18/5/213 Mechanisms of Drug Hypersensitivity Reactions Munir Pirmohamed NHS Chair of Pharmacogenetics Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Definitions and Manifestations Hypersensitivity an inappropriate immune response leading to an inappropriate immune response leading to tissue damage from an otherwise non toxic agent 1
18/5/213 Immune Mediated Adverse Drug Reactions Drug Hypersensitivity Off target toxicity Not predictable from the known pharmacology of a drug Skin is the organ most commonly involved Role of T cells Proliferation of T cells from hypersensitive patients but not non hypersensitive controls Mauri Hellweg et al. J Immunol 155, 462 72 1995. Prominent cutaneous infiltration of CD4+ and CD8+ T cells Yawalkar et al. Clin Exp Allergy 3, 847 55, 2. Drug induced liver injury Stevens Johnson syndrome Toxic epidermal necrolysis Uetrecht & Naisbitt Pharmacol Rev, 65:779 88, 213 Pichler, Naisbitt & Park J Allergy Clin Immunol, 127:74 81, 211 2
18/5/213 Genetic Restriction and Drug Hypersensitivity: Discovery of HLA allele associations Discovery of associations between HLA alleles and drug hypersensitivity represents an important advance Screening for HLA alleles during clinical practice effectively prevents reactions Abacavir Carbamazepine Mechanistic studies using samples from volunteers with HLA risk alleles may shed light on the immune pathogenesis Pharmacogenetics and Clinical Syndromes Abacavir HLA B * 571 OR = 132 hypersensitivity Flucloxacillin HLA B * 571 OR = 72 DILI Carbamazepine HLA B * 152 (Chinese) OR = 1 SYS/TEN Carbamazepine HLA A*311 (Japanese) OR = 11 Hypersensitivity Carbamazepine HLA A*311 (Caucasians) OR = 3 Hypersensitivity Lumiracoxib HLA DRB1*151 OR = 7 DILI HLA DQA1*12 Ximelagatran HLA DRB1*71 OR = 4.4 Lapatinib HLA BQA1*21 OR = 9. DILI Mallal, 28; Kindmark et al., 28; Daly et al., 29; Chung et al., 24; McCormack et al., 211; Singer et al., 21; Spraggs et al., 211 Delayed Hypersensitivity Reactions (1) Maculopapular Eruption (MPE) Commonest type of cutaneous eruption Drug withdrawal Symptomatic and supportive treatment 3
C es 18/5/213 Delayed Hypersensitivity Reactions (2) Hypersensitivity Syndrome Systemic manifestations estato s Fever, eosinophillia Variable extracutaneous involvement Liver commonest to be affected Anticonvulsant Hypersensitivity Syndrome: Clinical Characteristics Carbamazepine (n=35) Clinical Feature Pneumonitis Aplastic anaemia Thrombocytopenia Lymphadenopathy Liver toxicity Eosinophilia Fever Lamotrigine (n=34) Skin rash N C O NH 2 Carbamazepine C l C l N N 2 4 6 8 1 % of patients NH 2 N NH 2 Lamotrigine 4
18/5/213 Delayed Hypersensitivity Reactions (3) Stevens Johnson Syndrome 1 1% 1% of skin blistered Two mucous membranes involved 1% mortality Delayed Hypersensitivity Reactions (4) Toxic Epidermal Necrolysis >3% of skin blistered Two mucous membranes involved 3% mortality 5
18/5/213 Delayed Hypersensitivity Cutaneous Reactions Maculopapular exanthem Hypersensitivity syndrome Stevens Johnson Syndrome Severity Mortality Rarity Toxic Epidermal Necrolysis Phenotypic Heterogeneity Skin Liver Kidney Phenotypic heterogeneity common in drug allergies Same drug can cause different manifestations in different patients Within the same organ, clinical picture can vary Within different patients, a number of organs can be affected Important to have clear phenotypic criteria to allow for phenotype standardisation 6
18/5/213 Phenotype is Crucial Clinical Pharmacology and Therapeutics, June 211 Cellular pathophysiology of drug hypersensitivity reactions in skin: characterization of T cell clones Drug T cell CD4+ T cell Revised classification of drug hypersensitivity in skin Proliferation Maculopapular TCR exanthema Bullous Pustular MHC I & II Drug Drug presented antigen on MHC class presenting II cell Drug presented on MHC class I CD4+ > CD8+ CD8+ > CD4+ cytotoxicity (CD4+) cytotoxicity (CD8+) IL 5 > IFN gamma IFN gamma > IL 5 Tissue damage in skin Clonal Drug presented on MHC expansion class II CD4+ and CD8+ cytotoxicity (CD4+ & CD8+) IL 8, IL 5 and IFN gamma CD8+ T cell Anticonvulsant hypersensitivity Drug presented on MHC class I & II CD4+, CD8+ and CD4+CD8+ cytotoxicity (CD8+) IFN gamma, MIP1beta, RANTES CLA, CCR4, CCR8, CCR1 Pichler et al. Am J Clin Dermatol IL 5 3, 229 38, IL 13 22 Pichler Ann Intern Med 139, 683 93, 23 Naisbitt et al. Curr Allergy Asthma Rep 3, 22 9, IL 4 IFN γ 23 Naisbitt et al. Expert Opin perforin Drug Saf. 6, 19 24, 27 granzymeb IL 17 MIP 1β TNF alpha granulysin FasL Uetrecht & Naisbitt Pharmacol Rev, 65:779 88, 213 IL 1 IL 22 Pichler, Naisbitt & Park JACI, 127:74 81, 211 Th1/2/17/22 response Cytotoxicity 7
18/5/213 The Hapten Hypothesis Small compounds (<1Da) incomplete antigen Covalent binding Reactive chemical Cellular Soluble (serum) Protein conjugate Immune recognition Antigen presentation Cellular Response Immune recognition PI mechanism Pathways of T-cell activation Hapten mechanism TCR TCR Drug Reactive species MHC MHC Drug Reactive species Protein Reactive species Nature of the binding interaction Non-covalent Covalent 8
18/5/213 Abacavir Hypersensitivity: Translational Paradigm Discovery of HLA B*571 Replication Demonstration of cost effectiveness Randomised controlled trial (PREDICT 1) Implementation (UK from 26) Change in Peptide Repertoire 9
18/5/213 HLA B*57:1 and Abacavir Hypersensitivity Peptides from untreated cells standard peptide profile ABC treated cells show novel self peptides (2 25%) 25%) with Ile/Leu occupying C terminal anchor ptotein No change in peptide profile with closely related allotypes Illing et al, 213, Curr Opin Immunol Immunogenic HLA B*57:1 peptide complexes (after Illing et al, 213) 1
18/5/213 Patients with DRESS due to carbamazepine, allopurinol, sulfamethoxazole EBV, HHV 6 and HHV 7 reactivation in 76% Increased secretion of cutaneous homing markers, TNF α, IFNgamma CD8+ T cells recognised several EBV epitopes Culprit drugs triggered production of EBV from patient B cells?link between altered peptide repertoire and viral reactivation Nature Genetics, 29 52 cases NNT for abacavir 12 NNT for flucloxacillin 13,819 Illumina 1 million SNP array Strong (P=1 3 ) association with SNP in LD with HLA B*57:1 Collaboration between UK DILIGEN and SAEC 11
18/5/213 Why HLA B*57:1? Abacavir Flucloxacillin 1. 8.5 in 1 patients 2. Elderly and prolonged course Chance finding or is there a common mechanism? Flucloxacillin Binding to HSA Lys19 Lys212 12
18/5/213 Flucloxacillin Induced Liver Injury Flucloxacillin forms an antigen in patients Jenkins et al., 29 Lys19 Nature Genetics 29 MISSING LINK: Lys212 Albumin conjugates in Evidence to show that reactions 8/8 patients (Lys 19, 212) to flucloxacillin are edriven by drug specific activation of cytotoxic CD8+ T lymphocytes Association between the expression of HLA B*571 and liver injury 4/64 controls 63/74 cases HLA B*571 restricted activation of flucloxacillin responsive T cells: The immunogenetic basis for DILI Patient cohort: Six patients with clinically well-defined flucloxacillin-mediated liver injury Phase 1 study: LTT negative in all patients IFN-gamma ELIspot positive in 5/6 patients IFN γ ELIspot Patient 178 Patient 18 Ø Ø flucloxacillin Patient 178 flucloxacillin IFN γγ IL 13 granzymeb Ø Monshi et al., Hepatology, 57, 727 39, 213 flucloxacillin 13
18/5/213 HLA B*571 restricted activation of flucloxacillin responsive T cells: The immunogenetic basis for DILI Patient cohort: Six patients with clinically well-defined flucloxacillin-mediated liver injury Phase 1 study: LTT negative in all patients IFN-gamma ELIspot positive in 5/6 patients Phase 2 study: Characterization of drugspecific cytotoxic T-cells Generation and characterization of T-cell clones Role of the HLA risk allele in the immune response Monshi et al., Hepatology, 57, 727 39, 213 Proliferation (cpmx1 3 ) 14 12 1 8 6 4 2 14 12 1 8 6 4 2 8 6 4 2 CD4+ clone 1 2 CD4+ clone 1 2 CD8+ clone 1 2 12 1 8 6 4 CD8+ clone 2 1 2 3 25 2 15 1 5 CD8+ clone 1 2 Flucloxacillin (mm) ELIspot (sfu) 5 4 3 2 1 5 4 3 2 1 5 4 3 2 1 5 4 3 2 1 5 4 3 2 1 Cytotoxic T cell clones IFN γ perforin granzymeb IL 5.5 1 1.5 2.5 1 1.5 2.5 1 1.5 2.5 1 1.5 2.5 1 1.5 2 Flucloxacillin (mm) FasL IL 13 IFN γ flucloxacillin flucloxacillin flucloxacillin flucloxacillin flucloxacillin granzymeb FasL perforin IL 5 IL 13 HLA B*571 Restricted Activation of Flucloxacillin Responsive T cells: The Immunogenetic Basis for DILI Patient cohort: Six patients with clinically well-defined flucloxacillin-mediated liver injury Pilot study: LTT negative in all patients IFN-gamma ELIspot positive in 5/6 patients Phase 2 study: on-going Characterization of drugspecific cytotoxic T-cells Generation and characterization of T-cell clones Role of the HLA risk allele in the immune response Proliferation (cpmx1 3 ) Proliferation (cpmx1 3 ) 3 2 1 3 25 2 15 1 2 15 1 5 5 Phenotype of Clones Patient HLA B allele IFN gamma ELIspot No. Of Flu specific clones CD4+ CD8+ 1 571 2 5 571 + 3 35 6 442 / 551 + 7 1 2 HLA restriction with CD8+ clones No antibody + anti class I antibody + anti class II antibody 35 3 25 2 15 1 5 1 2 1 2 Flucloxacillin (mm) 8 6 4 2 4 3 2 1 1 2 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 Autologous 1 2 3 (1) (2) (3) (1) (2) (3) (3) (1) (4) (2) No 21 26 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 22 23 24 25 27 EBV s EBVs B*571 B*581 Other B alleles 14
18/5/213 Detection of Drug Specific T cell Responses in HLA Typed Drug Naïve Volunteers Dissemination of results HLA typed volunteer cohort READOUTS proliferation cytokine secretion cell surface markers Cell bank containing functional lymphocytes Provision of costimulatory signals through endogenous activators Proliferation assay against suspect chemicals Volunteer Demographics (N=4) Age Mean ±SD Range 29 ±1 years 18 6 years Gender Female Male 64% 36% 1 Fr requency 8 6 4 2 18 22 26 3 34 38 42 46 5 54 58 62 Age White Indian Other Chinese Black 15
18/5/213 HLA B*57:1 Restricted Activation of Flucloxacillin Responsive T cells: The Immunogenetic Basis for DILI Volunteer cohort: Frozen PBMC from 26 drug-naive individuals expressing HLA-B*571 CD14 selection B*57:1 PBMC 7 days Flucloxacillin DCs DCs 8 days IFN γ ELIspot Naive T cell selection Flucloxacillin 125 Flucloxacillin 25 5 Proliferation (cpm) 4 31 CD8+ 35 flucloxacillinresponsive 3 T cell clones 25 2 15 1 5 125 25 5 1 Flucloxacillin 2 3 Flucloxacillin: 3 patients Patient 1 Cholestatic hepatitis after 2 weeks of flucloxacillin Confirmed on biopsy T cell HLA B*57:1 Patient 2 Acute generalised exanthematous pustulosis Rechallenge with recurrence of reaction T cell IL8 HLA B*57:1 Patient 3 Anaphylaxis Positive skin and intradermal testing IgE HLA B*57:1 What factor(s) determine phenotype specificity? 16
18/5/213 Key knowledge gaps Frequency / Severity of Drug Hypersensitivity Chemistry Biology 1 = f 1 + f of drug 2 1of individual Relationship between drug protein binding and immunogenicity is not defined 2 The importance of genetic (HLA) predisposing factors (do they relate to the T cell response?) The immunological processes responsible for sensitization and tolerance are not defined 3 4 How T cells cause different forms of severe skin injury is not known Acknowledgements The University of Liverpool B Kevin Park Ana Alfirevic Dean Naisbitt Andrea Jorgensen Javier Vilar Dyfrig Hughes Stephane Bourgeois (Sanger Institute) Panagiotis Deloukas (Sanger Institute) Ann Daly and other DILIGEN members SERIOUS ADVERSE EVENT CONSORTIUM EPIGEN EUDRAGENE Funders: MRC, WT, DH, NIHR, EU FP7 17