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Introduction to pharmacogenomics and personalised medicine Jerzy Jankowski, MD Department of Clinical Pharmacology www.zfk.ump.edu.pl The Relationship Between Dose and Effect Pharmacotherapy – clinical problems Adverse Drug Reactions (ADRs) 56% of drugs that cause ADRs are metabolized by polymorphic phase I enzymes, of which 86% are CYP P450; only 20% of drugs associated with ADRs are substrates for non-polymorphic enzymes ADRs cause > 100 000 deaths/y in the USA Up to 7% of all hospital admissions in the UK and Sweden are due to ADRs ADRs cost the US society ~ US$ 100 billion Pharmacotherapy – clinical problems Efficacy 30-60% of subjects treated with drugs do not respond to drug therapy Pharmacogenomics Is the study of how an individual’s genetic makeup affects the body’s response to drugs. The key to creating personalised drugs with greater efficacy and safety Combines traditional pharmaceutical sciences with an understanding of common DNA variations in the human genome The most common DNA variations- SNPs Genetic polymorphisms Exist in a human population when allelic variants occure with a frequency of 1% or greater TYPES OF GENETIC VARIANTS single nucleotide polymorphisms ( SNPs ) 1 SNP every 300 – 1000 base pairs Insertions/deletions ( INDELS ) in comparison with SNPs, indels are much less frequent, especially in coding regions of genes Copy number variations ( CNVs ) – large segments of DNA ( gene duplications, gene deletions, gene inversions ) SINGLE NUCLEOTIDE POLYMORPHISM SNPs in the coding region cSNPs: - non-synonymous or missense (protein strucutre, stability, substrate affinty or introduce a stop codon) - synonymous or sense (transcript stability, splicing) Noncoding SNPs may occur in 3´ and 5´ UTR, in promoter or enhancer regions, in introns or in intergenic regions MOLECULAR MECHANISMS OF GENETIC POLYMORPHISMS ETHNIC DIVERSITY Polymorphisms differ in their frequencies within human populations Polymorphisms are classified as: - cosmopolitan ( present in all ethnic group ) - population specific ( or ethnic and race ) The presence of ethnic and race - specific polymorphisms are consistent with geographical isolation of human populations Pharmacogenomics SNPs are often linked to an individual’s response to a drug. Anticipated benefits of pharmacogenomics More powerful medicine Drugs more targeted to specific diseases, maximising therapeutic effects while decreasing damage to nerby healthy cells Better, safer drugs the First Time The best available drug therapy from the beginning; shorter recovery time; the likelikehood of adverse reactions is eliminated Anticipated benefits of pharmacogenomics More accurate methods of determinig appropriate drug dosages Current methods of basing dosages on weight and age will be replaced with dosages based on person’s genetics Advanced screening for disease Treatments can be introduced at the most appropriate stage to maximize their therapy Anticipated benefits of pharmacogenomics Better vaccines Made of either DNA or RNA, promise all the benefits of existing vaccines without all the risks Improvements in the drug discovery and approval process The cost and risk of clinical trials will be reduced by targeting only those persons capable of responding to a drug Decrease in the overall cost of health care The fate of a drug in the body Is affected by: Liberation Absorption Distribution Metabolism Excretion LADME PHASE I AND PHASE II REACTIONS IN DRUG DYSPOSITION Pathways of drug metabolism Phase I reactions: oxidation reduction hydrolysis Phase II reactions: glucuronidiation sulfation acetylation methylation PHASE I REACTIONS convert the parent drug to a more polar (water-soluble) and/ or more reactive product by unmasking or inserting a polar group such as -OH, -SH, -NH2 PHASE II REACTIONS increase water solubility by conjugation of the drug molecule with a polar moiety such as glucuronate, sulfate, acete, glutathione, glycine and methyl groups Both types of reaction convert relatively lipid- soluble original drug molecules into more water-soluble metabolites that are more easily excreted. Determinants of drug biotransformation biological: gender age renal and liver fun. disease- related fac. lifestyle: smoking alkohol consumtion diet drug -drug interaction inherited GENETIC CONTRIBUTION TO PK PARAMITERS 75 – 85% Determinants of drug biotransformation The potential risk factors of drug inefficacy and toxicity Differences in drug metabolism can lead to severe toxicity or therapeutic failure Determinants of drug biotransformation Of greater importance are inherited determinants that affect the kinetics and dynamics of numerous drugs. Pharmacokinetic variability Refers to variability in delivery of drug to, or removal from, key molecular sits of action that mediate efficacy and/or toxicity. Drug- metabolising enzymes (DMEs) and drug transportes (P-gp) are involved in this processes. Pharmacodynamic variability refers to variable drug effects despite equivalent drug delivery to molecular sits of action. This may reflect variability in the function of the drug targets (receptors or enzymes) Genetic variation in genes for DMEs, drug receptors (DR) and drug transportes (DT) is associated with variability in efficacy and toxicity of drugs The fate of drug in the body The majority of pharmacogenomic differences represent variability in drug metabolism Most of the remaining represent alternations in: Receptors Transporters Protein binding Pharmacogenetic differences in absorption or excrition of drugs are relatively uncommon Polygenic Determinants of Drug Effects The phenotypes of drug metabolism The extensive metabolizer (EM) (dominant trait; inherited as either homozygous or heterozygous for the wild- type allele (wt) The poor metabolizer (PM) recessive autosomal trait due to mutation and/or deletion of both allels; inherited as either homozygous or heterozygous for mutant allele The ultra- extensive metabolizer (UEM) recessive autosomal trait due to gene amplification Metabolism of Debrisoquin Cytochrome P450 (CYP450) enzymes The most important enzymatic system Biosynthesis and degradation of endogenous compounds (steroids, lipids, vitamines) Degradation of exogenous compounds (diet, environment, medications) Highly polymorphic Clasiffication of CYP450 enzymes Amino acid similarities Designated by a family number, a subfamily letter, a number of an individual enzyme within the subfamily and an asterisk followed by a number and a letter for each genetic (allelic) variant www.imm.ki.se/CYPalleles/ CYP 450 ENZYMES Cytochrome P450 (CYP450) enzymes 57 CYP450 genes CYP1, CYP2, CYP3 families appear to contribute to the metabolism of drugs These CYP enzymes are involved in approximately 80% of oxidative drug metabolism and account for 50% of the overall elimination of commonly used drugs Cytochrom P450 3A (CYP 3A) 3A4, 3A5, 3A7 and 3A43 isoenzymes in adults Chromosom 7q22.1 Probably the most important of all DMEs Aboundant in intestinal epithelium and in the liver 50% of the CYP450 activity in the liver Involved in the metabolism of more then half drugs that undergo oxidation Drug interactions involving inhibition of CYP 3A Drug interactions involving inhibition of CYP 3A The interaction between grapefruit juice nad CYP 3A substrates 250ml of this juice inhibits intestinal CYP 3A for 2448h. Grapefruit juice is contraindicated when drugs extensively metabolised by CYP 3A are used CYP 3A inhibition is reversible 2-3 days Drug interactions involving inhibition of CYP 3A The problem of drug interactions can be serious For exapmle: interaction of erythromycin and inhibitory drugs (nitroimidazole, diltiazem, verapamil, troleandomycin) When an orally administered drug undergoes extensive first-pass metabolism, its bioavailability in the face of CYP3A inhibition may increase severalfold, thus prolonging the presence of the drug in the body Cytochrom P450 3A (CYP 3A) Activity vary markedly among individuals of a given population Multiple genes are involved in its regulation Activity modulated by several factors including drugs Drug interactions may increase or reduce CYP 3A activity (expanding the range of variablility to about 400fold) Cytochrom P450 3A (CYP 3A) Variability in drug levels of this magnitude, potentially presents a major therapeutic problem in dosage optimization. For example: dosage of cyclosporine in patiens receiving also ketoconazole For example: dosage of cyclosporine in patients receiving also rifampin Drug interactions involving induction of CYP 3A The induction of CYP 3A significantlly decreases (up to 95%) the plasma levels of certain drugs administered concurrently CYP 3A activity is especially sesnitive to modulation Previously effective drug dosages become ineffective Drug interactions involving induction of CYP 3A The consequences of CYP 3A induction are not immediate Steady- state levels are reached in 2-3 weeks „Washing out” the induction effect also takes several weeks Effectivnes of drug therapy is reduced CYP 3A4 42 alleles ( to date ) Functional characterisations of most CYP 3A4 variants – a limited impact on protein expresion and activity CYP 3A4*1B and CYP 3A4*20 alter CYP function CYP 3A4*1B – 4% in Caucasians, 67% in Black sub. CYP 3A5 25 alleles ( to date ) CYP 3A5*3A – decreased enzymatic activity in vivo and in vitro Alleles *3B - *3L only in vitro CYP 3A7 AND CYP 3A43 7 alleles ( to date ) CYP 3A7*1A, *1B, *1C, *1D, *1E, *2, *3 5 alleles ( to date ) CYP 3A43*1A, *1B, *2A, *2B, *3 No significant role in drug metabolism CYP 3A CYP 3A genes are under the transcriptional control of nuclear receptors such as: - the pregnane X receptor ( PXR ) - the constitutive androstane receptor ( CAR ) - the hepatocyte nuclear factor-4α ( HNF4α ) CAR and HNF4α regulates the constitutive CYP3A PXR mediates the induction by exogenous comp. CYP 3A GENES REGULATION Genetic polymorphisms of nuclear receptors may influence CYP 3A gene expression and CYP activity Becouse of the simultaneous effect of nuclear receptors on CYP 3A genes, marked increases of CYP 3A activity are observed following the exposure to CYP 3A inducers Cytochrom P450 2D6 Plays a role in the metabolism of 100 most commonly used drugs 78 variants Mechanisms of gentic polymorphism: SNPs (insertions/ deletions) Complete gene deletion Gene duplication or multiplication CYP2D6 genetic polymorphism Designation Nature of mutation CYP2D6 *2 G1749C, silent mutation, C2938T, amino acid change, multiple copies result in increased activity 3.5 CYP2D6 *3 A2637 deletion in exon 5 2.7 CYP2D6 *4 G1934A, splice side defect, no activity 28.6 CYP2D6 *5 CYP2D6 gene deletion 11.6 CYP2D6 *6 T1795, deletion, premature stop codon 1.8 CYP2D6 *7 A3023C, amino acid change 1.5 CYP2D6 *8 G1846T CYP2D6 *9 3 bp deletion, loss of K281 <1.5 CYP2D6 *10 C188T, G4268T; amino acid substitutiondecreased catalytic activity <0.5 CYP2D6 *11 G971C, splice defect, no activity Allelic frequency (%) 1 <1 Cytochrom P450 2D6 The CYP 2D6 polymorphisms are associated with: The PM phenotype (complete lack of active proteine, a recesive trait): CYP 2D6*4 allele 70% in PMs CYP 2D6*5 allele 25% in PMs CYP 2D6*3 allele 3% in PMs Heterozygous individuals normal metabolic ratio (SNP) increased metabolic ratio (multipoint mutant allele) 35% of individuals with a heterozygous genotype Cytochrom P450 2D6 The UEM phenotype duplication/amplification of CYP2D6*2 (indentified in the heterozygous form) The amplified gene product has functionally the same but catalytically increased activity Cytochrom P450 2D6 Cytochrom P450 2D6 Clinical importance of CYP 2D6 polymorphism: The greater risk of adverese reactions in PMs (high plasma level of the affected drug) Lack of efficacy in UEMs (low plasma level of the affected drug) Cytochrom P450 2D6 CYP 2D6 inhibitors (qinidine, fluoxetine, paroxetine) are able to convert EMs into PMs. Phenomenon termed PHENOCOPYING Cytochrom P450 2D6 Clinical relevance of phenocopying Combination of SSRI and TAD (2-4 - fold increase in plasma level of tricyclic antidepressant; inhibition lasts for several weeks because of persistant inhibitory metabolites) Codeine – loss of analgesic efficacy Cytochrom P450 2D6 Inhibitory interactions don’t occur in persons with PM, who lack active enzyme. Cytochrom P450 2C19 Proton- pump inhibitors (omeprazole, lansoprazole ) diazepan, propranolol, clopidogrel) Chromosome 10q24.1 36 alleles ( to date ) CYP 2C19 polymorphism associated with either PMs or UEMs phenotype (CYP 2C19*17) CYP 2C19*2 CYP 2C19*3 account for more than 95% of cases of PM CYP 2C19 POLYMORPHISM CYP 2C19*2 – 681 G→A in exon 5 leading to a splice-defective site CYP 2C19*3 – 636 G→A in exon 4 leading to a premature stop codon Loss of function variants CYP 2C19*17 – 806 C→T in 5´ flanking region of the gene leading to an increased transcription and increased CYP 2C19 activity CYP 2C19 POLYMORPHISM CLINICAL SIGNIFICANCE Eradication of Helicobacter pylori with PPIs is more effective in CYP 2C19*2 and CYP 2C19*3 homozygotes and CYP 2C19 *2/*3 heterozygotes then in patients who are CYP 2C19*1 homozygotes Clopidogrel, an inactive prodrug, requires metabolisation and activation by hepatic CYPs including CYP 2C19. Loss-of-function alleles are associated with reduced effectiveness of the drug CYP 2C19 POLYMORPHISM CLINICAL SIGNIFICANCE CYP 2C19*17 allele is significantly associated with enhanced response to clopidogrel and increased risk of bleeding Cytochrom P450 2C9 Warfarin, tolbutamid, phenytoin, glipizide, losartan, NSAIDs Cytochrom P450 2E1 Metabolism and bioactivation of procarcinogens and some drugs Alternative system of metabolising ethanol Gene located on chromosom 10 with seven different loci Indentified two variants of the gene (C, c2) Clinical significance hasn’t yet been established PHASE II DRUG METABOLISM UDP-GLUCURONOSYLTRANSFERASE Prevent the accumulation of toxic lipophilic compounds and initiate their elimination through more hydrophilic vehicules Hydrophilic sugar moiety ( glucuronide ) from uridine diphosphate glucuronic acid Two families: UGT1A and UGT2 ( 2A, 2B ) UGT1A members are all encoded by a singel-gene locus on chromosome 2q37.1 UGT2B and UGT2A3 encoded by several likely duplicated genes located on 4q13.2 UDP-GLUCURONOSYLTRANSFERASE UGT1A family has the ability to conjugate several xenobiotics such as drugs and carcinogens UDP-GLUCURONOSYLTRANSFERASE UGT 1A1 has over 30 genetic variants One of the genetic variants of UGT 1A1 occurs in the TATA promoter region and has variable repeats of thymine-adenine ( TA ) The wild-type promoter ( UGT1A1*1 ) has 6 TA repeats UGT 1A1*28 variant has ( TA ) An increase in TA repeats within TATA region results in reduced UGT 1A1 activity UGT1A1*28 POLYMORPHISM AND TOXICITY OF IRINOTECAN An inverse relationship between SN-38 glucuronidation rates and frequency of diarrhea An association between UGT1A1*28 and pharmacokinetic values and toxicity endpoints with irinotecan therapy ( grade 4 neutropenia, grade 3 diarrhea ) 9-fold higher risk of developing grade 4 neutropenia in patients who were homozygous for the UGT1A1*28 THIOPURINE SMETHYLTRANSFERASE ( TPMT ) Thiopurine drugs mercaptopurine, azathioprine, thioguanine are all prodrugs converted to thioguanine nucleotide (TGN) metabolites Purine antimetabolites used as immunosuppresants and to treat neoplasias ( ALL of Chiledhood ) White populations can be separated into 3 groups based on the level of TPMT activity in their red cells TPMT activity is inherited as a monogenic autosomal co-dominant trait THIOPURINE SMETHYLTRANSFERASE ( TPMT ) The phenotypic test for the level of TPMT activity in red cells The genotypic tests based on DNA TPMT*3A is the most common variant allele (~4%) associated with very low levels of TPMT in whites TPMT*3A no observed in China, Korea, Japan P- glycoprotein (P- gp) pump The multidrug resistance pump A transmembrane protein A member of the ATP- binding cassette family of transport proteins Encoded by the multidrug resistance gene (MDR1/ABCB1) on chromosome 7q21.1 MDR1 (ABCB1) appears to be involved in drug transport and the development of drug resistance P- glycoprotein (P- gp) pump The apical surface of epithelial cell of the: small and large intestines biliary ductules ductules of the pancreas proximal tubules of the kidneys Choroid plexus The role of P-gp P-gp acts as a transmembrane efflux pump (removes drugs from the cell) Sites of expression of P-gp suggest a role in decreasing absorption from the gut secretion of endogenous and exogenous hydrophobic toxins List of P-glycoprotein substrates Steroid compounds: Anticancer agents: Aldosterone Progesterone Hydrocortisone Cortisol amethasone Corticosterone Dex Doxorubicin Daunorubicin Vinca alkaloids Actinomycin D Immunosuppressive agents: Cyclosporin FK 506 Methotrexate Protease inhibitors: Indinavir Nelfinavir Ritonavir Antibiotics: Erythromycin Cardiac drugs: Digoxin Rifampicin Quinidine Lovastatin Antihistamines: Terfenadine Others: Domperidone Loperamide Epotoside P- glycoprotein (P- gp) pump The same site of expression for both P-gp and CYP 3A4 in the small intestinal epithelial cells is the reason that P-gp: Can influence the intracellular levels of many CYP 3A substrates It may also affect the availability of those substrates to CYP 3A and thereby the extent of their CYP3A- mediated metabolism P- glycoprotein (P- gp) pump P- gp expression and function shows wide interindividual differences influcenced by both: Environmental Genetic factors (SNP in exon 26 3435CT, exon 21 2677GT) Assessment of Individual’s Polymorphism Phenotyping – administration of the respective drug, followed by determination of the metabolic ratio Genotyping – identification of specific DNA variations that result in a specific phenotype: over expresion (gene duplication) absence of active enzyme (null allele) formation of a mutant protein with ↓ activity (inactivating allele) Indications for genotyping signs of toxicity or therapeutic failure upon therapy administration Drugs selection that require genotyping before therapy introduction Drug of choice is a substrate for a polymorphic enzyme www.zfk.ump.edu.pl