Pharmacogenomics

Pharmacogenomics

1. Pharmacogenomics Dr Shinde Viraj Ashok Junior Resident Department of Pharmacology

2. Overview

3. Definitions Pharmacogenetics – study of genetic basis for variation in drug response Pharmacogenomics – makes use of genetic make up (genome) of an individual to choose drug therapy for responders and avoid giving such drug to non responders ( i.e tailoring of drug therapy on the basis of individuals genotype)

4. Allele – one of two alternative forms of gene that arise by mutation & are found at same gentic locus Each star (*) allele is defined as specific sqeuence variation(s) with in gene locus e.g single nucleotide polymorphisms Single nucleotide polymorphism – substitution of one DNA unit for another at a particular site

5. Sum of allelic activity score ranges from 0 & ≥ 3 and this used to define phenotypes as poor metabolisers = 0 intermediate metabolisers = 0.5 extensive metabolisers = 1 – 2 ultra rapid metabolisers = ≥ 2 CPIC – clinical pharmacogenetics implementation consortium

6. Pharmacogenomics Concept

7. Drug Response Individual Variability in Drug Therapy Factors Affecting Individual Drug Response Genetic Polymorphisms of Drug Targets Genetic Polymorphisms of Drug-Metabolizing Enzymes Genetic Polymorphisms of Drug Transporters Genetic Variables Affecting Adverse Drug Reactions

8. Genetic variations in enzymes Phase I enzymes : Biotransformation - Over 75% of prescribed drugs Polymorphisms of phase I enzymes Significantly affect blood levels of drugs Which in turn may alter response to many drugs

9. CYP2D6 Metabolism - up to one quarter of all drugs used clinically (ß blockers, antidepressants , antipsychotics & opioid analgesics) Highly polymorphic over 100 alleles defined ie , CYP2D6 alleles *3, *4, *5, and * 6 - non-functional ; alleles *10, *17, and *41 - reduced function; alleles *1 and *2 - fully functional.

10. Poor metabolisers(PMs) & Intermediate metabolisers (IMs) - likely to experience insufficient pain relief Ultra rapid metabolisers (UMs) – ↑ risk for side effects Due to higher systemic concentrations of morphine Eg - Drowsiness and respiratory depression CPIC guidelines recommend – EMs & IMs - standard starting doses with close monitoring, especially in IMs PMs and UMs - alternative agent e.g. morphine , non opoid analgesic

11. CYP2C19 Metabolize acidic drugs including Proton-pump inhibitors Antidepressants Antiepileptics Antiplatelet drugs Four clinical phenotypes - based on activity of CYP2C19 Poor metabolisers(PM) Intermediate metabolisers (IM) Extensive metabolisers(EM) Ultra rapid metabolisers(UM) Highly polymorphic over 30 alleles defined

12. Poor metabolisers(PM) phenotype Asians (~16%) Europeans and Africans (~2–5 %) Carriers of reduced function CYP2C19*2 alleles taking clopidogrel are at ↑ risk for serious adverse cardiovascular events

13. Clopidogrel - thienopyridine antiplatelet prodrug indicated for prevention of atherothrombotic events 85% - Hepatic esterases - Inactive carboxylic acid derivative 15 % two sequential cyp -mediated oxidation reactions (predominantly CYP2C19) active thiol metabolite - antiplatelet activity Genetic polymorphisms CYP2C19 gene – ↓ active metabolite formation & reduce drug’s antiplatelet activity associated with variability in response to clopidogrel Current clinical recommendations from Clinical Pharmacogenetics Implementation Consortium(CPIC) specific for acute coronary syndrome with percutaneous coronary intervention (PCI ) Standard starting doses are recommended in extensive metabolisers(EMs) and ultra rapid metabolisers (UMs) Use of an alternative antiplatelet agent in poor metabolisers (PMs)and intermediate metabolisers (IMs) Eg , prasugrel or ticagrelor

14. Dihydropyrimidine Dehydrogenase (DPD) Encoded by DPYD gene – First and rate-limiting step in pyrimidine catabolism Major elimination route for fluoropyrimidine chemotherapy agents Three non-functional alleles - rare - ie , DPYD*2A, * 13 & rs67376798 Allele * 2A most commonly observed allele & is often only variant tested in commercial genotyping platform

15. Example 5-fluorouracil (active compound)( 5-FU ) ; capecitabine & tegafur (oral prodrugs ) To treat solid tumors ( colorectal and breast cancer) Capecitabine & tegafur - Only 1–3% administered dose of prodrug is Converted to active cytotoxic metabolites, ie , 5-fluorouridine 5'monophosphate (5-FUMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) Effectively target rapidly dividing cancer cells and inhibit DNA synthesis Majority of an administered dose (~80%) is subjected to pyrimidine catabolism via DPD & is excreted in urine

16. Complete or partial deficiency of DPD can lead to ↑ Half-life of toxic metabolites F-UMP and f-dump ↑ risk for severe dose-dependent fluoropyrimidine toxicities Eg , myelosuppression , mucositis , neurotoxicity, hand-and-foot syndrome & diarrhoea CPIC guidelines recommend that Normal activity – standard dose Reduced activity – reduce initial dose 50% & titrate based on toxicity or on pharmacokinetic test results Complete deficiency – different non fluoropyrimidine anticancer drugs

17. PHASE II ENZYMES Conjugate endogenous molecules eg sulfuric acid , glucuronic acid & acetic acid, onto a wide variety of substrates in order to enhance their elimination from body Polymorphic phase II enzymes - ↓ drug elimination & ↑ risks for toxicities

18. Uridine 5'-Diphosphoglucuronosyl Transferase 1 (UGT1A1 ) Encoded by the UGT1A1 gene Individuals with UGT1A1*28 /*28 genotype – Associated with reduced expression of UGT1A1 enzyme ↑ risk for adverse drug reactions with UGT1A1 drug substrates Due to ↓ biliary elimination

19. Example Irinotecan – Indicated - treatment of metastatic carcinoma of colon or rectum Hepatic carboxylesterase enzymes → cytotoxic metabolite, SN-38 ( inhibits topoisomerase 1 ) Active SN-38 metabolite is responsible for Majority of therapeutic action Dose-limiting bone marrow and gastrointestinal toxicities Inactivation of SN38 occurs via polymorphic UGT1A1 enzyme and carriers of UGT1A1*28 variant – ↑ risk for severe life-threatening toxicities, eg , neutropenia and diarrhea , due to ↓ clearance of SN-38 metabolites

20. CPIC guidelines Normal activity -*1/*1 , *1/*28 – standard starting dose Reduced activity – *28/*28 - reduce starting dose by at least one dose level

21. Thiopurine S- Methyltransferase (TPMT) Attaches methyl group onto aromatic & heterocyclic sulfhydryl compounds Responsible for pharmacologic deactivation of thiopurine drugs Genetic polymorphisms in gene encoding TPMT - 3 clinical TPMT activity phenotypes High Intermediate & Low activity Associated with differing rates of inactivation of thiopurine drugs & altered risks for toxicities

22. Example Azathioprine, 6-mercaptopurine (6-MP) & 6-thioguanine (6- TG) Azathioprine (a prodrug of 6-MP) and 6-MP - treating immunologic disorders 6-MP and 6-TG ( Anti-cancer agents ) Activated by salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase ( HGPRTase ) to form 6thioguanine nucleotides (TGNs) 6thioguanine nucleotides (TGNs)- responsible for majority of therapeutic efficacy & bone marrow toxicity

23. CPIC guidelines Normal , high activity – Standard starting dose Intermediate activity – Start 30 – 70 % starting dose & titrate every 2-4 weeks with close clinical monitoring of tolerability eg TLC , LFTs Low activity – Malignant disease – drastic reduction of thiopurine doses Non malignant diseases – alternative non thiopurine immunosuppressive agents

24. Other enzymes G6PD Gene that encodes G6PD enzyme is Located on X chromosome Highly polymorphic over 180 genetic variants identified that result in enzyme deficiency Glucose 6-phosphate dehydrogenase (G6PD) First & rate-limiting step in pentose phosphate pathway Supplies a significant amount of reduced NADPH in body Red blood cells (RBCs )- Mitochondria are absent G6PD - exclusive source of NADPH Reduced glutathione - play critical role in prevention of oxidative damage

25. Under normal conditions – G6PD in RBCs is able to detoxify unstable oxygen species While working at just 2% of its theoretical capacity Individuals with G6PD deficiency (WHO classification) Defined as less than 60% enzyme activity are at ↑ risk for abnormal RBC destruction, ie , haemolysis due to ↓ antioxidant capacity under oxidative pressures

26. Classification of G6PD deficiency (WHO Working Group, 1989).

27. Examples Rasburicase Recombinant - urate - oxidase enzyme Initial management of uric acid levels in cancer patients receiving chemotherapy Manufacturer recommends that Patients at high risk (individuals of african or mediterranean ancestry) be screened prior to initiation of therapy Rasburicase not be used in G6PD deficiency

28. GENETIC VARIATIONS IN TRANSPORTERS Plasma membrane transporters Located on epithelial cells of many tissues, eg , intestinal, renal, and hepatic membranes, Mediate selective uptake and efflux of endogenous compounds and xenobiotics including many drug products Genetic differences in transporter genes alter drug disposition and response may ↑ risk for toxicities.

29. ORGANIC ANION TRANSPORTER (OATP1B1) Encoded by the SLCO1B1 gene – Transporter – Located on sinusoidal membrane (facing the blood) of hepatocytes Responsible for hepatic uptake of mainly weakly acidic drugs and endogenous compounds eg statins, methotrexate & bilirubin 40 non-synonymous variants ( nsSNPs ) of this transporter - some of which result ↓ transport function

30. Example Common variant rs4149056 in SLCO1B1, ↑ systemic exposure of simvastatin Identified to have single strongest association with simvastatin-induced myopathy For individuals receiving simvastatin with reduced OATP1B1 function (at least one non-functional Allele), CPIC recommends a lower simvastatin dose or an alternative statin

31. GENETIC VARIATIONS IN IMMUNE SYSTEM FUNCTION Genetic sources of variation - Pharmacodynamic genes (drug receptors and drug targets genes) Example - Polymorphism in HLA loci is associated with a predisposition to drug toxicity.

32. DRUG-INDUCED HYPERSENSITIVITY REACTIONS Worst hypersensitivity reactions are Liver injury Toxic epidermal necrosis (TEN) Stevens- johnson syndrome (SJ S) (Drugs and/or their metabolites form antigens) Drug classes associated sulfonamides nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics , steroids, antiepileptic agents & methotrexate

33. Example Abacavir - Hypersensitivity reactions - SJS (idiosyncratic ) Consistent with allele frequencies of HLA-B*57:01 Activated to carbovir triphosphate -reactive molecule - involved in immunogenicity of abacavir Mediated by activation of cytotoxic CD8 T cells Because of importance of abacavir in therapeutics, genetic testing of the HLA-B*57:01 biomarker associated with abacavir hypersensitivity has been rapidly incorporated into clinical practice

34. Flucloxacillin hypersensitivity reactions – Lead to drug-induced liver toxicity Highly significant association was identified with polymorphism linked to HLAB*57:01

35. IFNL3 ( Il-28B)(INTERFERON LAMBDA 3) rs12979860 variant near IFNL3 is considered strongest baseline predictor of a cure for patients with HCV-1 receiving PEG-IFN-a/ RBV Approximately two fold greater cure rates were observed in patients with a favourable genotype Favourable allele, rs12979860 variant, is inherited most frequently in Asians (~90%), and least frequently in Africans

36. POLYGENIC EFFECTS Polygenic influences – combinatorial effect of multiple genes on drug response, may more accurately describe individual differences with respect to clinical outcomes

37. Example CYP2C9 & VKORC1 on warfarin Allele CYP2C9*2 – Leads to reduced metabolism of CYP2C9 substrates, Including a 30–40% reduction in S-warfarin metabolism Allele CYP2C9*3 – Lowered affinity for many CYP2C9 substrates & More marked 80–90 % reduction in S-warfarin metabolism

38. Most important consequences of VKORC1 polymorphism (VKORC1-1639G>A) Reduced expression of VKORC1 in liver ↑ sensitivity to warfarin ↑ risk for excessive anticoagulation following standard warfarin dosages VKORC1-1639G>A polymorphism occurrence- Asian populations (~90%) & Africans (~10 %) Gene-based dosing may help - Optimize warfarin therapy management and minimize risks for adverse drug reactions

39. Application of Pharmacogenomics in drug development Drug target and pharmacogenomics Drug discovery starts with identification of a potential target at which drug can act Target can be an Enzyme in a vital pathway Receptor Transporter Protein in signal transduction or Any protein produced in a pathological condition After sequencing of human genome, number of drug targets was estimated to be around 8000, out of which 4990 could be actually acted upon - 2329 for antibodies & 794 for drug proteins

40. Examples: Drug Target C-KIT expression in GIST – Imatinib CCR5 -Chemokine C-C motif receptor on human T-cell – Maraviroc EGFR expression - Erlotinib HER2/ neu expression – Trastuzumab

41. Pharmacogenomics and clinical trials Incorporation of pharmacogenomic testing with clinical trials has multiple advantages Two most important concerns for new drug development are efficacy and safety

42. Availability of sophisticated pharmacogenetic tools - Attrition rate can be significantly reduced (reduction in loss of financial resources for drug development) Drug metabolized by polymorphic enzymes Can be identified during preclinical studies with in vitro methods Decision regarding continuation of trial can be made

43. Prediction of efficacy of drug Drugs designed with pharmacogenomic support have predetermined efficacy status Chance of a drug failing in preclinical & clinical studies due to absence of efficacy is minimized

44. Example , Drug trastuzumab – Anti-HER2 monoclonal antibody against metastatic breast cancer Found to be effective only in women over expressing HER2 protein during early clinical trials In subsequent trials - studies were done only on women found to be over expressing HER2 protein Approval for marketing - before starting therapy testing for HER2 over expression must be done

45. Pharmacogenomics – Used to identify target population that would benefit most from drug Example – Association between polymorphisms in apolipoprotein E (APOE ) cholesteryl ester transfer protein(CETP ) stromelysin-1 & ß-fibrinogen with progression of atherosclerosis , cardiovascular events & death People with such polymorphisms derived maximum benefit from HMG-CoA inhibitors , as compared to those without polymorphisms

46. Prediction of safety of drug During a clinical trial – Occurrence of a serious adverse event could jeopardize drug status Such an event would culminate in termination of clinical trial Drug toxicity Mainly due to ↑ plasma levels of drug Result of poor metabolizing capacity owing to genetic polymorphisms

47. Availability of high throughput genotyping methods, pharmacogenetic testing can be incorporated into inclusion criteria for selecting a subject for trial Poor metabolizers - Tend to attain higher plasma concentration of drug Higher incidence of toxicity If poor metabolizers are avoided in study occurrence of serious adverse events - reduced

48. Issues of concern in Pharmacogenomics Practical application in routine patient care is at present limited due to pre requirement of multiple drug specific genotyping screening which involves huge cost Will lead to ‘discrimination’ in medical therapy provided to a patient – treatment will be based which in turn correlated with ethnic and racial factors

49. Rare genotypes(orphan genotypes) – deprived of health care – may not enjoy insurance cover Pharmaceutical companies – will not be willing to invest in developing drugs for groups with less common genotype – creation of ‘therapeutic orphans’

50. Pharmacogenomics in India Department of Biotechnology, Ministry of Science and Technology, New Delhi Human Genetics and Genome Analysis Programme. Human Genome Diversity Project

51. Regulatory Guidance FDA: Guidance for Industry - Pharmacogenomic Data Submissions EMEA ( european medical agency )

52. Conlclusion Therapeutic response in many disease processes is genotype-specific and multifactorial Tailoring medication regimens based on patient genomes maximizes efficacy and compliance while avoiding adverse effects and drug-drug interactions

53. References Katzungs basic and clinical pharmacology 13 th edition Role of pharmacogenomics in drug discovery and development Department of Pharmacology, JIPMER , Puducherry , India, A . Surendiran , S.C. Pradhan , C. Adithan India, Department of Biotechnology, Ministry of Science and Technology, (2013) List of Ongoing Projects as on 30/05/2013

54. Examples: Drug Metabolism CYP2C19 and CYP2D6 Variants – Poor vs extensive metabolizers N- acetyltransferase - slow and fast acetylators Deficiency of dihydropyrimidine dehydrogenase ( DPD) activity – Capecitabine Glucose- phosphate dehydrogenase (G6PD) deficiency Rasburicase Thiopurine methyltransferase deficiency or lower activity – Azathioprine Homozygous UGT1A*28 allele - Irinotecan

55. Phenotyping and Genotyping Phenotyping Dose subjects with a compound or compounds that are metabolized to a product exclusively by the enzyme systems in question. Collect plasma or urine samples » Single time point » Over a period of time Analyze for model compound and metabolite Ratio of concentrations of compound and its metabolite is used to measure metabolic capacity for a specific P450. • Genotyping Collect blood (> 1 ml) Isolate DNA from nucleated blood cells. Amplify number of copies of DNA by the Polymerase Chain Reaction (PCR). Genotype by sequencing or probing.

56. Pharmacogenomics in Drug Development DNA samples taken for ADME genotyping in drug development Routine if one enzyme is known as the predominant route of metabolism . Compounds with narrow safety margin Reduce risk of developing concentration-dependent side effects when treated with standard doses Exclude poor metabolizers (if the parent drug is predominantly biologically active) Exclude ultra-rapid metabolizers (if metabolite is predominantly biologically active) Compounds with wide therapeutic window Dose adjustments based on pharmacogenomic tests. Increase opportunity for regulatory approval on subpopulation. Less important if compound and metabolite have similar activity . Troubleshooting Retrospective analysis in subjects with side effects or lack of therapeutic effect. Prediction of ethnic variation explaining profiles in different populations .

57. Labeling Regulations “If evidence is available to support the safety and effectiveness of the drug only in selected subgroups of the larger population with a disease , the labeling shall describe the evidence and identify specific tests needed for selection and monitoring of patients who need the drug.”

58. Examples of Pharmacogenomic Information in Drug Label

59. Pegylated interferon with ribavirin – PEG-IFN-a / RBV regimens - associated with many side effects and poor response , Clinical decisions of whether to initiate therapy are largely based on likelihood of sustained virologic response(SVR ) Europeans homozygous for favourable genotype ( IFNL3 rs12979860/rs12979860 ; SVR: 69%) are more likely to achieve SVR compared with the unfavourable genotype ( IFNL3 reference/reference or reference/rs12979860; SVR: 33% and 27%, respectively )

60. Vitamin K epoxide reductase complex subunit 1 (VKORC1 ) encoded by VKORC1 gene - Target gene of anticoagulant warfarin Rare genetic variants in coding region of VKORC1 may lead to bleeding disorders eg , multiple coagulation factor deficiency type 2A, or warfarin resistance

61. ICH Topic E15: Definitions for genomic biomarkers, pharmacogenomics, pharmacogenetics,genomic data and sample coding categories To ensure consistency in the terminology used by the different regions