Farmakogene*ka

Prof.dr. Amina Kozaric

24.10.2014
PMF
1

Deni*ons
Pharmacogenomics: Analysing en*re
genomes, across groups of individuals, to
iden*fy the gene*c factors inuencing
responses to a drug.
Pharmacogene-cs: Studying an individual's
gene*c make up in order to predict responses
to a drug and guide prescrip*on.

Objec*ves of Pharmacogene*cs

1. Iden*fy varia*on in response
2. Elucidate molecular mechanisms
3. Evaluate clinical signicance
4. Develop screening tests
5. Individualize drug therapy

Personalized Medicine!

Courtesy of Felix W. Frueh US FDA

Personalized Medicine!
Medicine is personal:!
We are all different.!
Some of our differences translate into how we react to drugs
as individuals.!
This is why personalized medicine is important to
everyone.!

Why does someone need twice the standard dose to

be effective?!
Why does this drug work for you but not me?!
Why do I have side-effects and you dont?!
Why do some people get cancer and others dont?!
Why is anecdotal information irrelevant to your own
health and treatment?!

Variability of Disease!

Variability of Disease!

Courtesy Felix W. Frueh

The Goal of Personalized Medicine!

The Right Dose of

The Right Drug for
The Right Indica*on for
The Right Pa*ent at
The Right Time.

What is DNA?

April 1953
Drs. James Watson and
Francis Crick
determined the
structure of DNA
(double helix)

April 1953

April 2003

Drs. James Watson and

Francis Crick
determined the
structure of DNA
(double helix)

Human Genome Project

determined the en-re DNA
sequence of a human
(3 billion le/ers)

What is Pharmacogenomics?

R
X

Pharmacogenomics?
Pharma = drug or medicine
Genomics = the study of genes

R
X

What is Pharmacogenomics?
Pharma = drug or medicine
Genomics = the study of genes

Personalized medicine tailored to your genes

R
X

Case Study Breast Cancer Pa-ents

Case Study Breast Cancer Pa-ents

Tumoricide

Case Study Breast Cancer Pa-ents

Case Study Breast Cancer Pa-ents

30%

No Eect/Hurt

Helped

Tumoricide

No Eect/Hurt

Helped

Tumoricide

Why?

How do scien-sts make personalized

medicine?

Your DNA

How do scien-sts make personalized

medicine?
You

Your cells

Its all about what makes

Your DNA
YOUR gene-c code UNIQUE

Picture credit: adapted from Riken Research: h[p://www.rikenresearch.riken.jp/eng/frontline/5514

Gene*c Code: DNA

DeoxyriboNucleic Acid (DNA) contains all the informa-on
necessary to make a complete organism
DNA is composed of a combination of 4 nucleotides

Gene*c Code: DNA

DeoxyriboNucleic Acid (DNA) contains all the informa-on
necessary to make a complete organism
DNA is composed of a combination of 4 nucleotides

A
Adenine

Gene*c Code: DNA

DeoxyriboNucleic Acid (DNA) contains all the informa-on
necessary to make a complete organism
DNA is composed of a combination of 4 nucleotides

Adenine

Thymine

Gene*c Code: DNA

DeoxyriboNucleic Acid (DNA) contains all the informa-on
necessary to make a complete organism
DNA is composed of a combination of 4 nucleotides

Adenine

Thymine

Cytosine

Gene*c Code: DNA

DeoxyriboNucleic Acid (DNA) contains all the informa-on
necessary to make a complete organism
DNA is composed of a combination of 4 nucleotides

Adenine

Thymine

Cytosine

Guanine

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
RNA

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
RNA
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleo-des called codons.

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleo-des called codons.

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1 Codon 2
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleo-des called codons.

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1 Codon 2
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleo-des called codons.

Amino acid 1 Amino acid 2

The Central Dogma: DNARNAProtein

DNA: A long double-stranded string of
nucleo-des that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1 Codon 2
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleo-des called codons.

Amino acid 1 Amino acid 2

Protein!

A small change in the gene sequence can result in a

very dierent protein
DNA:

ATG GTG CTG TCT CCT

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

Leu

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

Pro

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

Pro

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

DNA:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

Pro

ATG GTG CTG TCT ACT

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:

DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

Pro

ATG GTG CTG TCT ACT

Met

Val

Leu

Ser

Thr

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:
Words:

DNA:
Amino Acids/Protein:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

Pro

Tom and Sam are bad

ATG GTG CTG TCT ACT

Met

Val

Leu

Ser

Thr

A small change in the gene sequence can result in a

very dierent protein
DNA:
Amino Acids/Protein:
Words:

DNA:
Amino Acids/Protein:
Words:

ATG GTG CTG TCT CCT

Met

Val

Leu

Ser

Pro

Tom and Sam are bad

ATG GTG CTG TCT ACT

Met

Val

Leu

Ser

Thr

Tom and Sam are sad

A small change in the gene sequence can result in a

very dierent protein
DNA:

ATG GTG CTG TCT CCT

Amino Acids/Protein:
Words:

Val

Leu

Ser

Pro

Tom and Sam are bad

DNA:

ATG GTG CTG TCT ACT

Amino Acids/Protein:
Words:

Met

Met

Val

Leu

Ser

Thr

Tom and Sam are sad

Changes in DNA are called varia-ons or muta-ons

Variations in the DNA (genotype) can cause

observable changes (phenotype) in individuals

No Eect/Hurt

Helped

Tumoricide

Why does Tumoricide work

on some pa-ents but not
on others?

What are the reasons a person would

react dierently to drugs?
1. Having the receptor (protein) to recognize the drug
2. Other physiological traits that enable you to respond
to a drug
3. How your body processes the drugs a[er receiving it

Drugs and Receptors

Cell

Drugs and Receptors

Receptor
(Protein)

Cell

Drugs and Receptors

Receptor
(Protein)

Cell

Drugs and Receptors

Drug
(Ligand)

Receptor
(Protein)

Cell

Drugs and Receptors

Drug
(Ligand)

Receptor
(Protein)

Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors

Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors

Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors

Cell
Cell

Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors

Cell
Cell

Cell

Cell
Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors
Too Many
(hypersensi-ve)

Cell
Cell

Cell

Cell
Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors
Too Many
(hypersensi-ve)

Too Few
(hyposensi-ve)

Cell
Cell

Cell

Cell
Cell

Your DNA and Drugs

Varia*on in genes can cause varia*on in receptors
Too Many
(hypersensi-ve)

Too Few
(hyposensi-ve)

Cell
Cell

Cell

Mutated
(insensitive)

Cell
Cell

Where Drugs Fit In

Lock = Receptor Key = Drug

Why can some people taste PTC

and others cant?
PTC-
Receptor

PTC

Taste
cell
This tastes bi/er!
Phenylthiocarbamide

Why can some people taste PTC

and others cant?
PTC-
Receptor

PTC

Taste
cell
This tastes bi/er!

PTC

Non-
binding
PTC-
Receptor

Taste
cell
I dont taste anything!

Where does tas*ng PTC come from?

You have two copies of every
gene:
one from Mom
and
one from Dad

Where does tas*ng PTC come from?

You have two copies of every
gene:
one from Mom
and
one from Dad

Where does tas*ng PTC come from?

You have two copies of every
gene:
one from Mom
and
one from Dad

Where does tas*ng PTC come from?

You have two copies of every
gene:
one from Mom
and
one from Dad

Your two genes are the genotype

Where does tas*ng PTC come from?

You have two copies of every
gene:
one from Mom
and
one from Dad

Your two genes are the genotype

A gene can be dominant or recessive

Where does tas*ng PTC come from?

You have two copies of every
gene:
one from Mom
and
one from Dad

Your two genes are the genotype

A gene can be dominant or recessive
The expressed trait is a phenotype

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

Tt

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

Tt

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

Tt

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

Tt

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

Tt

For individuals with these genotypes,

what would their phenotypes be?

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

This tastes REALLY bi[er!

SUPERTASTER

Tt

Tas*ng PTC is dominant (T) over inability taste PTC which is

recessive (t)

TT

This tastes REALLY bi[er!

SUPERTASTER

Tt

This tastes
bi[er!

TASTER

Tas*ng PTC is dominant (T) over inability taste PTC

which is recessive (t)

TT

This tastes REALLY bi[er!

SUPERTASTER

Tt

This tastes
bi[er!

I dont taste anything!

TASTER

NON-TASTER

Drug receptor summary

Ability to taste PTC has a very strong gene-c component

PTC = chemical and Drugs = chemical

Dierences in ability to taste PTC is similar to
dierences in reac*ons to drugs
"
"

No Eect/Hurt

Helped

Tumoricide

Why?

Two Types of Breast Cancer

Y
Her2-

Her2+

Tumoricide is a personalized medica-on

Tumoricide only works for Her2+ breast tumors

No Eect/Hurt

Helped

Tumoricide

Her2-

Her2+

Screening for Her2+ Cells

Her2-

American Journal of Clinical Pathology. 2008;129(2):263-273

Her2+

Screening for Her2+ Cells

+
American Journal of Clinical Pathology. 2008;129(2):263-273

Tumoricide

= ?

Screening for Her2+ Cells

+
American Journal of Clinical Pathology. 2008;129(2):263-273

Tumoricide

Breast Cancer
1990

Surgery
Radia-on
Chemotherapy (drugs)

2012

Surgery
Radia-on
Chemotherapy
Specialized treatments (for certain types
of breast cancer)

http://www.ucdmc.ucdavis.edu/welcome/features/20080709_cancer_sweeney/index.html

What are the reasons a person would

react dierently to drugs?
1. Having the receptor (protein) to recognize the drug
2. Other physiological traits that enable you to respond
to a drug
3. How your body processes the drugs a[er receiving it

The presence of receptors inuence how we react to

drugs like Tumoricide or chemicals like PTC

Y
Her2-

Her2+

Tumoricide
Does Not Work

Tumoricide
Works!

The presence of receptors inuence how we react to

drugs like Tumoricide or chemicals like PTC

TT

This tastes REALLY bi[er!

SUPERTASTER

Tt

This tastes
bi[er!

TASTER

I dont taste anything!

NON-TASTER

Where are the PTC receptors?

What are taste buds?

Taste buds are found on papillae on your tongue

What are taste buds?

Taste buds are found on papillae on your tongue

Papillae

bumps on
your tongue

What are taste buds?

Taste buds are found on papillae on your tongue

Papillae

bumps on
your tongue
Taste buds cells are found on the papilla

What are taste buds?

Taste buds are found on papillae on your tongue
PTC receptors are found
on the taste buds

Papillae

bumps on
your tongue
Taste buds cells are found on the papilla

What are taste buds?

Taste buds are found on papillae on your tongue
PTC receptors are found
on the taste buds

Papillae

bumps on
your tongue

Nerve Cell
Transmits signal
to brain
Taste buds cells are found on the papilla

What are taste buds?

Taste buds are found on papillae on your tongue
PTC receptors are found
on the taste buds

Brain
Wow! This tastes
really bi[er

Papillae

bumps on
your tongue

Nerve Cell
Transmits signal
to brain
Taste buds cells are found on the papilla

Are there other traits that can allow a person to

more strongly taste PTC?

If a person has more taste buds, then he/she may

be able to taste the PTC more strongly.

Coun-ng the number of tongue papillae

5 papillae

20 papillae

35 papillae

Coun-ng the number of tongue papillae

5 papillae

20 papillae

35 papillae

Ideal graph representing the number of tongue papillae

related to the phenotype of PTC taste

These results support our hypothesis that the super-taster

has more papillae!

Ideal graph representing the number of tongue papillae

related to the phenotype of PTC taste

The number of papillae in the non-taster is variable.

Why would the number of papillae be variable in a nontaster?

What does it take to be a PTC Taster?

Two traits are important for determining PTC taste sensi-vity

1) PTC receptor genotypeDo y ou have the receptors that
enable you to taste PTC

What does it take to be a PTC Taster?

Two traits are important for determining PTC taste sensi-vity

1) PTC receptor genotypeDo y ou have the receptors that
enable you to taste PTC

2) The density of papillae on your tongue correlates to the

sensi-vity of tas-ng PTC

super-taster

taster

What are the reasons a person would react

differently to drugs?
1. Having the receptor (protein) to recognize the drug
2. Other physiological traits that enable you to respond to a
drug
3. How your body processes the drugs after receiving it

A Drugs Life

ADME
Absorp-on
Distribu-on
Metabolism
Excre-on

http://publications.nigms.nih.gov/medbydesign/chapter1.html

Metabolic enzymes
Enzymes
Metabolites

Drug

Liver

DNA variations in special proteins in the liver called

enzymes can influence a persons ability to metabolize
certain drugs

Adverse Drug Reactions (ADR)

Definition- unwanted, negative response to a
prescribed drug at normal doses and during normal
use
Examples?

Adverse Drug Reactions (ADR)

Definition- unwanted, negative response to a
prescribed drug at normal doses and during normal
use
Examples?
There are multiple causes for ADRs
environmental basis
genetic basis

Adverse Drug Reactions (ADR)

Definition- unwanted, negative response to a
prescribed drug at normal doses and during normal
use
Examples?
There are multiple causes for ADRs
environmental basis
genetic basis
Poor metabolizers can experience ADRs at normally
therapeutic drug doses

Case study: Nortriptyline

metabolism
Three women of the same height, weight, age, and
racial background are depressed and go to the doctor.
The doctor prescribes an antidepressant, Nortriptyline,
at a dose of 100 mg.

Person A has an adverse reaction

Person B nothing happens
Person C gets better

B
A

Case study: Nortriptyline

metabolism
Three women of the same height, weight, age, and
racial background are depressed and go to the doctor.
The doctor prescribes an antidepressant, Nortriptyline,
at a dose of 100 mg.

Person A has an adverse reaction

Person B nothing happens
Person C gets better

Why?

B
A

ADME of Nortriptyline
100mg Nortriptyline

Adverse reactionNothing happensGets better

How much active drug in blood?

ADME of Nortriptyline
100mg Nortriptyline

Adverse reaction
Nothing happensGets better

95mg

5mg

50mg

DNA variation influence drug

metabolism
Enzymes
A

Metabolites

Drug

Liver
Poor
Metabolizer
95mg

DNA variation influence drug

metabolism
Enzymes
B

Metabolites

Drug

Liver
Ultrarapid
Metabolizer
5mg

DNA variation influence drug

metabolism
Enzymes
C

Metabolites

Drug

Liver
Intermediate
Metabolizer
50mg

2012 - What do doctors do?

Poor Metabolizer

Ultrarapid Metabolizer

Decrease Dose

Increase Dose

Or change drug

Today
One-size-fits-all drugs
Current drug development system develops
drugs for the average patient
No simple way to determine who will respond
well and who will respond poorly
One size does NOT fit all!
Whats the solution?

Today
One-size-fits-all drugs
Current drug development system develops
drugs for the average patient
No simple way to determine who will respond
well and who will respond poorly
One size does NOT fit all!
Whats the solution?
Pharmacogenomics (PGx)
Personalized Medicine

April, 2050
You wake up feeling terrible, and you know it's time to
see a doctor. In the office, the physician looks you over,
listens to your symptoms, and decides to prescribe you
a drug.
But first, the doctor takes a look at your DNA.

TODAY vs. FUTURE

Today = Drugs are One-Size-Fits-All
Future = Drugs Specific for You!
More effective & minimizes side effects

Summary
Gene-c varia-on leads to phenotypic dierences and
dierences in how we all react to drugs.

Summary
Gene-c varia-on leads to phenotypic dierences and
dierences in how we all react to drugs.

1. Having the receptor (protein) to recognize the drug

PTC and HER2 receptors

Summary
Gene-c varia-on leads to phenotypic dierences and
dierences in how we all react to drugs.

1. Having the receptor (protein) to recognize the drug

PTC and HER2 receptors
2. Other physiological traits that enable you to respond to a
drug
Number of taste buds on tongue

Summary
Gene-c varia-on leads to phenotypic dierences and
dierences in how we all react to drugs.

1. Having the receptor (protein) to recognize the drug

PTC and HER2 receptors
2. Other physiological traits that enable you to respond to a
drug
Number of taste buds on tongue
3. How drugs are processed in the body
Enzymes in liver metabolize drugs

Pharamcogenomics
Using peoples gene-c informa-on for the right
drug at the right dose at the right -me!

R
X

Pharmacogenetics &
Pharmacogenomics!

Pharmacogene*cs: The role of gene*cs in

drug responses.
F. Vogel. 1959

Pharmacogenomics: The science that allows

us to predict a response to drugs based on an
individuals gene*c makeup.
Felix Frueh, Associate Director of Genomics, FDA

Courtesy Felix W. Frueh

Pharmacogenetics &
Pharmacogenomics"
http://www.pharmgkb.org/!
Pharmacogenetics: study of individual gene-drug
interactions, usually one or two genes that have
dominant effect on a drug response (SIMPLE
relationship)!
Pharmacogenomics: study of genomic influence on
drug response, often using high-throughput data
(sequencing, SNP chip, expression, proteomics COMPLEX interactions)!
PharmGKB Website:

http://www.pharmgkb.org/!

Purine Analogs: A Case Study in

Pharmacogenetics!
6-mercaptopurine, 6-thioguanine, azathioprine!

Used to treat lymphoblastic leukemia, autoimmune disease,

inflammatory bowel disease, after transplant!
Interferes with nucleic acid synthesis!
Therapeutic index limited by myelosuppression!
(treatment limited by immune suppression side effect)!

6-mercaptopurine!

6-thioguanine

azathioprine!

Metabolism of 6-MP!

LWang and RWeinshilboum, Oncogene 25, 1629-1638 (2006)!

Pharmacogenetics: A Case Study!

Courtesy of Michelle Whirl-Carillo!

Pharmacogenetics: A Case Study!

Courtesy of Michelle Whirl-Carillo!

Pharmacogenetics: A Case Study!

Thiopurine S-methyl Transferase Activity"

and Personalized Dosage!

Second Example: Codeine and

Cytochrome P450 CYP2D6
Codeine is a commonly used opioid
Codeine is a prodrug
It must be metabolized into morphine for activity

Cytochrome P450 allele CYP2D6 is the

metabolizing enzyme in the liver
7% of Caucasians are missing one copy of the
Cytochrome P450 CYP2D6 gene
codeine does not work effectively in these
individuals

Codeine and Morphine Metabolism!

Cytochrome Oxidase P450

Enzymes!
57 Different active genes!
17 Different families!
CYP1, CYP2 and CYP3 are primarily involved in
drug metabolism.!
CYP2A6, CYP2B6, CYP2C9 ,CYP2C19, CYP2D6,
CYP2E1 and CYP3A4 are responsible for
metabolizing most clinically important drugs!

Polymorphic!
Cytochrome!
P-450s!

2006 American Medical Association. All rights reserved

Effect of Metabolic Rate on Drug

Dosage!

2006 American Medical Association. All rights reserved

Warfarin: Significant Problems

for Rats!!

Warfarin: Significant Problems for Humans!!

Ranks #1 in total mentions of deaths for drugs causing adverse
events (from death certificates)
Ranks among the top drugs associated hospital emergency room
visits for bleeding
Overall frequency of major bleeding range from 2% to 16%
(versus 0.1% for most drugs)
Minor bleeding event rates in randomized control trials of new
anticoagulants has been as high as 29% per year.

Warfarin: Significant Problems for Humans!!

Case Report July 2, 2008!
Company director dies of brain hemorrhage
after heading a football!
Consultant neurosurgeon told the inquest
the warfarin effect was probably the cause of
the death!
It can happen to anyone!!

Other Warfarin Patients!

Case Report July 2, 2008!
Joseph Stalin!

Why Maintaining Warfarin"

Therapeutic Range is Critical!

European Atrial Fibrillation Trial Study Group, N Engl J Med 1995;333:5-10.!

Finding Doses to Maintain Therapeutic

Anticoagulation is Largely Trial and Error!

Warfarin Levels Depend on Two

Enzymes CYP2C9 & VKORC1!

Estimated Warfarin Dose (mg/day)

Based on Genotypes!

Frequency of VKORC1 Alleles"

in Various Populations!

Sconce et al. Blood 2005, Yuan et al. Human Mol Genetics 2005, Schelleman et al.
Clin Pharmacol Ther 2007, Montes et al Br J Haemat 2006!

Genetic Analysis Permits!

More rapid determination of stable therapeutic
dose.
Better prediction of dose than clinical methods
alone.
Applicable to the 70-75% of patients not in
controled anticoagulation centers.
Reduces between 4,500 and 22,000 serious
bleeding events annually.
Genetic testing now required by FDA

Another Anticoagulant Clopidogrel

(Plavix) and CYP2C19 Alleles!

"
!

23andMe!
Drug Response!
Reports!

What are Targeted Drugs?!

Often, drugs are only effective in specific subpopulations (responders).
Early identification of responders can have a
dramatic effect of treatment success.
Treatment of non-responders puts these
individuals at unnecessary risk of adverse
events, while providing no benefit.
Personalized Medicine allows the identification
of responders and non-responders for targeted
therapies.
This is happening today!

Personalized Drugs!
Hercep*n
Erbitux
Tarceva
Straeera

6-MP
An*virals

(breast cancer, target: Her2/neu)

(colorectal cancer, target: EGFR)
(lung cancer, target: EGFR)
(aeen*on-decit/hyperac*vity
disorder, Metabolism: P4502D6)
(leukemia, Metabolism: TPMT)
(i.e. resistance based on form of HIV)

etc. and the list is growing rapidly ...

FDA Requires Genetic Tests"

for Certain Therapies!

Courtesy of Michelle Whirl-Carillo!

Roche Chip for Cytochrome P450"

Genes: CYPC19 and CYP2D6 !

Xie and Frueh, Pharmacogenomics steps toward Personalized Medicine, Personalized Medicine 2005, 2,
325-337!

AMA Course on Pharmacogenomics

and Personalized Medicine!

http://ama.learn.com

2006 American Medical Association. All rights reserved

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%

154

Frequency

Normal Distribution

Activity

155

Polymorphic Distribution

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

156

GENETIC
POLYMORPHISMS
Pharmacokinetic
Transporters
Plasma protein binding
Metabolism

Pharmacodynamic
Receptors
Ion channels
Enzymes
Immune molecules

157

From: Evans
WE, Relling MV.
Pharmacogenom
ics: Translating
functional
genomics into
rational
therapeutics.
Science
286:487-491,
1999.
158

II. Genetic polymorphisms in drug

metabolizing enzymes

From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics

into rational therapeutics. Science 286:487-491, 1999.

A. Atypical Plasma Cholinesterase

SUCCINYLCHOLINE
+

(H3C)3NH2CH2C O C CH2CH2
choline

+
C O CH2CH2N(CH3)3
succinylmonocholine

Hydrolysis by pseudocholinesterase

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
160

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

% Inhibition

Typical

Atypical

100
80
60
40
20
0

normal enzyme inhibited > 70%

abnormal inhibited < 30%

-6

-4

log molar dibucaine conc.

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

161

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

162

B. Glucose-6-phosphate dehydrogenase activity

Effects >100 million worldwide
R-NH2

HMP Shunt
G-6-PD
Dependent

CYP
MPO
PGH Synthase

NADP+ or
GSSG(?)
NADPH
or GSH(?)

R-NOH

O2
R-NOH

HgbFe+2

R-NO

HgbFe+3

GSH
Semi-mercaptal
sulfinamide

R-NH2

ERYTHROCYTE
NAD+
MetHgb
Reductase

Reactive
Oxygen

NADH
Splenic
Sequestration

SOD
Catalase
GSH Peroxidase

Detoxification

Hemolytic
Anemia
163

Drugs and Chemicals Unequivocally

Demonstrated to Precipitate Hemolytic Anemia
in Subjects with G6PD Deficiency
Acetanilide
Nitrofurantoin
Methylene Blue
Sulfacetamide
Naphthalene
Sulfanilamide
Sulfamethoxazole

Primaquine
Nalidixic Acid
Sulfapyridine

164

INCIDENCE OF G6PD DEFICIENCY IN

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

8
0.7-3
165

INCIDENCE OF G6PD DEFICIENCY IN

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

166

C. N-ACETYLTRANSFERASE ACTIVITY

Distribution of plasma isoniazid concentration in 483 subjects

after and oral dose. Reproduced from Evans DAP. Br Med J 2:485, 1960.
167

NAT1*4

NAT2*4

PABA
PAS
SMX

PA
DDS
SMZ

AF

NAT2*5A

NAT2*6A

NAT2*7A

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

168

ETHNIC DIFFERENCES IN THE DISTRIBUTION OF

ACETYLATOR PHENOTYPE
Population
South Indians
Caucasians
Blacks
Eskimos
Japanese
Chinese

% Slow

% Hetero Fast

59
58.6
54.6
10.5
12
22

35.6
35.9
38.6
43.8
45.3
49.8

% Homo Fast
5.4
5.5
6.8
45.7
42.7
28.2

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

169

XENOBIOTICS SUBJECT TO
POLYMORPHIC ACETYLATION IN MAN
Hydrazines
Arylamines
isoniazid
dapsone
hydralazine
procainamide
phenylzine
sulfamethazine
acetylhydrazine
sulfapyridine
hydrazine
aminoglutethimide

Carcinogenic
Arylamines
benzidine
-naphthylamine
4-aminobiphenyl

Drugs metabolized to amines

sulfasalazine
nitrazepam
clonazepam
caffeine
170

ADVERSE EFFECTS TO
SULFASALAZINE IN PATIENTS WITH
INFLAMMATORY BOWEL DISEASE
SULFASALAZINE

H
N

COOH

O
S

N N

OH

COOH

H
N

O
S
O

SULFAPYRIDINE

NH2

H2 N

OH

5-AMINOSALICYLIC ACID

171

ADVERSE EFFECTS TO
SULFASALAZINE IN PATIENTS WITH
INFLAMMATORY BOWEL DISEASE
Frequency of side effect
Slow Acetylators Fast Acetylators
Side Effect
9
1
cyanosis
5
0
hemolysis
6
0
transient reticulocytosis

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

172

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.

% of pts with lupus

120
100
80
60
Slow Acetylators

40

Fast Acetylators
20
0
0

20

40

60

80

100

Duration of Therapy (months)

173

PROCAINAMIDE
O
(H3CH2C)2NH2CH2CHN

CYP450

NH2

NAT

O
(H3CH2C)2NH2CH2CHN

PROCAINAMIDE HYDROXYLAMINE

NHOH

(H3CH2C)2NH2CH2CHN

NH CCH3

N-ACETYLPROCAINAMIDE

174

Distribution of acetylator phenotype in control

subjects and those experiencing a sulfonamide
hypersensitivity reaction.
Rieder et al. Clin Pharmacol Ther 49:13-17, 1991.

Percentage of Subjects

100

Control
HS

80
60
40
20
0
SLOW

FAST

175

SMX-glucuronide

UDPGT

NH2

S
O

H
N
N
O

NAT1

CH3

N-acetyl-SMX

Sulfamethoxazole
(SMX)

CYP2C9
MPO
PGH SYNTHASE

Detox

Nitroso

SMX hydroxylamine

NAT1

O-acetylation

Covalent binding to
cellular macromolecules/
cytotoxicity

Hypersensitivity/
Adverse Reaction

Hydroxamic
acid
N,O-AT

Acetoxy ester

Detoxified metabolite

176

D. CYP2D6 ACTIVITY
OH

CYP2D6
N

C NH

C NH
NH2

NH2

4-HYDROXYDEBRISOQUINE

DEBRISOQUINE

N CH3

N CH3

CYP2D6
H

OH

OCH3

DEXTROMETHORPHAN

DEXTRORPHAN
177

178

DRUGS WHOSE METABOLISM COSEGREGATES WITH DEBRISOQUINE

alprenolol amitriptyline
codeine
desipramine
flecainide fluoxetine
metoprolol nortriptyline
propafenone

bufuralol
encainide
guanoxan
paroxetine
propranolol

clomipramine
ethylmorphine
imipramine
phenformin

179

Plasma metoprolol concentrations in poor (l) and extensive ()

metabolizers of debrisoquine after 200 mg of metoprolol tartrate
administered orally. Redrawn from Lennard MS, et al. NEJM 307:1558-1560, 1982.
180

Dose requirements for nortriptyline in patients with different CYP2D6

Phenotypes. From: Meyer U. Lancet 356:1667, 2000.
181

6-glucuronidation
H3CO

codeine-6-glucuronide

M-6-G
O

O-demethylation

CYP2D6

morphine

NCH3

M-3-G
normorphine

HO

CODEINE

N-demethylation

norcodeine

norcodeine6-glucuronide

182

Effect of Quinidine on the Analgesic Response

to Codeine in Extensive Metabolizers of
CYP2D6 (Phenotyped with Dextromethorphan)

20

EM

15

EM + Q

10
5
0

1 1.5 2 2.5
Time (hr)

EM

Pain
Threshold
(mA)

Morphine Conc
(nM)

Data from: Desmeules J, et al. Eur J Clin Pharmacol 41:23:26, 1991

10
8 EM + Q
6
4
2
0
0
1

Time (hr)
183

What is the cause of hypermetabolizers?

184

Debrisoquine phenotype in subjects

with different CYP2D6 genotypes
Genotype
CYP2D6wt/(CYP2D6L)2

# of
Subjects
9

Metabolic
Ratio
0.33

CYP2D6wt/CYP2D6wt

12

1.50

CYP2D6wt/CYP2D6(A or B)

2.14

CYP2D6B/CYP2D6B

48.84

(CYP2D6L)2 - gene duplication; CYP2D6A - single base deletion

CYP2D6B - multiple point mutations
Data from: Agundez JG et al. Clin Pharmacol Ther 57:265, 1995.

185

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

186

E. CYP2C9 ACTIVITY
Prescribed Daily Warfarin Dose and CYP2C9 Genotype
Warfarin Dose*
5.63 (2.56)
4.88 (2.57)
3.32 (0.94)
4.07 (1.48)
2.34 (0.35)
1.60 (0.81)

Genotype
*1/*1
*1/*2
*1/*3
*2/*2
*2/*3
*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.
187

F. THIOPURINE METHYLTRANSFERASE (TPMT)

6-mercaptopurine

6-methylmercaptopurine
SCH3

SH
N

TPMT

N
H

SAM

N
H

SAH

188

Frequency

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

189

190

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
CYP1A1 AA
CYP1A1 Aa/aa

Weight Reduction
252 g
520 g

GST1 AA/Aa
GST1 aa

285 g
642 g

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

191

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
methyltransferase
Methylation of the promoter
region of MGMT may silence
the gene
From: 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.
192

From: 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.
193

From: 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.
194

195

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.
196

Patients with efficacy

in clinical trials

Patients without efficacy

in clinical trials

Predictive of efficacy

Predictive of no efficacy

197