Pharmacogenetics and respiratory diseaseGenetic biomarkers for treatment of disease and targeting of treatments Dr John W Holloway

• Polymorphism in a gene either in a single nucleotide (SNP) or in a series of associated alleles

(haplotype) may cause variable responses to drug therapy:

• Pharmacodynamics – drug actions

• Pharmacokinetics – drug metabolism (e.g warfarin)

• Adverse effects

• Matching drug therapy to the patient genotype may improve drug efficacy or reduce adverse

events

Pharmacogenomics

Severe outcome (mortality, quality of life, expensive care)

Difficulty of monitoring drug levels or predicting response

Strong association between genotype and clinical outcome

Availability of cheap genotyping assay

High frequency of variant genotype

Veenstra & Higashi AAPS Pharmsci 2000;2(3):29

Characteristics favouring the cost-effectiveness of pharmacogenomics-based therapies

Pharmacogenomics I

Targeting of therapies

Determination of the likelihood of an individual responding to a particular therapy (pharmacogenetics) and individualised treatment plans

Sub classification of disease on the basis of genetics and targeting of specific therapies based on this classification

Szefler et al. JACI 2005115:233-242

8 week crossover trial of:

Oral montelukast (5-10 mg/d) versus Inhaled fluticasone (200

ug/d)

126 mild-moderate asthmatic children (aged 6-

17y)

Flu

ticason

e

su

peri

or

Mon

telu

kast

su

peri

or

FEV1: forced expiratory volume

Identifying sub-groups of responders will improve targeting of drug therapy

1. Beta-2 agonists

2. Theophylline

3. Glucocorticoids

4. Anti-muscarinic drugs

5. Leukotriene modifiers

β2-AR

Gs

AC

Beta-2 agonists:

Albuterol

Salmeterol

AC: adenylate cyclase

AR: adrenoceptor

AMP: adenosine monophosphate

Gs:G-protein

cyclic AMP

Bronchial smooth muscle

relaxation

Protein kinase

A

Chronic downregulation

β2-ARAcute

desensitisation

P

Smooth muscle cell membrane

Reduces chronic response to LABA?

Reduces acute response to rescue

medication?

Arg:39%

Glu:43%

Ile: 0.03%

Met: <0.01%

Beta-2 adrenocepto

r

Reihsaus et al. AJRCMB 1993;8:334-9, Liggett AJRCCM 1997;156:S156-S162

Hall, Chest 2006;130:1873-8

Frequencies:

Arg16>Gly36%

Gln27>Glu43%

Others: <0.1%

Transfected CH fibroblasts

Green et al. AJRCMB 1995;13:25-33

Beta-2 receptor down-regulation by isoprenaline

is:

• Decreased by Arg16->Gly

• Increased by Gln27->Glu(Isoprenalin

e)

Arg/Gly-16 polymorphism in 2AR receptor reduces FEV1 response to Beta-2 agonist in children

3

4

5

6

2

n = 38 n = 124 n = 103

Arg16/Arg16 Arg16/Gly16 Gly16/Gly16

p = 0.007

1

Odds

rati

o o

f >

15%

incr

ease

in

FEV

1 a

fter

180u

g s

alb

ute

rol

Martinez FD et al. J Clin Invest 1997; 100:3184-3188

Arg/Gly16 polymorphism in 2AR and response to regular albuterol use

Israel E et al. Am J Respir Crit Care Med 2000; 162:75-80

Gly16/Gly16 receptors are already down-regulated by endogenous catecholamines and

do not fall further

Gly16/Gly16 n=62

Albuterol

Arg16/Arg16 n=28

Beta-2 receptor expressio

n

Time

Albuterol as-needed

0

Israel E et al. AJRCCM 2000;162:75-80

Albuterol

Albuterol

Mild asthmatics with Arg16/Arg16 genotype show decline in morning peak flow during regular albuterol therapy (4 x daily for 16 weeks)

Israel E et al. The Lancet 2004;364(9444):1505-12

ADRB2 polymorphisms and Long Acting Beta Agonists (LABAs)

Arg16/Arg16 individuals may also have impaired responses to LABAs such as Salmeterol

Wechsler ME et al. AJRCCM 2006;173:519-26Palmer CN et al. Thorax 2006;61:940-44

Treatment Genotype Total exacerbationsTotalOR(p values)

Not on salmeterol GlyGly 48 (33%) 971451.26(p = 0.149)*

ArgGly 61 (35%) 117178ArgArg 25 (41% 3459Total 132 248382

On regular salmeterol GlyGly 26 (41%) 37631.79(p = 0.020)*

ArgGly 39 (51%) 3776ArgArg 16 (64%) 925Total 81 83164

183 mild-moderate asthmatics Salmeterol 100ug/d plus fluticasone (200ug/d)

No differences between codon 16

genotypes in:

• FEV1, morning PEF

• Symptom scores

• Rescue medication

Genetic influence on receptor desensitisation may still be important in patients who receive only beta-2 agonists and over-use them.

Bleecker ER et al. JACI 2006;118:809-16Bleecker ER et al. Lancet 2007;370:2118-25

Inhaled corticosteroids may protect against receptor desensitisation in patients taking long-acting beta-2 agonists

• Eight SNPS in 5’-UTR (frequencies 33-67%)

• Commonest haplotype (-20C, -47C, -367C, -468G) reduces transcription in COS-7 cells

Scott et al.1999 BJP 126:841-4 ; Drysdale et al.2000 PNAS 97(19):10483-8; Moore et al.2000 ARCCM 162:2117-24; Weiss et al.2006 Pharmacogenomics J 6:311-26

ADRB25’ BU

P

NF-

IL6

SP

2 3’1 exon, 1200bp

Chr 5q31.32

-102

3

G -654

G

-468

C

-367

T

-47

T

-20

T

-134

3

A-142

9

T

A G A A G C CC

• BUP (Beta Upstream Protein): suppresses 2-AR expression

• Arg19>Cys enhances expression and resists desensitisation

• Further SNPS in 3’-UTR

• Highly complex and predictability may be poor

16 27

34

164

Beta-2 adrenoceptor gene (ADRB2)

Interaction between steroid treatment and -agonist response: Adenylyl Cyclase 9 polymorphism

β2-AR

Gs

AC

Beta-2 agonists:

Albuterol

Salmeterol

cyclic AMP

Bronchial smooth muscle

relaxation

Protein kinase

A

Tantisira KG et al. Hum Mol Genet 2005;14:1671-77

• 436 asthmatic children randomised to budesonide vs. placebo for 4 years (CAMP)

•Met772 AC9 SNP carriers show significantly better acute response to albuterol after steroid treatment

1. Beta-2 agonists

2. Theophylline

3. Glucocorticoids

4. Anti-muscarinic drugs

5. Leukotriene modifiers

CRH hypothalam

us

CRHR1

ACTH (adrenocorticotrophic

hormone from pituitary)

Cortisol adrenals

Glucocorticoid drug

Anti-inflammatory

effectsTantisira et al. Hum Mol Gen 2004;13:1353-9

GR

rs242941

T allele freq ~30%

• Defects in CRHR1 may reduce endogenous suppression of allergic inflammation

• May enhance effect of GC drug therapy

• Three SNPs in CRHR1 enhanced FEV1 response to inhaled GC for 6-8 weeks in >1100 adult &

pediatric asthmatics

Corticotrophin-releasing hormone receptor-1 (CRHR1)

T-bet (TBX21) polymorphism predicts response to inhaled corticosteroid

Tantisira et al. PNAS 2004;101:18099-104

rs2240017

Q allele freq ~4.5%

TBX21

Enhances Th1 gene transcription

(e.g. IFN-)

SuppressesTh2 transcription

(IL4, IL5)

• H33Q polymorphism of T-bet increases induction of Th1 gene transcription

• May enhance effect of GC drug therapy

• H33Q SNP in T-bet enhanced improvement in PC20 in response to inhaled GC over 4 years in >700

pediatric asthmatics

1. Beta-2 agonists

2. Theophylline

3. Glucocorticoids

4. Anti-muscarinic drugs

5. Leukotriene modifiers

Arachidonate

5-lipoxygenase (5-LO)

LTC4/D4/E4

LTA4

LTC4 synthase

CysLT1 receptor

montelukast

pranlukast

zileuton

FLAP

Phospholipase A2

MRP1

CysLT2 receptor

LTB4

BLT1, BLT2 receptors

LTA4 hydrolase

Sp1/Egr-1 tandem repeats in ALOX5 promoter

(-176 to -147)

1

2

3 4

5

Normal allele:5 (82%)

Variant alleles: 3 (3%)

4 (15%)

6 (<1%)

Frequency

In KH et al. J Clin Invest. 1997;99:1130-7

5-Lipoxygenase (ALOX5)

Kalayci et al. Allergy 2006;61:97-103

mRNA

Wild-typeVariants

Variants (non5/non

5)

Wild-type (5/5)

Variant 5-LO alleles reduce 5-LO mRNA transcripts and leukotriene C4 synthesis in human eosinophils

• Heterozygotes (n=40) have same response as

wildtype, so variant 5-LO alleles are recessive.

• Variant 5-LO homozygotes (6% of patients) show no response to LT modifier.

Drazen et al. Nature Genetics 1999;22:168-70

n=64

n=64

n=10

5-LO genotype and bronchodilator response to an oral 5-LO inhibitor (ABT-761) for 12 weeks in asthmatics

Lima et al. AJRCCM 2004;173:379-85Klotzman et al. Pharmacogenet Gen 2007;17:189-196

Lima et al. 2004

• 252 asthmatics receiving montelukast for >6 months

• 29 polymorphisms in 5-LO, LTA4H, LTC4S, MRP1, CysLTR1

• 5-LO and MRP1 SNPs linked to FEV1 (p<0.05)

• LTC4S -444A/C and a SNP in LTA4H linked to exacerbations (-76%)

• 5-LO tandem GC repeat variants: reduced exacerbations (-73%)

Montelukast pharmacogenetic studies

Pharmacogenomics II

Identification of novel pharmacological targets

Identification of novel asthma susceptibility genes leading to new pharmacological targets and pathways for novel therapeutics

Genetic variability and Leukotriene B4

Arachidonate5-

lipoxygenase (5-LO)

LTC4/D4/E4

LTA4

LTC4 synthas

eCysLT1 receptor

montelukast

pranlukast

zileuton

FLAP

Phospholipase A2

MRP1

CysLT2 receptor

LTB4

BLT1, BLT2 receptors

LTA4 hydrolase

Helgadottir A et al. Nat Genet. 2004;36:233-9, Nat Genet. 2006;38:68-74

increased LTB4 in cardiovascular disease (stroke, MI). Genetic variation in FLAP (ALOX5AP) and LTA4H associated with

increased LTB4 production and risk of disease

Genetic association suggests a role for Leukotriene B4 in asthma

dbSNP reference

Gene Location

Alleles MAF Asthma No. Z-score p -value

SG13S25 5’UTR G/A 0.106 104 0.639

SG13S114 Intron 1 T/A 0.331 216 +2.349 0.018*

rs3803277 Intron 2 C/A 0.451 246 0.144

SG13S89 Intron 3 G/A 0.043 47 0.157

rs4468448 Intron 4 C/T 0.247 213 0.184

SG13S32 Intron 4 C/A 0.479 238 0.201

SG13S41 Intron 4 A/G 0.067 69 +2.681 0.007*

SG13S35 3’UTR G/A 0.079 83 0.466

ALOX5AP

Holloway JW et al. The role of LTA4H and ALOX5AP polymorphism in Asthma and Allergy Susceptibility. Allergy 2008 (In Press)

Genetic association suggests a role for Leukotriene B4 in asthma

LTA4H

dbSNP reference

Gene Location

Alleles MAF Asthma No. Z-score p -value

rs1978331 Intron 11

T/C 0.417 227 -2.095 0.036*

rs17677715 Intron 6

T/C 0.195 163 0.216

rs2540482 5’UTR A/G 0.224 181 0.248

rs2660845 5’UTR A/G 0.260 199 0.853

rs2540475 5’UTR C/T 0.216 181 0.303

Holloway JW et al. The role of LTA4H and ALOX5AP polymorphism in Asthma and Allergy Susceptibility. Allergy 2008 (In Press)

Arachidonate

5-lipoxygenase (5-LO)

LTC4/D4/E4

LTA4

LTC4 synthase

CysLT1 receptor

montelukast

pranlukast

zileuton

FLAP

Phospholipase A2

MRP1

CysLT2 receptor

LTB4

BLT1, BLT2 receptors

LTA4 hydrolase

Theophylline PDE4 family

CYP 1A2

Not known

Possible

Anti-muscarinics

M2 receptor Not known

M3 receptor Unlikely

Leukotriene modifiers

ALOX5 (5-LO)

Yes (GC tandem repeats)

LTC4S Probable (-444A/C)

MRP1, LTA4H, CysLTR1/2FLAP

Possible

Unlikely

Updated from Hall & Sayers, 2007 ERJ 29:1239-45

Drug class Candidate gene Pharmacogenetic effect

SUMMARY

Glucocorticoids GR Possible (Asp363Ser)

CRHR1 Possible

TBX21 Possible

Beta-2 agonists ADRB2 Probable (Arg16Gly) but clinical relevance unclearAC9Possible

US Human Genome Program

Complexity of finding gene variations that affect drug response

- 10 million SNPs in human genome & infinite haplotypes

- Linking specific genes to drug responses

Limited drug alternatives for many diseases

Disincentives for drug companies to fragment their market with multiple versions of drugs, particularly for

small sub-groups

Extra complexity for drug prescribers and dispensers

Barriers to Pharmacogenomics Progress

Pharmacological treatment of disease

33

Health maintenanc

e(diet, lifestyle)

ONE SIZE FITS ALL

Risk factor

(biomarker, symptoms)

Generalised treatment

Now:

Future:

Predisposition

Early detection

Personalised, early, effective

therapies

PERSONALISED

AcknowledgementsUniversity of Southampton

Tony SampsonIan SayersStephen HolgateSheila BartonMatthew Rose-ZerilliSonia MallShu YeSalman Siddiqui

University of NottinghamIan Sayers

Ajou University, S KoreaHae-Sim Park

Asthma UKMedical Research Council

Merck & Co.Genome Therapeutics Corporation