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Genetical determination Genetical determination of drug action. of drug action.
Phenotyping and genotyping Phenotyping and genotyping in optimizing patient therapy.in optimizing patient therapy.
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Pharmacogenetics
The study of the role of inheritance in individual variation in response to
xenobiotics, including drugs.
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Pharmacogenomics
The study of the genome and its products (including RNA and protein) as it relates to drug discovery, development and
funktion.
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Pharmacogenomics
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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
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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.
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Evans, W. E., Johnson, J. A., Annu. Rev. Genomics Hum. Genet., 2001, 2: 9 - 39.
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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
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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 )
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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.
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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.
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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
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2. Favism - Glucose-6-phosphate dehydrogenase (G6PD) activity
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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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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.
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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.
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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
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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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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
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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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Ultrarapid metabolizers Ultrarapid metabolizers Extensive metabolizers Extensive metabolizers Poor metabolizers Poor metabolizers
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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
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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
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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
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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
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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.
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7. Other important polymorphisms:7. Other important polymorphisms:
• CYP2C9,• CYP2C19, • DPD,• TYMS, • RYDR.
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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
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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
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TPMTGenetic PolymorphismClinical Consequences
• Low TPMT– Increased thiopurine toxicity– Increased risk for secondary neoplasm
• High TPMT– Decreased therapeutic effect
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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
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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
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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
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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