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Pharmacogenomics: Basics and its applications Dr. C.Adithan Pharmacogenomic Laboratory Department of Pharmacology JIPMER, Pondicherry Outline of the talk: Pharmacogenomics: Definition Polymorphism SNP Consequences of Polymorphism Therapeutic importance Bio-informatics Present Scenario in drug therapy Optimal therapy for major illness: Still elusive Schizophrenia: 30 % do not respond Hypertension: 27 % adequately controlled ADR: 1 lakh patients die every year in USA Cost of drug development : $ 500 – 700 million for each drug …… 80% fails in clinical trial This can be improved by At Giving the right drug Right Dose To the Right Patient At the Right Time Patient specific selection of medication and their dosage PHARMACOGENOMICS Right Drug Right Gene Individualized medicine Pharmacogenetics: Study of the effect of variation in a single gene Pharmacogenomics: Study of the effect of variation in multiple genes DNA sequence of all human beings is 99.9% identical Our DNAs differ by 0.1%. Does it make a difference ? Yes 0.1% difference translates into 3 million separate “spelling” differences in a genome of 3 billion bases What is Genetic Polymorphism? A genetic polymorphism is any mutant or variant gene that occurs with a frequency of more than 1% in the normal population POLYMORPHISMS SNP • Missense • Nonsense • Silent • Frameshift • Splice site INSERTIONS DELETIONS • Missense • Nonsense • Frameshift • Missense • Nonsense • Frameshift Single nucleotide polymorphisms (SNP) ……..G G T A A C T T G …... ……..G G C A A C T T G …... Most common, Incidence 1 per 300 - 600bp 10q24.2 Chromosome Example: Nucleotide position 430 CYP2C9 gene 5’ C A G C T G C A A C T 3’ CYP2C9*1 CYP2C9*2 Normal Decreased enzyme enzyme activity activity Consequences of polymorphisms Drug metabolism Disease susceptibility Drug transport Polymorphisms Adverse Drug Reaction Receptor sensitivity Responders/ Non-responders DRUG METABOLISM Molecular mechanisms that can alter drug metabolism Primary CYP Enzymes in Drug Metabolism % of total enzyme % of drugs metabolised 1A2 5% 1A2 19% 2C9 2C19 19% 3A4/5 42% 2C9 2C19 26% 2E1 10% 3A4/5 51% 2D6 24% 2D6 3% 2E1 1% Phase - I enzymes known to have polymorphism CYP2C9: Phenytoin, warfarin, NSAIDs etc CYP2C19: Omeprazole, proguanil, diazepam CYP2D6: More than 60 drugs CYP2E1: Ethanol CYP1A6: Nicotine Mutant alleles of Phase I enzymes CYP 450 gene Mutant Alleles CYP2C9*1 *2, *3, *4, *5, *6 CYP2C19*1 *2, *3, *4, *5, *6, *7, *8 CYP2D6*1 *1XN, *2XN, *3,*4,*5, *6 *9,*10,*17 Substrates Warfarin, losartan phenytoin, tolbutamide Proguanil, Imipramine, Ritonavir, nelfinavir, cyclophosphamide Clonidine, codeine, promethazine, propranolol, clozapine, fluoxetine, haloperidol, amitriptyline Red: Absent; Blue: Reduced; Green: Increased activity Phase II enzymes known to have polymorphism NAT2: Isoniazid, hydralazine, GST: D-Penicillamine TPMT: Azathioprine, 6-MP Pseudocholinesterase: Succinyl choline UGT1A1: Irinotecan Mutant alleles of Phase II enzymes Gene NAT2 GST TPMT UGT1A1 Mutant Alleles *2, *3, *5, *6,*7, *10,*14 M1A/B, P1 M1 null, T1 null Substrates Isoniazid, hydralazine, D-penicillamine *1,*2,*3A,C, *4-*8 Azathioprine, 6-MP *28 Irinotecan Red: Absent; Blue: Reduced; Potential consequences of polymorphic drug metabolism Extended pharmacological effect Adverse drug reactions Drug toxicity Increased effective dose Lack of prodrug activation Metabolism by alternative, deleterious pathways Exacerbated drug-drug interactions CYP2D6 – Implications for Poor metabolisers Decreased elimination of parent compound. Beta blockers: metoprolol, timolol Antidepressants: nortriptyline, clomipramine Decreased prodrug activation: Codeine, encainide Decreased elimination of active metabolite: imipramine Decreased elimination of parent compound & active metabolite: Amitriptyline & nortriptyline CYP2D6 Vs Starting dose of nortriptyline Normal CYP2D6 : 150 mg/day Mutant CYP2D6 : 10-20 mg/day CYP2C9 Vs Phenytoin maintenance dose Genotype CYP2C9 *1/*1 CYP2C9 *1/*2 CYP2C9 *2/*3 Mean dose (mg/d) 314 mg/d 193 mg/d 150 mg/d Why diazepam metabolism is slower in Asians compared to Caucasians? Because Asians have high frequency of mutant alleles CYP2C19 Genotype Allele Diazepam t1/2 EM CYP2C19 *1/*1 20 hours PM CYP2C19 *2/*2 84 hours CYP2C19 Vs Treatment of H.Pylori Omeprazole 20 mg/day and amoxicillin 2gm/day Genotype Allele Cure rate Wild type CYP2C19 *1/*1 29 % Htz Mutant CYP2C19 *1/*2 60 % Hmz Mutant CYP2C19 *2/*2 100 % Due to higher concentration of omeprazole DRUG TRANSPORTERS There are 7 different ABC transporters MDR1 is important among them. MDR1 encodes a P-glycoprotein that mediates ATP-dependent efflux of drugs. Expressions of P-glycoprotein in different tissues Molecular genetics of MDR1/ P-gp • • • • • Gene : MDR1 Gene symbol: ABCB1 Chromosome location: 7q21.1 No of Exons : 28 No of SNP detected : 29 Exon 19 SNP - Intron 9 SNP - Promoter 1 SNP - Polymorphism in Exon 26 (C3435T), Exon 21(G2677T/A) significantly affect P-gp expression Allele frequencies of MDR1 exon 26, 21 polymorphism in Various ethnic populations Sl No Population 3435 C T 2677 G T A 1. Caucasian 48% 52% 55% 42% 03% 2. Africans 81% 19% ND 3. Orientals 55% 45% 41% 45% 14% 4. Indians 38% 62% 33% 61% 06% ND ND ND- Not Done Substrates of P-glycoprotein Category Substrates of P-gp Anti-cancer agents Actinomycin D, Vincristine,etc Cardiac drugs Digoxin, Quinidine etc HIV protease inhibitors Ritonavir, Indinavir etc Immunosuppressants Cyclosporine A, tacrolimus etc Antibiotics Erythromycin,levofloxacin etc Lipid lowering agents Lovastatin, Atorvastatin etc RECEPTOR SENSITIVITY Receptor Sensitivity/Effect 1 receptor gene Arg389Gly Subjects with Gly 389 have reduced sensitivity to beta-blockers Subjects with Gly 49 have increased Ser49Gly sensitivity to beta-blockers 2 receptor gene Response to salbutamol is 5.3 fold Arg16Gly lower in Gly16 asthmatics. Subjects with Glu27 have strong Gln27Glu resistance to beta 2 agonists RESPONDERS & NON-RESPONDERS Disease Asthma Atherosclerosis Smoking cessation Gene and Allele/ Polymorphism Genotype ALOX5 Promoter region CETP TaqIB CYP2B6 C1459T mut B2/B2 Gln27Glu Respond poorly to antileukotriene treatment with ABT761 Poor response to treatment with pravastatin TT Greater craving for cigarettes and higher relapse rates Glu27 Better response to carvedilol treatment 2 AR gene Heart failure Effect ADVERSE DRUG REACTIONS CYP2C9 polymorphism and phenytoin toxicity Ataxia, nystagmus, drowsiness, gingival hyperplasia grade II Phenytoin dose = 300 mg/day Plasma phenytoin level =33.2 µg/ml Genotype : CYP2C9*3/*3 This girl may develop side effects to Warfarin Acenocoumarol Losartan Irbesartan Glipizde Glibenclamide Tolbutamide Ibuprofen Flurbiprofen Diclofenac All are metabolised by CYP2C9 enzyme CYP2C9 and ADR of Warfarin Subjects who are carriers of at least one mutant allele (*2 or *3) are 4 times more susceptible to bleeding complications in spite of low dose administration TPMT polymorphism induced ADR Patients having TPMT*2, *3A and *3C alleles have low enzyme activity They are at risk for excessive toxicity, especially fatal myelosuppression, even at standard dose of azathioprine, mercaptopurine and thioguanine DISEASE SUSCEPTIBILITY Genetic polymorphism & disease susceptibility Disease Gene Polymorphism Allele/ Genotype AGT M235T T allele BP ACE ACEI/D DD risk AT1R A1166C C risk β1 AR Arg389Gly Arg389 risk CETP TaqIB B2/B2 risk Hypertension Atherosclerosis Effect Disease Acute MI Alzheimer’s disease Cancer Gene Allele/ Genotype Effect CYP2C9 eNOS *3 T786C susceptibility to AMI. ApoE ε2 ε 4/ ε4 Reduced risk Poor prognosis GST M1 Null T1 Null NAT NAT2 *10 susceptibility to lung and bladder cancer susceptibility to colorectal cancer Bio-informatics in Pharmacogenomics Designing PCR-RFLP experiment (a method of genotyping): Steps involved in PCR-RFLP are: DNA extraction PCR – amplification of specific sequence Restriction digestion of amplified product Electrophoresis of digested product- genotype Validation of genotype - sequencing Bio-informatics tools used 1. Retrieval of genomic sequences 2. Identification of site of mutation on gene of interest 3. Design of PCR primers 4. Selection of the restriction endonucleases 5. PCR method validation by sequencing 6. Analysis and comparisons of electropherograms Retrieval of genomic sequence: Nucleic acid databases: GenBank (NIH Genetic Sequence Database) http://www.ncbi.nlm.nih.gov EMBL – Europe’s primary nucleotide sequence resource in collaboration with GenBank and DDBJ http://www.ebi.ac.uk/embl/ DNA Data Bank of Japan http://www.ddbj.nig.ac.jp/ Identification of site of mutation CYP allele nomenclature committee http://imm.ki.se/cyp/ UCSC Genome Browser http://www.genome.ucsc.edu/ Ensembl Genome Browser http://www.ensembl.org/ Vega Genome Browser http://vega.sanger.ac.uk/ Designing primers for PCR Free interactive primer design tools: Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi WebPrimer http://seq.yeastgenome.org/cgi-bin/web-primer GeneFisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/ SNPBox http://www.snpbox.org Selection of restriction enzyme NEB Cutter http://tools.neb.com/NEBcutter2/index.php Restriction Mapper http://www.restrictionmapper.org/ WebCutter http://firstmarket.com/cutter/cut2.html Analysis of Electropherogram Chromas http://www.technelysium.com.au/chromas_lite.html Finch TV http://geospiza.com/finchtv/ BioEdit http://www.mbio.ncsu.edu/BioEdit/page2.html What next ? Patient requires Treatment Examination by the Physician Genomic testing Traditional investigations EXPERT SYSTEM Decision making by Physician, assisted by an Expert System (interactive interpretation) Prescribes individualized drug treatment ..And what many thought would not happen has already happened Basel, 25 June 2003 Roche Diagnostics Launches the AmpliChip CYP450 in the US, the World’s First Pharmacogenomic Microarray for Clinical Applications Personalized Medication in the Future Gene Chip Analysis SMART CARD Person’s name Xenobio GeneChip GENOME (Confidential) In the future (? years), doctors will be able to select the best drug to treat your disease and the appropriate dose based on knowledge of your specific genetic makeup! “Here is my sequence” DNA from 5ml of blood ONGOING / FUTURE PROJECTS • Epilepsy (CYP2C9,C19, and MDR1) • Upper-aerodigestive cancers (GSTM1,GSTT1,GSTP1, CYP1A1 and CYP2E1) • Coronary heart disease (ApoE, eNOS, CETP and CYP2C8,C9,J2) • Hypertension (M235T, ACEI/D, T174M, A1166C, Arg389Gly, Glu298Asp) Collaborating Departments Medicine Cardiology Neurology Radiotherapy ENT Surgery Psychiatry CTVS Tuberculosis Biochemistry Thank You Patient requires Treatment Examination by the Physician Traditional investigations Genomic testing EXPERT SYSTEM Decision making by Physician, assisted by an Expert System (interactive interpretation) Prescribes individualized drug treatment