat CD25 PE antibodies. Isolated naïve T cells were resuspended in RPMI 1640 medium
(supplemented with 10% FBS and antibiotics) at 10-20 X 106 cells/mL, and then an equal
volume of FBS containing 20% DMSO was added to the cell suspension dropwise on ice. The
cells were stored at -80 o
C until use.
2. T Cell Co-culture with BMDCs. Immature DCs were harvested on day 8 of differentiation
from bone marrow cells and resuspended at a concentration of 4 X 105
cells/mL. These cells
were seeded at 500 µL per well in 24-well plates. Naïve T cells were thawed, and the RPMI
1640 medium was added dropwise to make 10 times the initial volume. After washing, cells
were resuspended at 2 X 106
cells/mL and added to BMDCs at 1 mL per well. 10 µL of the stock
solutions of NVP, 12-OH-NVP, or 12-OH-NVP sulfate (12.5 mg/mL dissolved in DMSO) were
diluted with 490 µL of cell culture medium and then added to the T cell/BMDCs mixture
resulting in a final drug concentration of 62.5 µg/mL and a total culture volume of 2 mL. The
mixture was incubated for 7-8 days at 37 o C in a 5% CO2 atmosphere.
3. Carboxyfluorescein Succinimidyl Ester (CFSE) Staining and T Cell Restimulation. After
the co-culture period, the cells were labeled with CFSE and re-stimulated before T cell
proliferation was assessed. Specifically, the stimulated T cell/APC mixtures were harvested and
CFSE staining was carried out according to the manufacturer’s instructions (Invitrogen, Canada).
The cells were then resuspended in 1 mL of medium, and 100 µL was aliquoted to 96-well
plates. Another batch of immature BMDCs prepared in advance were resuspended at 4 X 105
cells/mL and added to the T cell/APC cell mixture at a volume of 50 µL per well. Various
concentrations of NVP, 12-OH-NVP, and 12-OH-NVP sulfate diluted in 50 µL of medium were
added to the culture and incubated for 72 hours at 37 oC in a 5% CO2 atmosphere.
PMA/Inomycin-stimulated cells were used as a positive control. At the end of restimulation
period, the proliferation of T cells was tested using flow cytometry. Specifically, anti-rat αβ-
TCR+ antibody was used to label the T cells, and the proliferation was measured by the reduction
in CFSE staining.
Flow Cytometry. A PBS buffer containing 3% FBS was used as the medium for flow
cytometry. Cells were first incubated with an antibody against the Fc binding receptor for 10
96
minutes at room temperature to reduce nonspecific binding. After that, monoclonal antibodies or
suitable isotype controls were aliquoted to the appropriate wells and incubated at room
temperature for 20 minutes. The cells were washed twice and finally resuspended in 200 µL of
the same buffer. Samples were processed immediately with a FACS Canto II flow cytometer
(BD Biosciences) with CellQuest software. FlowJo (Tree Star, Inc., Ashland, OR) was used to
analyze the samples.
ELISA. Rat ELISA kits for IL-1β, IL-12p40, and TNF-α were purchased from R&D system
(Minneapolis, MN). Experiments were performed following the manufacturer’s instructions.
Stability of 12-OH-NVP Sulfate. The sulfate of 12-OH-NVP was synthesized as described by
Chen et al. [59]. The stability of 12-OH-NVP sulfate was determined by high-performance liquid
chromatography (HPLC) (SHIMADZU LC-10AS). The column was an Ultracarb 100X2 mm, 5
micron, ODS(30), and the mobile phase consisted of 12% acetonitrile in aqueous 2 mM
ammonium acetate/1% acetic acid with a pH of 3.7 and a flow rate of 0.4 mL/minutes. To test
the stability in cell culture, immature BMDCs harvested on day 8 of differentiation were
stimulated with 62.5 µg/mL of 12-OH-NVP sulfate. Aliquots of supernatant were diluted 10X
with the mobile phase and analyzed with HPLC at various time points. A parallel experiment
was also carried out to determine the stability of the sulfate in the culture medium in the absence
of cells. In both studies, 12-OH-NVP was used for a control incubation to represent complete
hydrolysis.
97
5.3. RESULTS
Activation of RAW264.7 Cells by NVP and Its Metabolites. Examples of the flow cytometry
dot plots and a histogram are shown in Figure 26. The dot plots showed that there was an
increased expression of CD40 on the 12-OH-NVP sulfate-treated cells compared to that of the
DMSO vehicle control cells (65.4% versus 15.9%). The histogram also showed that the mean
fluorescence intensity (MFI) was greater for the 12-OH-NVP sulfate-treated cells than for the
DMSO control cells. The dose response curves in Figure 27 showed that both the percentage of
CD40-expressing cells and the expression level per cell (measured by MFI) increased with
increasing drug concentrations. Among the three substances, 12-OH-NVP seemed to better
activate RAW 264.7 cells at the concentrations of 32 and 64 ug/mL compared to NVP and 12-
OH-NVP sulfate.
One problem with RAW 264.7 cells was that the baseline levels of activation markers increased
throughout the experiments (Figure 28). This was independent of the solvent because even in the
absence of DMSO, the cells express higher levels of CD40 at higher passages (results not
shown). There is literature evidence that over-passaged cells may exhibit reduced or altered
functions as a result of “selective pressures and genetic drift”, and therefore they no longer
represent reliable models of their original source material [134].
98
A. B.
C. D.
E.
Figure 26. Expression of CD40 on RAW 264.7 cells after a 24 hours stimulation with 12-
OH-NVP sulfate.
A) unstained cells, B) cells incubated with 0.5% DMSO and stained with CD40 antibody as a
negative control, C) cells incubated with 125 µg/mL 12-OH-NVP sulfate in 0.5% DMSO and
stained with CD40 antibody, D) cells incubated with 2 µg/mL LPS as a positive control, E)
Histogram of CD40 expression: grey (unstained control), blue (DMSO negative control), red
(125 µg/mL 12-OH-NVP sulfate), green (LPS positive control).
99
A.
0
20
40
60
80
**
**
**
**
**
NVP 12-OH-NVP 12-OH-NVP
Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
% C
D40
+C
ells
B.
0
5000
10000
15000
**
***
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
CD
40 M
FI
Figure 27. Expression of CD40 on RAW264.7 cells in response to various concentrations of
NVP, 12-OH-NVP, or its sulfate metabolite expressed in two different ways.
A) percent CD40 positive cells; B) the number of CD40 molecules on cells as measured by MFI.
The data represent the mean ± SE of 4 experiments. Unpaired t test, p < 0.05. The control (solid
lines) represent cells treated with 0.5% DMSO (N=4).
100
Figure 28. Precent CD40 positive cells in “aging” RAW264.7 cells.
The data represent the average of 2 experiments. Cells were treated with 0.5% DMSO for 24
hours.
dmso effect
8 10 12 14 160
10
20
30
40
50
Cell Passage
% C
D40
+C
ells
101
Activation of THP-1 cells by NVP and Its Metabolites. There was an increase in percentage
of cells that express CD86 with increasing concentrations of all three drugs. Surprisingly, the
MFI of MHC II expression decreased when the concentration of all three substances increased.
No significant change was observed for CD40 and CD80 expression (Figure 30).
102
CD80 CD86 CD40 MHC II
Fluorescence Intensity
Figure 29. Representative dot plots and histograms of surface marker staining of THP-1
cells stimulated by various substances.
Un
stai
n
DM
SO
N
VP
(6
2.5
µg
/mL
) L
PS
Count
103
A. B.
0.0
0.5
1.0
1.5
NVP 12-OH-NVP 12-OH-NVP
Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
CD
40
MF
I
0.0
0.5
1.0
1.5
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
MH
C I
I M
FI
C. D.
0.0
0.5
1.0
1.5
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
% o
f C
D8
0 c
ell
s
0.0
0.5
1.0
1.5
* *
*
*
* *
**
*
*
**
*** *
**
NVP 12-OH-NVP 12-OH-NVP
Sulfate
*
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
% o
f C
D8
6 c
ell
s
Figure 30. Change in cell surface marker expression on THP-1 cells in response to NVP or
its metabolites.
Cells incubated with 0.5% of DMSO were used as the baseline (the dash lines). Data represent
the ratio of treated versus DMSO control samples for various surface marker expression. A) the
intensity of CD40 expression measured by MFI, B) the intensity of MHC II expression
measured by MFI, C) percent CD80+
cells, D) percent CD86+
cells. The data represent the
average of 3 experiments ± SE. Unpaired t test, p < 0.05.
104
Activation of BMDCs by NVP and Its Metabolites.
1. Phenotype of BM-derived Cells. Figure 31. shows that the majority (>90%) of bone
marrow-derived cells express CD11c, which suggests that they are DCs. The presence of other
cell types, such as granulocytes and nature killer cells (CD161+), macrophages (pan-
macrophage), and T cells (αβ-TCR) were minimum with percentages less than <0.5%, <5%, and
<2%, respectively.
2. Change in Cell Surface Marker Expression. The dose-response curve (Figure 33) shows
that CD40 was significantly upregulated with an increasing concentration of all three substances
with 12-OH-NVP being the most potent. On the other hand, MHC II was down regulated with
increasing drug concentration; the same trend was also observed for CD80.
The production of several cytokines by BMDCs was measured by ELISA. Figure 34 shows that
there may be a small increase in IL-12p40 and TNF-α levels at higher concentrations of all three
substances; however, the differences were not statistically significant.
3. Covalent Binding of NVP and Its Metabolites to BMDCs (Western Blot By Amy
Sharma). In order to determine the mechanism of the effect of 12-OH-NVP on BMDCs, we
investigated the covalent binding of these substances to BMDCs. Figure 35 showed that at both
24 and 72 hours, the sulfate metabolite binds strongly to proteins extracted from BMDCs; there
was also a bit of binding with 12-OH-NVP. No binding was observed for NVP and DMSO
samples.
105
MH
C I
I
A.
CD11c
B. C. D.
Figure 31. Phenotype of bone marrow-derived cells.
Bone marrow cells were differentiated in the presence of 5 ng/mL recombinant rat GM-CSF and
5 ng/mL IL-4 for 6 – 8 days before use. A) cells stained with MHC II and CD11c antibodies
(blue dots) versus unstained cells (red dots), B) cells stained with CD161 antibody, C) cells
stained with pan-macrophage antibody, D) cells stained with αβ-TCR antibody.
106
CD80 CD86 CD40 MHC II
Fluorescence Intensity
Figure 32. Representative dot plots and histograms of surface marker staining on BMDCs
stimulated by various substances.
Un
stai
n
DM
SO
S
ulf
ate
(62
.5 µ
g/m
L)
LP
S
Count
107
A. B. Relative MFI of CD40 (all studies N=6)
0.0
0.5
1.0
1.5
2.0
* *
**
*
*
**
*
*
**
*
*
*
NVP 12-OH-NVP 12-OH-NVP
Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
CD
40
MF
I
0.5
1.0
1.5
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
MH
C I
I M
FI
C. D.
0.0
0.5
1.0
1.5
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
% C
D8
0 c
ell
s
0.0
0.5
1.0
1.5
NVP 12-OH-NVP 12-OH-NVP
Sulfate
1 2 4 816
32 64
125 1 2 4 8
16
32 64
125 1 2 4 8
16
32 64
125
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
% C
D8
6 c
ell
s
Figure 33. Change in cell surface marker expression on BDMCs in response to NVP or its
metabolites.
Cells incubated with 0.5% of DMSO was used for the baseline (the dash lines). Data represent
the ratio of treated versus DMSO control samples for various surface marker expression. A) the
intensity of CD40 expression measured by MFI (N=6), B) the intensity of MHC II expression
measured by MFI (N=3), C) percent CD80 positive cells (N=3), D) percent CD86 positive cells
(N=3). The data represent the average ± SE. Unpaired t test, p < 0.05.
108
A.
0
10
20
30
40
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 8 16
32 64
1250 1 2 4 8 16
32 64
1250 1 2 4 8 16
32 64
1250
Drug Concentration (g/mL)
pg/m
L
B.
0
100
200
300
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 8 16
32 64
1250 1 2 4 8 16
32 64
1250 1 2 4 8 16
32 64
1250
NVP
12-OH-NVP
12-Sulphate-NVP
Drug Concentration (g/mL)
pg/m
L
C. TNF-
0
50
100
150
NVP 12-OH-NVP 12-OH-NVP Sulfate
1 2 4 8 16
32 64
1250 1 2 4 8 16
32 64
1250 1 2 4 8 16
32 64
1250
NVP
12-OH-NVP
12-Sulphate-NVP
Drug Concentration (g/mL)
pg/m
L
Figure 34. Cytokine production by drug-stimulated BMDCs at the end of a 24 hour
incubation.
The data represent the average ± SE of 5 experiments. A) IL-1β, B) IL-12 p40, C) TNF-α.
109
Figure 35. Covalent binding of DMSO, NVP, 12-OH-NVP, and its sulfate to BMDCs after
a 24 or 72 hour incubation.
62.5 µg/mL of each of the three substances in 0.5% DMSO were incubated with 10 million
BMDCs. Immature BMDCs were generated from naïve female BN rats and harvested on day 8
of differentiation. 40 µg/lane for 24 hour samples and 35 µg/lane for 72 hour samples were
loaded.
110
BMDCs and RAW264.7 Cells Stimulated by D-penicillamine or Isoniazid. D-penicillamine
and isoniazid can activate macrophages by direct binding to the aldehyde groups on the cell
surface; therefore, they may serve as “positive control” drugs for the experiment. Figure 36
shows that the level of CD80 expression on BMDCs increased at the highest concentration of D-
penicillamine but not with isoniazid. The level of CD40 expression is lower than that of the
control at all concentrations. In the parallel experiment where RAW264.7 cells were used, MHC
II was upregulated with an increasing concentration of D-penicillamine but not with isoniazid
(Figure 37). That was also the only change observed.
111
A. B.
0.0
0.5
1.0
1.5
D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
0.1
0.4
1.2
3.7 11
33
100
300
0.1
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
CD
40
M
FI
0.0
0.5
1.0
1.5
D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
0.1
0.4
1.2
3.7 11
33
100
300
0.1
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
MH
C II
M
FI
C. D.
0.0
0.5
1.0
1.5
*
D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
0.1
0.4
1.2
3.7 11
33
100
300
0.1
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
% C
D8
0 c
ell
s
0.0
0.5
1.0
1.5
D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
0.1
0.4
1.2
3.7 11
33
100
300
0.1
Drug Concentration (g/mL)
rati
o o
f tr
eat
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ers
us
co
ntr
ol
cell
s in
% C
D8
6 c
ell
s
Figure 36. Change in cell surface marker expression on BMDCs in response to D-
penicillamine or isoniazid.
Data represent the ratio of treated versus control samples in various surface marker expressions.
Cells without the addition of any substance were used as control (the dash line). A) the intensity
of CD40 expression measured by MFI, B) the intensity of MHC II expression measured by MFI,
C) percent CD80+ cells, D) percent CD86
+ cells. The data represent the average of 3
experiments ± SE. Unpaired t test, p < 0.05.
112
A. B.
0.0
0.5
1.0
1.5
D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
0.1
0.4
1.2
3.7 11
33
100
300
0.1
Drug Concentration (g/mL)
rati
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ers
us
co
ntr
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cell
s in
CD
40
M
FI
0.0
0.5
1.0
1.5
2.0
D-penicillamine Isoniazid
* *
* *
0.4
1.2
3.7 11
33
100
300
0.1
0.4
1.2
3.7 11
33
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0.1
Drug Concentration (g/mL)
rati
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cell
s in
MH
C I
I M
FI
C. D.
0.0
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1.0
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D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
0.1
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33
100
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0.1
Drug Concentration (g/mL)
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cell
s in
% C
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D-penicillamine Isoniazid
0.4
1.2
3.7 11
33
100
300
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Drug Concentration (g/mL)
rati
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cell
s in
% C
D8
6 c
ell
s
Figure 37. Change in cell surface marker expression on RAW264.7 cells in response to D-
penicillamine or isoniazid.
Data represent the ratio of treated versus controlsamples in various surface marker expressions.
Cells without the addition of any substance were used as baselines (the dash lines). A) the
intensity of CD40 expression measured by MFI, B) the intensity of MHC II expression measured
by MFI, C) percent CD80 cells, D) percent CD86 cells. The data represent the average of 3
experiments ± SE. Unpaired t test, p < 0.05.
113
T Cell Activation Assay.
1. Confirmation of the of Naïve T Cell Phenotype. Although the direct effects that we detected
on APCs were minimal, NVP or its metabolites might have an effect on their ability to activate T
cells. In this study, naïve T cells were used instead of total CD4+ T cells because the latter
contains natural FoxP3+ Treg cells resulting in a weaker response [135]. Figure 38 shows that
the purity of isolated CD4+CD25
- cells was about 92%.
2. T Cell Proliferation Measured by CFSE Staining. Figure 39 shows sample dot plots of the
CFSE staining of αβ-TCR+ cells in the cell culture and the corresponding dose response curve.
The results suggest that there was no difference in proliferation between drug-stimulated T cells
and the DMSO-treated control cells (the dashed lines).
114
A. B.
Figure 38. Phenotype of CD4+CD25
- cells isolated from the spleen and ALNs of naïve
female BN rats using magnetic beads and column.
A) unstained control sample, B) isolated cells stained with anti-rat CD4 and CD25 antibodies. C
D25
CD4 Unstained
115
A. B.
CFSE
C. D.
CFSE
E.
0.5 1 2 4 8 16 32 64 1280
1
2
3
4
NVP
12-OH-NVP
12-OH-NVP Sulfate
Drug Concentration (g/mL)
% p
roli
fera
ting c
ell
s
Figure 39. Proliferation of αβ-TCR+ cells measured by CFSE staining.
T cells were re-stimulated by BMDCs in the presence of NVP or its metabolites and stained with
αβ-TCR+ antibody. A) unstained control (no CFSE), B) DMSO control cells stained with
CFSE, C) 12-OH-NVP sulfate (62.5 µg/mL) stimulated cells stained with CFSE, D)
PMA/inomycin stimulated cells stained with CFSE, E) Percent of αβ-TCR+ cells that
proliferated as a function of drug concentration (dashed line represents the DMSO control). The
data represent the average of 3 experiments ± SE.
116
Stability of 12-OH-NVP Sulfate. The stability of 12-OH-NVP sulfate was determined using
HPLC. Figure 40 showed two representative chromatographs of 12-OH-NVP and its sulfate
after 24 hours of incubation with BMDCs. The retention time was 6 minutes for the sulfate and
10 minutes for 12-OH-NVP. At 24 hours, there was no detectable 12-OH-NVP formation in the
sulfate sample (A) and no detectable sulfate formation in the 12-OH-NVP sample (B).
To quantify the amount of 12-OH-NVP and its sulfate in the cell culture system, the area under
the curve (AUC) of the corresponding peak on the chromatograph was used. Figure 41 showed
the AUC of the 12-OH-NVP sulfate in cell culture (B) and medium only (A) over time. Both
showed that there was no significant change in AUC of 12-OH-NVP and the sulfate over time
regardless of the presence of BMDCs.
117
A.
B.
B.
Figure 40. Chromatographs of (A) 12-OH-NVP sulfate and (B) 12-OH-NVP after a 24
hour incubation with immature BMDCs generated from naïve female BN rats.
118
A.
B.
Figure 41. Quantification of 12-OH-NVP sulfate in cell culture medium in the presence or
absence of BDMCs over time.
(A) Change of AUC of 12-OH-NVP sulfate or 12-OH-NVP peaks when incubated with cell
culture medium over 8 days, (B) AUC of 12-OH-NVP sulfate or 12-OH-NVP peaks when
incubated with BMDCs for 24 hours. Each line in the above two figures represent one
incubation sample with either the sulfate or the hydroxyl metabolite.
119
5.4. DISCUSSION
In this study, we investigated the response of three types of APCs originating from three
different species to NVP and/or its metabolites. Specifically, RAW264.7 cells are a mouse
macrophage cell line with a BALB/c background. Previous studies from our lab showed that
they can be activated by D-penicillamine, isoniazid, and hydralazine [78, 80]. Although we do
not have a mouse model for NVP-induced skin rash because they do not have the required
sulfotransferase in the skin, the initial steps of activation of APCs may be the same. The use of
BMDCs generated from female BN rats may be the best cell type to vigorously test our
hypotheses given that the animal model was developed in these animals. However, it was also
desirable to test a human cell line; therefore, THP-1 cells were also studied. THP-1 is a human
acute monocytic leukemia cell line, which has been used as a DC surrogate to study the
maturation of DCs and their role in protein haptenation [136]. Given the origin of this cell line,
it may better represent what occurs in humans.
A surprising finding was that 12-OH-NVP appeared to have greater activity than 12-OH-NVP
sulfate even though we know that formation of the sulfate is required for the induction of the
skin rash, and even NVP had some activity. Different cells responded differently; specifically, an
upregulation of CD40 was observed with RAW264.7 cells and BMDCs but not with THP-1
cells; the latter had an upregulation of CD86 instead. Despite the fact that different surface
molecules were changed with different cell types, all three: NVP, 12-OH-NVP, and 12-OH-NVP
sulfate, appeared to activate APCs to some degree.
One interesting observation was the significant decrease of MHC II expression level in both
BMDCs and THP-1 cells. First it seemed strange because the upregulation of MHC II was
expected to be a marker of APC activation; however, there are studies that suggest that at early
time points, the internalization of surface MHC II after antigen exposure indicates antigen
processing [132, 133, 137].
D-penicillamine and isoniazid were used as “positive control” comparator drugs because they
were reported to induce a production of IL-6 and other cytokines by RAW264.7 cells in previous
studies [80]. From these studies it appears that only D-penicillamine induced an upregulation of
surface markers regardless of the cell type.
120
The fact that 12-OH-NVP activated APCs more than the sulfate contradicts the hypothesis that it
is the chemical reactivity of the sulfate metabolite that is responsible for APC activation. What
is more important than upregulation of surface markers is the ability of the APCs to active T
cells. Other studies with SMX found that it was the reactive metabolite that led to T cell
proliferation and cytokine production [138]. However, we were not able to detect T cell
proliferation in our studies.
The observation that 12-OH-NVP appeared to have greater activity than 12-OH-NVP sulfate
raises the question of whether APCs can metabolize NVP or 12-OH-NVP. From the literature,
gene expression of metabolic enzymes (cytochromes P450 and sulfotransferase) in THP-1 cells
is very low or non-detectable [139, 140]; there is little data for rat BMDCs. However, peripheral
blood-derived DCs (mostly human) were shown to express the majority of cytochromes P450,
myeloperoxidase, and cyclooxygenases, and the inhibition of these enzymes abrogated the
increase in co-stimulatory molecule expression and decreased the level of drug-protein adduct
formation [46, 141-143]. Therefore, it appears that biotransformation of the parent drug and/or
metabolites could contribute to the observed dose-response pattern. In addition, we found that
nearly half of the 12-OH-NVP sulfate was hydrolyzed back to its hydroxyl form in the first half
hour following in vivo administration (Maria Novalen’s thesis, 2010). Therefore, another
possible explanation for the lack of response for sulfate could be that most of it was hydrolyzed
back to 12-OH-NVP during the incubation. However, the stability test showed that there was no
detectable 12-OH-NVP formation in the sulfate samples, nor was sulfate found in the incubations
with 12-OH-NVP. The finding with covalent binding of NVP metabolites to skin proteins
suggested that only 12-OH-NVP sulfate binds to skin homogenates and forms adducts in the
epidermis, and the topical treatment of 1-phenyl-1-hexanol, a sulfotransferase inhibitor,
prevented covalent binding where it was applied and it also prevented the rash where it was
applied [68, 69]. This is the definitive evidence that 12-OH-NVP sulfate is responsible for the
skin rash, not 12-OH-NVP. We performed similar covalent binding studies with the BMDCs,
and the results showed that there was a high degree of binding with the sulfate, and there was
also significant binding with 12-OH-NVP. Although there was no sulfate detected in the 12-OH-
NVP samples as determined by HPLC, it is possible that a trace amount is formed inside the cells
and covalent binds to them. An experiment was performed with the addition of 1-phenyl-1-
hexanol to block the sulfation and covalent binding to see if it prevents the activation by 12-OH-
121
NVP [144]. However, 1-phenyl-1-hexanol itself led to activation of BMDCs and induced a
strong response of BMDCs when added to the cell culture. The covalent binding data suggest
that some of the 12-OH-NVP is converted to the sulfate; however, this is insufficient to explain
the observation that the concentration of 12-OH-NVP required to activate APCs was less than
that of the sulfate metabolite.
The APC activation results are complicated, but they do not rule out the possibility that the
reactive metabolite can directly activate APCs. A systemic immune response is likely to be a
complicated mechanism that involves several key factors, i.e. release of the danger signals,
protein haptenation, and activation of APCs. The covalent binding of 12-OH-NVP sulfate may
be necessary but not sufficient to induce a skin rash. It is possible that in addition to the covalent
binding of 12-OH-NVP sulfate formed in the skin, NVP and 12-OH-NVP also contribute to
induction of the immune response. In fact, a recent paper suggested that tricyclic molecules such
as carbamazepine and oxcarbazepine can directly activate APCs through toll-like receptor 4
[145], and this may also apply to NVP, which contains a tricyclic structure. It is also possible
that certain types of sulfate-protein adducts are necessary to activate APCs. The fact that the
sulfate was formed in the epidermis, and the sulfotransferase inhibitor prevented the local skin
rash suggests that the responding APCs reside in the epidermis, which is primarily composed of
keratinocytes. Keratinocytes are considered non-professional APCs, and in fact the recent
finding in our group suggest that they might be involved in inflammasome activation and
contribute to NVP-induced skin rashes [73, 146].
122
5.5. CONCLUSION
NVP-induced skin rash in female BN rats is one of the very few valid animal models of an IDR
that occurs in humans, and it has brought us many insights into the mechanisms of these
unpredictable drug reactions. However, with more studies, the mechanism appears to become
more complicated. This is a reaction orchestrated by various immune cells, and although it is
possible that some in vitro systems can model certain parts of the immune reaction, essentially
the only way to rigorously test mechanistic hypotheses is with in vivo studies.
123
CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS
124
6.1. Summary
IDRs represents a significant threat to the pharmaceutical industry; however, to date we have not
been able to effectively prevent or treat them due to limited understanding of the mechanisms
involved. While using valid animal models is essentially the only way to conduct mechanistic
studies, the development of such models has also been extremely difficult, and the discovery of
such models is often by luck.
In the late 1990’s to early 2000’s, our lab found that NVP causes a skin rash in female BN rats
that is similar to NVP-induced rashes in humans. Followed by a few years of validation of the
dose, strain, sex and characteristics of the rash in BN rats, the animal model was finally
developed by Shenton et al., and it has been used to rigorously test many hypotheses of IDRs
since then [54]. The early studies focused on characterizing the disease and confirming the
involvement of the immune system. Specifically, the onset of disease is delayed on primary
exposure but accelerated upon rechallenge, depletion of CD4+ T cells leads to decreased
incidence, and adoptive transfer of CD4+ T cells transferred the sensitivity. Overwhelming
evidence suggests that this reaction is immune-mediated – specifically – CD4+ T cell mediated
[54, 55, 61]. The question then became which metabolite causes the rash and how is the immune
system orchestrated to mediate the rash?
The first milestone to fully answer the question of what species is responsible for the rash was
established by Jie Chen et al. in 2008. With several years of effort, they were able to
demonstrate that the NVP-induced skin rash is not caused by NVP itself, but requires 12-
hydroxylation of NVP, presumably because the 12-hydroxy metabolite is further converted to a
more reactive sulfate in the skin. This conclusion was based on experiments in which the 12-
methyl hydrogens were replaced by deuterium, which decreases 12-hydroxylation and rash, but
does not affect other properties of the drug. Furthermore, treatment with 12-OH-NVP also led to
a rash [59]. The outline of the NVP biotransformation and confirmation of the culprit metabolic
pathway forms the stepping-stone for subsequent mechanistic studies.
Chapter 2 of the current work focused on testing the PI hypothesis, and it showed that T cells
from sensitized animals responded to the parent drug much better than to the 12-OH-NVP
metabolite, even though it had been demonstrated that the 12-OH-NVP pathway was responsible
for the rash, not the parent drug [59]. Specifically, CD4+ T cells isolated from sensitized animals
125
proliferate and produce cytokines when incubated with NVP, but the response was minimal
when other metabolites were used. This result showed that there is a clear disconnect between
what induces the immune response and what the T cells respond to, and it also suggested the
basis of PI hypothesis is wrong.
In addition to the PI hypothesis, other major hypotheses are also under investigation in our lab
using this unique IDR model. In 2007, our lab has successfully produced an anti-NVP antibody
serum that could be used to detect covalent binding, and that allowed subsequent work to test the
hapten hypothesis. The hapten hypothesis suggests that the hapten-protein conjugate is
responsible for inducing an immune response, and we speculate that the target organ is most
likely where this immune reaction is initiated, in this case the skin. While another student, Amy
Sharma, was mainly involved in characterizing the covalent binding and determining subsequent
pathological events, I investigated if NVP/its metabolite could induce a rash if directly applied to
skin (Chapter 3). It was found that although topical treatment of a low dose of NVP induced a
skin rash with an accelerated onset in NVP-sensitized animals, a 6-week treatment with 12-OH-
NVP in naïve animals did not lead to any significant histological changes, nor did it induce a
skin rash. The fact that topical treatment lead to a skin rash in sensitized animals suggested that,
at least on rechallenge, the immune response is initiated in the skin.
Chapter 4 is composed of several independent co-administration studies intended to manipulate
the immune system and see what effect they have on NVP-induced IDRs. However, none of
these modulators that seemed to change the incidence/severity of NVP-induced IDR is “clean” –
they all have more than one effect. Based on the BSO and RA experiments, in which both
reagents seemed to have an impact on the metabolism of NVP and resulted in lower drug levels
in the plasma, one cannot conclude the effect of any modulator on drug-induced adverse
reactions without testing its effect on metabolism.
Studies in Chapter 5 aimed to address the initial steps of how a reactive metabolite leads to an
immune response at the cellular level. Similar studies have been done with SMX to demonstrate
that the reactive metabolite can activate APCs by upregulating co-stimulatory molecule
expression, which in turn activates T cells and induces an immune response. Our results showed
that different co-stimulatory markers were upregulated for different APCs; however, the
upregulation of co-stimulatory markers did not lead to T cell activation.
126
6.2. Implications and Future Directions
The most important implication of the current work is its impact on use of the LTT in clinical
settings. For a long time it has been well accepted that the LTT can be used to determine which
drug caused a hypersensitivity reaction, and it has been used as a diagnostic tool for IDRs.
Although the LTT can differentiate the likely drug involved, our research demonstrated that the
“cause” and “response” of an immune reaction are not necessarily the same, and the LTT should
not be used to determine if it is the parent drug or the metabolite that is responsible for an IDR.
Our research set out to test the validity of PI hypothesis in the NVP model, and it revealed that
the foundation of this hypothesis is not valid. The finding was unexpected, but it again
addressed the importance of using an animal model for mechanistic studies; this hypothesis
simply could not be tested in humans. It also demonstrated that every assumption in science has
to be carefully validated regardless of how reasonable it sounds and how well accepted it is.
Although the basis of the PI hypothesis is not valid, it does not mean that the hypothesis itself is
wrong. It may be valid for drugs that do not appear to form reactive metabolites such as
ximelagatran and lamotrigine [39, 147]. In fact, ximelagatran causes elevated levels of serum
alanine aminotransferase in some patients and was shown to form a labile bond to MHC
molecules [104]. The fact that none of the metabolites or the parent drug seem to be reactive and
form any specific covalent binding to proteins suggested that the immune response may be
initiated through a PI type of interaction.
The topical treatment set out to test if the IDR is initiated locally. Although the treatments did
not successfully induce a skin rash on primary exposure, it also does not directly prove the
hypothesis wrong. It appears that penetration of the 12-hydroxy metabolite into the skin was not
sufficient to lead to covalent binding, which is observed with oral administration of 12-OH-NVP
(Amy Sharma, unpublished observation). Nevertheless, the fact that topical NVP induced a skin
rash in sensitized animals is interesting, and we suspect there is an autoimmune component in
this IDR. For instance, the rash induced by topical NVP upon rechallenge seems to be a
systemic reaction even when minimal doses were used. There are also autoantibodies present in
the sera of rechallenged animals, which further supports the autoimmune component (Amy
Sharma, unpublished observations). Further characterization of the cellular mechanisms as well
127
as the autoantibodies induced on rechallenge may help to further elucidate the mechanisms
involved.
It was unfortunate that the upregulation of APC co-stimulatory markers did not lead to T cell
activation. Such results are difficult to interpret because the reasons for failing to induce T cell
activation are hard to determine. Recently, Amy Sharma (the student who used this model to
determine if the Hapten Hypothesis was operative) found that NVP sulfate forms protein adducts
in the epidermis of the skin isolated from female BN rats that had been treated with NVP, and
this metabolite is responsible for the rash [68, 69]. This is the biggest breakthrough of our over a
decade of IDR research using the NVP model, and it is also the first time to directly prove that a
reactive metabolite is responsible for inducing an IDR by using an animal model. The fact that
the sulfate was formed in the epidermis, and a topical sulfotransferase inhibitor prevented the
local skin rash suggests that the responding APCs reside in the epidermis, which is primarily
composed of keratinocytes. Meanwhile, results of our danger signal studies also suggested that
they might be involved in inflammasome activation and contribute to NVP-induced skin rashes
[73]. Inflammasomes are large intracellular multiprotein complexes, which comprise a
nucleotide-binding oligomerization domain receptors (NOD-like receptor or NLRs). They are
expressed in many cells including keratinocytes and play a critical role in IL-1β and IL-18
production [148]. The NLRP3 inflammasome has been shown to be involved in contact
hypersensitivity, where covalent binding of reactive chemical species are formed and activate the
inflammasome [149]. It is possible that 12-OH-NVP sulfate also acts this way, i.e. by inducing
an immune response via inflammasome activation. Our group has shown that the gene
expression of tripartite motif containing 63 (TRIM63) and death-associated protein kinase 1
(DAPK1), both involved in inflammasome activation, were significantly upregulated in the rat
skin following NVP treatment [73], which suggests that inflammasome is involved in the
induction of NVP-induced skin rash. Testing this hypothesis is an important future direction of
this research.
Keratinocytes are considered non-professional APCs, and therefore were not the focus of our
APC activation research. However, it is possible that they also contribute to the antigen
presentation process in addition to the professional APCs. Given that the drug-modified proteins
are formed in the epidermis, it is reasonable to speculate that cells resident in the skin, possibly
keratinocytes or Langerhans cells, may be the key APCs that lead to T cell activation. This
128
would also explain why the activated APCs did not lead to T cell activation – because they
cannot form the proper epitope recognized by T cells and pass on the “danger message”. In
contact hypersensitivity, dendritic cells and Langerhans cells migrate to the draining lymph
nodes and the spleen once being activated by antigens. The signals are then presented to T cells
in the secondary lymphoid organs and an immune response may be elicited. On the other hand,
T cells expressing skin homing receptors could also be attracted by skin residential cells
expressing the corresponding ligands and migrate to the local inflammatory site, then induce an
immune response. [111, 112]. This may also be applied to NVP-induced skin rash and can be
tested. Predicting which drug candidates will cause IDRs and who will have an IDR has been a
big challenge and will continue to be a problem without a thorough understanding of their
mechanisms. NVP-induced skin rash in female BN rats, being one of the very few animal
models of IDRs, has allowed us to test hypotheses that are not possible to do in human subjects,
and this model has provided tremendous information that sheds some light on the mechanisms of
IDRs in humans. There are other drugs that cause serious drug rashes that also have the potential
to form reactive sulfate metabolites in the skin. Testing whether these drugs also form reactive
sulfate metabolites is also an important future direction of this research. However, it is worth
noting that each IDR is different, and the NVP model may not necessarily represent other drug-
induced skin rashes. In fact, the mechanisms of IDRs are so complex that it took us over 10
years to prove that a reactive metabolite is responsible for this IDR. Yet, it may take even longer
to fully understand how the immune reaction is initiated and how to prevent IDRs, but the only
way to progress is to rigorously test hypotheses and that requires developing valid animal
models.
129
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APPENDIX Appendix 1. Supplemental Data of APC activation by 12-OH-NVP sulfate.
Fluorescence Intensity Intensity of MHCII expression:Sulphate-NVP VS DMSO (0.4%)
0.1250.25 0.5 1 2 4 8 16 32 64 1280
50
100
150
200
Experiment 2
Experiment 1
Drug Concentration (g/mL)
rati
o o
f tr
eat
ed v
ers
us
co
ntr
ol
cell
s in
MH
C I
I M
FI
Appendix 1. Histograms and dose-response curve of MHC II expression on APCs in response to
12-OH-NVP sulfate. APCs were concentrated from the ALNs of naïve female BN rats using
magnetic beads and incubated with 12-OH-NVP sulfate dissolved in 0.5% DMSO for 24 hours at
37 ºC, 5% CO2. A) representative histograms. Green: LPS stimulated cells (positive control),
Red: DMSO stimulated cells (negative control), Blue: 12-OH-NVP sulfate stimulated cells (50
µg/mL). B) dose-response curve.
Count
A.
B.
