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Basic Immunology 101
Amy Sharma
Ph.D. Candidate
Uetrecht Laboratory
Leslie Dan Faculty of Pharmacy, University of Toronto
Q. Why does your immune
system exist?
Immunology Overview

Immune system is like a double edged
sword

Key players of the immune system

Humoral versus Cellular Immunity
I. Cells of the immune system


T-lymphocytes (Thymus derived ‘T’ cells)
Key role in cell-mediated immunity
• co-ordinate and regulate immune
responses through cytokine activation,
antibody stimulation, etc



Constitute ~60-70% of lymphocytes in
circulating blood
Many different sub-types
Identified by T-cell receptor
I. Cells of the immune system


B-lymphocytes (Bone marrow derived ‘B’ cells)
Key role in humoral immunity:
• produce antibodies against antigens
• act as antigen-presenting cells (APCs)
• develop into memory B cells after activation
by antigen interaction

Constitute ~10-20% of lymphocytes in
circulating blood
I. Cells of the immune system


Macrophages (“Big-eaters”)
Key role in immunity in general:
• main type of APC (process and present
antigen to CD4+ Th-cells)
• phagocytose and kill microbes coated by
antibody and/or complement
• produce cytokines, regulating T and B
cell function
I. Cells of the immune system


Dendritic cells (“Potent” APC)
Key role: link between adaptive and
cell mediated immunity
• process antigen and present peptide
fragments to other cells of the immune
system goes on to regulate T and B
cell responses
I. Cells of the immune system


Natural Killer ‘NK’ cells (“LGL’s”)
Key role in cell-mediated immunity
• contain azurophilic granules thus capable
of lysing tumor cells, virus infected cells,
etc, without previous sensitization

Constitute ~10-15% of lymphocytes in
circulating blood
Innate Immunity (cellular immunity)


Mediated by lymphocytes
Does not involve antibodies (antigen nonspecific)

Cellular immunity protects the body by:
• activating macrophages, NK cells, and
cytotoxic T-cells
• stimulating cytokine secretion, influencing
the function of other immune cells
Humoral Immunity


Mediated by soluble antibody proteins
(antigen specific)
Humoral immunity protects the body
by:
• antigen presentation, discriminating
recognition of “non-self” versus “self”
• the generation of antibody responses
• the development of immune memory
Idiosyncratic Drug Reactions
What are They, Why & How Do We Study Them?
Amy Sharma
Ph.D. Candidate
Uetrecht Laboratory
Leslie Dan Faculty of Pharmacy, University of Toronto
Overview
I.
II.
III.
IV.
V.
VI.
VII.
Adverse Drug Reactions (ADRs)
Idiosyncratic Drug Reactions (IDRs)
Characteristics of IDRs
Proposed Mechanism of IDRs
Drugs Known to Induce IDRs
Studying IDRs
Future Directions
I. Adverse Drug Reactions
I. Adverse Drug Reactions

The World Health Organization definition:
“any noxious, unintended, and undesired
effect of a drug, which occurs at doses used
in humans for prophylaxis, diagnosis, or
therapy”

ADRs are common
•
•
2,216,000 hospitalized patients/year
experienced a serious ADR and 106,000/year
died from an ADR
Fatal ADRs rank 4th to 6th in leading causes of
death in US (Bond CA et al. Pharmacotherapy 2006)
I. Adverse Drug Reactions
I. Adverse Drug Reactions
Adverse drug reactions can be divided into five basic types:

Type A (augmented):
• Can be predicted from the pharmacology of the drug
• Are typically dose-dependent

Type C (chemical), D (delayed) and E (end of treatment)

Type B:
• Cannot be predicted on the basis of the known
pharmacology of the drug
• Also known as idiosyncratic adverse reactions
• Can affect almost any organ system
II. Idiosyncratic Drug Reactions

Rare & unpredictable reactions
•
•
•

Do not occur in most patients at any dose
•


Incidence: 1/103 - 1/106 patients
25% of all ADRs
Still very prevalent because of the number of drugs
involved and the number of people taking these drugs
No simple dose-response relationship
Effects not related to pharmacological
properties of the drug
Can be very severe
•
most serious ADRs in drug therapy
III. Characteristics of IDRs

Organs affected:
Liver
Skin
(cholestatic liver)


(mild-severe rash)
Bone marrow / Blood
cells
(aplastic anemia;
agranulocytosis)
Most thought to be immune-mediated
Detected during the late stage of development or when drug is
released on to market


May lead to withdrawal
Significant financial burden
III. Risk Factors for IDRs:
Don’t have a good understanding of who will
develop IDRs.

Age - Incidence increases with age

Concomitant challenge – increase risk for HIV
patients

Ethnic background – Incidence of clozapineinduced agranulocytos is 20% in a Jewish hospital
vs. <1% elsewhere

Gender - female >> male
IV. Mechanisms of IDRs
If we can understand how drugs induce IDRs we can:


Scan for drugs that have high risk of causing IDRs early
in the drug development process, and avoid later losses
to both patients and manufacturers
Devise therapy that prevents IDRs in patients
(administer concomitant therapy)
There is circumstantial evidence that indicates a
potential role of reactive metabolites (RMs) in
development of IDRs
IV. Step 1: Reactive Metabolite Formation

Drug Metabolism:
• Process whereby therapeutically active drugs are
converted to a more soluble form (metabolites) and
are cleared by renal or biliary excretion

Reactive Metabolites (RMs) and Covalent Binding
• During metabolism, usually through P450 oxidation,
drugs can form RMs (chemically reactive species)
that can covalently bind to endogenous proteins or
other macromolecules
Reactive Metabolites

Reactive metabolites are electrophiles
or free radicals
•
•
•
•
Sulfates/sulfonates
Epoxides/arene oxides
Michael Acceptors
Nitroso amines
IV. Where Does Metabolism Occur?
Metabolizing enzymes are present in the following organs:
Cytochrome P450, Sulphotransferases,
Peroxidases
White blood cells (macrophages and
neutrophils) that become activated
to kill bacteria, and do so by releasing
oxidants such as H2O2 and HOCl.
IV. Where Does Metabolism Occur?
Once formed, reactive metabolites tend to bind to
nucleophilic groups on proteins or macromolecules near
the site of their formation. Thus, toxicity most often
occurs at sites of RM formation, especially if RM is highly
reactive!
Example – Clozapine:
 Clozapine is oxidized to a RM in both the liver and
neutrophils. The main toxic effects of clozapine are
liver and neutrophil toxicity (hepatotoxicity and
agranulocytosis).
IV. Step 2: Immune Response

Basic paradigm in Immunology
• To discriminate against pathogens, the immune system
learns to recognize self from non-self. In this way,
autoimmunity is avoided and immune responses are
mounted against foreign invaders.

Hapten Hypothesis
• Once drug is covalently bound to a host protein it forms
a novel antigen known as the hapten-carrier complex.
Host immune system then perceives the modified
endogenous protein as foreign, and mounts an
immune response against it.
IV. Hapten Hypothesis Detailed
Step 1 – Reactive Metabolite Formation
Step 2 – T-cell activation and Initiation of an
Immune Response
IDR
IV. T-cell Activation
IV. IDR Characteristics that Indicate
Immune Involvement
1. Reaction takes several weeks to develop
2. Once the drug is removed, reaction clears quickly
3. On re-exposure the time to onset is shorter than on
first exposure
4. In some reactions anti-hapten antibodies or
antibodies against self-tissues are found (e.g., in
patients with halothane-induced hepatitis anti-hapten
antibodies have been found)
Not all IDRs have these characteristics
V. Clinical Evidence in Support of
Hapten Hypothesis

Penicillin-induced anaphylaxis

Aminopyrine-induced agranulocytosis

Halothane-induced hepatitis
V. Penicillin-Induced Anaphylaxis
-lactam ring
O
C C
H2
S
NH
N
O
Benzylpenicillin
CH3
CH3
COOH
O
C C
H2
S CH
NH
3
O C HN
CH3
NH
COOH
lys
Protein
 Covalent binding due to spontaneous ring opening
 IgE antibodies were detected in patients with anaphylactic
reaction
 Re-exposure can be life-threatening
V. Aminopyrine-Induced Agranulocytosis
N N
N
N N
Myeloperoxidase/H2O2/Cl-
CH3
O
CH3
CH3
CH3
CH3
O
CH3
CH3
N
CH3
Dication intermediate
 Associated with a high risk of agranulocytosis (~1%)
 Reactive dication formed by neutrophil-derived hypochlorous acid
could be responsible for the IDR
 Onset of symptoms (fever, sore throat and infections) in 1 week - 1
month
 Drug-specific Abs
 Re-challenge results in rapid drop in neutrophil count as well as their
bone marrow precursors
V. Halothane-Induced Hepatitis
Protein
F
F
Br
C
F
C Cl
H
Halothane
P4502E1
F
F
O
C
C Cl
F
Trifluoroacetyl
Chloride
F
F
O
C
C NH
F
RM covalently
bound to protein
 Halothane is oxidized by P450 to form trifluroacetyl chloride, which
can bind to proteins
 20% of patients develop asymptomatic elevation of liver
transaminases (AST, ALT)
 leads to the development of hepatitis
 hepatitis rarely occurs on first exposure, which suggests that
sensitization is required
 Serum of affected patients contain antibodies against native hepatic
proteins as well as trifluoroacetylated proteins (hapten-carrier complex)
V. Drugs Known to Cause IDRs
Felbamate
antiepileptic
Nevirapine
HIV drug (NNRTI)
D-Penicillamine
anti-rheumatic
Clozapine
antipsychotic
Carbamazepine
anticonvulsant
V. Felbamate
O
Idiosyncratic reactions:
CNH 2
O
Aplastic Anemia and Liver Toxicity
Reactive Metabolite: Yes
Protein Binding: Probable
Animal Model: No
O
CNH 2
O
Phenylacrolein (Michael Acceptor)
H
O
V. Nevirapine
Idiosyncratic reaction:
N
N
N
Severe Skin Rash, Liver Toxicity
N
Reactive Metabolite: quinone
methide
O
H
CH3
Protein Binding: Yes; epidermis
Animal Model: Yes; Skin rash
in the female Brown Norway rat
N
N
N
N
Quinone Methide
O
CH2
..
NH2-Protein
V. Nevirapine skin rash
Human skin in response to NVP
treatment
Female rat skin in response to NVP treatment
V. D-Penicillamine
Idiosyncratic reaction:
Autoimmunity, lupus
Reactive Metabolite: None,
parent drug can bind to proteins
through the thiol group
H2N
COOH
C CH3
HS
CH3
Protein Binding: Yes
Animal Model: Yes; Autoimmunity
in the male Brown Norway rat
Forming mixed disulfides
V. Clozapine
Idiosyncratic reaction:
Agranulocytosis, Liver Toxicity,
Cardiac Toxicity
CH3
N
N
N
Cl
N
H
Reactive Metabolite: Yes
CH3
N
Protein Binding: Yes
Animal Model: No
N
N
Cl
Nitrenium Ion
N
+
V. Carbamazepine
Idiosyncratic reaction:
Anticonvulsant hypersensitivity
syndrome (fever, rash, multiorgan involvement etc.)
N
O
NH2
Reactive Metabolite: Yes
Protein Binding: Yes
Animal Model: No
O
N
Iminoquinone
VI. Methods
Ideally want to illustrate each step for each drug:
1. Metabolism
2. Reactive Metabolite Formation
3. Protein Binding in Target Tissue(s)
4. Immunogenicity of Hapten
5. Immune Response  IDR
VI. Step 1: Metabolism – Microsomes
Excise liver
Centrifuge
100,000 x g
cytosol
microsomes
Mince liver in
sucrose buffer
Homogenize
Centrifuge
10,000 x g
S9 fraction
nuclei, cell
membrane
mitochondria
VI. Step 1: Metabolism – Microsomes
a) Buffer (physiological
conditions, salt/ pH7.4)
b) Microsomes
c) Drug
d) NADPH generating
system (NADP+, G6PD,
G6P)
Incubate at
37C
Analyze reaction
mixture by HPLC or
LC/MS
Confirm identity of
products with NMR
(require pure
metabolite)
VI. Step 1: Metabolism - Neutrophils
Obtain (human /
rat) blood
Sediment RBCs
with dextran
Upper solution is placed
on top of a ficoll solution
(density gradient)
Plasma, WBCs
RBCs
Pour off upper
layers, use
remaining
neutrophils
lymphocytes
neutrophils
Ficoll
Centrifuge
~1000 rpm
VI. Step 1: Metabolism - Neutrophils
a) Buffer (physiological
conditions, salt/ pH7.4)
b) Neutrophils
c) Drug
d) Neutrophil activator
(PMA)
Incubate at
37C
Analyze reaction
mixture by HPLC or
LC/MS
Confirm identity of
products with NMR
(require pure
metabolite)
VI. Step 2: RM Formation
Complete same experiments as when looking at
metabolism but with an additional step
Reactive metabolite may be so reactive that it is not
detected on the HPLC chromatogram
Must add GSH or NAC to the reaction mixture to trap the
reactive metabolite in a stable form that can be detected by
HPLC and later identified by LC/MS and NMR



SG
N
N
N
N
N
N
N
2B6
N
O
H
N
N
+
N
N
O
OH
N
CH3
O
H
N
N
CH3
O
H
CH3
OH
GSH
N
O
CH3
H
VI. Step 3: Protein Binding in Target
Tissues

Require an antibody that recognizes the reactive metabolite
(the hapten)

Must prepare antigen by linking the reactive metabolite to an
immunogenic carrier protein e.g., KLH

Immunize rabbits with this antigen

Sera obtained from the blood of these rabbits is polyclonal, and
contains antibodies against the hapten
VI. Step 3 Cont’d

Complete in vivo and in vitro studies

in vitro studies are similar to metabolism studies

in vivo studies involve administering the drug to
animals (rats or mice)
In Vivo:
Administer
drug to rats
or mice
Isolate and
homogenize target
tissues
VI. Step 3 Cont’d
Take tissues from either in vitro or in vivo experiment and
perform Western blot analysis to detect covalent binding
of reactive metabolites to proteins:
•
•
•
•
Run the protein sample on an SDS polyacrylamide
gel
Transfer separated proteins from gel to nitrocellulose
membrane
Blot membrane with an antibody against the HAPTEN
Visualize antibody binding with a detection system;
presence of covalent adducts will thus be elucidated
VI. Animal Models in Study of IDRs




Basically impossible to run prospective clinical trials
• Unpredictable nature of IDRs
• Ethics
Reactions likely involve differences in
metabolism/detoxification of reactive metabolites,
various aspects of the immune system and perhaps
other systems
Can not effectively study such complex systems
in vitro
Lack of animals because IDRs are just as idiosyncratic
in animals as it is in humans
VI. Nevirapine Animal Model
Nevirapine
Idiosyncratic skin rash in ~17% of HIV
patients in clinical trials
Skin lesions in some strains of rats:
keratinocyte death, sloughing of skin
Nevirapine-Induced Skin Rash in the Female Brown Norway Rat
Animal
Model?
YES!
VI. Hopes for the Future
To elucidate the
mechanism(s)
 predictability
 morbidity and
mortality
Summary
 IDRs are serious and potentially life-threatening ADRs
 Quite often formation of drug RMs triggers IDRs
 RMs are most often formed in liver, bone marrow
(peripheral neutrophils), skin and lungs
 Once formed, RMs bind to nearby tissue entities, inducing
immune response and triggering IDRs
Discuss:

Compare and contrast the use of
animal models versus in vitro tests in
the study of IDRs.