Pharmakogenetik und Pharmakogenomik

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1 Pharmakogenetik und Pharmakogenomik Prof. Dr. habil. Egon Amann 8. Dezember 2010 Profilmodul: Grundlagen und Anwendungen der Genom- und Proteomforschung WS 2010/2011 Frank Vitzthum und Egon Amann

2 Gliederung der Vorlesung: Was ist Pharmakogenetik / Pharmakogenomik? (Definitionen und Einleitung) Konzepte und Beispiele (CYP450 / TPMT) Fallbeispiel: Alzheimersche Krankheit (ApoE) Technologische Voraussetzungen (DNA-Chips) Diskussion: Beginn einer individualisierten Medizin?

4 Adverse Drug Reactions: Major Health Threat, Major Costs 1 in 100 Fatal, unexpected drug reaction 1 in in 10,000 Car crash Murder 1 in 100,000 Plane Crash AIDS Infection in Transfusion 1 in a million Lightning Strike 1 in 10 million Source: Consumer Report, 9/99

5 Medical Justification for Patient Stratification In USA, fourth to fifth leading cause of death in hospitalized patients is adverse drug reactions (> 2 million Americans hospitalized each year for adverse drug reactions; 100,000 of which die) - JAMA 79: (1998). Adverse effects with deadly consequences occur in 0.3 % of hospitalized patients; in 6.7 % with severe consequences : 131 approved drugs had to be taken off the market because of severe side effects. Only % of the patients benefit from approved drugs. 80 % of drug candidates fail in clinical trials.

6 Genotye- Phenotyperelationship of the CETP gene (reduction of cholesterol)

7 Pharmakogenomik und Pharmakogenetik Pharmakogenomik (pharmacogenomics) ist die direkte Anwendung des Wissens aus dem Human-Genomprojekt in der Medizin. Sie versucht, das Maß der Wirkung - auch der Nebenwirkung eines Medikamentes im Kontext der genetischen Konstitution eines Patienten zu verstehen und vorherzusagen. Die pharmakogenomische Forschung widmet sich der Identifizierung und dem Verständnis neuer Zielmoleküle für die medikamentöse Intervention sowie dem Verständnis genetischer Faktoren, welche die individuelle Reaktion auf ein Medikament bestimmen. Letzteres wird durch den Begriff der Pharmakogenetik (pharmacogenetics) beschrieben.

8 Pharmacogenomics NIH Definitions The comprehensive compilation of information about genomic sequences and sequence variants, and the application of this information to understanding individual variations in drug response. Pharmacogenetics The effect of inheritance on individual variations in response to drugs and xenobiotics including individual differences in therapeutic efficacy or adverse drug reactions. U.S. National Institutes of Health, 1998

9 More definitions (I) Pharmocogenomics Use of genomics to target areas for drug development. Pharmacogenetics Use of genomics to target subpopulations for increased drug efficacy or decreased risk. Source: PAREXEL

10 More definitions (II) Pharmocogenomics: The hybridization of functional genomics and molecular pharmacology. > Find correlations between therapeutic response to drugs and the genetic profiles of patients. Toxicogenomics: The hybridization of functional genomics and molecular toxicology. > Find correlations between toxic responses to toxicants and the genetic profiles of patients exposed to them. Source: PAREXEL

11 More definitions (III) Pharmocogenetics The search for genetic polymorphisms (genetic deviations in drug metabolizing enzymes, drugs, receptors, transport proteins, etc) a major reason for the diversity in drug response. The identification of subpopulations for which a drug works better or for which the new drug creates a serious health hazard. Source: PAREXEL

12 More definitions (IV) Pharmocogenetics The analysis of geneticallycaused variations in: Pharmacodynamics > What the drug does to the body (i.e., receptor binding/expression, enzyme inhibition, signal transduction Pharmacokinetics > What the body does to the drug (i.e., drug metabolizing enzymes) Source: W. Pschyrembel, 257th ed., 1998; Iyer and Ratain, 1998

13 Detailed definitions (I) Pharmacogenomics Differential effects of compounds in vivo or in vitro on gene expression, among the entirety of expressed genes. Assessed by expression profiling. A tool for compound selection / drug discovery. Many drugs (i.e., early-stage compounds) one genome (i.e., normative genome; database, technology platform). Focus: compound variability. Source: Klaus Lindpaintner, 2002

14 Detailed definitions (II) Pharmacogenetics Differential effects of a drug in vivo in different patients, dependent on the presence of inherited gene variants. Assessed primarily by genetic (SNP) and genomic (expression) approaches. A concept to provide more patient / disease-specific health care. One drugs many genomes (i.e, different patients). Focus: compound variability. Source: Klaus Lindpaintner, 2002

16 Genetic Diversity The human population is genetically diverse with thousands of alleles at most gene loci. But diversity is also limited in that most genes have only a handful (2-5) of common (e.g., > 10 %) variants in their coding regions, the vast majority of alleles are exceedingly rare. Source: Urs A. Meyer, 1998

17 Evolutionary concept of genetic polymorphism Genetic polymorphisms provides variability in the population so that a change in the environment (e.g., exposure to chemicals, infectious agents, nutritional compounds, etc.) can elicit a change in the population, increasing its chance for survival. Source: Urs A. Meyer, 1998

18 History of Pharmacogenetics Source: Urs A. Meyer, 1998

19 An important acronym in Pharmacokinetics and Pharmacology ADME = Adsorption, Distribution, Metabolism, Excretion

20 Drug Metabololizing Enzymes Transform small lipid-soluble molecules. Substrate specificities are limited and overlapping. Occur in multiple isoenzymes or isoforms. Provide capacity to metabolize and detoxify countless substances in the diet and the environment. Frequently show genetic polymorphisms. Source: Urs A. Meyer, 1998

21 Drug metabolizing enzymes Source: Urs A. Meyer, 1998

22 Most drug metabolising enzymes exhibit clinically relevant genetic polymorhisms Source: W.E. Evans, M.V. Relling, Science 286, 487 (1999)

23 Source: Urs A. Meyer, 1998

24 Genetic polymorphism of xenobiotic metabolizing enzymes: Phenotyping and genotyping tests Source: Urs A. Meyer, 1998

25 Die Cytochrom (CYP) P-450 Superfamilie (I) Eisenthiolat-Proteine / enzymatisch: Monooxygenase. Lokalisierung: Glattes endoplasmatisches Retikulum. Verantwortlich für die oxidative Metabolisierung, vor allem in der Leber. Phase I drug metabolizer. > 30 verschiedene P-450-Moleküle sind im Menschen beschrieben. Alle zeigen Unterschiede in katalytischer Aktivität und Regulation. Nomenklatursystem: CYP1A2 > Isoform 2 der Subfamilie A der 1-CYP-Familie

26 Die Cytochrom (CYP) P-450 Superfamilie (II) Mehr als die Hälfte (175 von 315 = 56 %) aller in den USA häufig verschriebenen Arzneimittel werden von CYP P-450-Proteinen metabolisiert. Allein das Isoenzym CYP2D6 metabolisiert 25 dieser häufig verwendeten Arzneimittel. Die bekannten Polymorphismen des CYP2D6-Gens beeinflussen bis zu 17 % der Mitglieder einzelner ethnischer Gruppen. Source: R.J. Bertz, G.R. Grannemann, Clinical Pharmacokinetics 32, 210 (1997)

27 Structure of CYP2D6*2xN Alleles

28 Debrisoquine / Sparteine Debrisoquine: 3,4-dihydro-2(H)-isoquinolinecarboxamidine (sulfate); oxidative metabolism of this drug is used to assay and model drug metabolism in general, especially genetic, ethnic, and individual pharmacokinetic differences An adrenergic neuron-blocking drug. A substrate for a polymorphic cytochrome P-450 enzyme. Persons with certain isoforms of this enzyme are unable to properly metabolize this and many other clinically important drugs. They are commonly referred to as having a debrisoquin 4-hydroxylase polymorphism. Debrisoquine acts on the nerves to lower blood pressure by preventing the action of noradrenaline, a chemical which is released by nerve cells in order to transmit messages between nerves. After being released and producing its effect, it is taken back up into the nerve cells. Debrisoquine is taken up into the nerve cells by the same mechanism as noradrenaline. It therefore replaces the stores of noradrenaline in the nerves. Thus, when a signal is sent between nerves, debrisoquine is released instead of noradrenaline. As debrisoquine has no effect on nerves, the message is not passed on any further. This prevents the nerves sending signals to the muscles in the walls of the blood vessels. As a result, these muscles relax and the blood vessels widens. Sparteine: A quinolizidine alkaloid isolated from several Fabaceae, including Lupinus, Spartium and Cytisius. It has been used as an oxytocic and an anti-arrhythmia agent. It has also been of interest as an indicator of CYP2D6 genotype. Source:

29 Debrisoquinine / Sparteine Polymorphism (CYP2D6) Phenotype Extensive (EM, %) Poor (PM, 1-10 %) Intermediate (IM, 5-9 %) Ultra rapid (UM, 2-7 %) Genotypes 48 Mutations, 53 alleles of CYP2D6 gene. 4 common alleles represent > 95 % of variant alleles Source: Urs A. Meyer, 1998

30 Thiopurin-S-Methyltransferase (TPMT) Die Behandlung der kindlichen Leukämie und des Morbus Crohn mit Thiopurinen (6-Mercaptopurin, Azathiopurin) kann bei Vorliegen einer bestimmten allelen Variante des TPMT- Gens zu schweren Blutbildstörungen auch mit tödlichem Ausgang führen. TPMP metabolisiert Thiopurine. Ist die TPMT-Aktivität im Falle der allelen Variante erniedrigt, kommt es zur verminderten Metabolisierung der Thiopurine, dadurch zu einem entsprechendem Anstieg des Plasmaspiegels, mit Folge einer schweren Vergiftung. Durch Genotypisierung können Patienten mit erniedrigter TPMT-Aktivität identifiziert werden. Geringere Arzneimitteldosen können bei diesen Patienten die lebensgefährliche Vergiftung vermeiden. Source: R.M. Weinshilboum, 1999

31 Human TPMT polymorphisms Source: Otterness et al.

32 Human TPMT polymorphisms Source: Urs A. Meyer, 1998

33 Typical distribution of metabolizers Source: Urs A. Meyer, 1998

34 Gene dose effect and clinical consequences Source: Urs A. Meyer, 1998

35 Daily dose of Nortriptyline in relation to phenotype

36 Polygenic determinants of drug effects Source: W.E. Evans, M.V. Relling, Science 286, 487 (1999)

38 Alzheimer s disease: Some Characteristics Alzheimer s is the fourth leading cause of death in North America and costs the U.S. $ 93 billion in direct and indirect costs. There are two common forms of Alzheimer s: - 50 % E4 positive, 50 % E4 negative These two forms respond differently to drug treatments: - Drugs-designed to stimulate the cholinergic system tend to work well in non-e4 - Drugs that are non-cholinergic work well in E4 Patient stratification is fast becoming the standard of care Source: AACC

39 Limited benefits in current Alzheimer s patient treatment schemes (Cognex) Some patients may receive clinical meaningful benefit from Tacrine (2 points on a 70-point scale) Nausea, vomiting, diarrhea are expected Liver toxicity in % of patients In a clinical trial, 72 % of patients who took the highest dose had to stop taking the drug

40 Alzheimer s Patient Stratification The effects of Cognex (tacrine) vary widely from improving to worsening the condition. Improvement / (Deterioration) Improvement in 38 Alzheimer's Patients with Cognex Test for Presence of apoe4 Negative Positive Improvement / (Deterioration) Improvement / (Deterioration) Improvement in 19 apoe2/e3 Alzheimer's Patients with Cognex Improvement in 19 apoe4 Alzheimer's Patients with Cognex Sources: AACC; JAMA 279, 1200 (1998)

41 Alzheimer s patient stratification scheme Yes No Cognex Analysis of the apoe Gene E4 Form? No Cognex

43 Identifying genes influencing polygenic drug responses a-c, Three genome-wide approaches gene expression analysis, genome-wide scans and proteomics. d, e, The conventional candidate gene approach, based on clinical pharmacology studies of proteins and pathways known to be involved in a drug s pharmacokinetic or pharmacodynamic response. Source: W.E. Evans, M.V. Relling, Nature, 429, 464 (2004)

44 Evidence linking candidate gene poymorphisms to phenotype can come from several sources a, In vitro screens of human tissues. b, In vitro functional studies of expressed alleles. c, Clinical phenotype-genotype studies. Source: W.E. Evans, M.V. Relling, Nature 429, 464 (2004)

45 Molecular diagnostics of pharmacogenomic traits Source: W.E. Evans, M.V. Relling, Science 286, 487 (1999)

46 Der Roche / Affymetrix AmpliChip Quelle:

47 Der Roche AmpliChip CYP 450 hat verschiedene Oligonukleotid- Sonden zur -Bestimmung von P4502D6 und -2C19 Polymorphismen Quelle:

49 Lessions Genetic polymorphisms of drug metabolizing enzymes are common and give rise to subpopulations with Altered drug kinetics A higher risk for toxicity, or A higher risk for undermedication Source: Urs A. Meyer, 1998

50 Drei Anwendungsgebiete phramakogenomischer Erkenntnisse Identifizierung neuer Zielmoleküle für die Entwicklung von Medikamenten (drug target). Therapeutische Berücksichtigung genetischer Unterschiede in Arzneimittel-metabolisierenden / Toxizitäts-Verursachenden Enzymen (drug metabolism). Diagnose und therapeutische Berücksichtigung Krankheits-Assoziierter Gene (disease pathway)

51 Pharma Development Dilemma Patient stratification will become the standard of care for many drugs in 5-10 years. No Clinically Significant Response Increasing Degree of Clinical Effectiveness Adverse Reactions Death Adverse Reactions Do Not Treat Number of Patients Clinically Effective Treat Non-Responders Do Not Treat Therapeutic Response