1

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2

Rx + =

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Differences in genetic constitution

Rx + =

3

PHARMACOGENETICS

The study of genetically controlled variations in drug response

4

I. Key Concepts and Terms

Monogenic: due to allelic variation at a single gene

Polygenic: due to variations at two or more genes

Polymorphic: frequently occurring monogenic variants occurring at a frequency >1%

5

Normal DistributionFr

eque

ncy

Freq

uenc

y

ActivityActivity

6

Polymorphic Distribution

From Pratt WB,Taylor P. Fig 7-5b

7

GENETIC POLYMORPHISMS

Pharmacokinetic Pharmacodynamic•Transporters•Plasma protein binding•Metabolism

•Receptors•Ion channels•Enzymes•Immune molecules

8

From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999.

9From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999.

II. Genetic polymorphisms in drug metabolizing enzymes

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A. Atypical Plasma Cholinesterase

•a rapid acting, rapid recovery muscle relaxant - 1951•usual paralysis lasted 2 to 6 min in patients•occasional pt exhibited paralysis lasting hrs•cause identified as an “atypical” plasma cholinesterase

Hydrolysis by pseudocholinesterase

choline succinylmonocholine

O C CH2CH2

O

(H3C)3NH2CH2C CO

O CH2CH2N(CH3)3+ +

SUCCINYLCHOLINE

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•Atypical plasma cholinesterase has 1/100 the affinity for succinylcholine as normal enzyme•occurs in 1:2500 individuals•tested clinically via the abilityof dibucaine to inhibit esterase hydrolysis of benzoylcholine

Adapted from: Pharmac Ther 47:35-60, 1990.

normal enzyme inhibited > 70%abnormal inhibited < 30%

12

•Atypical plasma cholinesterase has 1/100 the affinity for succinylcholine as normal enzyme•occurs in 1:2500 individuals•tested clinically via the abilityof dibucaine to inhibit esterase hydrolysis of benzoylcholine•Family studies indicate variability in plasma cholinesterase activity consistent with 2 allelic, autosomal, codominant genes•other variant forms exist as well

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B. Glucose-6-phosphate dehydrogenase activityEffects >100 million worldwide

R-NH2 CYP MPOPGH Synthase

R-NOH

ERYTHROCYTE

R-NOH

O2

HgbFe+2

R-NO HgbFe+3

Reactive Oxygen

NADH

NAD+MetHgbReductase

NADPHor GSH(?)

NADP+ or GSSG(?)HMP Shunt

G-6-PDDependent

SODCatalaseGSH Peroxidase

Detoxification

SplenicSequestration

Hemolytic Anemia

GSHSemi-mercaptal

sulfinamide

R-NH2

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Drugs and Chemicals Unequivocally Demonstrated to Precipitate Hemolytic Anemia

in Subjects with G6PD Deficiency

Acetanilide Nitrofurantoin PrimaquineMethylene Blue Sulfacetamide Nalidixic AcidNaphthaleneSulfanilamide SulfapyridineSulfamethoxazole

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INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS

Ethnic Group Incidence(%)Ashkenazic Jews 0.4Sephardic Jews Kurds 53 Iraq 24 Persia 15 Cochin 10 Yemen 5 North Africa <4

Iranians 8Greeks 0.7-3

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INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS

Ethnic Group Incidence(%)Asiatics Chinese 2 Filipinos 13 Indians-Parsees 16 Javanese 13 Micronesians <1

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C. N-ACETYLTRANSFERASE ACTIVITY

Distribution of plasma isoniazid concentration in 483 subjectsafter and oral dose. Reproduced from Evans DAP. Br Med J 2:485, 1960.

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NAT1*4 NAT2*4 NAT2*5A NAT2*6A NAT2*7A

PABAPASSMX

PADDSSMZ

AF Modified from Grant DM. Pharmacogenetics 3:45-52, 1993

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ETHNIC DIFFERENCES IN THE DISTRIBUTION OF ACETYLATOR PHENOTYPE

Population % Slow % Hetero Fast % Homo Fast

South Indians 59 35.6 5.4Caucasians 58.6 35.9 5.5Blacks 54.6 38.6 6.8Eskimos 10.5 43.8 45.7Japanese 12 45.3 42.7Chinese 22 49.8 28.2

From: Kalo W. Clin Pharmacokinet 7:373-4000, 1982.

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XENOBIOTICS SUBJECT TO POLYMORPHIC ACETYLATION IN MAN

Hydrazines isoniazid hydralazine phenylzineacetylhydrazine hydrazine

Arylamines dapsone procainamide sulfamethazine sulfapyridineaminoglutethimide

Carcinogenic Arylamines benzidine-naphthylamine4-aminobiphenyl

Drugs metabolized to aminessulfasalazine nitrazepamclonazepam caffeine

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ADVERSE EFFECTS TO SULFASALAZINE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

N N

COOH

OHS

O

O

HNN

SULFASALAZINE

NH2S

O

O

HNN

H2N

COOH

OH

SULFAPYRIDINE 5-AMINOSALICYLIC ACID

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ADVERSE EFFECTS TO SULFASALAZINE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

Data from: Das et al. N Engl J Med 289:491-495, 1973.

Side Effect cyanosis hemolysistransient reticulocytosis

Frequency of side effectSlow Acetylators Fast Acetylators

9 15 06 0

23

0

20

40

60

80

100

120

0 20 40 60 80 100

Duration of Therapy (months)

% o

f pts

with

lupu

s

Slow AcetylatorsFast Acetylators

Relationship Between Onset of Lupus Syndrome in Fast and Slow Acetylators Receiving Procainamide.

Data from: Woosley RL, et al. N Engl J Med 298:1157-1159, 1978.

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NH2C

O

(H3CH2C)2NH2CH2CHN

PROCAINAMIDE

NHC

O

(H3CH2C)2NH2CH2CHN CCH3

O

NHOHC

O

(H3CH2C)2NH2CH2CHN

NATCYP450

PROCAINAMIDE HYDROXYLAMINE N-ACETYLPROCAINAMIDE

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Distribution of acetylator phenotype in control subjects and those experiencing a sulfonamide

hypersensitivity reaction.Rieder et al. Clin Pharmacol Ther 49:13-17, 1991.

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NAT1 N-acetyl-SMXUDPGTSMX-glucuronide

CYP2C9MPOPGH SYNTHASE

SMX hydroxylamineNitrosoDetox

Covalent binding tocellular macromolecules/

cytotoxicity

Hypersensitivity/Adverse Reaction

O-acetylation

Acetoxy ester

NAT1 HydroxamicacidN,O-AT

Detoxified metabolite

Sulfamethoxazole(SMX)

NO

NH2 S

O

O

HN

CH3

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D. CYP2D6 ACTIVITY

N C NHNH2

N C NHNH2

CYP2D6

OH

DEBRISOQUINE4-HYDROXYDEBRISOQUINE

H

N CH3

OCH3

H

N CH3

OH

DEXTROMETHORPHAN DEXTRORPHAN

CYP2D6

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DRUGS WHOSE METABOLISM CO-SEGREGATES WITH DEBRISOQUINE

alprenolol amitriptyline bufuralol clomipraminecodeine desipramine encainide ethylmorphineflecainide fluoxetine guanoxan imipraminemetoprolol nortriptyline paroxetine phenforminpropafenone propranolol

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Plasma metoprolol concentrations in poor () and extensive () metabolizers of debrisoquine after 200 mg of metoprolol tartrate administered orally. Redrawn from Lennard MS, et al. NEJM 307:1558-1560, 1982.

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Dose requirements for nortriptyline in patients with different CYP2D6Phenotypes. From: Meyer U. Lancet 356:1667, 2000.

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O-demethylationmorphine

N-demethylationnorcodeine

6-glucuronidationcodeine-6-glucuronide

M-6-G

M-3-G

normorphine

norcodeine-6-glucuronide

CYP2D6

H3CO

NCH3

HO

O

CODEINE

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Effect of Quinidine on the Analgesic Response to Codeine in Extensive Metabolizers of

CYP2D6 (Phenotyped with Dextromethorphan)Data from: Desmeules J, et al. Eur J Clin Pharmacol 41:23:26, 1991

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What is the cause of ‘hypermetabolizers’?What is the cause of ‘hypermetabolizers’?

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Debrisoquine phenotype in subjects with different CYP2D6 genotypes

Genotype # of Subjects

Metabolic Ratio

CYP2D6wt/(CYP2D6L)2 9 0.33

CYP2D6wt/CYP2D6wt 12 1.50

CYP2D6wt/CYP2D6(A or B) 9 2.14

CYP2D6B/CYP2D6B 6 48.84

(CYP2D6L)2 - gene duplication; CYP2D6A - single base deletionCYP2D6B - multiple point mutations

Data from: Agundez JG et al. Clin Pharmacol Ther 57:265, 1995.

36From: Dalen P, et al. Clin Pharmacol Ther 63:444-452, 1998.

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E. CYP2C9 ACTIVITYPrescribed Daily Warfarin Dose and CYP2C9 Genotype

Warfarin Dose* Genotype5.63 (2.56) *1/*14.88 (2.57) *1/*23.32 (0.94) *1/*34.07 (1.48) *2/*22.34 (0.35) *2/*31.60 (0.81) *3/*3

*Data presented as mean (SD) daily dose in mg

From: Higashi MK, et al. Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. JAMA 287:1690-1698, 2002.

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F. THIOPURINE METHYLTRANSFERASE (TPMT)

N

N NH

N

SH

N

N NH

N

SCH3

TPMT

SAM SAH

6-mercaptopurine 6-methylmercaptopurine

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TPMT Activity

Freq

uenc

y

Distribution of Thiopurine Methyl-transferase Activity. Reproduced from: Weinshelboum RM, Sladek SL. Am J Hum Genet 32:651-662, 1980.

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41

G. GENETIC POLYMORPHISMS, MATERNAL SMOKING AND LOW BIRTH WEIGHT (LBW)65% of all infant deaths occur among LBW infants, while LBW infants account for 7.6% of all live births

Reduction in birth wgt among smoking women

Genotype Weight Reduction CYP1A1 AA 252 g CYP1A1 Aa/aa 520 g

GST1 AA/Aa 285 g GST1 aa 642 g

Data from: Wang X, et al. JAMA 287:195-2002, 2002.

42From: Esteller M, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. NEJM 243:1350-1354, 2000.

Why are some gliomas resistant to nitrosourea alkylating agents?

Evidence suggests this may be the result of an epigenetic phenomenon – one that does not involve a change in DNA sequence.

MGMT – methylguanine-DNA methyltransferaseMethylation of the promoter region of MGMT may silence the gene

43From: Esteller M, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. NEJM 243:1350-1354, 2000.

44From: Esteller M, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. NEJM 243:1350-1354, 2000.

45

46

Future Role of SNPs and Pharmacogenetics

SNP - Single Nucleotide Polymorphisms

……. G G T A A C T G …………. G G C A A C T G …...

AS of February 2001, 1.42 million SNPs had been identified in the human genome.

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Patients with efficacyin clinical trials

Patients without efficacyin clinical trials

Predictive of efficacy

Predictive of no efficacy