Genetical determination Genetical determination of drug action. of drug action.

Phenotyping and genotyping Phenotyping and genotyping in optimizing patient therapy.in optimizing patient therapy.

Pharmacogenetics

The study of the role of inheritance in individual variation in response to

xenobiotics, including drugs.

Pharmacogenomics

The study of the genome and its products (including RNA and protein) as it relates to drug discovery, development and

funktion.

Pharmacogenomics

Human Genetics• SNPs• Haplotypes• Sequencing

Expression Profiling• Specific transcript levels• Total RNA profiling

Proteomics• Specific biochemical

markers• Protein profiling

Phenotype• Drug response

• Disease

Prediction

Pharmacogenomics

Both pharmacogeneticspharmacogenetics and ppharmacogenomicsharmacogenomics deal with the genetic basis underlying variable drug response in individual patients.

The traditional pharmacogeneticpharmacogenetic approach relies on studying sequence variations in single candidate genes suspected to be responsible of affecting drug efficacy and safety.

Evans, W. E., Johnson, J. A., Annu. Rev. Genomics Hum. Genet., 2001, 2: 9 - 39.

GENETIC GENETIC POLYMORPHISMSPOLYMORPHISMS

PharmacokineticPharmacokinetic PharmacodynamicPharmacodynamic•TransportersTransporters

•MetabolismMetabolism

•ReceptorsReceptors•Ion channelsIon channels•EnzymesEnzymes•Immune moleculesImmune molecules

Genetic mechanisms relatively well known

Genetic mechanisms mostly yet unknown

TransportTransportersers

Best known genetically determined transporter system is ABCB1 (ATP Binding Cassette Proteins B1),

former known as Multi Drug Resistance 1 (MDR1).

- Main product: P-glycoprotein

O U T

M E M B R A N E

IN1 85

2 0 0

1 0 0

3 00

4 00

5 00

6 00

7 00

1

11 0 0

1 2 8 0

AT P S IT E AT P S IT E

8 0 09 0 0

1 00 0 1 2 0 0

M D R 1 v s . m d r1 b (m o u s e )

P-glycoproteinP-glycoprotein

P-glycoprotein is a cellular efflux pump encoded by the ABCB1 (MDR1) gene.P-glycoprotein is expressed on the apical membrane of epithelial cells in the intestine, liver, kidney, testes, blood-brain barrier and adrenals.P-glycoprotein is responsible for the resistance of tumor cells to multiple chemotherapeutic agents.

P-glycoproteinP-glycoproteinP-glycoprotein plays a role in the absorption, distribution and elimination of numerous drugs:

• digoxin, chinidine, celiprolol, talinolol, verapamil • doxorubicin, daunorubicin, etoposid, irinotecan,

paklitaxel, vinblastin• ciclosporin, tacrolimus • aldosteron, hydrocortisone, dexametasone • morphine, metadone • rifampicine • cephazoline, kolchicine, atorvastatin, erythromycin,

fluphenasin, lovastin, perphenazine, ondansetron, phenytoin, tamoxiphen, tioridazine, loperamid.

Metabolism:

1. Atypical Plasma Cholinesterase(Butyrylcholinesterase)

•a rapid acting, rapid recovery muscle relaxant - 1951•usual paralysis lasted 2 to 6 min in patients•occasional pt exhibited paralysis lasting 2 to 6 hrs•cause identified as an “atypical” plasma cholinesterase, which has 1/100 the affinity for succinylcholine•occurs in 1:2500 individuals – gene BCHE

O C CH2CH2

O

(H3C)3NH2CH2C CO

O CH2CH2N(CH3)3+ +

SUCCINYLCHOLINE

2. Favism - Glucose-6-phosphate dehydrogenase (G6PD) activity

Drugs and Chemicals Unequivocally Demonstrated to Precipitate Hemolytic Anemia

in Subjects with G6PD Deficiency

Acetanilide Nitrofurantoin PrimaquineMethylene Blue Sulfacetamide Nalidixic AcidNaphthaleneSulfanilamide SulfapyridineSulfamethoxazole

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

3. N-ACETYLTRANSFERASE ACTIVITY (NAT2)

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

ETHNIC DIFFERENCES IN THE DISTRIBUTION OF ACETYLATOR PHENOTYPE

Population % Slow % Heterozygotes % Homozygotes Fast Fast

South Indians 59 35.6 5.4Caucasians 58.6 35.9 5.5Blacks 54.6 38.6 6.8

Chinese 22 49.8 28.2Japanese 12 45.3 42.7Eskimos 10.5 43.8 45.7

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

XENOBIOTICS SUBJECT TO POLYMORPHIC ACETYLATION (NAT2)

IN MAN Hydrazines isoniazid hydralazine phenylzineacetylhydrazine hydrazine

Arylamines dapsone procainamide sulfamethazine sulfapyridineaminoglutethimide

Carcinogenic Arylamines benzidineβ-naphthylamine4-aminobiphenyl

Drugs metabolized to aminessulfasalazine nitrazepamclonazepam caffeine

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

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

4. CYP2D6 ACTIVITY

N C NH

NH2

N C NH

NH2

CYP2D6

OH

DEBRISOQUINE 4-HYDROXYDEBRISOQUINE

H

N CH3

OCH3

H

N CH3

OH

DEXTROMETHORPHAN DEXTRORPHAN

CYP2D6

O-demethylationmorphine

N-demethylationnorcodeine

6-glucuronidationcodeine-6-glucuronide

M-6-G

M-3-G

normorphine

norcodeine-6-glucuronide

CYP2D6

H3CO

NCH3

HO

O

CODEINE

Ultrarapid metabolizers Ultrarapid metabolizers Extensive metabolizers Extensive metabolizers Poor metabolizers Poor metabolizers

Substrates

AntidepressantsAmitriptylineImipramineNortriptilineDoxepinFluoxetine (Prozac) Paroxetine

Antipsychotics HaloperidolDroperidolRisperidon

Beta blockers MetoprololTimololCarvedilol

CYP2D6Effect among poor metabolizers

Central Nervous System toxicity (tremors, CNS depression)

Lower doses effective

Loss of cardioselectivityADR in respiratory systemHypotesion

Substrates

Narcotics Codeine

Tramadol

Dextrometorphane

5-HT3 antagonists:OndansetronTropisetron

Other Perhexyline

CYP2D6Effect among poor metabolizers

Loss of efficacy

Increase of ADR

Lower doses effective

Hepatotoxicity, neurotoxicity

Importance of CYP2D6 genotype: Cardiac Arrhythmia Suppression Trial (CAST)

• A. Encainide (CYP2D6 dependent)• B. Flecainide (CYP2D6 dependent)• C. Moricizine

Expected reduction in mortality due to treatment = 30%

(No difference among active treatments)Required sample size = 4400 patients

CAST – main results after 10 months

Regime N CHD deaths at

10 months (%)

All deaths at

10 months

(%)

RR CHD death (95% CI)

RR all deaths (95% CI)

Placebo 725 9 (1.2) 22 (3.0) 1.0 1.0

Flecainide or encainide

730 33 (4.5)33 (4.5) 56 (7.7)56 (7.7) 3.63.6(1.7 to (1.7 to

8.5)8.5)

2.52.5 (1.6 to (1.6 to

4.5)4.5)

Cause: lethal flecainide toxicity among CYP2D6 poor metabolizers

6. 6. THIOPURINE S-METHYLTRANSFERASETHIOPURINE S-METHYLTRANSFERASE ((TPMTTPMT))

Thiopurine S-methyltransferase (TPMT; S-adenosyl-L-methionine thiopurine S-methyltransferase) catalyzes thiopurine S-methylation, an important metabolic pathway for thiopurine drugs such as azathioprine and 6-mercaptopurine.

Weinshilboum and Sladek found trimodality for level among 298 randomly selected subjects: 88.6% had high enzyme activity; 11.1% had intermediate activity and 0.3% had undetectable activity. This distribution conforms to Hardy-Weinberg expectations for a pair of autosomal codominant alleles for low and high activity, TPMT-L and TPMT-H, with frequencies of 0.059 and 0.941, respectively.

7. Other important polymorphisms:7. Other important polymorphisms:

• CYP2C9,• CYP2C19, • DPD,• TYMS, • RYDR.

0 5 10 15 200

5

10

Human RBC TPMT298 Unrelated Adults

TPMT Activity (Units/ml RBC)

TPMTL/TPMTL

TPMTL/TPMTH

TPMTH/TPMTH

1 2 3 4 5 6 7 8 9 10

G460A A719G

TPMT*1

TPMT*3A

Selected Human TPMT Alleles

Ala154Thr Tyr240Cys

(wild type)

TPMT Pharmacogenetics(A)

(B)

% o

f Sub

ject

s Pe

r 0.

5 U

nits

of A

ctiv

ity

Correlation Between Genotype and ToxicityCorrelation Between Genotype and Toxicity

Evans et al J. Clin. Onc. 2001

Toxicity –fever, chills, nausea, vomiting, anorexia, diarrhea, rash, hepatotoxicity, bone marrow suppression. Severe myelotoxicity is potentially fatal and occurs early or occasionally later in treatment

TPMTGenetic PolymorphismClinical Consequences

• Low TPMT– Increased thiopurine toxicity– Increased risk for secondary neoplasm

• High TPMT– Decreased therapeutic effect

Pharmacogenetic diagnostics:A. Phenotyping

patient receives model substance (sparteine, debrisoquine for CYP2D6, sulphadimidine, isoniazid, caffeine for NAT2)-> 4-8 hr blood/urine collection -> evaluatng unchanged drug/metabolites ratio (Metabolic Ratio - MR) in blood or urine

B. Genotyping

Mutation/Mutation/SNPSNP databases: databases:

•dbSNP http://www.ncbi.nlm.nih.gov/SNP/index.html

•Human Genome Variation Database (HGVbase) http://hgvbase.cgb.ki.se/

TSC: The SNP Consortiumhttp://snp.cshl.org/

B. Genotyping:

Step 1:Step 1:

Finding of clinically Finding of clinically revelant mutations revelant mutations

- Sequencing- Sequencing

PCR/RFLPPCR/RFLPPolymerase Chain Reaction/Restricted Fragment Length PolymorphismPolymerase Chain Reaction/Restricted Fragment Length Polymorphism

http://www.ornl.gov/sci/techresources/Human_Genome/publicat/hgn/v10n3/images/megabaces.jpg

Step 2: Step 2: identification of patient genotypeidentification of patient genotype

GeneChip® Probe ArraysGeneChip® Probe Arrays

11-18µm11-18µm

Millions of copies of a specificMillions of copies of a specificoligonucleotide probeoligonucleotide probe

Image of Hybridized Probe ArrayImage of Hybridized Probe Array

> 1M different> 1M differentcomplementary probes complementary probes

Single stranded, Single stranded, labeled RNA targetlabeled RNA target

Oligonucleotide probeOligonucleotide probe

* **

**

1.28cm1.28cm

GeneChipGeneChip Probe ArrayProbe ArrayHybridized Probe CellHybridized Probe Cell