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Basic Immunology 101

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Basic Immunology 101 Amy Sharma Ph.D. Candidate Uetrecht Laboratory Leslie Dan Faculty of Pharmacy, University of Toronto – PowerPoint PPT presentation

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Title: Basic Immunology 101


1
Basic Immunology 101
  • Amy Sharma
  • Ph.D. Candidate
  • Uetrecht Laboratory
  • Leslie Dan Faculty of Pharmacy, University of
    Toronto

2
Q. Why does your immune system exist?
3
Immunology Overview
  • Immune system is like a double edged sword
  • Key players of the immune system
  • Humoral versus Cellular Immunity

4
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

5
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

6
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

7
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

8
I. Cells of the immune system
  • Natural Killer NK cells (LGLs)
  • 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

9
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10
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

11
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12
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

13
Idiosyncratic Drug ReactionsWhat are They, Why
How Do We Study Them?
  • Amy Sharma
  • Ph.D. Candidate
  • Uetrecht Laboratory
  • Leslie Dan Faculty of Pharmacy, University of
    Toronto

14
Overview
  1. Adverse Drug Reactions (ADRs)
  2. Idiosyncratic Drug Reactions (IDRs)
  3. Characteristics of IDRs
  4. Proposed Mechanism of IDRs
  5. Drugs Known to Induce IDRs
  6. Studying IDRs
  7. Future Directions

15
I. Adverse Drug Reactions
16
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)

17
I. Adverse Drug Reactions
18
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

19
II. Idiosyncratic Drug Reactions
  • 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

20
III. Characteristics of IDRs
  • 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

Liver (cholestatic liver)
Skin (mild-severe rash)
21
III. Risk Factors for IDRs
  • Dont 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. lt1 elsewhere
  • Gender - female gtgt male

22
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

23
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

24
Reactive Metabolites
  • Reactive metabolites are electrophiles or free
    radicals
  • Sulfates/sulfonates
  • Epoxides/arene oxides
  • Michael Acceptors
  • Nitroso amines

25
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.

26
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).

27
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.

28
IV. Hapten Hypothesis Detailed
Step 1 Reactive Metabolite Formation
Step 2 T-cell activation and Initiation of an
Immune Response
IDR
29
IV. T-cell Activation
30
IV. IDR Characteristics that Indicate Immune
Involvement
Not all IDRs have these characteristics
31
V. Clinical Evidence in Support of Hapten
Hypothesis
  • Penicillin-induced anaphylaxis
  • Aminopyrine-induced agranulocytosis
  • Halothane-induced hepatitis

32
V. Penicillin-Induced Anaphylaxis
  • Covalent binding due to spontaneous ring opening
  • IgE antibodies were detected in patients with
    anaphylactic reaction
  • Re-exposure can be life-threatening

33
V. Aminopyrine-Induced Agranulocytosis
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

34
V. Halothane-Induced Hepatitis
  • 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)

35
V. Drugs Known to Cause IDRs
Felbamate
antiepileptic
Nevirapine
HIV drug (NNRTI)
D-Penicillamine
anti-rheumatic
Clozapine
antipsychotic
Carbamazepine
anticonvulsant
36
V. Felbamate
Phenylacrolein (Michael Acceptor)
37
V. Nevirapine
Quinone Methide
38
V. Nevirapine skin rash
Human skin in response to NVP treatment
Female rat skin in response to NVP treatment
39
V. D-Penicillamine
Forming mixed disulfides
40
V. Clozapine
Nitrenium Ion
41
V. Carbamazepine
Iminoquinone
42
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
43
VI. Step 1 Metabolism Microsomes
Mince liver in sucrose buffer
Homogenize
Excise liver
Centrifuge 100,000 x g
Centrifuge 10,000 x g
cytosol
S9 fraction
microsomes
nuclei, cell membrane mitochondria
44
VI. Step 1 Metabolism Microsomes
45
VI. Step 1 Metabolism - Neutrophils
46
VI. Step 1 Metabolism - Neutrophils
47
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

48
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

49
VI. Step 3 Contd
  • 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)

50
VI. Step 3 Contd
  • 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

51
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

52
VI. Nevirapine Animal Model
53
VI. Hopes for the Future
54
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

55
Discuss
  • Compare and contrast the use of animal models
    versus in vitro tests in the study of IDRs.
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