farmakogenetika

Farmakogene*ka

Prof.dr. Amina Kozaric 24.10.2014

PMF

1

Defini*ons

•  Pharmacogenomics: Analysing en*re genomes, across groups of individuals, to iden*fy the gene*c factors influencing 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.

2

Objec*ves of Pharmacogene*cs

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

3

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 don’t?!•  Why do some people get cancer and others don’t?!•  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 2003 Human Genome Project

determined the en-re DNA sequence of a human

(3 billion le/ers)

April 1953 Drs. James Watson and

Francis Crick determined the structure of DNA (double helix)

What is Pharmacogenomics?

R X

Pharma = drug or medicine Genomics = the study of genes

R X

Pharmacogenomics?

Personalized medicine tailored to your genes

Pharma = drug or medicine Genomics = the study of genes

R X

What is Pharmacogenomics?

Case Study – Breast Cancer Pa-ents


Tumoricide

16


17


30%

18

Helped No Effect/Hurt

Tumoricide


Tumoricide

Why?

Your DNA

How do scien-sts make personalized medicine?

You Your cells

Your DNA

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

How do scien-sts make personalized medicine?

It’s all about what makes YOUR gene-c code UNIQUE

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




A T

Adenine Thymine




A T C

Adenine Thymine Cytosine




A T C G

Adenine Thymine Cytosine Guanine

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

Gene

The Central Dogma: DNAàRNAàProtein


Gene

RNA: A single-‐stranded copy of one gene.

RNA



Gene



RNA

Protein: Proteins are composed amino acids. Amino acids are made from triplets of nucleo-des called codons.


Gene




Codon 1


Gene




Codon 1 Codon 2


Gene


Amino acid 1 Amino acid 2



Codon 1 Codon 2


Gene


Amino acid 1 Amino acid 2

Protein!



Codon 1 Codon 2

A small change in the gene sequence can result in a very different protein

ATG GTG CTG TCT CCT DNA:


ATG GTG CTG TCT CCT

Met

DNA:

Amino Acids/Protein:


ATG GTG CTG TCT CCT

Met

DNA:

Amino Acids/Protein: Val


ATG GTG CTG TCT CCT

Met Leu

DNA:



ATG GTG CTG TCT CCT

Met Leu Ser

DNA:



ATG GTG CTG TCT CCT

Met Leu Ser Pro

DNA:



ATG GTG CTG TCT CCT

Met Leu Ser Pro

DNA:


ATG GTG CTG TCT ACT DNA:


ATG GTG CTG TCT CCT

Met Leu Ser Pro

DNA:


ATG GTG CTG TCT ACT

Met Leu Ser Thr

DNA:



ATG GTG CTG TCT CCT

Met Leu Ser Pro

DNA:


Words: Tom and Sam are bad

ATG GTG CTG TCT ACT

Met Leu Ser Thr

DNA:



ATG GTG CTG TCT CCT

Met Leu Ser Pro

DNA:



ATG GTG CTG TCT ACT

Met Leu Ser Thr

DNA:


Tom and Sam are sad Words:


ATG GTG CTG TCT CCT

Met Leu Ser Pro

DNA:



ATG GTG CTG TCT ACT

Met Leu Ser Thr

DNA:


Tom and Sam are sad Words:

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

Variations in the DNA (genotype) can cause observable changes (phenotype) in individuals


Tumoricide

Why does Tumoricide work on some pa-ents but not

on others?

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 a[er receiving it

Drugs and Receptors

Cell

Drugs and Receptors

Cell

Receptor (Protein)

Drugs and Receptors

Cell

Drug (Ligand) Receptor

(Protein)

Your DNA and Drugs Varia*on in genes can cause varia*on in receptors

Cell


Cell

Cell

Cell


Cell

Cell

Cell

Cell

Cell


Cell

Cell

Cell

Too Many (hypersensi-ve)

Cell

Cell


Cell

Cell

Cell


Too Few (hyposensi-ve)

Cell

Cell


Cell

Cell

Cell

Cell

Cell


Too Few (hyposensi-ve)

Mutated (insensitive)

Where Drugs “Fit” In

Lock = Receptor Key = Drug

Taste cell

“This tastes bi/er!”

PTC-‐Receptor PTC

Why can some people taste PTC and others can’t?

Phenylthiocarbamide

Taste cell

“This tastes bi/er!”

PTC-‐Receptor PTC

Why can some people taste PTC and others can’t?

Taste cell

“I don’t taste anything!”

Non-‐binding PTC-‐

Receptor

PTC

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

You have two copies of every gene:

one from Mom and

one from Dad

Where does tas*ng PTC come from?

•  Your two genes are the genotype •  A gene can be dominant or recessive


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


one from Mom and

one from Dad


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

TT Tt [


TT Tt [

For individuals with these genotypes, what would their phenotypes be?

TT

“This tastes REALLY bi[er!”

SUPERTASTER


Tt [

“This tastes bi[er!” TASTER

TT


SUPERTASTER


Tt [


TT


Tt [

“I dont taste anything!”

NON-‐TASTER


SUPERTASTER

Drug receptor summary

Ability to taste PTC has a very strong gene-c component PTC = chemical and Drugs = chemical

Differences in ability to taste PTC is similar to

differences in reac*ons to drugs ""


Tumoricide

Why?

Two Types of Breast Cancer

•  Tumoricide is a personalized medica-on •  Tumoricide only works for Her2+ breast tumors

Y Her2-‐ Her2+

80


Tumoricide

Her2-‐ Her2+

Screening for Her2+ Cells

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

Her2-‐ Her2+



+ Tumoricide = ?



+ 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




3.  How your body processes the drugs a[er receiving it

The presence of receptors influence how we react to drugs like Tumoricide or chemicals like PTC

Tumoricide Works!

Tumoricide Does Not Work

Her2-‐ Her2+

Y

TT Tt [


SUPERTASTER


“I dont taste anything!”

NON-‐TASTER

The presence of receptors influence how we react to drugs like Tumoricide or chemicals like PTC

Where are the PTC receptors?

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

Papillae




PTC receptors are found on the taste buds

Papillae




Papillae


Nerve Cell Transmits signal

to brain Taste buds cells are found on the papilla



Papillae


Nerve Cell Transmits signal

to brain

Brain Wow! This tastes

really bi[er


If a person has more taste buds, then he/she may be able to taste the PTC more strongly.

Are there other traits that can allow a person to more strongly taste PTC?

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 non-

taster?

What does it take to be a PTC Taster? Two traits are important for determining PTC taste sensi-vity

1) PTC receptor genotype—Do you 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 genotype—Do you 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

taster super-taster




3.  How your body processes the drugs after receiving it

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

ADME

•  Absorp-on •  Distribu-on

•  Metabolism

•  Excre-on

A Drug’s Life

Metabolic enzymes

Liver

DNA variations in special proteins in the liver called enzymes can influence a person’s ability to metabolize

certain drugs

Drug Metabolites Enzymes

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



•  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…

A

B

C

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…

A

B

C

Why?

ADME of Nortriptyline

100mg Nortriptyline

Adverse reaction Nothing happens Gets better

A B C

How much active drug in blood?

ADME of Nortriptyline

100mg Nortriptyline

Adverse reaction Nothing happens Gets better

A B C

95mg 5mg 50mg

DNA variation influence drug metabolism

Liver


A

Poor Metabolizer

95mg


Liver


B

Ultrarapid Metabolizer

5mg


Liver


C

Intermediate Metabolizer

50mg

2012 - What do doctors do?

A B C

Decrease Dose Increase Dose

Or change drug

Poor Metabolizer Ultrarapid Metabolizer

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! • What’s 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! • What’s 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 differences and

differences 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




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 people’s 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-thioguanine azathioprine!6-mercaptopurine!

Metabolism of 6-MP!

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

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!

© 2006 American Medical Association. All rights reserved

Polymorphic!Cytochrome!P-450s!

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 “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!

Personalized Drugs!•  Hercep*n (breast cancer, target: Her2/neu) •  Erbitux (colorectal cancer, target: EGFR) •  Tarceva (lung cancer, target: EGFR) •  Straeera (aeen*on-‐deficit/hyperac*vity

disorder, Metabolism: P4502D6) •  6-‐MP (leukemia, Metabolism: TPMT) •  An*virals (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!

I. Key Concepts and Terms

154

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%

Normal Distribution

155

Freq

uenc

y

Activity

Polymorphic Distribution

156

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

157

GENETIC POLYMORPHISMS

Pharmacokinetic Pharmacodynamic • Transporters • Plasma protein binding • Metabolism

• Receptors • Ion channels • Enzymes • Immune molecules

158

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

II. Genetic polymorphisms in drug metabolizing enzymes

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

O C CH2CH2

O

(H3C)3NH2CH2C COO CH2CH2N(CH3)3

+ +

SUCCINYLCHOLINE

160

A. Atypical Plasma Cholinesterase

• 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

Hydrolysis by pseudocholinesterase

choline succinylmonocholine

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

020406080

100

-6 -4log molar dibucaine conc.

% In

hibi

tion

Typical Atypical

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

normal enzyme inhibited > 70% abnormal inhibited < 30%

162

• 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

163

B. Glucose-6-phosphate dehydrogenase activity Effects >100 million worldwide

R-NH2 CYP MPO PGH Synthase

R-NOH

ERYTHROCYTE

R-NOH

O2 HgbFe+2

R-NO HgbFe+3

Reactive Oxygen

NADH

NAD+ MetHgb Reductase

NADPH or GSH(?)

NADP+ or GSSG(?) HMP Shunt

G-6-PD Dependent

SOD Catalase GSH Peroxidase

Detoxification

Splenic Sequestration

Hemolytic Anemia

GSH Semi-mercaptal

sulfinamide

R-NH2

164

Drugs and Chemicals Unequivocally Demonstrated to Precipitate Hemolytic Anemia

in Subjects with G6PD Deficiency

Acetanilide Nitrofurantoin Primaquine Methylene Blue Sulfacetamide Nalidixic Acid Naphthalene Sulfanilamide Sulfapyridine Sulfamethoxazole

165

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 8 Greeks 0.7-3

166

INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS

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

167

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.

168

NAT1*4 NAT2*4 NAT2*5A NAT2*6A NAT2*7A

PABA PAS SMX

PA DDS SMZ

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

169

ETHNIC DIFFERENCES IN THE DISTRIBUTION OF ACETYLATOR PHENOTYPE

Population % Slow % Hetero Fast % Homo Fast

South Indians 59 35.6 5.4 Caucasians 58.6 35.9 5.5 Blacks 54.6 38.6 6.8 Eskimos 10.5 43.8 45.7 Japanese 12 45.3 42.7 Chinese 22 49.8 28.2

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

170

XENOBIOTICS SUBJECT TO POLYMORPHIC ACETYLATION IN MAN

Hydrazines isoniazid hydralazine phenylzine acetylhydrazine hydrazine

Arylamines dapsone procainamide sulfamethazine sulfapyridine aminoglutethimide

Carcinogenic Arylamines benzidine β-naphthylamine 4-aminobiphenyl

Drugs metabolized to amines sulfasalazine nitrazepam clonazepam caffeine

171

ADVERSE EFFECTS TO SULFASALAZINE IN PATIENTS WITH

INFLAMMATORY BOWEL DISEASE

N N

COOH

OHS

O

O

HNN

SULFASALAZINE

NH2S

O

O

HNN

H2N

COOH

OH

SULFAPYRIDINE 5-AMINOSALICYLIC ACID

172

ADVERSE EFFECTS TO SULFASALAZINE IN PATIENTS WITH

INFLAMMATORY BOWEL DISEASE

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

Side Effect cyanosis hemolysis transient reticulocytosis

Frequency of side effect Slow Acetylators Fast Acetylators

9 1 5 0 6 0

173

0

20

40

60

80

100

120

0 20 40 60 80 100

Duration of Therapy (months)

% o

f pts

with

lupu

s

Slow AcetylatorsFast Acetylators

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.

174

NH2C

O

(H3CH2C)2NH2CH2CHN

PROCAINAMIDE

NHC

O

(H3CH2C)2NH2CH2CHN CCH3

O

NHOHC

O

(H3CH2C)2NH2CH2CHN

NATCYP450

PROCAINAMIDE HYDROXYLAMINE N-ACETYLPROCAINAMIDE

175

0

20

40

60

80

100

SLOW FAST

Perc

enta

ge o

f Sub

ject

s ControlHS

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

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

176

NAT1 N-acetyl-SMX UDPGT SMX-glucuronide

CYP2C9 MPO PGH SYNTHASE

SMX hydroxylamine Nitroso Detox

Covalent binding to cellular macromolecules/

cytotoxicity

Hypersensitivity/ Adverse Reaction

O-acetylation

Acetoxy ester

NAT1 Hydroxamic acid N,O-AT

Detoxified metabolite

Sulfamethoxazole(SMX)

NO

NH2 S

O

O

HN

CH3

177

D. CYP2D6 ACTIVITY

N C NH

NH2

N C NH

NH2

CYP2D6

OH

DEBRISOQUINE4-HYDROXYDEBRISOQUINE

H

N CH3

OCH3

H

N CH3

OH

DEXTROMETHORPHAN DEXTRORPHAN

CYP2D6

179

DRUGS WHOSE METABOLISM CO-SEGREGATES WITH DEBRISOQUINE

alprenolol amitriptyline bufuralol clomipramine codeine desipramine encainide ethylmorphine flecainide fluoxetine guanoxan imipramine metoprolol nortriptyline paroxetine phenformin propafenone propranolol

180

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.

181

Dose requirements for nortriptyline in patients with different CYP2D6 Phenotypes. From: Meyer U. Lancet 356:1667, 2000.

182

O-demethylation morphine

N-demethylation norcodeine

6-glucuronidation codeine-6-glucuronide

M-6-G

M-3-G

normorphine

norcodeine- 6-glucuronide

CYP2D6

H3CO

NCH3

HO

O

CODEINE

183

Effect of Quinidine on the Analgesic Response to Codeine in Extensive Metabolizers of

CYP2D6 (Phenotyped with Dextromethorphan) Data from: Desmeules J, et al. Eur J Clin Pharmacol 41:23:26, 1991

0

5

10

15

20

Mo

rph

ine

Co

nc

(nM

)

0 1 1.5 2 2.5

Time (hr)

EMEM + Q

02468

10

Pa

in

Th

resh

old

(m

A)

0 1 2

Time (hr)

EMEM + Q

184

What is the cause of ‘hypermetabolizers’?

Debrisoquine phenotype in subjects with different CYP2D6 genotypes

Genotype # of Subjects

Metabolic Ratio

CYP2D6wt/(CYP2D6L)2 9 0.33

CYP2D6wt/CYP2D6wt 12 1.50

CYP2D6wt/CYP2D6(A or B) 9 2.14

CYP2D6B/CYP2D6B 6 48.84

185

(CYP2D6L)2 - gene duplication; CYP2D6A - single base deletion CYP2D6B - multiple point mutations

Data from: Agundez JG et al. Clin Pharmacol Ther 57:265, 1995.

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

E. CYP2C9 ACTIVITY

187

Prescribed Daily Warfarin Dose and CYP2C9 Genotype

Warfarin Dose* Genotype 5.63 (2.56) *1/*1 4.88 (2.57) *1/*2 3.32 (0.94) *1/*3 4.07 (1.48) *2/*2 2.34 (0.35) *2/*3 1.60 (0.81) *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.

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F. THIOPURINE METHYLTRANSFERASE (TPMT)

N

N NH

N

SH

N

N NH

N

SCH3

TPMT

SAM SAH

6-mercaptopurine 6-methylmercaptopurine

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TPMT Activity

Freq

uenc

y

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

G. GENETIC POLYMORPHISMS, MATERNAL SMOKING AND LOW BIRTH WEIGHT (LBW)

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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 Weight Reduction CYP1A1 AA 252 g CYP1A1 Aa/aa 520 g GST1 AA/Aa 285 g GST1 aa 642 g

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

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.

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

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194


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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.

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Patients with efficacy in clinical trials

Patients without efficacy in clinical trials

Predictive of efficacy

Predictive of no efficacy

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