PHARMACOGENETIC

Dr. dr. H. Mgs Irsan Saleh, M.Biomed

Dept. of Pharmacology Faculty of Medicine
Univ. of Sriwijaya

INTRODUCTION
New field within clinical
pharmacology (30-40 years)
Patients respond differently to a
given therapeutic agent even if they
have the same illness
The same dose of a given drug in
some patients causes very different
plasma levels and different
therapeutic response

Drugs costs are escalating..

Drug efficacy is questioned..

Percentage of non-responders

Drug safety is questioned..

Vioxx..
Celebrex..
Bextra..

What next??

1999 IOM Report (USA)

Estimated cost of adverse drug reactions:
US $15 billion/year in the USA alone
Estimated number of drug-related hospitalizations:
~1,000,000 per year in the USA
~50,000 - 100,000 toxicity-related death per year
Kohn et al (eds): To Err is Human. Building a Safer Health System.
Committee on Quality of Health Care in America. (1999)
Institute of Medicine. National Academy Press: Washington, DC.

UK ADRs Study
6.5% of new hospital admissions to internal medicine wards
are directly related to ADRs!
(1,225 admissions out of 18,820 during six months)
4% of bed occupancy - directly due to ADRs
Annual UK cost: EUR 706 million
(direct hospitalization costs - actual costs much higher!)
Women: 59% of ADRs (while only 52% of admissions)
Pirmohamed et al (July 2004) Adverse drug reactions as cause of admission to
hospital: prospective analysis of 18 820 patients. BMJ. 329:15-19.

All patients with same diagnosis

Remove:
(1) non-responders
(2) toxic responders

Treat:
Responders and Patients
Not Predisposed to Toxicity

Which is more urgent?

What should our priorities
be?

Pharmacogenetics
The study of
genetically determined
interindividual differences in
therapeutic response to drugs
and
susceptibility to adverse
effects

History
510 BC Pythagoras some people develop haemolytic anaemia after eating fava
beans
1902 Garrod genetic factors direct chemical transformations
1932 Snyder phenylthiourea nontasting is inherited as an autosomal recessive
trait
1957 Motulsky first demonstration of the relationship between adverse drug
reaction and genetically determined variation
1959 Vogel pharmacogenetics: the hereditary basis of variability in drug effects
1960 Evans speed of INH acetylation is under genetic control
1962 Kalow abnormal form of serum cholinesterase causes adverse reactions to
succinylcholine
1977 Mahgoub polymorphism of CYP2D6 causes adverse effects to debrisoquine

Aim of pharmacogenetic
studies
Identify and categorize the genetic factors that
underlie the differences and apply this in clinical
practice
Rational, individual therapy
Screening for those patients who carry the genes
which place them at risk in case of certain
therapies
Discovering which drugs are potentially dangerous
for carriers of a given polymorphism
Establishing the frequency of pharmacogenetic
phenotypes

 Pharmacogenetics: study of genetically determined interindividual

differences in response to drugs
Pharmacogenomics: use of genome based techniques in drug
development

The differences in the response to a given drug can be attributed to

two major factors that are under genetic influence:
Pharmacokinetic: genetically based differences in the processes
influencing bioavailability
Pharmacodynamic: genetically based differences in the proteins at
which the drug acts

Polymorphism

Genetic variation occuring with a frequency of 1% or more in

the population
SNP (Single Nucleotide Polymorphism):
most frequent type
difference in a single base of the genomic sequence
usually 1/1000 base
most does not influence the structure or function of
proteins
SNP can occur
In exons (may alter the structure of proteins and may lead to
functional consequences)
In introns (may influence splicing)
In the regulatory regions (may influence expression of the gene)

Polymorphism
Insertion/deletion polymorphism:
insertion or deletion of a few nucleotides
Variable number tandem repeats:
variation in the number of times a
sequence of several hundred base pairs
is repeated
Simple tandem repeats
(microsatellites): 2-4 nucleotides
repeated a variable number of times

Transkripsi, Translasi dan Sintesis

Protein

Polymorphism
Pharmacogenetic traits are mainly
polygenic (influenced by several genes,
the effect may be additive or interactive)
multifactorial (both genetic and
environmental factors contribute)
Some pharmacogenetic traits are
monogenic

 Genotype:: structure encoding for the given

characteristics
Phenotype : the manifestation of the genotype, which
can be observed and can be influenced by other factors:
Other gene products
Environment
Acquired characteristics
Frequency of genetic polymorphisms differs greatly
among ethnic groups
The functional relevance of a given polymorphism can
vary across ethnic groups

DETERMINATION
Determination of genotype: PCR
Determination of phenotype:
determination of metabolic rate (level of original
drug/metabolit in urine)
after administration of a given dose of the drug,
pharmacokinetic parameters are measured (halflife,
clearance, plasma levels)

Distribution of phenotypes in the population:

Multimodal (usually bi- or trimodal) distribution
indicates determination by a single gene
having polymorphic variants
Unimodal distribution indicates polygenic
multifactorial inharitance, or monogeic
inheritance but no polymorphism

 The function is usually bi- or trimodal

indicating two or three phenotypes
Enhanced/extensive metaboliser:
intensive metabolisation, resulting in low
plasma concentration of the drug
usually heteozygote or homozygote
dominant
Intermedier metaboliser
Poor metaboliser or nonmetaboliser:
Slow or no metabolisation of the drug
resulting in high plasma concentration for
an extended time
Usually homozygote recessive

Types of adverse effects of

drug therapy

Predictability
Dose
dependence
Frequency
Mortality

Augmented:
related to the
therapeutic effect

Bizarre: related
to
pharmacogeneti
cs

frequent
rare

rare
frequent

 The observed differences in therapeutic

response and susceptibility to adverse
reactions are due to alterations of
pharmacokinetic and pharmacodynamic
processes
Absorption
Distribution
Protein binding
METABOLISM
Disposition

Inheritance of the activity of

metabolic enzymes
Monogenic inheritance
one gene is responsible for encoding the
enzyme
mutant enzyme variants may cause defects in
metabolism, leading to different genotypes
within the population
usually autosomal recessive in case of the
allele carrying the reduced enzyme function

Polygenic inheritance
several genes are responsible for encoding
the enzyme

GENETIC VARIANCE
(POLYMORPHISM) IN DRUG
METABOLISM ENZYME

Pharmacogenetics of drug
metabolism
Drug metabolism is crucial in determining therapeutic and adverse
effects
Genetic factors play an important role in individual differences of drug
metabolism

Phase I
Oxidation, reduction, hydroxilation, dealkylation, etc.
Aim: introduce a new functional group
Cytochrome P450 enzymes in hepatocytes

Phase II
Conjugation with glucuronic acide, glutathione, acetate, etc
Aim: to increase water solubility
Ususally in the cytosol

Lampe JW. Am J Clin Nutr 1999;

70(Suppl):475S-90S

Diet
Drug
Chemical
Endogenous compound (eg. steroid hormones)

PHASE I REACTIONS
Oxidation
Hydroxylation
Reduction
Dehalogenation

Cytochrome P450 enzymes

Intermediate
compounds
PHASE II REACTIONS
Sulfation
Glucoronidation
Glutathione conjugation
Acetylation
Amino acid conjugation
Methylation

Damage to DNA, RNA,

proteins and lipids

Sulfotransferases
UDP-glucoronosyltransferases
Glutathione S-transferases
Acetyltransferases

Detoxified
conjugated
products

Excretion

II. Genetic polymorphisms in

drug metabolizing enzymes

From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational
therapeutics. Science 286:487-491, 1999.
28

From: Evans WE,

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

29

Nelson et al., Pharmacogenet 1996; 6:1-42

CYP450 NOMENCLATUR
CYP450 SUPERFAMILY
FAMILIES CYP PLUS ARABIC NUMERAL
(> 40% HOMOLOGY OF AMINO ACID SEQUENCE).
eg: CYP1
SUBFAMILY ADDITIONAL CAPITAL LETTER
AND ARABIC NUMERAL (> 55% HOMOLOGY OF AMINO
ACID SEQUENCE). eg: CYP1A2
ITALICS FOR GENE (CYP2D6) AND NORMAL (CYP2D6)
FOR ENZYME

SYNTHESIS OF CYP450 IN CELLS

THE CYP450 GENE REGULATION

XENOBIOTIC (DRUGS OR ENVIRONMENTAL
CHEMICALS) CAN SWITCH ON GENE TRANSCRIPTION
ENDOGENOUS TRANSCRIPTION FACTORS CAN
SWITCH ON OR SWITCH OFF GENE TRANSCRIPTION
ENZYME ACTIVITY MAY BE MODIFIED BY SIGNAL
FROM THE 3-UTR.
3

5
promoter

Open reading frame

UTR

WHY DOES CYP450 MATTER?

THE MAJOR SYSTEM FOR DRUG METABOLISM
SOURCE OF INTER-INDIVIDUAL VARIABILITY
IN DRUG METABOLISM
EXPLAINS SOME SPECIFIC TOXIC EFFECTS
OF DRUG
EXPLAINS MANY DRUG INTERACTIONS

INTER-INDIVIDUAL VARIABILITY
IN METABOLISM BY CYP450
ENVIRONMENTAL FACTORS
GENETIC FACTORS

POLYMORPHISM

POLYGENIC DETERMINANTS OF DRUG EFFECTS

DRUG METABOLISM GENOTYPES

Drug Concentration (%)

100

m/m

80
60

wt/m

40

wt/wt

20
0
0

10

15

Time (hours)

20

25

HUMAN CYP450 FAMILIES

SUBSTRATES

ISOFORM
CYP1, CYP2, CYP3

DRUGS, XENOBIOTICS

CYP4,CYP5, CYP6

FATTY ACIDS,
PROSTAGLANDINS,
THROMBOXANES

CYP7, CYP11,
CYP17, CYP19, CYP21, CYP24,
CYP27, CYP39, CYP46, CYP51

STEROID HORMONES

Nebert DW and Russel DW, Lancet 2002;360:1155-1162

with modification.

HUMAN CYP450 ISOFORM

CYP1A2

CYP2C9

SUBSTRATES
caffeine
R-warfarin
theophylline

phenytoin
S-warfarin

INHIBITORS
ciprofloxacin
erythromycin
INDUCERS
phenytoin, rifampin,
phenobarbital, ritonavir, smoking

fluconazole
sulphaphenazole

HUMAN CYP450 ISOFORM

CYP2C19

CYP2D6

CYP3A4

SUBSTRATES
diazepam
mephenytoin
omeprazole

codeine
desipramine
flecainide

cyclosporin
diltiazem
simvastatin
verapamil

haloperidol
quinidine

diltiazem
erythromycin
ketoconazole

INHIBITORS
omeprazole

CONCLUSION
CYP450 RESPONSIBLE FOR WIDE VARIETY OF DRUG
METABOLISM REACTIONS
INTER INDIVIDUAL VARIABILITY HAS
ENVIRONMENTAL AND GENETIC COMPONENTS
ISOFORM GIVE A BASIS FOR:
(a) UNDERSTANDING AND PREDICTING DRUG INTERACTIONS
(b) UNDERSTANDING TOXIC SIDE EFFECTS

POLYMORPHISM OF CYP2D6
FIRST DESCRIBED IN EARLY 1980S FOR
DEBRISOQUINE AND SPARTEIN
POPULATION SPLIT INTO EXTENSIVE (EM),
POOR (PM) AND VERY EXTENSIVE (UEM)
METABOLISERS
MOLECULAR BASIS IS MUTATIONS OR DELETIONS
IN THE CYP2D6 GENE
CYP2D6A, CYP2D6B, CYP2D6D AND CYP2D6T
IDENTIFIED ENZYME ACTIVITY

POLYMORPHISM OF CYP2D6
POLYMORPHISM IS EVIDENT IN DIFFERENT

POPULATION eg 7% OF CAUCASIANS ARE PM

PHENOTYPE, BUT ONLY 1% IN ORIENTALS
AFFECTS WIDE RANGE OF DRUGS: CODEINE,
IMIPRAMINE, DESIPRAMINE, FLECAINIDE,
HALOPERIDOL, METOPROLOL, NORTRIPTYLINE,
RISPERIDONE, THIORIDAZINE, PERPHENAZINE,
ZUCLOPENTHIXOL.

EM if homozygous for this allele

PM if homozygous for this site

Structure of the
CYP2D6-8 gene locus
D8

D7

D8

D7

D8

D7

D6
D7

D6
D6

Allelic XbaI
fragments

Wild type (wt) or

mutant alleles

29 kb

D6-wt

44 kb

D6-wt

29 kb

D6-wt

29 kb

D6-A

29 kb

D6-B

29 kb

D6-C

44 kb

D6-B

16 + 9 kb

D6-B

Unknown mutation
A deletion
Splice site mutation

AGA deletion

D8

D7

D7

D6

Splice site mutation

Splice site mutation
D6 deletion

11.5 kb

No DNA amplification

Kalow W and Grant. Pharmacogenetics. In:The metabolic and molecular bases of inherited disease, 1995:293-326

CYP2D6 AND CODEINE

CODEINE
MORPHINE

NORCODEINE
CODEINE-6-GLUCURONIDE

MORPHINE-3/6GLUCURONIDES

NORCODEINE-6GLUCURONIDE

Sindrup and Brosen, Pharmacogenet 1995; 5:335-336

CYP2D6 AND HALOPERIDOL

DOPAMINE 2 RECEPTOR OCCUPANCY IS RELATED
TO HALOPERIDOL PLASMA CONCENTRATION
HIGHEST PLASMA HALOPERIDOL CONCENTRATION
IN A PM PATIENT
SUGGESTED THAT PMs MAY BE AT GREATER RISK
OF EXTRAPYRAMIDAL SIDE EFFECTS

Hyberg et al., Am J Psychiatr 1995; 152:173-176

CYP2D6 AND HALOPERIDOL

PLASMA HALOPERIDOL (nmol/L/mg)

-5

PM

EM

-2

-1
0

12

16

20

24

28

32

HOURS AFTER DOSE

Lierena et al.; Ther Drug Monit 1992; 14:261-264