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  3. 3. INTRODUCTION • Drug Response :Environmental factors and genetic factors. • Pharmacogenetic disorders(plasma cholinesterase deficiency, acute intermittent porphyria, drug acetylation deficiency and aminoglycoside ototoxicity. • Pharmacogenomic tests:Tests for variations in human leukocyte antigen (HLA) genes. • Genes influencing drug metabolism. • Drug targets such as the epidermal growth factor receptor HER2, tyrosine kinase inhibitors and the main target for warfarin, vitamin K epoxide reductase (VKOR).
  4. 4. Exogenous & Endogenous factors contribute to variation in drug response
  5. 5. CONTD………….. • every year about 2 million people are hospitalized for drug adverse reactions. And every year 100,000 people die because of these reactions. • This makes it the 6th leading cause of death worldwide • 49% of adverse drug reactions associated with drugs that are substrates for polymorphic drug metabolizing enzymes. • Interindividual variation :can be pharmacokinetic/pharmacodynamic/ idiosyncratic. • If not taken into account, can result in lack of efficacy or unexpected side effects • Twin studies:very useful to explore genetic basis of drug response variation.
  6. 6. • Pharmacogenetic contribution to pharmacokinetic parameters. t1/2 of antipyrine is more concordant in identical in comparison to fraternal twin pairs. Bars show the t1/2 of antipyrine in identical (monozygotic) and fraternal (dizygotic) twin pairs. (Redrawn from data in Vesell and Page, 1968.)
  7. 7. • PHARMACOGENETICS = Pharma and genetics • Pharma the Greek word i.e. PHARMACON, related to Drugs. • Genetics related to genes / genome • The study of the genetic basis for variation in drug response. • PHARMACOGENOMICS: Surveying the entire genome to assess multigenic determinants of drug response. • PERSONALISED MEDICINE: Individualising drug therapy in light of genomic information.
  8. 8. • To use genetic information specific to an individual patient to preselect a drug that will be effective and not cause toxicity. • Better than relying on trial and error supported by physical clues. • USFDA :Addition of pharmacogenomics labelling information to the package inserts of over 50 drugs.
  9. 9. PERSONALIZED MEDICINE Understanding human genome Simpler methods identify genetic information Genetic information specific to individual Preselect effective drug No toxicity No trial & error
  10. 10. REVIEW OF ELEMENTARY GENETICS Definitions: a gene is the basic instruction—a sequence of nucleic acids (DNA or, in the case of certain viruses RNA), while an allele is one variant of that gene. Referring to having a gene for a disease for example, sickle-cell disease is caused by a mutant allele of a haemoglobin gene. • An allele is an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome
  11. 11. CONTD……….. • Mutations :Heritable changes in the base sequence of DNA. • Occur during crossing over of DNA during Meiosis. • Polymorphism :Variation in the DNA sequence that is present at an allele frequency of 1% or greater in a population. • Arise initially because of a mutation. • If nonfunctional stable. • If disadvantageous die out during subsequent generations . • Two major types: single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) • cosmopolitan or population (or race and ethnic) specific. • 95% of the genome is intergenic, most polymorphisms are unlikely to directly affect the encoded transcript or protein. • Most pharmacogenetic traits are multigenic rather than monogenic.
  12. 12. MARKERS OF GENETIC VARIATION Types of Polymorphisms • Single Nucleotide Polymorphism (SNP): GAATTTAAG GAATTCAAG • Insertion/Deletion: GAAATTCCAAG GAAA[ ]CCAAG
  13. 13. CONTD…………… • SNPs occur every 100–300 bases along the 3 billion base human genome. • The greatest number of DNA variations associated with diseases or traits are missense and nonsense mutations, followed by deletions.
  14. 14. HISTORY Time line of genomic discoveries
  15. 15. CONTD……… • First pharmacogenetic examples to be discovered was glucose-6-phosphate dehydrogenase (G6PD) deficiency. • Albinism and ‘Inborn errors of metabolism’ in the early part of the 20th century by Archibald Garrod, a British physician who initiated the study of biochemical genetics. • In the 1950s Walter Kalow discovered atypical cholinesterase while studying suxamethonium sensitivity. • Detected by a blood test that measures the effect of the inhibitor dibucaine . • Malignant Hyperpyrexia:Mutation of the Ryanodine receptor, located on sarcoplasmic reticulum mediate the release of calcium ions resulting in a drastic increase in intracellular calcium thus, muscle contraction . • Triggered by exposure to certain drugs used for general anesthesia (Halothane etc)
  16. 16. CONTD…………. • Acute intermittent prophyria . • use of sedative, anticonvulsant or other drugs in patients with undiagnosed porphyria can be lethal. CYP inducer i.e. barbiturates, griseofulvin, carbamazepine, estrogen can precipitate acute attacks in susceptible individuals. • fast acetylators’ and ‘slow acetylators’ of Isoniazid. • The N – acetyl transferase (NAT) enzyme is controlled by two genes, (NAT 1) and (NAT 2) of which NAT2 A and B are responsible for clinically significant metabolic polymorphism. • Fast:peripheral neuropathy • Slow:hepatotoxicity, • Aminoglycoside ototoxicity:Mitochondrially inherited. • 1970s and 1980s:debrisoquine &(CYP2D6) deficiency was isolated.
  17. 17. EFFECTS OF GENES ON DRUG RESPONSE • PHARMACOKINETIC: • Too much/not enough drug @site of action. I. Metabolism II. Transporters III. Plasma protein binding 1. Thiopurine drugs (Tioguanine, Mercaptopurine and its prodrug Azathioprine) and TPMT(Thiopurine-S-methyltransferase) activity:Bone marrow and liver toxicity. • About 1 in 300 Caucasians and African-Americans are TPMT- deficient 2. 5-Fluorouracil (5-FU) and DPYD(dihydropyrimidine dehydrogenase ) activity:Decreased metabolism leukocytopenia, stomatitis, diarrhea, nausea and vomiting. 3. Tamoxifen AND CYP2D6: 4. Irinotecan AND UGT1A1*28: In Gilbert’s syndrome,50 fold reduction in irinotecan metabolism and such patients can be at risk of toxicity.
  18. 18. 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
  19. 19. CYP450 CONTENT IN HUMAN LIVER Low levels of P4502D6 & P4502C19 P4502D6 Other P4501A2 P4502C19 P4502E1 P4502B6 P4502A6 P4502C9 P4502C8 P4503A4
  20. 20. MUTANT ALLELES OF PHASE I ENZYMES 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, fluoxetine, haloperidol, amitriptyline Red: Absent; Blue: Reduced; Green: Increased activity
  21. 21. MUTANT ALLELES OF PHASE II ENZYMES 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;
  22. 22. GENETIC POLYMORPHISM BASED ON DRUG METABOLIZING ABILITY PHENOTYPE GENOTYPE EFFECTS 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 attendant effects. D. ultrarapid metabolizers (UM) (2 – 7%) gene amplification . drug failure
  23. 23. GENETIC VARIATION IN DRUG RECEPTOR: (i)ATP binding cassette (ABC) family : I. multi drug resistance gene also classified as ABCB 1 i.e. (ABCB1/MDR1), :MDR1 encodes a P-glycoprotein (an energy-dependent transmembrane efflux pump) II. ABCC1, ABCC2, uric acid transporter (ABCG2), III. breast cancer resistance protein BCRP also classified ABCG2 i.e. (BCRP/ABCG2). (ii) The solute transporter superfamily (SLC): I. organic anion transport polypeptide (SLC 21/OATP), II. organic cation transporter SLC 22 OCT), III. zwitterion/cation transporter (OCTNs), IV. folate transporter(SLC19A1), V. neurotransmitter transporter (SLC6,SLC17,&SLC18) VI. serotonin transporter (5HTT). • Important roles in the GI absorption,biliary and renal elimination and distribution to target sites of their substrates.
  24. 24. 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 Dipeptide transporter, organic anion and cation transporters, and L-amino acid transporter. Other Polymorphic Drug Transporters
  25. 25. PHARMOCODYNAMIC • • Receptors • Ion channels • Enzymes • Immune molecules • Drug target-related genes. 1. TRASTUZUMAB AND HER2 receptor:EGF antagonist that binds Human epidermal growth factor receptor 2—HER2. 2. DASATINIB, IMATINIB AND BCR-ABL1 receptor: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. . Combined (metabolism and target)gene tests: Warfarin and CYP2C9 & VKORC1(vitamin K epoxide reductase ) genotyping:
  26. 26. G-protein Coupled Receptors (GPCR):Over 50% of all drug targets have G-protein coupled receptors (GPCR). Genes of GPR has more coding regions than non – GPCR genes making them more important for pharmacological investigations. GABAA Receptor Mutation in GABAA receptor ion channel:diminished protection of anti epileptic drugs. Insulin Receptor(INSR):Mutation of the gene encoding the receptor will result in poor response particularly in type 2 diabetes.Also contribute to genetic susceptibility to the polycystic ovarian syndrome. B2 Receptor:Patients with B2 receptor arginine genotype experience poor asthma control with frequent symptoms and a decreasing scores of poor exploratory volume compared with those with glycine genotype.17% of whites and 20% of blacks carry the arginine genotype
  27. 27. Neurotransmitter Transporters: SLC6, SLC17 and SLC18 families. •sites of action of various drugs of abuse e.g cocaine, amphetamine and other clinically approved drugs like desipramine, reserpine, benztropine and tiagabine. •Genetic variation may affect the efficacy of such drugs. Ion Channels:KCNJ10, KCNJ3, CLCN2, GABRA1, SCN1B and SCN1A. •Some polymorphism of this channel has been linked to idiopathic generalized epilepsy. •The 5-HT3 receptor is a ligand-gated ion channel composed of five subunits. • five different human subunits are known; 5-HT3A-E, which are encoded by the serotonin receptor genes HTR3A, HTR3B, HTR3C, HTR3D and HTR3E, respectively. •Functional receptors are pentameric complexes of diverse composition. •Different receptor subtypes seem to be involved in chemotherapy-induced nausea and vomiting (CINV), irritable bowel syndrome and psychiatric disorders. • 5-HTR3A and HTR3B polymorphisms may also contribute to the etiology of psychiatric disorders and serve as predictors in CINV and in the medical treatment of psychiatric patients.
  28. 28. IDIOSYNCRATIC 1. ABACAVIR AND HLAB*5701:severe rashes. 2. ANTICONVULSANTS AND HLAB*1502:severe life-threatening rashes including Stevens Johnson syndrome and toxic epidermal necrolysis . 3. CLOZAPINE AND HLA-DQB1*0201: agranulocytosis
  29. 29. PHARMACOGENOMIC BIOMARKERS AS PREDICTORS OF ADVERSE DRUG REACTIONS Gene Relevant Drug TMPT 6-mercaptopurines UCT1A1*28 Irinotecan CYP2C0 and VKORC1 Warfarin CYP2D6 Atomoxetine; Venlafaxine; Risperidone; Tiotropium bromide inhalation; Tamoxifen; Timolol Maleate; Fluoxetine HCL; Olanzapine; Cevimeline hydrochloride; Tolterodine; Terbinafine; Tramadol; Acetamophen; Clozapine; Aripiprazole; Metoprolol; Propranolol; Carvedilol; Propafenone; Thioridazine; Protriptyline HCl; Tetrabenazine; Codeine sulfate; Fiorinal with Codeine; Fioricet with Codeine CYP2C19 Omperazole HLA-B5701 Abacavir HLA-B1502 Carbamazepine G6PD Deficiency Rasburicase; Dapsone; Primaquine; Chloroquine MDR1 Protease inhibitors ADD1 Diuretics Ion channel genes QT prolonging antiarrhythmics CRHR1 Inhaled steroids
  30. 30. Polymorphism-Modifying Diseases and Drug Responses: • MTHFR polymorphism, for example, is linked to homocysteinemia, which in turn affects thrombosis risk. • . polymorphisms in ion channels (e.g., HERG, KvLQT1, Mink, and MiRP1) affect risk of cardiac dysrhythmias, accentuated in the presence of a drug prolonging QT interval(macrolide antibiotics, antihistamines. • Polymorphisms in HMG-CoA reductase degree of lipid lowering following statins and degree of positive effects on high-density lipoproteins among women on HRT.
  31. 31. PHARMACOGENETICS AND DRUG DEVELOPMENT • Pharmacogenomics may contribute to a “smarter” drug development process – Allow for the prediction of efficacy/toxicity during clinical development – Make the process more efficient by decreasing the number of patients required to show efficacy in clinical trials – Decrease costs and time to bring drug to market. • Genome-wide approaches hold promise for identification of new drug targets and therefore new drugs. • To identify which genetic subset of patients is at highest risk for a serious ADR, and to avoid testing the drug in that subset of patients. • Usually dosing alteration done,NOT drug preclusion.
  32. 32. THERAPEUTIC DRUGS AND CLINICALLY AVAILABLE PHARMACOGENOMIC TESTS: • Tests for • (a) variants of different human leukocyte antigens (HLAs),strongly linked to susceptibilities to several severe idiosyncratic reactions; • (b) genes controlling aspects of drug metabolism; • (c) genes encoding drug targets • Mostly use germline DNA, that is, DNA extracted from any somatic, diploid cells, usually white blood cells or buccal cells (due to their ready accessibility). • Usually made on venous blood samples which contain chromosomal and mitochondrial DNA in white blood cells • The genomic tests are performed on DNA from samples of the tumour obtained surgically.
  33. 33. CONTD……… • amplification of the relevant sequence(s) and molecular biological methods, often utilising chip technology, to identify the various polymorphisms • BUT, relatively few are used routinely in patient care. • Because genomic variability is so common (with polymorphic sites every few 100 nucleotides), "cryptic" or unrecognized polymorphisms may interfere with oligonucleotide annealing, thereby resulting in false positive or false negative genotype assignments. • It is important to select polymorphisms that are likely to be associated with the drug-response phenotype.
  34. 34. LIMITATIONS OF PHARMACOGENETICS • Complex targeting due to multiple gene involvement • Difficult and time consuming to identify small variations in genes • Interaction with other drugs and environment to be determined
  35. 35. PHARMACOGENETICS IN CLINICAL PRACTICE • . The development has been slowed by various scientific, commercial, political and educational barriers. • 3 major types of evidence that should accumulate in order to implicate a polymorphism in clinical care. A. Screens of tissues from multiple humans linking the polymorphism to a trait; B. Complementary preclinical functional studies indicating that the polymorphism is plausibly linked with the phenotype; C. Multiple supportive clinical phenotype/genotype studies • Ideal example:Impact of the polymorphism in TPMT on mercaptopurine dosing in childhood leukemia.
  36. 36. CONTD……… • Most drug dosing takes place using a population "average" dose of drug. • Much more hesitation from clinicians to adjust doses based on genetic testing. • Broad public initiatives,i.e.NIH-funded Pharmacogenetics and Pharmacogenomics Knowledge Base provide useful resources to permit clinicians to access information on pharmacogenetics. • Complexity of dosing will be likely to increase substantially in the postgenomic era.
  37. 37. Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB) • Publicly accessible knowledge base – • Goal: establish the definitive source of information about the interaction of genetic variability and drug response 1. Store and organize primary genotyping data 2. Correlate phenotypic measures of drug response with genotypic data 3. Curate major findings of the published literature 4. Provide information about complex drug pathways 5. Highlight genes that are critical for understanding pharmacogenomics
  38. 38. ..what many thought would not happen has already happened Roche Diagnostics Launches the AmpliChip CYP450 in the US, - the World’s First Pharmacogenomic Microarray for Clinical Applications
  39. 39. CONCLUSION • Nonetheless, the potential utility of pharmacogenetics to optimize drug therapy is great. • Advantage They need only be conducted once during an individual's lifetime. • With continued incorporation of pharmacogenetics into clinical trials, the important genes and polymorphisms will be identified. • Refinement of dosing in the context of drug interactions and disease influences. • More precise ‘personalised’ therapeutics for several drugs and disorders.
  40. 40. S M A R T C A R D Person’s name GENOME Personalized medicine (Confidential) “Here is my sequence”
  42. 42. Thank You for your Attention!