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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