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.