Genetic Insights: Polymorphic Drug Enzymes in Smoking Behavior

1. 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

2. Polymorphic Drug Metabolizing EnzymesCYP2A6 and CYP2B6 Genetic polymorphisms • Ethnic variation • Association with nicotine metabolism • Association with smoking behavior Therapeutic approaches • Traditional NRTs • Novel treatments

3. Nicotine removal (i.e. metabolism) Nicotine intake (i.e. smoking)  80% NICOTINECOTININE CYP2A6 Nicotine Dependent Individuals Adjust Smoking Behavior to Maintain Nicotine Levels

4. Centromere Telomere Chromosome 19 Gene cluster CYP2A and CYP2B 350bp 2A6 2A7 2B7 2B6 94% homologous

5. activity

6. Frequencies of CYP2A6 variant alleles vary among ethnic groups WHO statistics African Americans Caucasians Chinese Japanese Canadian Natives

7. Genetic Variation in CYP2A6 alters nicotine and cotinine plasma levels Nicotine 4 mg base, oral Japanese subjects (Xu et al., 2001) Nicotine inactive Cotinine *4/*4 reduced *1/*1 plasma (ng/ml) *1/*4 *1/*4 *1/*1 *4/*4 active Time (min) Time (min) People with 1 or 2 inactive (*4) alleles have significantly higher NIC (2.3x, 2.8x) and lower COT (7x, 12x)

8. CYP2A6 slow metabolizers are at lower risk for becoming tobacco dependent • 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 Caucasians OR 0.46 (0.22-0.95) P = 0.034 Slow metabolizer frequency (%) Non-smokers Smokers TND (N=334) TD (N=365) Tyndale et al, 2001

9. CYP2A6 Genotype Alters Smoking (n=296 Caucasians) Carbon Monoxide Levels (ppm)* Plasma Cotinine Levels (ng/ml) P<0.05 P<0.05 *1/*1 (n=277) *1/null (n=14) *1/dup (n=5) *1/*1 (n=277) *1/null (n=14) *1/dup (n=5) Rao et al., 2000 *dose and timing not controlled

10. Summaryof 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)

11. 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)

12. 6 4 2 0 Nicotine Induces Increase in Brain Levels of CYP2B6 Enzyme Brain Stem Protein *** ** Relative Density Units ** Brain Stem Protein Saline 0.1 0.3 1.0 mg/kg Nicotine S Miksys et al., Biochem Pharmacol 59(12): 1501-1511, 2000.

13. 17 14 13 12 11 10 9 8 7 6 5 20 23 16 17 15 21 22 24 Nonsmokers Smokers CYP2B6 is found at higher levels in specific brain regions of smokers, compared with nonsmokers Non-Smokers (n=10) Smokers (N=14) Density Units 4 2 0 FC TC CG OC HC EC CD PT NA GP SN CV CH

14. Interactions between environment and genotype on Hippocampal CYP2B6 P=0.03 x3.9 P=0.07 x2.5 2 CYP2B6 Denisty P=0.07x1.7 1 0 Miksys, Lerman, Shields, Mash, Tyndale, 2003 Wt Wt Mut Mut NS SMK NS SMK

15. Bupropion Male Female n=88 n=109 n=106 n=123 P<.05 for sex x geno x trt CYP2B6 and Smoking Cessation Lerman, Shields et al. Pharmacogenetics, 2002 Placebo Male Female

16. QUIT P<.05

17. 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? Summary: CYP2B6

18. Therapeutic Implications

19. CYP2A6 inhibitors & oral nicotine pills (4 mg) increasebioavailability & decreasedesire to smoke in deprived smokers during 90 minute ad-lib period Mean plasma nicotine Mean of “desire to smoke” 8 0 p = 0.0001 p = 0.0001 7 -5 6 -10 5 -15 4 Mean (ng/ml), baseline adjusted -20 Mean Score, baseline adjusted 3 -25 2 -30 1 p = 0.007 p = 0.02 -35 0 -40 Placebo Methox- salen Tranyl- cypromine Placebo Methox- salen Tranyl- cypromine Sellers & Tyndale, 2000

20. 40 p = 0.01 p = 0.01 30 20 Mean (±SEM) (min) 10 0 2A6 In & Nic 2A6 In & Plac Plac & Nic Plac & Plac CYP2A6 inhibitors & oral nicotine pills (4 mg) leads to reduction in smoking Latency Between Cigarettes Number of cigarettes smoked decreased Total cigarette puffs decreased Time between cigarettes increased Sellers & Tyndale, 2000

21. Inhibition of CYP2A6 Increases Nicotine from Nicorette (4mg) n=11 *1/*1 genotype 52% Increase in Nicotine Concentration with Methoxsalen 35 * 30 * * 25 * * 20 15 Day 3 Nicotine (ng/mL) 10 5 0 8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 Time Methoxsalen Placebo

22. 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

23. Acknowledgements! University of Toronto: Rachel Tyndale, Sharon Miksys, Ewa Hoffman, Chun Xu, Yushi Rao, Bo Xu, Edward Sellers University of Pennsylvania : Janet Audrain, Paul Wileyto, Angela Pinto, Vyga Kaufmann, Sue Kucharski, Mike Burdick, Freda Patterson Georgetown University: Peter Shields Brown University: Ray Niaura UCSF: Neal Benowitz Tyndale Lab: Canadian Research Chair in Pharmacogenetics NIDA: DA06889 Centre for Addiction and Mental Health CIHR: MT-14173; MT-14719, training grant and doctoral awards NCIC: 010271, Ontario Mental Health Foundation, Nicogen Res.