basic immunology 101
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Basic Immunology 101
Amy SharmaPh.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 non-
specific) 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
Questions
List 5 types of immune cells discussed in lecture and name the primary functions of each.
List the two “arms” of the immune system and describe the function of each arm.
Idiosyncratic Drug ReactionsWhat are They, Why & How Do We Study Them?
Amy SharmaPh.D. Candidate Uetrecht Laboratory
Leslie Dan Faculty of Pharmacy, University of Toronto
Overview
I. Adverse Drug Reactions (ADRs)II. Idiosyncratic Drug Reactions (IDRs)III. Characteristics of IDRsIV. Proposed Mechanism of IDRsV. Drugs Known to Induce IDRsVI. Studying IDRsVII. Future Directions
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
I. Adverse Drug ReactionsAdverse 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
Rare & unpredictable reactions • 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
Do not occur in most patients at any dose• No simple dose-response relationship
Effects not related to pharmacological properties of the drug
Can be very severe• most serious ADRs in drug therapy
II. Idiosyncratic Drug Reactions
Organs affected:
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. Characteristics of IDRs
Liver
(cholestatic liver) Skin
(mild-severe rash)
Bone marrow / Blood cells
(aplastic anemia; agranulocytosis)
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 clozapine-induced agranulocytos is 20% in a Jewish hospital vs. <1% elsewhere
Gender - female >> male
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. Mechanisms of IDRs
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
IV. Step 1: Reactive Metabolite Formation
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,
PeroxidasesWhite blood cells (macrophages and
neutrophils) that become activated to kill bacteria, and do so by releasing oxidants such as H2O2
and HOCl.
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. Where Does Metabolism Occur?
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. Step 2: Immune Response
IV. Hapten Hypothesis Detailed
IDR
Step 1 – Reactive Metabolite Formation
Step 2 – T-cell activation and Initiation of an Immune Response
IV. T-cell Activation
Not all IDRs have these characteristics
1. Reaction takes several weeks to develop
3. On re-exposure the time to onset is shorter than on first exposure
2. Once the drug is removed, reaction clears quickly
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)
IV. IDR Characteristics that Indicate Immune Involvement
Penicillin-induced anaphylaxis
Aminopyrine-induced agranulocytosis
Halothane-induced hepatitis
V. Clinical Evidence in Support of Hapten Hypothesis
Covalent binding due to spontaneous ring opening IgE antibodies were detected in patients with
anaphylactic reaction Re-exposure can be life-threatening
V. Penicillin-Induced Anaphylaxis
CH2
C NHO
NO
S CH3
CH3
COOH
CH2
C NHO
C HN
S CH3
CH3
COOHNHlys
Protein
O
Benzylpenicillin
-lactam ring
N N
O
CH3
CH3
NCH3CH3
N N
O
CH3
CH3
NCH3CH3
Myeloperoxidase/H2O2/Cl-
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. Aminopyrine-Induced Agranulocytosis
Dication intermediate
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. Halothane-Induced Hepatitis
Trifluoroacetyl Chloride
RM covalently bound to protein
C CF
FF
Br
H
Cl C C
F
F
F
OP4502E1
Halothane
Protein
Cl NHC C
F
F
F
O
Felbamate
Clozapine
Carbamazepine
D-Penicillamine
Nevirapine
antiepileptic
HIV drug (NNRTI)
antipsychotic
anticonvulsant
anti-rheumatic
V. Drugs Known to Cause IDRs
V. Felbamate
O
O
CNH2
CNH2O
O
OH
Idiosyncratic reactions:Aplastic Anemia and Liver Toxicity
Animal Model: No
Reactive Metabolite: Yes
Protein Binding: Probable
Phenylacrolein (Michael Acceptor)
V. Nevirapine
N
N
NN
O HCH3
Idiosyncratic reaction:Severe Skin Rash, Liver Toxicity
Animal Model: Yes; Skin rash in the female Brown Norway rat
Reactive Metabolite: quinone methide
Protein Binding: Yes; epidermis
NN
N
N
OCH2
NH2-Protein..Quinone Methide
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
Animal Model: Yes; Autoimmunity in the male Brown Norway rat
Reactive Metabolite: None, parent drug can bind to proteins through the thiol group
Protein Binding: Yes
H2N
C
COOH
HS CH3
CH3
Forming mixed disulfides
V. Clozapine
N
NN
Cl
H
NCH3
N
NN
Cl
NCH3
+
Idiosyncratic reaction: Agranulocytosis, Liver Toxicity, Cardiac Toxicity
Animal Model: No
Reactive Metabolite: Yes
Protein Binding: Yes
Nitrenium Ion
V. Carbamazepine
N
NH2O
N
O
Idiosyncratic reaction: Anticonvulsant hypersensitivity syndrome (fever, rash, multi-organ involvement etc.)
Reactive Metabolite: YesProtein Binding: Yes
Animal Model: No
Iminoquinone
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. Methods
VI. Step 1: Metabolism – Microsomes
Excise liverMince liver in sucrose buffer Homogenize
Centrifuge 100,000 x g
Centrifuge 10,000 x g
nuclei, cell membrane mitochondria
S9 fractionmicrosomescytosol
VI. Step 1: Metabolism – Microsomes
Incubate at 37C
Analyze reaction mixture by HPLC or LC/MS
Confirm identity of products with NMR (require pure metabolite)
b) Microsomes
c) Drug
d) NADPH generating system (NADP+, G6PD, G6P)
a) Buffer (physiological conditions, salt/ pH7.4)
VI. Step 1: Metabolism - Neutrophils Obtain (human /
rat) bloodSediment RBCs
with dextran Upper solution is placed on top of a ficoll solution
(density gradient)
Centrifuge ~1000 rpm
Pour off upper layers, use remaining neutrophils
RBCs
Plasma, WBCsFicoll
neutrophilslymphocytes
VI. Step 1: Metabolism - Neutrophils
Incubate at 37C
Analyze reaction mixture by HPLC or LC/MS
Confirm identity of products with NMR (require pure metabolite)
d) Neutrophil activator (PMA)
b) Neutrophils
c) Drug
a) Buffer (physiological conditions, salt/ pH7.4)
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
N
N
NN
OH
CH3
N
N
NN
OH
CH3
OHN
N
NN
OH
CH3
ON
N
NN
OH
CH3
OH
SG
+
2B6
GSH
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
Animal Model?
YES!
Nevirapine-Induced Skin Rash in the Female Brown Norway Rat
VI. Hopes for the Future
To elucidate the mechanism(s)
predictability
morbidity and mortality
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
Summary
Questions
Compare and contrast the use of animal models versus in vitro tests in the study of IDRs.
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