Idiosyncratic Drug Reactions What are They, Why How Do We Study Them

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

2. Overview Adverse Drug Reactions (ADRs) Idiosyncratic Drug Reactions (IDRs) Characteristics of IDRs Proposed Mechanism of IDRs Drugs Known to Induce IDRs Studying IDRs Future Directions

3. I. Adverse Drug Reactions

4. 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

5. I. Adverse Drug Reactions

6. I. Adverse Drug Reactions ADRs can be divided into two basic types: Type A: Can be predicted from the pharmacology of the drug Are typically dose-dependent Type B: Cannot be predicted on the basis of the known pharmacology of the drug Also known as idiosyncratic adverse reactions

7. 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

8. 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

10. 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

11. 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

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

14. Generation of Halothane Reactive Metabolite and Covalent Binding to Protein IV. Example of Covalent Binding

15. 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.

16. Once formed, reactive metabolites tend to bind to the proteins or macromolecules near the site of their formation. Thus, toxicity most often occurs at sites of RM formation. 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?

17. 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

19. IV. IDR Characteristics that Indicate Immune Involvement

20. Penicillin-induced anaphylaxis Aminopyrine-induced agranulocytosis Halothane-induced hepatitis V. Clinical Evidence in Support of Hapten Hypothesis

23. V. Aminopyrine-Induced Agranulocytosis

24. V. Drugs Known to Cause IDRs

25. V. Felbamate

26. V. Nevirapine

27. V. D-Penicillamine

28. V. Clozapine

29. V. Carbamazepine

31. VI. Step 1: Metabolism � Microsomes

32. VI. Step 1: Metabolism � Microsomes

33. VI. Step 1: Metabolism - Neutrophils

34. VI. Step 1: Metabolism - Neutrophils

35. 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

36. 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

37. 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)

38. 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

39. VI. Step 4: Hapten Immunogenicity

40. VI. Animal Models in Study of IDRs

41. VI. Nevirapine Animal Model

42. VI. Hopes for the Future

43. Summary