Pharmacogenetics by dr.mahi

IT HAS DETAILED EXPLANATION ABOUT PHARMACOGENETICS AND GENOMICS AND THEIR USES IN PRESENT TRENDS

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2. PHARMACOGENETICS Dr. Mahendranath , PG 1 st year, Dept of Pharmacology . 2

3. INDEX INTRODUCTION . HISTORY OF PHARMACOGENETICS PHARMACOGENETICS,PHARMACOGENOMICS &’PERSONALISED MEDICINE.’ FACTORS FOR VARIATION IN DRUG RESPONSE 5. PRINCIPLES OF PHARMACOGENETICS TYPES OF PHARMACOGENETIC VARIATIONS PHARMACOGENETICS IN PRACTICE REFERENCE 9 . CONCLUSION 3

4. PHARMACOGENETICS :- I s the study of inherited genetic differences in drug metabolic pathways which can affect individual responses to drugs both in terms of therapeutic effect as well as adverse effects. Genetic differences in a SINGLE gene. E.g.: peripheral neuritis in slow acetylators and hepatotoxicity in fast acetylators who are under ISONIAZID treatment. 4

5. PHARMACOGENOMICS :- Study of the role of the genome in drug response. It analyzes how the genetic makeup of an individual affects response to drugs. It is useful to choose a particular drug to the responders and avoid unnecessary usage of drugs in non responders and avoid using in persons with adverse drug reactions. So it is useful in tailoring the drug therapy on the basis of individual genotype 5

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8. HISTORY 8 The history of pharmacogenetics stretches as far back as 510 B . C . when PYTHAGORAS noted that ingestion of FAVA BEANS resulted in a potentially fatal reaction (Hemolytic Anemia and oxidative stress) in some, but not all, individuals . Interestingly, this identification was later validated and attributed to deficiency of 6GDP in the 1950s and called favism.

9. HISTORY OF PHARMACOGENETICS FREDRICH VOGEL  Word PHARMACOGENETICS coined (at first In 1959 ) In 1964 he established the journal HUMAN GENETICS SIR ARCHIBALD GARROD  The role of genetics in response to Drugs. He wrote book The Incidence of Alkaptonuria . GARRODS TETRAD-   ALKAPTONURIA INBORN ERRORS OF ALBINISM METABOLISM CYSTINURIA PENTOSURIA. 9

10. Time line of genomic discoveries 10

11. (1950 - 1990) & (1990 AND T HEREAFTER ) In 195 3  watson and crick DNA double helix . MOTULSKY drug gene interactions in drug efficacy. Chronic myelogenous Leukemia(CML)  Its association with chromosomal defects (Philadelphia chromosome ) in 1960 by PETER NOWELL & JENET ROWLEY in university of pennsylvania . In 1961 EVANS and CLARKE published 1 st paper on pharmacogenetics . The inheritance pattern of responses to some of the drugs were found during this period. Until 1990, about100 of properties polymorphic and monomorphic pharmacogenetics were identified. 11

12. History of pharmacogenetics N- acetyltransferase polymorphism  Racial distribution and depends on the latitude of countries. Polymorphism discovery in hemoglobin  Sickle cell disease the SNP of HFE gene  hemochromatosis Apolipoprotein E=ApoE  Cardiovascular and Alzheimer's disease , Factor’s gene 5 and prot hr ombin gene  thrombosis Methylene Tetra Hydro Folate Reductase = ( MTHFR )  Venous thromboembolism 12

13. Factors contributing to variation in drug response Diet Age Gender Lifestyle Circadian & seasonal variation Exercise Comorbidities Renal and hepatic function Genetic factors 13

14. Human genome has 30,000 genes. Each gene has several thousands of nucleotides. Each person inherits 2 copies of genes one from each parent. Any two individuals DNA is 99.9% identical 3 billion nucleotides. Variation is seen in >1% of population called polymorphism Of that most common is SNP BASICS IN GENETICS Between 2 people (except identical twins) the rate of genetic variation (individuality) is about 0.1% [0.1% of 3 billion = 3 million base pair differences] 14

15. genotype : pair of alleles a person has at a region of the chromosome phenotype : outward manifestation of a genotype. monogenic : due to allelic variation at a single gene polygenic : due to variations at two or more genes 15

16. Mutation : difference in the DNA code that occurs in less than 1% of population › Often associated with rare diseases  Cystic fibrosis, Albinism , Huntington’s disease . Polymorphism :- difference in the DNA code that occurs in more than 1% of the population › A single polymorphism is less likely to be the main cause of a disease › Polymorphisms often have no visible clinical impact 16

17. GENETIC POLYMORPHISMS Single nucleotide ( polymorphisms (SNPs ) Coding, nonsynonymous C C G – Pro C A G – Gln Coding, synonymous CCG – Pro CC A – Pro Non coding Promoter/intronic Transcript stability/splicing Indels ( smaller) Insertions/deletions Tandem repeats Copy number variations (larger) Gene duplications Large deletions 17

18. SNPs 18 A single nucleotide polymorphism (SNP) , is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g. >1 %). 75 % 23 % 2% *

19. SNPs types 19 SNPs usually occur in non-coding regions more frequently than in coding regions. Non-coding SNPs in promoters/enhancers are in 5′ and 3′ untranslated regions may affect gene transcription /gene splicing. for   example, a common genetic variant due to an SNP in one  of the coagulation factors, known as factor V Leiden, is  the commonest form of inherited  thrombophilia.

20. These mutations may have no effect on enzyme activity(normal) Lead to enzyme activity with Decreased activity Absent activity Duplications lead to increased enzyme activity Wild or normal activity enzymes (75 – 85%) of population Intermediate metabolizers (10 -15%) Poor metabolizers (5 – 10%) Ultra-rapid metabolizers (2 – 7%) of population – multiple genes 20

21. GENETIC POLYMORPHISM BASED ON DRUG METABOLIZING ABILITY P H EN O TYP E GEN O TYP E EFF E C T S A. extensive or normal drug metabolizers (EM) (75 – 85%) homozygous or heterozygous for wild type allele. Normal metabolism.No dose modification needed. B.intermediate metabolizer phenotype (IM) (10 - 15%) heterozygous for the wild type allele may require lower than average drug dose for optimal therapeutic response. C. poor metabolizers (PM) (5 – 10%) mutation or deletion of both alleles accumulation of drug substrates in their systems with toxic effects . D. ultrarapid metabolizers (UM) (2 – 7%) gene amplification /gene duplication. drug failure 21

22. DRUG T A R GETS DRUG T R A N SPO R T E RS DRUG ME T A B OLI Z ING ENZYMES PHARMACOKINETICS PHARMACODYNAMICS Variability in Efficacy / T ox i city T rans p ort e rs Plasma protein binding Metabolising enzymes Receptors Ion channels Enzymes Immune molecules 22

23. Polymorphisms Drug metabolism Adverse Drug Reaction Disease su s c e pt i b i l i ty Receptor s e n siti v ity Drug transport Responders/ No n - r e spo n ders Consequences of polymorphisms 23

24. PHARMACOKINETIC VARIATIONS OXIDATION ACETYLATION SUCCINYLCHOLINE HYDROLYSIS AMINOGLYCOSIDE OTOTOXICITY 24

25. OXIDATION (phase 1 ):- most of drugs are lipophilic compounds eliminated by oxidation catalyzed by cytochrome p 450 enzyme present in liver. Total number of cyp450 genes in human consist of 57 CYP genes and 29 pseudo genes. 95% of all drug oxidation occurs in 5 CYP enzymes. 25

26. http://www.doctorfungus.org/t hedrugs/images/antifung_2.gif 26

27. DRUG METABOLIZING ENZYMES Phase I: biotransformation reactions: oxidation, hydroxylation, reduction, hydrolysis Phase II: conjugation reactions—to increase their water solubility and elimination from the body. The reactions are glucuronidation, sulation,acetylation, glutathione conjugation 27

28. 1A2 19% 2D6 3% 2E1 10% 3 A4/5 42% 2C9 2C19 26% 2D6 24% 2 E 1 1% 3 A 4 / 5 51% 2C9 2 C 19 19% Primary CYP Enzymes in Drug Metabolism % of total enzyme % of drugs me t abolis e d 1 A 2 5% 28

29. CYP2 D 6 29 Source of sparteine / debrisoquine oxidation polymorphism 7-9 % caucasian population referred as poor metabolizers They don’t express the enzyme they have mutation on the long arm of chromosome 22. CYP2D6 show marked allelic heterogenecity 80 known variants of SNP are reported. It oxidizes tricyclic antidepressants , antipsychotics ,SSRI, antiarrhythmics , beta adrenoreceptor blockers,phenformin and opiates. Poor hydroxylators have dose related toxicity like Lactic acidosis with phenformin,CNS toxicity with nortriptyline Extensive metabolizers have duplication of CYP2D6 allele and have therapeutic failure. AR

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31. CYP2D6 :- - Tricyclic antidepressants Poor metabolisers – high plasma concentration – toxic effects (tardive dyskinesia) Rapid metabolisers – low plasma concentrations – therapeutic failure Codeine (as analgesic) Poor metabolisers – therapeutic failure Rapid metabolisers - toxic effects of morphine is seen. 31

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33. CYP2C19 MEDIATOR OF BIOTRANSFORMATION OF TERITIARY AMINE TRICYCLIC ANTIDEPRESSANTS METABOLIZES MEPHENYTOIN,PPI,CLOPIDOGRIL,BIOACTIVATION OF PROGUANIL,DIAZEPAM. 3-5% EUROPEANS AND 15-20 % ASIANS ARE POOR METABOLIZERS. POOR METABOLIZERS :- CLOPIDOGRIL IS IN INACTIVE FORM(15%) OMEPRAZOLE HAS 100% CURE RATE FAILURE OF PROGUANIL METABOLISM TO CYCLOGUANIL SO LOSS OF PROTECTION FROM MALARIA. IMPAIRED MEPHENYTOIN METABOLISM 33

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35. CYP2 C 9 35 Major enzyme catalyzing the biotransformation of warfarin, phenytoin, fluvastatin and several NSAIDS,tolbutamide and other oral antidiabetic drugs. patients with either “CYP2C9*2 or CYP2C9*2 variant require lower warfarin maintenance dose” . The risk for bleeding doubled in these patients, as they metabolize warfarin slower than the wild-type patient.

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37. CYP 450 gene Mutant Alleles Substrates CYP2C9*1 *2, *3, *4, *5, *6 Warfarin, losartan phenytoin, tolbutamide CYP2C19*1 *2, *3, *4, *5, *6, *7, *8 Proguanil, Imipramine, Ritonavir, nelfinavir, cyclophosphamide CYP2D6*1 *1XN, *2XN, *3,*4,*5, *6 *9,*10,*17 Clonidine, codeine, promethazine, propranolol, clozapine, f l uoxet i n e , halope r ido l , amitriptyline MUTANT ALLELES OF PHASE I ENZYMES Red : Absent; Blue : Reduced; Green : Increased activity 37

38. ACETYLATION Several drugs acetylated by hepatic NAT2 of the enzyme N- acetyltransferase . The difference between fast and slow acetylators depends on the amount of hepatic N- acetyltransferase . Fast acetylators are autosomal dominant slow are recessive Drugs that undergo acetylation are isoniazid, hydralazine, procainamide,phenelzine,dapsone,sulfamethoxypyradizine In slow acetylators there is enhanced response to treatment but increased drug toxicity. Hence slow acetylators require lower doses. 38

39. Pharmacogenetic variations. Acetylation Polymorphism of N-acetyl transferas e Acetylation of Isoniazid Fast acetylators High N- acetylase Eskimos,japanese hepatotoxicity slow acetylators Low N- acetylase Egyptians,swedes , mediterranian jews peripheral neuropathy So pyridoxine( vit B6) is added with isoniazid Therapy 39

40. Succinylcholine hydrolysis Psuedocholi n est Succinylcholine Atypical Psue d o cho linesterase Sleep apnoea Doesn’t metabolize succinylcholine rapidly so levels of succinylcholine and Continue to produce neuromuscular blockade for several hours. Results in respiratory paralysis need prolonged ventilation. 40

41. Gene Mutant Alleles Substrates NAT2 *2, *3, * 5, *6,*7, *10,*14 Isoniazid, hydralazine, GST M1A/B, P1 M1 null, T1 null D-penicillamine TPMT *1, *2,*3A,C , *4-*8 Azathioprine, 6-MP UGT1A1 *28 Irinotecan Red : Absent; Blue : Reduced; MUTANT ALLELES OF PHASE II ENZYMES 41

42. Gene product Drugs Responses affected CYP2C9 Warfarin, Tolbutamide, Phenytoin, NSAIDs Anticoagulant effect of warfarin CYP2C19 Omeprazole, clopidogrel, mephenytoin, propranolol Peptic ulcer response to omeprazole, Cardio-vascular events after clopidogrel CYP2D6 Beta blockers, codeine, antidepressants, tamoxifen antipsychotics, debrisoquine, Codeine efficacy, Tardive dyskinesia from antipsychotics CYP3A4/A5/A7 Macrolides, cyclosporine, tacrolimus, CCBs, etc Efficacy of immunosuppressive effect of tacrolimus UGT1A1 (UDP gl u c u r on o s yl transferase) Irinotecan , bilirubin Irinotecan toxicity Thiopurine methyl transferase (TPMT) Mercaptopurine, thioguanine, azathioprine Thiopurine toxicity and efficacy Dihydropyramidine dehydrogenase Fluorouracil, capacitabine 5-fluorouracil toxicity 42

43. PHARMACOGENETIC VARIATION IN DRUG RESPONSE DUE TO ENZYME DEFICIENCY : RED CELL ENZYME DEFECT. Glucose – 6 – phosphate dehydrogenase deficiency(G-6-PD) : Deficiency in RBC’s Sex – linked recessive trait( X – linked) Africans, American negroes, Mediterranean Jews, middle east and south east races. Drugs having oxidising properties can cause haemolytic anaemia in persons having G-6-PD deficiency . Reduced NADPH production & glutathione accumulates. Eg: Primaquine, Sulphonamides, Dapsone, Nitrofurantoin, Quinine, Chloroquine, Quinidine , nalidixic acid, doxorubicin . HEINZ BODIES IN BLOOD FILM. 43

44. GLUTATHIONE REDUCTASE DEFICIENCY Autosomal dominant. Directly cause a deficiency of reduced glutathione and hemolysis will result from effects of oxidizing agents METHAEMOGLOBIN REDUCTASE DEFICIENCY In normal individuals methaemoglobin is reduced to haemoglobin . In methaemoglobin reductase deficiency, methaemoglobin is accumulated and causes impairement of oxygen delivery to the tissues and causes hypoxaemia On exposure to oxidant drugs the condition worsens. 44

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46. Porphyria is due to enzymatic abnormality in haem biosynthetic pathway. In acute intermittent porphyria basic defect is in the gene HMBS for enzyme porphobilinogen deaminase,which catalyses the production of porphyrins , the precursor of haem . The reduction in haem synthesis switches ALA SYNTHASE,with excess production of ALA , porphobilinogen and its metabolite causing ACUTE INTERMITTENT PORPHYRIA. Drugs that induce CYP 450 precipitate porphyria CYP 450 is a haeme containing enzyme, induction of CYP450 demands more haem production exaggerating ALA synthase response and overproduction of porphobilinogen and its metabolite products. Drugs that are unsafe in porphyria are barbiturates,carbamazepine,phenytoin,isoniazid,dapsone,diclofenac. ACUTE INTERMITTENT PORPHYRIA 46

47. Management of acute attack of porphyria : No specific measures High intake of carbohydrates inhibits ALA synthase activity and a high carbohydrate diet will not do any harm . H ematin ( Hemin ) IV 3-4mg /kg/day for 3-4 days has been used as a specific therapy . 47

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49. MALIGNANT HYPERTHERMIA Fatal complication of general anaesthetics with haolthane , methoxyflurane , succinylcholine. RYNODINE RECEPTOR mutation in sarcoplasmic reticulum. Excessive release of calcium into cytoplasm trigerred by anaesthetics . AD. CLINICAL FEATURES:- ACUTE RISE IN TEMPERATURE MUSCLE STIFFNESS TACHYCARDIA & TACHYPNOEA SWEATING,CYANOSIS. 49

50.  streptomycin and gentamicin are primarily vestibulotoxic , whereas amikacin , neomycin, dihydrosterptomycin , and kanamicin are primarily cochleotoxic . Cochlear damage can produce permanent hearing loss, and damage to the vestibular apparatus results in dizziness, ataxia, and/or nystagmus . Aminoglycosides appear to generate free radicals within the inner ear, with subsequent permanent damage to sensory cells and neurons, resulting in permanent hearing loss. Two mutations in the mitochondrial 12S ribosomal RNA gene have been previously reported to predispose carriers to aminoglycoside-induced ototoxicity. As aminoglycosides are indispensable agents both in the treatment of infections and Meniere's disease, a great effort has been made to develop strategies to prevent aminoglycoside ototoxicity . Anti-free radical agents, such as salicylate, have been shown to attenuate the ototoxic effects of aminoglycosides. AMINOGLYCOSIDE OTOTOXICITY 50

51. INSULIN RESISTANCE Different mutations in the insulin receptors alpha subunit were proposed in Different families ( Ark-i , Atl , Minn .) Based on phenotype , cellular insulin binding and insulin receptor structure. Arrhythmogenic effects of antiarrhythmic drugs Torsade de pointes is associated with long QT syndrome. AD . Drugs like clarythromycin,levofloxicin,haloperidol with are QT prolonging medications when given with cyp450 inhibitors like FLUOXETINE, CIMITIDINE and also grape fruit. Genetic abnormality in potassium channel function has been attributed to torsade de pointes. 51

52. RESISTANCE TO DRUG EFFECTS VITAMIN D RESISTANT RICKETS:- There are 3 varieties of rickets that are resistant to effects of vitamin D. FAMILIAL HYPOPHOSPHATAMIC RICKETS : impaired phosphate reabsorption in the kidney. 2) TYPE II VITAMIN D DEPENDENT RICKETS: impaired tissue sensitivity to vitamin D and decreased receptor binding. RICKETS IN FANCONI SYNDROME: failure in tubular reabsorption of phosphate 52

53. http://mostgene.org/2009_conference/personalized_meds_Gettig.pdf 53

54. Clinically available Pharmacogenomic tests 54 54

55. 1 ) HLA GENE TESTS:- ABACAVIR & HLAB*5701 ANTICONVULSANTS & HLAB*1502 CLOZAPINE & HLA-DQ 1*0201 2 ) DRUG METABOLISM RELATED GENE TEST: - THIOPURINE & TPMT 5-FLUOROURACIL (5-FU) & DPYD TAMOXIFEN & CYP2D6 IRINOTECAN & UGT1A1*28 Various type of test s 55 55

56. DRUG TARGET RELATED GENE TEST Trastuzumab & HER 2 DASATINIB, IMATINIB & BCR-ABL 1 COMBINED (METABOLISM & TARGET) GENE TEST WARFARIN & CYP2C9 + VKORC 1 GENOTYPING 56 56

57. IDIOSYNCRATIC ABACAVIR AND HLAB*5701: severe rashes . ANTICONVULSANTS & HLAB*1502 : severe life-threatening rashes including Stevens Johnson syndrome and toxic epidermal necrolysis 3. CLOZAPINE AND HLA DQB1*0201 : agranulocytosis . 57

58. DRUG METABOLISM RELATED GENE TESTS. Fluorouracil is a chemotherapy agent that belongs to the drug class of fluoropyrimidines .(CAPECITABINE & TEGAFUR) F luorouracil is used in the palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas. The  DPYD  gene encodes dihydropyrimidine dehydrogenase (DPD), an enzyme that catalyzes the rate-limiting step in fluorouracil metabolism. Individuals who carry at least one copy of non function  DPYD  variants, such as  DPYD*2A , may not be able to metabolize fluorouracil at normal rates, and are at risk of potentially life-threatening fluorouracil toxicity, such as bone marrow suppression and neurotoxicity . The prevalence of DPD deficiency in Caucasians is approximately 3%-5%. The FDA-approved drug label for fluorouracil states that “rarely, unexpected, severe toxicity associated with 5-fluorouracil has been attributed to deficiency of dipyrimidine dehydrogenase activity ”. 58

59. The FDA also states that fluorouracil therapy should be discontinued promptly whenever one of the following signs of toxicity appears: Stomatitis or esophageal pharyngitis at the first visible sign, Leukopenia (WBC under 3500) or a rapidly falling white blood count, Vomiting , intractable Diarrhea, frequent bowel movements, or watery stools Gastrointestinal ulceration and bleeding Thrombocytopenia (platelets under 100,000) Hemorrhage from any site TOXICITY 59

60. Likely phenotype Functional definition Genetic definition Example diplotypes Normal metabolizer Fully functional DPD enzyme activity Combinations of normal function and decreased function alleles DPYD*1/*1 Intermediate metabolizer (~3–5% of patients) Decreased DPD enzyme activity (activity between normal and poor metabolizer) Combinations of normal function, decreased function, and/or no function alleles *1/*2A ;  *1/*13 ; or *1/ rs67376798 Poor metabolizer  (~0.2% of patients) Little to no DPD enzyme activity Combination of no function alleles and/ or decreased function alleles *2A/*2A; 13/*13;  *2/*13 ; or  rs67376798 /  rs67376798 60

61. The  TPMT  gene encodes enzyme thiopurine S- methyltransferase . TPMT is one of the main enzymes involved in the metabolism of thiopurines , such as azathioprine,6 mercaptopurine , 6 thiogunanine . TPMT activity is inherited as a co-dominant trait, as the  TPMT  gene is highly polymorphic with over 40 reported variant alleles. The wild-type  TPMT*1  allele is associated with normal enzyme activity. Individuals who are homozygous for  TPMT*1  (TPMT normal metabolizers) are more likely to have a typical response to azathioprine and a lower risk of myelosuppression . This accounts for the majority of patients (~86–97%). Individuals who are TPMT poor (approximately 0.3%) or intermediate (approximately 3–14%) metabolizers carry variant  TPMT  alleles that encode reduced or absent enzyme activity. Incresed levels of active metabolite Thioguanine nucleotide and causes myelosuppression and hepatotoxicity THIOPURINE AND TPMT POLYMORPHISM 61

62. Phenotype Phenotype details TPMT Genotype Examples of diplotypes Therapeutic recommendations for azathioprine Homozygous wild-type (“normal”) High enzyme activity. Found in ~86–97% of patients. Two or more functional  TPMT  alleles *1/*1 Start with normal starting dose (e.g., 2–3 mg/kg/d) and adjust doses of azathioprine based on disease-specific guidelines. Allow 2 weeks to reach steady state after each dose adjustment. Heterozygous Intermediate enzyme activity.  Found in ~3–14% of patients. One functional  TPMT  allele plus one nonfunctional  TPMT  allele *1/*2 *1/*3A *1/*3B *1/*3C *1/*4 If disease treatment normally starts at the “full dose”, consider starting at 30–70% of target dose (e.g., 1–1.5 mg/kg/d), and titrate based on tolerance. Allow 2–4 weeks to reach steady state after each dose adjustment. Homozygous variant Low or deficient enzyme activity. Found in ~1 in 178 to 1~3736 patients. Two nonfunctional  TPMT  alleles *3A/*3A *2/*3A *3C/*3A *3C/*4 *3C/*2 *3A/*4 Consider alternative agents. If using azathioprine start with drastically reduced doses (reduce daily dose by 10-fold and dose thrice weekly instead of daily) and adjust doses of azathioprine based on degree of myelosuppression and disease-specific guidelines. Allow 4–6 weeks to reach steady state after each dose adjustment. Azathioprine is the likely cause of myelosuppression . 62

63. Case study A 72 year old male with metastatic colorectal cancer was prescribed an anticancer drug Irinotican 180mg/m 2 , as an intravenous infusion, which was repeated every 2weeks, along with several other chemotherapeutic agents. Liver function and renal function were normal. Blood samples were drawn. 63

64. After the treatment cycle, the patient experienced very severe neutropenia and diarrhea . Plasma levels of SN-38 , the active metabolite of irinotecan, were 4fould higher than those found in most patients. The irinotecan dose was reduced by 50%. Plasma levels of SN-38 were lower but still more than twice normal. However after 2 nd cycle, there was no neutropenia and only grade 1 diarrhea. CT and MRI scan showed partial response to the chemotherapy. 64

65. Case study answer Irinotecan is metabolized to the active cytotoxic molecule SN-38, which is also responsible for toxicity. Inactivation of SN-38 occurs via the polymorphic UGT1A1 enzyme. Carriers of the UGT1A1*28 variant have reduced enzyme activity . SN-38 G is inactive form 65

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67. Denomination Variants 14 Allele frequency (ethnicity) 15 , 16 Expression level Enzymatic activity Clinical consequence UGT1A1*1 (TA) 6 TA Common allele 100% 100% None TATA box polymorphisms UGT1A1*28 c.–39_–40 ins TA: (TA) 7 TA 29–45% (Caucasians); 42–51% (Africans); 16% (Asians) Reduced Reduced Gilbert’s syndrome, Crigler–Najjar syndrome 17 Polymorphisms in the promoter region UGT1A1*60 c.–3279 T>G 23–39% (Caucasian); 15% (African Americans); 17% (Asians) Reduced Unchanged Gilbert’s syndrome, Crigler–Najjar syndrome 18 Polymorphisms in exon 1 UGT1A1*6 c.211 G>A p.Gly71Arg 15–20% (Asians) Unchanged Reduced Gilbert’s syndrome, Crigler–Najjar syndrome 19 UGT1A1*27 c.686 C>A p.Pro229Gln 5–28% (Asians) Unchanged Reduced Gilbert’s syndrome, Crigler – Najjar syndrome 19 67

68. The cytochrome P450 2D6 (CYP2D6) is an enzyme known to metabolize drugs. Genetic polymorphisms have been grouped as nonfunctional, reduced function, functional, and multiplication alleles. Individuals carrying these alleles are presumed to correspond to poor, intermediate, extensive, and ultrarapid metabolizers (UM), respectively . Tamoxifen has been shown to be metabolized by CYP2D6 to the more potent metabolite endoxifen . Poor metabolizers (PM) of tamoxifen have lower levels of endoxifen and poorer clinical outcomes as compared to extensive metabolizers. DRUG METABOLISM RELATED GENE TESTING 68

69. B.DRUG TARGETS The action of HALOPERIDOL depends on its ability to bind to the DOPAMINE d2 Receptor site. 63% population who has large number of dopamine receptor shows better response with haloperidol. When HER2 gene is over expressed in breast tissue extra protein receptors are produced on the cell surface They trigger the cell to grow and divide out of control and becomes cancerous. 20-30% breast cancer women express HER2 protein . TRASTUZUMAB works by binding to the receptor site on the cell surface thereby limiting cell proliferation and prevent cancer to grow. 69

70.   Dasatinib  is a dual BCR/ABL and  Src  tyrosine kinase inhibitor  used in  haematological  malignancies  characterised  by the presence  of a Philadelphia chromosome, namely chronic myeloid  leukaemia   (CML) and some adults with acute lymphoblastic  leukaemia  (ALL).  The Philadelphia chromosome results from a  translocation defect btwn (9  and  22) swap  places; part  of a ‘breakpoint   cluster  region’  ( BCR) in chromosome   22  links  to  the  ‘Abelson-1’ (ABL) region of chromosome 9. A mutation (T315I) in  BCR/ABL confers resistance to the inhibitory effect of  dasatinib  and  patients with this variant do not benefit from this drug.  Pharmacogenetic  testing is also being evaluated for  imatinib associated  with rearrangements in the gene for  platelet-derived growth factor receptor or for BCR-ABL. DASATINIB/IMATINIB and BCR-ABL-1 70

71. W arfarin (brand name Coumadin) is an anticoagulant (blood thinner ). Warfarin acts by inhibiting the synthesis of vitamin K-dependent clotting factors and is used in the prevention and treatment of various thrombotic disorders. Warfarin is a drug with narrow therapeutic index; thus, a small change in its plasma levels may result in concentration dependent adverse drug reactions or therapeutic failure. Therefore , the dose of warfarin must be tailored for each patient according to the patient’s response, measured as INR (International Normalized Ratio), and the condition being treated . Values of INR in normal individual < 1, 2-3 in patients on warfarin. If INR is high risk of bleeding If INR is low risk of thrombosis. WARFARIN VKORC1 & CYP2C9 POLYMORPHISM 71

72. Phenotype/diplotype Recommendation CYP2C9 IM Use 65% of the standard initial dose CYP2C9 PM Use 20% of the standard initial dose CYP2C9*1/*2 No action is required for this gene-drug interaction. CYP2C9*1/*3 Use 65% of the standard initial dose CYP2C9*2/*2 Use 65% of the standard initial dose CYP2C9*2/*3 Use 45% of the standard initial dose CYP2C9*3/*3 Use 20% of the standard initial dose VKORC1 C/T No action is required for this gene-drug interaction VKORC1 T/T Use 60% of the standard initial dose 72

73. P G S H E C A N R R E E M T E A I N C C I O N G 73

74. PHARMACOGENITIC SCREENING TESTS AmpliChip CYP450 Detects polymorphism in drug metabolizing enzymes (DMEs) such as CYP2D6, CYP2C19 Affymetrix DMET Detects polymorphism in DMEs – CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 A5 & A7 and transporters PHARMAChip Detects polymorphisms in CYP450 enzymes and in genes that code for drug receptors, transporters and other targets Therascreen Kit For use of afatinib in non-small-cell lung cancer Cobas EGFR Mutation Test For use of erlotinib in non-small-cell lung cancer 74

75. AMPLICHIP 75 Determine the genotype of the patient in terms of two CYP450 enzymes: 2D6 and 2C19 FDA approved the test on Dec 24, 2004. The Amplichip CYP450 test is the first FDA approved pharmacogenetic test. 75

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79. DNA Test in India DNA Test Cost in India Paternity DNA Test Price (Father + 1 Child) in India ₹ 13,382 Paternity DNA Test Price (Father + 2 Children) in India ₹ 19,823 Maternity DNA Test Price (Mother + 1 Child) in India ₹ 13,382 COST OF TEST IN INDIA 79

80. MET H ODOL O GY 80 WBCs/ Buccal cells *PharmGKB 80

81. Pharmacogenetics & Drug development 81 81

82. 82 GOALS OF PHARMACOGENETICS

83. Potential Benefits of Pharmacogenetics Improve Drug Choices: Each year, ~100,0000 people die of adverse reactions to medicine & ~2 million are hospitalized Pharmacogenitics will predict who's likely to have a negativ e or positive reaction to a drug Safer Dosing Options Testing of Genomic Variation Improve Determination of Correct Dose for Each Individual 83

84. Improvement in Drug Development: Permit pharmaceutical companies to determine in which populations new drugs will be effective Decrease Health Care Costs Reduce number of deaths & hospitalizations due to adverse drug reactions Redu c e p u r c h ase o f dr u gs w h ich are ineffe c t i ve i n c e r ta in in d iv i d u al s du e to genetic variations Speed Up Clinical Trials for New Drugs Potential Benefits of Pharmacogenetics 84

85. Barriers of Pharmacogenomics 42 1. Complexity of finding gene variations that affect drug response.  Millions of SNPs must be identified and analyzed to determine their involvement in drug response 2 . Confidentiality, privacy and the use and storage of genetic information

86. Barriers of Pharmacogenomics ... 3. Educating healthcare providers and patients Complicates the process of prescribing and dispensing drugs Physicians must execute an extra diagnostic step to determine which drug is best suited to each patient 43

87. Barriers of Pharmacogenomics .. 44 4. Disincentives for drug companies to make multiple pharmacogenomic products Most pharmaceutical companies have been successful with their “one size fits all” approach to drug development For small market- Pharmaceutical companies hundreds of millions of dollars on pharmacogenomic based drug development.

88. Understanding human genome Simpler methods identify genetic information Genetic information specific to individual Preselect effective drug PERSONALIZED MEDICINE No t o xicity No trial & error 88

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90. Per s on a lized medicine S M A R T C A R D Person’s name GENOME (Confidential) “Here is my sequence” 90

91. The Goal of Personalized Medicine  The Right Dose of  The Right Drug for  The Right Indication for  The Right Patient at  The Right Time. 91

92. Clinomics 47 Clinomics is the study of genomics data along with its associated clinical data. As personalized medicine advances, clinomics will be a bridge between basic biological data and its effect on human health.

93. Scope of Pharmacogenomics 49 93

94. Drug development and approval In vitro studies Preclinical testing Animal testing Clinical trials Average years 1 to 5 years 2 to 10 years 1 year IND NDA Post-marketing surveillance (Phase 4) Phase 1 – normal volunteers: safety, pharmacokinetics Phase 2 – selected patients: therapeutic efficacy, dose range Phase 3 – large populations of selected patients: therapeutic efficacy, safety in double blind studies Long-term toxicity studies 94

95. › www.pharmgkb.org Goal: establish the definitive source of information about the interaction of genetic variability and drug response . Store and organize primary genotyping data Correlate phenotypic measures of drug response with genotypic data Curate major findings of the published literature Provide information about complex drug pathways Highlight genes that are critical for understanding pharmacogenomics  Publicly accessible knowledge base KNOWLEDGE BASE 95

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97. BIBLIOGRAPHY THE PHARMACOLOGICAL BASIS OF THERAPEUTICS ,GOODMAN & GILMAN,12 TH EDITION,2011,PAGE 145-165 . RANG & DALE’S PHARMACOLOGY,7 TH EDITION,2012,PAGE 132-137 . POSTGRADUATE TOPICS IN PHARMACOLOGY,RITUPARNA MAITI PAGE 193 -2 02 . 97

98. Pharmacogenomics has great potential to optimize drug therapy. Newer molecular diagnostic test will have to be develop to detect polymorphisms. Pharmacotherapeutics decisions will soon become fundamental for diagnosing the illness & guiding the choice & dosage of medications. CON C LUSION 48 98

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