1. Drug Metabolism and Pharmacogenetics Brendan Stamper University of Washington Dept. of Medicinal Chemistry

2. What is Medicinal Chemistry? Medicinal Chemistry is a scientific discipline involved with designing, synthesizing and developing pharmaceuticals suitable for therapeutic use Highly interdisciplinary science combining genetics, molecular biology, biochemistry, organic chemistry, pharmacology, toxicology

3. Outline Background –Drug Metabolism –Pharmacogenetics Examples –CYP2D6 Codeine DDI Scenario (fluoxetine) –ALDH2 Ethanol DDI Scenario (acetaminophen)

4. Basic Vocabulary Lipophilicity vs Hydrophilicity –Lipophile: “Fat-lover” –Hydrophile: “Water-lover” Xenobiotic: a foreign chemical substance

5. More Basic Vocabulary Metabolism: chemical reactions that occur in living organisms Enzyme: a biomolecule that catalyzes a chemical reaction XenobioticMetabolite Enzyme Inducer Inhibitor

6. Today’s Focus We will focus on the enzymatic conversion of lipophilic xenobiotics to more water soluble metabolites...... and how these processes are influenced by genetic predisposition WHY DO WE CARE?

7. Lipophilic xenobiotics can be potentially dangerous because they can easily permeate lipid cell membranes and accumulate within cells By converting lipophilic xenobiotics to hydrophilic metabolites we can facilitate elimination

8. Today’s Focus We will focus on the enzymatic conversion of lipophilic xenobiotics to more water soluble metabolites...... and how these processes are influenced by genetic predisposition (i.e. Can we expect xenobiotic metabolism to be consistent from person-to-person?) NO

9. Variability in Dose-Response “If it were not for the great variability among individuals, medicine might as well be a science and not an art” William Osler 1849-1919 What factors are responsible for this variability?

10. Drug Response Age Gender Race Diet Occupational Exposure Genetics DiseaseStress Variability in Dose-Response

11. Pharmacogenetics Definition: The study of genetic variation that gives rise to variability in drug response (Optimize efficacy and limit toxicity) Drug Response Age Gender Race Diet Occupational Exposure Genetics DiseaseStress

12. How do we predict optimal dose for most efficacious response Step 1: Understand the mechanism of drug action at the molecular level Step 2: Understand how genetic variations affect drug action Step 3: Rational choice of drug and dosage

13. DNA Is Like a Language DNA ATGC Codon Gene Chromosome Genome English Abcdef... Word Sentence Chapter Book Like language, DNA changes over time

14. Polymorphism Polymorphism: Change in DNA sequence that occurs in more than 1% of the population Allele: An alternative form of a gene (i.e. site of sequence variation) SNP (Single Nucleotide Polymorphism) Gene: ATG-GGA-TGC-TAA met-gly-cys-STOP SNP: ATG-GCA-TGC-TAA met-ala-cys-STOP Impact of new allele –Alter protein function –Alter protein structure or stability –No consequence

15. CYP2D6 and Codeine Example #1

16. Codeine Analgesic Prodrug CYP2D6-mediated bioactivation critical for analgesic effect –200mg codeine is equivalent to 30mg morphine (~10%) CYP2D6 More LipophilicMore Hydrophilic

17. CYP2D6 Drug metabolizing enzyme Member of the P450 family (Cytochrome P450 2D6) Common substrates –Beta-blockers (Metoprolol) –SSRIs (Fluoxetine) –Opiods (Codeine) –SERMs (Tamoxifen) Highly polymorphic enzyme –Over 100 reported Rowland et al, JBC (2006) 281:7614-7622

18. Allele Kinetic Data Kinetic plot of product formation versus substrate concentration for metabolic turnover of codeine catalyzed by highly purified recombinant CYP2D6 isoforms in vitro. Yu et al, JPET (2002) 303:1291-1300

19. CYP2D6 Genotypes 1.Ultra Metabolizers 2.Extensive Metabolizers 3.Intermediate Metabolizers 4.Poor Metabolizers

20. Poor vs Extensive Metabolizers Individual plasma concentration of codeine and morphine in 14 extensive (filled) and 14 poor (open) metabolizers after an oral dose of codeine. Poulsen et al, Eur J Clin Pharmacol (1996) 51:289-295

21. How can we sort the population into the different CYP2D6 metabolizing groups?

22. Urinary Metabolic Ratio of CYP2D6 Substrate Dose patients with CYP2D6 substrate (codeine) Collect urine sample that contains substrate and metabolite Calculate ratio of substrate over metabolite Substrate (codeine) Metabolite (morphine) = High (poor metabolizer) Low (extensitve metabolizer)

23. Roden et al, Ann Intern Med 2006; 145:749-757 Urinary Metabolic Ratio of CYP2D6 Substrate Intermediate Metabolizers

24. Outcomes of CYP2D6 Allelic Variations Zanger et al, Naunyn-Schiedeberg’s Arch Pharmacol (2004) 369: 23-37 No analgesic effect Pain relief ‘Overdose’ effect Slight analgesic effect Null allele Decreased function allele Fully functional allele Expected plasma concentration-time curve with therepeutic window indicated by the boxed area

25. Codeine Pharmacogenetics Green: Intermediate and Extensive Metabolizers Purple: Ultra Metabolizers Orange: Poor Metabolizers

26. Who are most affected by CYP2D6 polymorphisms?

27. Ethnic Variation in CYP2D6 Mutation Frequencies VariantPhenotypeCaucasianAsianAfrican Ethiopian/ Saudi CYP2D6*2xNUM1-5%0-2%2%10-16% CYP2D6*4PM12-21%1%2%1-4% CYP2D6*10IM1-2%51%6%3-9% CYP2D6*17IM0%ND34%3-9% CYP450 allele nomenclature committee database: http://www.imm.ki.se/cypalleles

28. DDI Scenario: Codeine and Fluoxetine +

29. Drug-Drug Interactions: Codeine + Fluoxetine Codeine CYP2D6 Morphine Fluoxetine (inhibitor) X Zanger et al, Naunyn-Schiedeberg’s Arch Pharmacol (2004) 369: 23-37

30. Summary: CYP2D6 & Codeine Codeine is a prodrug (requires metabolism) CYP2D6 metabolizes codeine to morphine CYP2D6 is a highly polymorphic enzyme Populations can be separated into different metabolic sub-groups –UMs: ‘Overdose’ analgesic effect –EMs/IMs: Predicted analgesic effect –PMs: No analgesic effect Urinary sampling can enable the pre-sorting of different metabolizers Co-treatment with fluoxetine: EMs to PMs

31. ALDH2 and Ethanol Example #2

32. Ethanol Low dose: Muscle relaxant, euphoria impaired judgment High dose: CNS depressant, impaired sensory/motor function Toxic when BAC > 400mg/dL (0.4%)

33. Ethanol Metabolism Alcohol to aldehyde to carboxylic acid Ethanol: CNS depressant Acetaldehyde: Vasodilator –Flushing –Hangover effects Acetic Acid: Relatively harmless ADHALDH More LipophilicMore Hydrophilic

34. ALDH2 Aldehyde dehydrogenase 2 Mitochondrial enzyme Homotetramer Substrates: aldehydes Cofactor: NAD + Catalyzes the oxidation of aldehydes Polymorphic enzyme Larson et al, JBC (2005) 280:30550-30556

35. ALDH2 Genotypes ALDH2*1/*1Wild-type Homozygous ALDH2*1/*2Heterozygous ALDH2*2/*2Mutant Homozygous *2 allele = E487K mutation

36. ALDH Crystal Structure Violet & Blue: NAD + -bound ALDH2*1 Red: NAD + -bound ALDH2*2 Larson et al, JBC (2005) 280:30550-30556 *2 Mutation: E487K

37. Kitagawa et al, FEBS Letters (2000) 476:306-311 ALDH2 Activity Among Differing Genotypes Comparison of substrate specific activities of human liver ALDH2 derived from three ALDH genotypes MALD AALD BALD PALD

38. ALDH2 Influence on AALD Blood Levels Following Ethanol Ingestion Ginsberg et al, Reg Toxicol Pharmacol (2002) 36, 297-309 ADH ALDH2*1/*1 No Flushing No Hangover Effects Some Flushing Some Hangover Effects Flushing Hangover Effects ALDH2*1/*2ALDH2*2/*2 X X X

39. Who is Affected? Ginsberg et al, Reg Toxicol Pharmacol (2002) 36, 297-309 ALDH2 polymorphism by ethnic group

40. How do you treat an acetaldehyde overdose?

41. DDI Scenario: (Ethanol and Acetaminophen) + Analgesic/AntpyreticCNS Depressant

42. Drug-Drug Interaction: Ethanol and Acetaminophen CYP2E1 Glutathione adducts (Detoxification) Protein adducts (Toxicity) Ethanol (inducer) X Cell Death Liver Damage NAPQI

43. Toxic Scenario 1.Chronic alcohol abuser induces CYP2E1 2.‘Activated’ CYP2E1 forms more toxic metabolite (NAPQI) 3.Increased levels of NAPQI can lead to glutathione depletion 4.Increased protein adduct formation leading to cell death and liver damage If a you are a chronic alcohol abuser, use ibuprofen instead of acetaminophen to treat your hangover.

44. Summary: ALDH2 & Ethanol Ethanol metabolism occurs in two steps –ADH: Ethanol to acetaldehyde (toxic metabolite) –ALDH2: Acetaldehyde to acetic acid (detoxification step) Three common ALDH2 genotypes –ALDH2*1/*1: No flushing or hangover effects –ALDH2*1/*2: Some flushing and hangover effects –ALDH2*2/*2: Flushing and hangover effects ALDH2*2 most prevalent in Asian populations Chronic alcohol abusers should not take acetaminophen to treat their hangovers

45. Things to Think About Drug metabolizing enzymes tend to metabolize lipophilic compounds into hydrophilic compounds (absorption to excretion) Your genotype impacts how you metabolize drugs Pharmacogenetics can be used to optimize therapy Science is interdisciplinary Genetics - Molecular Biology - Biochemistry - Organic Chemistry - Pharmacology - Toxicology

46. Thank you!