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.