emerging evidence for the role of polymorphic drug metabolizing enzymes in smoking behavior and...
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Emerging Evidence for the Role of
Polymorphic Drug Metabolizing Enzymes in Smoking Behavior
and Treatment
Caryn Lerman & Rachel Tyndale University of Pennsylvania & University of Toronto
Polymorphic Drug Metabolizing EnzymesCYP2A6 and CYP2B6
Genetic polymorphisms
» Ethnic variation
» Association with nicotine metabolism
» Association with smoking behavior
Therapeutic approaches
» Traditional NRTs
» Novel treatments
Nicotine Dependent Individuals Adjust Smoking Behavior to Maintain Nicotine Levels
80%80%
NICOTINENICOTINE COTININECOTININE
CYP2A6CYP2A6
Nicotine intake(i.e. smoking)
Nicotine removal (i.e. metabolism)
Chromosome 19Chromosome 19
2A6 2A7
Centromere Telomere
2B7 2B6
94% homologous
Gene cluster CYP2A and CYP2B 350bp
CYP2A6 AllelesCYP2A6 Alleles
activity*7 & *8 mutations*10TATA box*9
normalPoint mutation*8 activityPoint mutation*7?Point mutation*6absentPoint mutation*5absentdeletion*4A-*4D?2A6/2A7 hybrid*3absentPoint mutation*2normalGene conversion*1Bnormalnone*1A
EnzymeEffectAlleles
activity
*1 x 2 Gene duplication activity
Frequencies of CYP2A6 variant alleles vary among ethnic groups
WHO statistics0%
10%
20%
30%
40%
50%
60%
1 2 3 4 5
*10 Lower
*9 Lower
*7 Lower
*5 Inactive
*4 Inactive
*2 Inactive
African
Americans
Caucasians
Canadian Natives
Chinese
Japanese
Genetic Variation in CYP2A6 alters nicotine and cotinine plasma levels
Nicotine 4 mg base, oral Japanese subjects (Xu et al., 2001)
People with 1 or 2 inactive (*4) alleles have significantly higher NIC (2.3x, 2.8x) and lower COT (7x, 12x)
0
10
20
30
40
50
60
0 50 100 150 200 250 300 350 400
*4/*4
*1/*4
*1/*1
Time (min)
Cotinine
0
5
10
15
20
25
0 50 100 150 200 250 300 350 400
*4/*4
*1/*4
*1/*1
pla
sm
a (
ng
/ml)
Time (min)
Nicotine inactive
reduced
active
CYP2A6 slow metabolizers are at lower risk for becoming tobacco dependent
Slo
w m
etab
oli
zer
freq
uen
cy (
%)
0
2
4
6
8
10
1
CaucasiansCaucasians
Non-smokers SmokersNon-smokers Smokers TND (N=334) TD (N=365) TND (N=334) TD (N=365)
Caucasians with an inactive alleles (*2, *4, *10) or low activity alleles (*7) are “slow” nicotine metabolizers
Allelic variants less frequent in smokers vs non-smokers: Odds Ratio = 0.46 ( 95% CI: 0.22-0.95)
indicates slow nicotine inactivators are less likely to become smokers
OR 0.46 (0.22-0.95)P = 0.034
Tyndale et al, 2001
265
217
378
0
100
200
300
400
500
23
14
20
0
5
10
15
20
25
30
CYP2A6 Genotype Alters Smoking (n=296 Caucasians)
*1/dup(n=5)
*1/*1(n=277)
*1/null (n=14)
Carbon Monoxide Levels (ppm)* Plasma Cotinine Levels (ng/ml)
*1/dup(n=5)
*1/*1(n=277)
*1/null (n=14)
P<0.05P<0.05
Rao et al., 2000 *dose and timing not controlled
Summary of 2A6 Epidemiological Data
CYP2A6 ( activity variant)
» Decreased nicotine metabolism
» Decreased risk for tobacco dependence
» Decreased smoking rate and exposure
» Increased success quitting (Gu et al., 2000)
Human Brain CYP2B6 (and rat CYP2B1)
Alters Local Drug and Metabolite concentrations
– Inactivate Drugs: Nicotine (central metabolic tolerance?)
– Activate Drugs: Bupropion (greater efficacy?)
– Endogenous Substrates: metabolizes Testosterone
Mutagenicity and Genotoxicity– Activate tobacco-smoke procarcinogens (i.e. NNK)
Re
lativ
e D
ens
ity U
nits
Brain Stem Protein
Saline 0.1 0.3 1.0
Brain Stem Brain Stem ProteinProtein
mg/kg Nicotine
0
2
4
6
****
***
S Miksys et al., Biochem Pharmacol 59(12): 1501-1511, 2000.
Nicotine Induces Increase in Brain Levels of CYP2B6 Enzyme
Den
sity
Uni
ts
CYP2B6 is found at higher levels in specific brain regions of smokers, compared with nonsmokers
0
2
Non-Smokers (n=10) Non-Smokers (n=10) Smokers (N=14)Smokers (N=14)
FC TC CG OC HC EC CD PT NA GP SN CV CH
4
17 14 13 12 11 10 9 8 7 6 5 20 23 16 17 15 21 22 24
Nonsmokers Smokers
Interactions between environment and genotype on
Hippocampal CYP2B6
1
Wt Wt Mut Mut NS SMK NS SMK
P=0.07x1.7
2
0
P=0.03 x3.9
P=0.07 x2.5
CY
P2
B6
D
en
isty
Miksys, Lerman, Shields, Mash, Tyndale, 2003
CYP2B6 and Smoking Cessation
56
3838
19
0
10
20
30
40
50
60
70
WT
MUT
Placebo
Male Female
64
5042
54
Male Female
Bupropion
n=88 n=109 n=106 n=123P<.05 for sex x geno x trt
Lerman, Shields et al. Pharmacogenetics, 2002
Cravings by Genotype and Treatment
Session
0 1 2 3 4 5 6
Cra
vin
g
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Wild/Plac
Mut/Plac
Wild/Drug
Mut/Drug
QUIT
P<.05
Summary: CYP2B6
CYP2B6( activity variant)» Lower brain 2B6 levels» Decreased induction of enzyme by smoking » (Slower nicotine clearance &increased tolerance)? » Increased cigarette cravings» Decreased success quitting» Therapeutic application?
Therapeutic Therapeutic ImplicationsImplications
Mean of “desire to smoke”
Placebo Methox-salen
Tranyl-cypromine
0
8p = 0.0001p = 0.0001
1
2
3
4
5
6
7 p = 0.0001p = 0.0001
Me
an
(n
g/m
l), b
ase
line
ad
just
ed
Me
an
(n
g/m
l), b
ase
line
ad
just
ed
Mean plasma nicotine
CYP2A6 inhibitors & oral nicotine pills (4 mg) increase bioavailability & decrease desire to smoke in deprived smokers during 90 minute ad-lib period
p = 0.007p = 0.007 p = 0.02p = 0.02
-40
-35
-30
-25
-20
-15
-10
-5
0
Me
an
Sco
re, b
ase
line
adj
ust
ed
Me
an
Sco
re, b
ase
line
adj
ust
ed
Placebo Methox-salen
Tranyl-cypromineSellers & Tyndale, 2000
CYP2A6 inhibitors & oral nicotine pills (4 mg) leads to reduction in smoking
Number of cigarettes smoked decreased
Time between cigarettes increased
Total cigarette puffs decreased
Latency Between Cigarettes
2A6 In& Nic
2A6 In& Plac
Plac & Nic
Plac& Plac
0
10
20
30
p = 0.01p = 0.01
p = 0.01p = 0.0140M
ea
n (
±SE
M)
(min
)
Sellers & Tyndale, 2000
0
5
10
15
20
25
30
35
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
Time
Day
3 N
ico
tin
e (n
g/m
L)
Methoxsalen Placebo
* ** *
*
52% Increase in Nicotine Concentration with Methoxsalen
Inhibition of CYP2A6 Increases Nicotine from Nicorette (4mg)
n=11 *1/*1 genotype
Therapeutic Implications
Systemic Inhibition of 2A6*
• Increase nicotine from tobacco & decrease smoking
• Increase nicotine from NRTs & improve efficacy
Induction of Brain 2B6?
*Sellers & Tyndale, 2000
Acknowledgements!
University of Toronto: Rachel TyndaleRachel Tyndale, Sharon Miksys, Ewa Hoffman, Chun Xu, Yushi Rao, Bo Xu, Edward SellersUniversity of Pennsylvania : Janet Audrain, Paul Wileyto, Angela Pinto, Vyga Kaufmann, Sue Kucharski, Mike Burdick, Freda PattersonGeorgetown University: Peter ShieldsBrown University: Ray NiauraUCSF: Neal Benowitz
Tyndale Lab: Canadian Research Chair in PharmacogeneticsNIDA: DA06889Centre for Addiction and Mental HealthCIHR: MT-14173; MT-14719, training grant and doctoral awardsNCIC: 010271, Ontario Mental Health Foundation, Nicogen Res.