MATA KULIAH DASAR UMUM

BIOLOGI MOLEKULER

PHARMACOGENETIC
Edhyana Sahiratmadja, dr. PhD.

Dept. Biokimia & Biologi Molekuler

Fakultas Kedokteran
Universitas Padjadjaran
Human Genome
DNA Organization
 Gene Structure

 Most of the genes consist of; short coding

sequences or exons are interrupted by a longer
intervening noncoding sequence or introns;
although a few genes in the human genome
have no introns.
The Central Dogma of Molecular Biology

 DNA molecules serve as templates for either

complementary DNA strands during the
process of replication or complementary RNA
during the process of transcription.

 RNA molecules serve as a template for

ordering amino acids by ribosomes during
protein synthesis.
The Central Dogma of Molecular Biology
Application of Molecular Biology

 Research
 Diagnosis
 Transplantation
 Paternity
 Forensic analysis
 Gene therapy
 Drug Design
 ……
Genomics, Bioinformatics & Medicine
http://biochem158.stanford.edu/

Pharmacogenomics
http://biochem158.stanford.edu/Drug-Development.html

Doug Brutlag 2011

The Proposition of the Usual Dose

“The dose makes the poison”

Paracelsus
(1493-1541)
 The Problem of Variability

“Variability is the law of life, and

as no two faces are the same,
so no two bodies are alike,
and no two individuals react alike,
and behave alike under the
abnormal conditions which we
know as disease.”
Sir William Osler
(1849-1919)
Courtesy of Felix W. Frueh US FDA
Doug Brutlag 2011
Paradox of Modern Drug Development

Clinical trials provide evidence of efficacy

and safety at usual doses in populations

Physicians treat individual patients who

can vary widely in their response to drug
therapy
How Can The Causes of Variability be
Unraveled?

Pharmacogenomics: systemic genomic analysis in

populations of treated subjects to identify variants
that predict drug response including the
occurrence of adverse reactions
 Pharmacogenomics Strategy
Applied to the Practice of Medicine

GCCCGCCTC

GCCCACCTC

From McLeod and Evans, Ann Rev of Pharmacol and Toxicol, 2001: 41,101-121
 Personalized Medicine

• Medicine is personal:
o We are all different.
o Some of our differences translate into how we react to drugs
as individuals.
o 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 don’t?
• Why do some people get cancer and others don’t?
• Why is anecdotal information irrelevant to your own
health and treatment?

Doug Brutlag 2011

 Is Medicine a Science or an Art?

If it were not for the great variability among

individuals, medicine might well be a science,
not an art.
o Sir William Osler, Physician 1892
o Johns Hopkins School of Medicine
o Johns Hopkins Hospital
o Father of modern medicine

Doug Brutlag 2011

 The Goal of Personalized Medicine

• The Right Dose of

• The Right Drug for
• The Right Indication for
• The Right Patient at
• The Right Time.

Courtesy Felix
DougW. Frueh
Brutlag 2011
Pharmacogenetics & Pharmacogenomics

• Pharmacogenetics: The role of genetics in

drug responses.
o F. Vogel. 1959

• Pharmacogenomics: The science that allows

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

Courtesy Felix
DougW. Frueh
Brutlag 2011
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)

o PharmGKB Website: http://www.pharmgkb.org/

Courtesy of MichelleDoug
Whirl-Carillo
Brutlag 2011
 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

Doug Brutlag 2011
Pharmacogenetics: A Case Study

Courtesy of MichelleDoug
Whirl-Carillo
Brutlag 2011
Pharmacogenetics: A Case Study

Courtesy of MichelleDoug
Whirl-Carillo
Brutlag 2011
Pharmacogenetics: A Case Study

Courtesy of MichelleDoug
Whirl-Carillo
Brutlag 2011
 Metabolism of 6-MP

L Wang and R Weinshilboum, Oncogene 25, 1629-1638 (2006)

Doug Brutlag 2011
Thiopurine S-methyl Transferase Activity
and Personalized Dosage

Eichelbaum et al., Annu. Rev. Med. 2006.57:119-137.

Doug Brutlag 2011
Warfarin: Significant Problems for Rats!

Doug Brutlag 2011

 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.

Doug Brutlag 2011

 Warfarin: Significant Problems for Humans!

• Case Report July 2, 2008

o Company director dies of brain
hemorrhage after heading a football
o Consultant neurosurgeon told the
inquest the warfarin effect was
probably the cause of the death
o It can happen to anyone!

• Other Warfarin Patients

• Case Report July 2, 2008
o Joseph Stalin

Doug Brutlag 2011

 Why Maintaining Warfarin
Therapeutic Range is Critical

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

Doug Brutlag 2011
 Finding Doses to Maintain Therapeutic
Anticoagulation is Largely Trial and Error

Doug Brutlag 2011

Warfarin Levels Depend on Two
Enzymes – CYP2C9 & VKORC1

Doug Brutlag 2011

 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

Doug Brutlag 2011

 What are Targeted Drugs?

• Often, drugs are only effective in specific

“sub-populations” (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!
Doug Brutlag 2011
Doug Brutlag 2011
 Personalized Drugs

• Herceptin (breast cancer, target: Her2/neu)

• Erbitux (colorectal cancer, target: EGFR)
• Tarceva (lung cancer, target: EGFR)
• Strattera (attention-deficit/hyperactivity
disorder, Metabolism: P4502D6)
• 6-MP (leukemia, Metabolism: TPMT)
• Antivirals (i.e. resistance based on form of
HIV)

• etc. and the list is growing rapidly ...

Doug Brutlag 2011

FDA Requires Genetic Tests
for Certain Therapies

Courtesy of MichelleDoug
Whirl-Carillo
Brutlag 2011
 Roche Chip for Cytochrome P450
Genes: CYPC19 and CYP2D6

Xie and Frueh, Pharmacogenomics steps toward Personalized Medicine, Personalized Medicine 2005, 2, 325-337
Doug Brutlag 2011
 Treatment of Pulmonary
Tuberculosis
• Approximately 20％ of the patients
treated with Rifampicin and INH
develop hepatotoxicity.
• Metabolic enzyme of INH is NAT2.
• Rifampicin strongly induces many
drug metabolizing enzymes.
40
Metabolic pathways of isoniazid
CONHNH2 Rifampicin
NAT2
Acetylation
N Enzyme
induction
CONHNHCOCH3 Isoniazid
(INH) Hydrolysis
Acetyl-
N isoniazid
COOH
Hydrolysis

N Acetylation
NH2NHCOCH3
Acetylhydrazine (AcHz) NAT2 NH2NH2
NAT2 Isonicotinyl Hydrazine (Hz)
glycine
(NHCOCH3)2 Hepatotoxic
Diacetylhydrazine 41
Mutation of N-acetyltransferase 2

A→C 759 C→T

434
T→C G→A A→C G→A 803 A→G
111 191 341 499 845 A→C

* ** * * * * * * ****
190 282 481 590 857
C→T C→T C→Ｔ G→A G→A

ＳＮＰｓ
42
Frequency of the NAT2 genotypes in patients

Genotype n %

NAT2*4 / *4 46 40.3 RA type (40.3%)

NAT2*4 / *5 4 3.5
NAT2*4 / *6 34 29.8 IA type (46.5%)
NAT2*4 / *7 15 13.2
NAT2*5 / *5 0 0.0
NAT2*5 / *6 2 1.8
NAT2*5 / *7 0 0.0 SA type (13.2%)
NAT2*6 / *6 7 6.1
NAT2*6 / *7 4 3.5
NAT2*7 / *7 2 1.8
114 100.0 43
Incidence of INH-Rifampicin (RFP) induced
hepatotoxicity and NAT2 genotype

Total
(n=114)

RA type
(n=46)

IA type
(n=53) *
*
SA type
(n=15)
* P<0.01
0 20 40 60 80 100
Incidence (%)
normal hepatotoxicity
criteria: serum AST and/or ALT > 1.5 x upper limits of normal44
and 2 x before administration.
 Case in SA type
Case 1（female 69years)
Serum aminotransferase (IU/L)
350

300

250 drug monitoring

AST
200 ALT
150

100
NAT2*6/*6 (SA type)
50

0
0 10 20 30 40 50 60 70 80 90
INH Day
(mg/day) 400 100 200
RFP 発疹のため投与中止
(mg/day) 450 450 300
検出菌数
2+ 1+ ± 45
（蛍光法）
Plasma INH concentration-time profile
observed in patient of case1
7
INH concentration (mg/mL)

6
400mg/day
5 200mg/day
4
Reduced

0
0 5 10 15
Time after administration (hr) 46
 Cases in RA type
Case 5 (male 62years)

: NAT2*4/*4 (RA type)

3

INH conc. (mg/mL)

Poor Response 2
Case 5
Mean of RA type
INH 400mg/day (po) (400 mg/day)
+RFP+SM 1

0 5 10 15
INH 400mg/day(po) Time after administration (hr)
+200mg/day(inhalation)
Cured +RFP+SM
47
 INH-RFP induced hepatotoxicity
INH＋RFP

slow acetylator intermediate acetylator rapid acetylator

(SA type) (IA type) (RA type)

Liver Continue
Toxicity
Increase dose
Change regim en

Delayed
Cured 48

RFP:rifampicin
 Plasma INH concentration-time profile

Patients (n )：114
INH concentration (mg/mL)

10 Sparse plasma (n )：278

(1 – 4 points/person)

1 Dose: INH 200 mg

at steady state
0.1 RAtype
Same dose IAtype
0.01 SAtype
but different
plasma concentrations
Therapeutic range
0.001
0 5 10 15 Minimal inhibitory
Time after administration (hr) concentration49(MIC)
 Simulation of dose adjustment of INH
based on NAT2 genotype (preliminary)

Present Simulated
INH conc. (mg/mL)

Css
3.0 max

0.2 min

0 12 24 hr 0 12 24 hr

All types RA-type 500 mg x 2 /day

I A-type 250 mg x 2 /day
200 mg x 2 /day 50
SA-type 100 mg x 2 /day
Clinical trial for genotype based chemotherapy against
pulmonary tuberculosis

◆ Multi-center prospective randomized clinical trial

◆ Rationalized dosing of isoniazid based on NAT2 gene
polymorphism
◆ Safety, Efficacy, Pharmacoeconomics

Patients with pulmonary tuberculosis

NAT2 genotyping NAT2

Gene chip

RA-type IA-type SA-type

51
Modern Rendition of Paracelsus

“The dose makes the poison, but

differently for genetically
different individuals.”

Paracelsus
(1493-1541)
Application of Molecular Biology

 Research
 Diagnosis
 Transplantation
 Paternity
 Forensic analysis
 Gene therapy
 Drug Design
 ………………………..what we consume n eat
………….. NUTRIGENOMIC
Application of Molecular Biology
Everywhere

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