Drug Metabolism and Pharmacogenetics

Brendan Stamper

University of Washington

Dept. of Medicinal Chemistry

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

Outline

• Background– Drug Metabolism– Pharmacogenetics

• Examples– CYP2D6

• Codeine• DDI Scenario (fluoxetine)

– ALDH2• Ethanol• DDI Scenario (acetaminophen)

Basic Vocabulary

• Lipophilicity vs Hydrophilicity– Lipophile: “Fat-lover”– Hydrophile: “Water-lover”

• Xenobiotic: a foreign chemical substance

More Basic Vocabulary

• Metabolism: chemical reactions that occur in living organisms

• Enzyme: a biomolecule that catalyzes a chemical reaction

Xenobiotic MetaboliteEnzyme

Inducer

Inhibitor

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?

• 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

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

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 Osler1849-1919

What factors are responsible for this variability?

DrugResponse

Age

Gender

Race

DietOccupational

Exposure

Genetics

DiseaseStress

Variability in Dose-Response

Pharmacogenetics

• Definition: The study of genetic variation that gives rise to variability in drug response (Optimize efficacy and limit toxicity)

DrugResponse

Age

Gender

Race

DietOccupational

Exposure

Genetics

DiseaseStress

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

DNA Is Like a Language

• DNA• ATGC• Codon• Gene• Chromosome• Genome

• English• Abcdef . . .• Word• Sentence• Chapter• Book

Like language, DNA changes over time

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-STOPSNP: ATG-GCA-TGC-TAA met-ala-cys-STOP

• Impact of new allele– Alter protein function– Alter protein structure or stability– No consequence

CYP2D6 and Codeine

Example #1

Codeine• Analgesic• Prodrug• CYP2D6-mediated bioactivation critical for

analgesic effect– 200mg codeine is equivalent to 30mg morphine (~10%)

CYP2D6

More Lipophilic More Hydrophilic

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

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

CYP2D6 Genotypes

1. Ultra Metabolizers

2. Extensive Metabolizers

3. Intermediate Metabolizers

4. Poor Metabolizers

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

How can we sort the population into the different CYP2D6

metabolizing groups?

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)

Roden et al, Ann Intern Med 2006; 145:749-757

Urinary Metabolic Ratio of CYP2D6 Substrate

IntermediateMetabolizers

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

Codeine Pharmacogenetics

Green: Intermediate and Extensive MetabolizersPurple: Ultra MetabolizersOrange: Poor Metabolizers

Who are most affected by CYP2D6 polymorphisms?

Ethnic Variation in CYP2D6 Mutation Frequencies

Variant Phenotype Caucasian Asian AfricanEthiopian/

Saudi

CYP2D6*2xN UM 1-5% 0-2% 2% 10-16%

CYP2D6*4 PM 12-21% 1% 2% 1-4%

CYP2D6*10 IM 1-2% 51% 6% 3-9%

CYP2D6*17 IM 0% ND 34% 3-9%

CYP450 allele nomenclature committee database: http://www.imm.ki.se/cypalleles

DDI Scenario:Codeine and Fluoxetine

+

Drug-Drug Interactions:Codeine + Fluoxetine

CodeineCYP2D6

Morphine

Fluoxetine(inhibitor)

X

Zanger et al, Naunyn-Schiedeberg’s Arch Pharmacol (2004) 369: 23-37

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

ALDH2 and Ethanol

Example #2

Ethanol

• Low dose: Muscle relaxant, euphoria impaired judgment

• High dose: CNS depressant, impaired sensory/motor function

• Toxic when BAC > 400mg/dL (0.4%)

Ethanol Metabolism

• Alcohol to aldehyde to carboxylic acid• Ethanol: CNS depressant• Acetaldehyde: Vasodilator

– Flushing– Hangover effects

• Acetic Acid: Relatively harmless

ADH ALDH

More Lipophilic More Hydrophilic

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

ALDH2 Genotypes

• ALDH2*1/*1 Wild-type Homozygous

• ALDH2*1/*2 Heterozygous

• ALDH2*2/*2 Mutant Homozygous

• *2 allele = E487K mutation

ALDH Crystal

Structure

Violet & Blue: NAD+-bound ALDH2*1Red: NAD+-bound ALDH2*2

Larson et al, JBC (2005) 280:30550-30556

*2 Mutation: E487K

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

ALDH2 Influence on AALD Blood Levels Following Ethanol Ingestion

Ginsberg et al, Reg Toxicol Pharmacol (2002) 36, 297-309

ADH ALDH2*1/*1

No FlushingNo Hangover Effects

Some FlushingSome Hangover Effects

FlushingHangover Effects

ALDH2*1/*2ALDH2*2/*2

X

X

X

Who is Affected?

Ginsberg et al, Reg Toxicol Pharmacol (2002) 36, 297-309

ALDH2 polymorphism by ethnic group

How do you treat an acetaldehyde overdose?

DDI Scenario:(Ethanol and Acetaminophen)

+

Analgesic/AntpyreticCNS Depressant

Drug-Drug Interaction:Ethanol and Acetaminophen

CYP2E1

Glutathione adducts(Detoxification)

Protein adducts(Toxicity)Ethanol

(inducer)

X

Cell Death

Liver Damage

NAPQI

Toxic Scenario1. 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.

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

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

Thank you!