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AN INVESTIGATION OF NICOTINE METABOLISM IN MICE:
THE IMPACT OF PHARMACOLOGICAL INHIBITION AND GENETIC INFLUENCES
ON NICOTINE PHARMACOLOGY
By
Eric Chun Kit Siu
A thesis submitted in conformity with the requirements for the degree of
Doctor of Philosophy
Graduate Department of Pharmacology
University of Toronto
©Copyright by Eric Chun Kit Siu 2010
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AN INVESTIGATION OF NICOTINE METABOLISM IN MICE: THE IMPACT OF PHARMACOLOGICAL INHIBITION AND GENETIC INFLUENCES
ON NICOTINE PHARMACOLOGY Eric Chun Kit Siu
Doctor of Philosophy, 2010 Graduate Department of Pharmacology
University of Toronto
ABSTRACT
INTRODUCTION: Smoking is one of the single greatest causes of numerous
preventable diseases. We were interested in developing an animal model of nicotine
metabolism that can be used to examine the effects of potential CYP2A6 inhibitors on
nicotine metabolism and nicotine-mediated behaviours. Pharmacogenetic studies have
demonstrated that in humans, smoking behaviour is associated with rates of nicotine
metabolism by the CYP2A6 enzyme. Mouse CYP2A5 shares structural and functional
similarities to human CYP2A6 and has been implicated in nicotine self-administration
behaviours in mice, therefore the mouse represents a potential animal model for
studying nicotine metabolism. METHODS: We characterized nicotine and cotinine
metabolism in two commonly used mouse strains (DBA/2 and C57Bl/6). We also
examined the association between nicotine self-administration behaviours and nicotine
metabolism, and the impact of direct manipulation (i.e. inhibition) of nicotine metabolism
on nicotine pharmacodynamics (hot-plate and tail-flick tests) in mice. Finally, we studied
the effect of selegiline (a known cytochrome P450 mechanism-based inhibitor) on
nicotine metabolism in mice and in human CYP2A6. RESULTS: Nicotine metabolism in
mice in vitro was mediated by CYP2A5, and this enzyme was responsible for over 70%
and 90% of the metabolism of nicotine to cotinine and cotinine to 3-hydroxycotinine as
shown by immuno-inhibition studies, respectively. A polymorphism in CYP2A5 between
mouse strains, known to alter the probe substrate coumarin’s metabolism, did not affect
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nicotine metabolism but dramatically altered cotinine metabolism. Nicotine self-
administration behaviour in mice was associated with level of hepatic CYP2A5 proteins
and rates of nicotine metabolism in male mice. In inhibition studies, the CYP2A5/6
inhibitor methoxsalen inhibited both in vitro and in vivo nicotine metabolism in mice and
substantially increased the anti-nociceptive effect of nicotine. Finally, selegiline was
found to be an inhibitor of CYP2A5 decreasing nicotine metabolism in vitro and in vivo in
mice. Moreover, we showed that selegiline is a mechanism-based inhibitor of CYP2A6
inhibiting nicotine metabolism irreversibly. CONCLUSION: The above data suggested
that the mouse model may be suitable for examining the impact of inhibition (and genetic
variation) on nicotine metabolism and nicotine-mediated behaviours and may potentially
be used to screen for novel inhibitors of nicotine metabolism.
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ACKNOWLEDGEMENTS
As author of this thesis, I would like to thank those individuals who contributed their time and effort to the creation of this work. First, I would like to thank my supervisor, Professor Rachel Tyndale, for her guidance and support throughout my graduate training. I would also like to thank the past and present colleagues in the laboratory who shared their technical experience, particularly those who contributed to my various projects. These include Ewa Hoffmann, Sharon Miksys, Helma Nolte, Raj Sharma, Zhao Bin, and Linda Liu. I would also like to thank Howard Kaplan for his statistical assistance. I would like to acknowledge the support of my fellow graduate students in the laboratory, particularly Jill Mwenifumbo and Nael Al Koudsi. I am grateful to Dr. Denis Grant, Dr. Paul Fletcher, and Dr. Jack Uetrecht for sharing their knowledge and insight for the successful completion of my projects. I would also like to thank Dr. Dieter Wildenauer and Dr. Imad Damaj for their collaborations on my various projects. I am mostly graciously indebted to OGS, CIHR TUSP, CIHR STPTR, and CTCRI for the financial support that has insured my continuing success in this program. I am also very grateful to the support of Mr. and Mrs. Binh Chow through the years of my stay in Toronto. Finally, I am forever indebted to my loving parents Simon and Maggie Siu and my fiancé Teresa Tsui, which without their inspiration and support the completion of this thesis would not have been possible.
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TITLE PAGE Page i
ABSTRACT Page ii-iii
ACKNOWLEDGEMENTS Page iv
TABLE OF CONTENTS Page v-ix
LIST OF PUBLICATIONS Page x-xi
SUMMARY OF ABBREVIATIONS Page xii
LIST OF TABLES Page xiii
LIST OF FIGURES Page xiv-xv
STATEMENT OF RESEARCH PROBLEM Page 1
CHAPTER 1 INTRODUCTION Page 3
Section 1 Nicotine Metabolism Page 3
Section 1.1 Nicotine Metabolism in Mice Page 3
Section 1.2 Nicotine Pharmacokinetics in Mice Page 3
Section 1.3 Mouse Nicotine Metabolizing Enzymes Page 4
Section 1.3.1 Mouse Cytochrome P450 2A5 (CYP2A5; P450Coh; Page 4 Coumarin Hydroxylase)
Section 1.3.2 Mouse Flavin-containing Monoxidase 1 (FMO1) Page 6
Section 1.3.3 Mouse Aldehyde Oxidase 1 (AOX1) Page 7
Section 1.4 Nicotine Metabolism in Humans Page 8
Section 1.5 Nicotine Pharmacokinetics in Humans Page 8
Section 1.6 Human Nicotine Metabolizing Enzymes Page 11
Section 1.6.1 Human Cytochrome P450 2A6 (CYP2A6; Page 11 Coumarin 7-hydroxylase)
Section 1.6.2 Human Cytochrome P450 2A13 (CYP2A13) Page 14
Section 1.6.3 Human Cytochrome P450 2B6 (CYP2B6) Page 15
Section 1.6.4 Human Aldehyde Oxidase 1 (AOX1) Page 17
Section 1.6.5 Human Flavin-containing Monooxygenase 3 (FMO3) Page 18
Section 1.6.6 Human Uridine Diphosphate (UDP)-Glucuronosyl- Page 19 Transferase (UGT)
Section 2 Pharmacological Treatments for Smoking Cessation Page 21
Section 2.1 Primary Treatments Page 21
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Section 2.1.1 Nicotine Replacement Therapies (NRTs) Page 21
Section 2.1.2 Bupropion Page 22
Section 2.1.3 Varenicline Page 23
Section 2.2 Secondary and Investigative (Non-approved) Page 25 Smoking Cessation Therapies
Section 2.2.1 Nortriptyline Page 25
Section 2.2.2 Clonidine Page 25
Section 2.2.3 Serotonergic Agents Page 26
Section 2.2.3.1 Buspirone Page 26
Section 2.2.3.2 Selective Serotonin Reuptake Inhibitors (SSRIs) Page 27
Section 2.2.4 Naltrexone Page 27
Section 2.2.5 Rimonabant (SR141716) Page 28
Section 2.2.6 Nicotine Vaccines Page 30
Section 2.2.7 Monoamine Oxidase Inhibitors (MAOIs) Page 30
Section 2.2.7.1 Selegiline (l-deprenyl; Eldepryl®) Page 32
Section 2.2.7.1.1 Selegiline Pharmacodynamics Page 32
Section 2.2.7.1.2 Selegiline Metabolism Page 33
Section 2.3 The Inhibition of Nicotine Metabolism and its Page 34 Application to Smoking Cessation
Section 2.3.1 Inhibitors of Human CYP2A6 Page 34
Section 2.3.2 Inhibitors of Mouse CYP2A5 Page 35
Section 2.3.3 Mechanism-Based Inhibition Page 36
Section 2.3.3.1 Methoxsalen (8-methoxypsoralen; 8-MOP): Page 37 A CYP2A5 and CYP2A6 MBI
Section 2.3.3.2 Selegiline as a MBI Page 37
Section 2.3.4 Improving Smoking Cessation through CYP2A6 Page 38 Inhibition
Section 2.3.5 Reduction of Tobacco-Specific Nitrosamine Page 40 Activation through Human CYP2A6 Inhibition
Section 3 Nicotine Pharmacological Behavioural Models Page 42
Section 3.1 Nicotine Self-Administration Page 42
Section 3.2 Analgesic Effect of Nicotine and Application in Page 43 Nicotine Studies
Section 4 Outline of Chapters Page 45
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Section 4.1 Chapter 2: Characterization and Comparison of Page 45 Nicotine and Cotinine Metabolism In Vitro and In Vivo in DBA/2 and C57BL/6 Mice
Section 4.2 Chapter 3: Nicotine Self-Administration in Mice is Page 46 Associated with Rates of Nicotine Inactivation by CYP2A5
Section 4.3 Chapter 4: Inhibition of Nicotine Metabolism by Page 47 Methoxysalen: Pharmacokinetic and Pharmacological Studies in Mice
Section 4.4 Chapter 5: Selegiline (L-Deprenyl) is a Mechanism- Page 48 based Inactivator of CYP2A6 Inhibiting Nicotine Metabolism in Humans and Mice
CHAPTER 2 CHARACTERIZATION AND COMPARISON OF Page 50 NICOTINE AND COTININE METABOLISM IN VITRO AND IN VIVO IN DBA/2 AND C57BL/6 MICE
Abstract Page 51
Introduction Page 52
Materials and Methods Page 54
Results Page 59
Discussion Page 73
Significance of Chapter Page 79
CHAPTER 3 NICOTINE SELF-ADMINISTRATION IN MICE IS Page 80 ASSOCIATED WITH RATES OF NICOTINE INACTIVATION BY CYP2A5
Abstract Page 81
Introduction Page 82
Materials and Methods Page 84
Results Page 88
Discussion Page 99
Significance of Chapter Page 104
CHAPTER 4 INHIBITION OF NICOTINE METABOLISM BY Page 105 BY METHOXSALEN: PHARMACOKINETIC AND PHARMACOLOGICAL STUDIES IN MICE
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Abstract Page 106
Introduction Page 107
Materials and Methods Page 109
Results Page 113
Discussion Page 128
Significance of Chapter Page 134
CHAPTER 5 SELEGILINE (L-DEPRENYL) IS A MECHANISM- Page 135 BASED INACTIVATOR OF CYP2A6 INHIBITING NICOTINE METABOLISM IN HUMANS AND MICE
Abstract Page 136
Introduction Page 137
Materials and Methods Page 141
Results Page 147
Discussion Page 157
Significance of Chapter Page 163
CHAPTER 6 GENERAL DISCUSSION Page 164
Section 5 Metabolism Topics Page 164
Section 5.1 In Vitro Metabolism Page 164
Section 5.1.1 In Vitro Nicotine Metabolism Page 164
Section 5.1.2 In Vitro Cotinine Metabolism Page 167
Section 5.1.3 Effect of a CYP2A5 Polymorphism on Substrate Page 167 Metabolism
Section 5.2 In Vivo Metabolism Page 168
Section 5.2.1 Biomarker of Nicotine Metabolism Page 170
Section 5.3 Mice (CYP2A5) as a Model to Study CYP2A6 Page 171 Inhibition
Section 5.3.1 Methoxsalen Page 172
Section 5.3.2 Selegiline Page 173
Section 6 Factors Affecting Nicotine Intake Page 176
Section 6.1 Impact of Genetic Variations Page 176
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Section 6.1.1 Variations in Nicotine Metabolism Page 176
Section 6.1.2 Non-Nicotine Metabolism Variations Page 177
Section 6.2 Non-Genetic Factors Page 179
Section 7 Sex-Dependent Association between Nicotine Page 180 Metabolism and NSA
Section 8 Effect of Chronic Nicotine Treatment on Nicotine Page 182 Metabolism
Section 9 Future Directions Page 183
Section 9.1 Selegiline Page 184
Section 9.2 Mouse Model of Nicotine Metabolism Page 184
Section 10 Conclusion Page 187
REFERENCES Page 189
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LIST OF PUBLICATIONS Research Articles Grieder, T.E., Sellings, L.H., Vargas-Perez, H., Ting-A-Kee, R., Siu, E.C., Tyndale, R.F., and van der Kooy, D., 2009. Dopaminergic signaling mediates the motivational response underlying the opponent process to chronic but not acute nicotine. Neuropsychopharmacology In press. Shram, M.J., Siu, E., Li, Z., Tyndale, R.F., and Le, A.D., 2008. Adolescent and adult rats respond differently to the aversive effects of mecamylamine-precipitated nicotine withdrawal. Psychopharmacology (Berl) 198(2):181-90. Siu, E.C. and Tyndale R.F., 2008. Selegiline (L-deprenyl) is a mechanism-based inactivator of CYP2A6 inhibiting nicotine metabolism in humans and mice. J Pharmacol Exp Ther. 324(3):992-9. Siu, E.C. and Tyndale, R.F., 2007. Characterization and comparison of nicotine and cotinine metabolism in vitro and in vivo in DBA/2 and C57Bl/6 mice. Mol Pharm. 71(3):826-34. Damaj, M.I., Siu, E.C., Sellers, E.M., Tyndale, R.F. and Martin, B.R., 2007. Inhibition of nicotine metabolism by methoxysalen: pharmacokinetic and pharmacological studies in mice. J Pharmacol Exp Ther. 320(1):250-7. Siu, E.C., Wildenauer, D.B., and Tyndale, R.F., 2006. Nicotine self-administration in mice is associated with rates of nicotine inactivation by CYP2A5. Psychopharmacology (Berl). 184(3-4):401-408. Siu, E., Carreno, B.M., and Madrenas, J., 2003. TCR subunit specificity of CTLA-4-mediated signaling. J Leukoc Biol. 74(6):1102-1107. Darlington, P.J., Baroja, M.L., Chau, T.A., Siu, E., Ling, V., Carreno, B.M., and Madrenas, J., 2002. Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation. J Exp Med. 195(10):1337-1347. Review Article Siu, E.C. and Tyndale, R.F.,2007. Non-nicotinic therapies for smoking cessation. Annu Rev Pharmacol Toxicol. 47:541-64. Conference Presentations Siu, E.C., Wildenauer, D.B., and Tyndale, R.F., Effect of genetic variation on the nicotine metabolizing enzyme (CYP2A5) on nicotine self-administration behaviours in mice. Society for Research on Nicotine and Tobacco's 12th Annual Meeting. Orlando, FL, 2006.
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On-line Publications Siu, E.C., Al Koudsi, N., Ho, M.K., and Tyndale, R.F., 2006. Smoking, quitting and genetics. The Doctor Will See You Now (http://www.thedoctorwillseeyounow.com/articles/behavior/smoking_14/). Siu, E.C., Al Koudsi, N., Ho, M.K., and Tyndale, R.F., 2006. New frontiers in the treatment and management of smoking cessation. Cyberounds (http://www.cyberounds.com/conf/psychiatryneuroscience/2006-08-11/index.html). Abstracts 1. Siu, E.C. and Tyndale, R.F., Selegiline (l-deprenyl) inhibits nicotine metabolism in
mice and humans and is a mechanism-based inhibitor of CYP2A6. Society for Research on Nicotine and Tobacco's 14th Annual Meeting and the The 4th Annual Invitational Symposium for Research to Inform Tobacco Control. Portland, OR, 2008.
2. Siu, E.C. and Tyndale, R.F., Contrasting in vivo and in vitro metabolism of nicotine
and cotinine in DBA/2 and C57Bl/6 mice. Visions in Pharmacology Symposium. Toronto, ON, 2007.
3. Siu, E.C. and Tyndale, R.F., Contrasting in vivo and in vitro metabolism of nicotine and cotinine in DBA/2 and C57Bl/6 mice. Society for Research on Nicotine and Tobacco's 13th Annual Meeting. Austin, TX, 2007.
4. Siu, E.C. and Tyndale, R.F., In vivo and in vitro metabolism of nicotine and cotinine in DBA/2 and C57Bl/6 mice. The 3rd Annual Invitational Symposium for Research to Inform Tobacco Control. Toronto, ON, 2006.
5. Siu, E.C., Boutros, A., Wildenauer, D.B., and Tyndale, R.F., Genetic variation in Cyp2a5-mediated nicotine metabolism correlates with differential oral nicotine consumption in male mice. Visions in Pharmacology Symposium. Toronto, ON, 2006.
6. Siu, E.C., Wildenauer, D.B., and Tyndale, R.F., Effect of genetic variation on the nicotine metabolizing enzyme (CYP2A5) on nicotine self-administration behaviours in mice. The 2nd Annual Invitational Symposium for Research to Inform Tobacco Control. Toronto, ON, 2005.
7. Siu, E.C., Boutros, A., Miksys, S., Wildenauer, D.B., and Tyndale, R.F., Effect of genetic variation and chronic nicotine exposure on nicotine (CYP2A5) and ethanol (CYP2E1) metabolizing enzymes in mice. Society for Research on Nicotine and Tobacco's 11th Annual Meeting and the 7th Annual SRNT European Conference. Prague, Czech Republic, 2005.
8. Siu, E.C., Miksys, S., Klein, L.C., Blendy, J.A., and Tyndale, R.F., Effects of in vivo nicotine treatment on mouse hepatic CYP2A5 and CYP2E1 level. Visions in Pharmacology Research Symposium. Toronto, ON, 2004.
9. Siu, E.C., Boutros, A., Miksys, S., Wildenauer, D.B., and Tyndale, R.F., An Investigation into the effect of nicotine exposure and genetic variation on ethanol (CYP2E1) and nicotine (CYP2A5) inactivating enzymes in mice. The 1st Annual Invitational Meeting of Tobacco Control Investigators and Trainees. Toronto, ON, 2004.
10. Madrenas, J., Baroja, M.L., Carreno, B.M., Chau, L.A., Chau, T.A., Darlington, P.J., Ling, V., Siu, E.C., Regulation of CTLA-4-mediated signalling. Keystone Symposium on Lymphocyte Activation and Signaling. Steamboat Spring, CO, 2004.
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SUMMARY OF ABBREVIATIONS
%MPE maximum possible effect
β-PEA phenylethylamine 3-HC 3’-hydroxycotinine 8-MOP 8-methoxypsoralen Ala alanine AOX aldehyde oxidase AUC area under the concentration CI confidence interval CL clearance Cmax maximum concentration COT cotinine COU coumarin CYP cytochrome P450 DES desmethylselegiline ED
50 effective dose 50%
F bioavailability FAD flavin adenine dinucleotide FMO flavin-containing monoxidase HLM human liver microsome HNC high nicotine consumption
HNF-4α hepatic nuclear factor-4 alpha IC50 inhibition concentration 50% L-AMP L-amphetamine LC liquid chromatography L-MAMP L-methamphetamine LNC low nicotine consumption MAOI monoamine oxidase inhibitor MBI mechanism-based inhibitor MLM mouse liver microsome NADPH nicotinamide adenine dinculeotide phosphate NCO nicotine C-oxidation NIC nicotine NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone NRT nicotine replacement therapy NSA nicotine self-administration OR odds ratio QTL quantitative trait loci SEL selegiline SNP single nucleotide polymorphism SSRI selective serotonin reuptake inhibitor T1/2 half-life Tmax time at maximum concentration UGT uridine diphosphate-glucuronosyl-transferase UTR untranslated region Val valine
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LIST OF TABLES Table 1. In vitro nicotine metabolism (C-oxidation) parameters, page 61 Table 2. Pharmacokinetic parameters of plasma nicotine and cotinine in mice
treated with nicotine (1 mg/kg, s.c.), page 63 Table 3. Pharmacokinetic parameters of plasma cotinine and 3’-hydroxycotinine in
mice treated with cotinine (1 mg/kg, s.c.), page 68 Table 4. In vitro cotinine hydroxylation parameters, page 70
Table 5. Average nicotine consumption (µg/day; mean ± SD) in F2 male and female mice in a two-bottle choice paradigm, page 90
Table 6. Estimated in vitro NCO kinetic parameters of male LNC and HNC mice
(mean ± SD), page 95 Table 7. Concentrations (mean ± SD) of nicotine, cotinine, and 3-hydroxycotinine
(ng/mL) from the in vitro NCO activity assays (at 30 µM nicotine), page 96 Table 8. Kinetic parameters of in vitro nicotine C-oxidation activities of adult male
ICR mouse liver microsomes, page 115 Table 9. Pharmacokinetic parameters of nicotine in adult male ICR mouse after
pretreatment with methoxsalen, page 119 Table 10. Blockade of methoxsalen’s effects on nicotine acute pharmacological
responses after s.c. administration in mice, page 124 Table 11. Pharmacokinetic parameters of plasma nicotine in mice treated
intraperitoneally with nicotine (1 mg/kg) or nicotine plus selegiline (1 mg/kg), page 152
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LIST OF FIGURES
Figure 1A. Metabolism and in vivo disposition of nicotine and metabolites in humans, page 9
Figure 1B. Comparison of nicotine metabolism in humans and mice by known
enzymes, page 10 Figure 2. In vitro metabolism of nicotine to cotinine in DBA/2 and C57Bl/6 mice,
page 60 Figure 3. Plasma nicotine and cotinine concentrations following nicotine treatment,
page 62 Figure 4. Characterization of 3’-hydroxycotinine in mice by HPLC and LC/MS/MS,
page 65 Figure 5. Plasma cotinine and 3’-hydroxycotinine concentrations following cotinine
injections, page 67 Figure 6. In vitro metabolism of cotinine to 3’-hydroxycotinine in DBA/2 and C57Bl/6
mice, page 69 Figure 7. Antibody inhibits in vitro nicotine and cotinine metabolism in DBA/2 and
C57Bl/6 mice, page 72 Figure 8. Selectivity and linearity of CYP2A5 immunoblotting for mouse liver
microsomes, page 89 Figure 9. CYP2A5 levels are higher in male high nicotine consuming mice, page 91 Figure 10. Optimization of nicotine-C-oxidation assay, page 92 Figure 11. Nicotine-C-oxidation activity in male high nicotine consuming mice is
higher compared to the low nicotine consuming mice, page 94 Figure 12. CYP2A4/5 levels and in vitro NCO activities do not differ between female
LNC and HNC mice, page 97 Figure 13. In vitro nicotine C-oxidation activity of ICR mice, page 114 Figure 14. A representative Dixon plot of inhibition of cotinine formation by ICR
mouse microsomes in presence of varying concentrations of methoxsalen, page 116
Figure 15. Time course of nicotine and cotinine plasma concentration in mice
pretreated with methoxsalen, page 118
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Figure 16. Time-course of methoxsalen effects on nicotine-induced (A) antinociception and (B) hypothermia after s.c. administration of 10 mg/kg in mice, page 120
Figure 17. Dose-response enhancement of methoxsalen after s.c. administration in
mice, page 121 Figure 18. Blockade of methoxsalen’s effects on nicotine-induced antinociception in
the tail-flick test after s.c. administration in mice, page 123 Figure 19. Effects of methoxsalen on the time-course of nicotine’s effects in (A) the
tail-flick test, (B) the hot-plate assay and (C) body temperature in mice, page 125
Figure 20. Relationship between nicotine-induced antinociception in the tail-flick test
and plasma levels of nicotine in the ICR mice after methoxsalen treatment, page 127
Figure 21. Selegiline and its metabolites, page 139 Figure 22. Selegiline and its metabolites inhibited nicotine metabolism, page 148 Figure 23. Pre-incubation of MLMs, but not CYP2A5, with selegiline increased
inhibition of nicotine metabolism, page 150 Figure 24. Selegiline decreased nicotine metabolism in vivo in DBA/2 mice, page
151 Figure 25. Pre-incubation of human hepatic microsomes and CYP2A6 with selegiline
increased inhibition of nicotine metabolism, page 154 Figure 26. Selegiline inhibited CYP2A6-mediated nicotine metabolism in a time-,
concentration-, and NADPH-dependent manner, page 155
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STATEMENT OF RESEARCH PROBLEM
According to the World Health Organization, over one billion men and 250 million women
smoke daily (WHO, 2007). Smoking is the leading preventable cause of morbidity and
mortality worldwide and accounts for almost five million deaths annually (Ezzati and
Lopez, 2004). The primary causes of mortality from smoking are cardiovascular disease,
lung cancer, and chronic obstructive pulmonary disease (Ezzati and Lopez, 2004).
Currently there are several approved therapies available for smoking cessation that
include nicotine replacement products (patch, gum, nasal spray, lozenges), bupropion,
and the recently marketed varenicline (Cahill et al., 2007; Siu and Tyndale, 2007b; Stead
et al., 2008). We have been interested in understanding the effects of compounds that
inhibit nicotine metabolism, and ultimately their therapeutic roles in smoking cessation.
The human cytochrome P450 2A6 (CYP2A6) is responsible for the primary metabolism
of nicotine and it is genetically and functionally variable (Mwenifumbo and Tyndale, 2007;
Nakajima et al., 1996b). Pharmacogenetic studies have demonstrated that among
smokers, slow metabolizers of nicotine (reduced CYP2A6 function) smoke fewer
cigarettes and have higher rates of quitting compared to normal nicotine metabolizers
(Malaiyandi et al., 2006; Patterson et al., 2008; Schoedel et al., 2004). More importantly,
preliminary studies have shown that inhibitors of CYP2A6 are effective in decreasing
nicotine metabolism as well as reducing smoking behaviours (Sellers et al., 2002;
Sellers et al., 2000; Tyndale et al., 1999). Considering the potential of CYP2A6 inhibitors
in smoking cessation, development and characterization of an animal model of nicotine
pharmacokinetics and pharmacodynamics will be important in studying these novel
inhibitors and their specificities, efficacies, toxicity profiles, and effects on nicotine
pharmacokinetics and pharmacodynamics. Furthermore, an animal model of nicotine
metabolism may be useful to examine the impact of genetic variation (e.g. the effects of
2
increased or decreased nicotine metabolism) on nicotine pharmacology and or nicotine-
induced behaviours.
In this thesis we investigated the mouse as an experimental model on the basis
that the mouse CYP2A5 is highly identical in amino acid sequence to the human
CYP2A6 and a preliminary quantitative trait loci analysis has suggested the possible
involvement of CYP2A5 in oral nicotine self-administration behaviours in mice
(Wildenauer et al., 2005). Furthermore, the rat CYP2A enzymes do not appreciately
metabolize nicotine (Nakayama et al., 1993), therefore, the mouse may represent a
useful small animal model of nicotine metabolism and pharmacodynamics.
3
CHAPTER 1 INTRODUCTION
Section 1 Nicotine Metabolism
Section 1.1 Nicotine Metabolism in Mice
Early in vitro mouse studies focused on the metabolism of nicotine and the distribution of
metabolites in tissue preparations using thin-layer chromatography but did not provide
the identification of various nicotine metabolites other than cotinine (Hansson et al., 1964;
Stalhandske, 1970; Stalhandske et al., 1969). Later, it was found using mouse (DBA/Jax)
hepatocytes, nicotine is metabolized primarily to cotinine, as well as nornicotine and
nicotine N-oxide (cotinine – 26.4%, nornicotine – 10.3%, and nicotine N-oxide 8.9% of
measurable metabolites formed) (Kyerematen et al., 1990), suggesting the involvement
of cytochromes P450 (C-oxidation) and flavin monooxygenases (N-oxidation). One study
found that neither nicotine nor cotinine were N-glucuronidated in vitro (Ghosheh and
Hawes, 2002), suggesting a potential lack of involvement of UGTs in nicotine
metabolism in mice.
Section 1.2 Nicotine Pharmacokinetics in Mice
The majority of in vivo nicotine disposition kinetic studies in mice were limited in details
(Hatchell and Collins, 1980; Mansner and Mattila, 1977). Nicotine and its metabolites are
mainly found in the liver and kidney, but they are also distributed in other tissues such as
bronchi, nasal mucosa, salivary gland, stomach, spleen, pancreas, intestine, bone,
gallbladder, and adrenal medulla (Waddell and Marlowe, 1976). A more comprehensive
study examining nicotine pharmacokinetics in three strains of mice (C3H/Ibg [C3H],
DBA/2Ibg [DBA/2], C57BL/6Ibg [C57BL/6]) showed that following a single intraperitoneal
injection, plasma nicotine levels peaked at around 5 minutes followed by rapid (~6-7 min)
4
and monophasic elimination (Petersen et al., 1984). This elimination half-life is
considerably faster than rats (~0.92-1.1 hr) (Lesage et al., 2007; Miller et al., 1977) and
humans (~1.6-2.8 hr) (Benowitz and Jacob, 1993; Benowitz et al., 2006b). In mice,
nicotine is present in the brain and liver, and its elimination from these organs is similar
(~6-9 min) compared to plasma (Petersen et al., 1984). Cotinine is the major nicotine
metabolite in plasma. It is formed rapidly but has a significantly slower elimination half-
life compared to nicotine. Its rate of elimination differs between strains (e.g. 39.8±2.0 min
for DBA/2, 20.1±2.3 min for C57Bl/6 and 24.8±2.6 min for C3H) (Petersen et al., 1984).
Similar to nicotine, the rate of cotinine elimination in the brain and liver approximate that
of the plasma. Nicotine N-oxide is a minor metabolite compared to cotinine; however, its
plasma elimination half-life is longer than nicotine but shorter than cotinine (Petersen et
al., 1984).
Section 1.3 Mouse Nicotine Metabolizing Enzymes
Section 1.3.1 Mouse Cytochrome P450 2A5 (CYP2A5; P450Coh; Coumarin
Hydroxylase)
CYP2A5 was originally identified as coumarin hydroxylase due to its exclusive capability
to hydrolyze coumarin to 7-hydroxycoumarin in mice (Lang et al., 1989). Recently it was
reported that the initial step in nicotine to cotinine conversion in mice is the 5’-oxidation
of the pyrrolidine ring of nicotine to the ∆5’(1’)-iminium ion which is mediated by CYP2A5;
this is followed by its conversion to cotinine by cytosolic aldehyde oxidase (Gorrod and
Hibberd, 1982; Murphy et al., 2005b). The affinity for nicotine, as measured by the total
formation of ∆5’(1’)-iminium ion and cotinine, by cDNA-expressed CYP2A5 is ~8 µM
(Murphy et al., 2005b). In humans, cotinine 3’-hydroxylation is thought to be exclusively
5
mediated by CYP2A6 (Nakajima et al., 1996a; Yamanaka et al., 2004); however, the
enzyme(s) responsible for this pathway in mice is unknown.
The mouse Cyp2a5 transcript is found in several tissues including liver, kidney,
lung, olfactory mucosa, brain, small intestines, but not in the heart or spleen (Su and
Ding, 2004). The tissue-specificity of the protein is not well characterized but coumarin
hydroxylase activity was found to be the highest in the liver followed by kidney and lung
(Kaipainen and Lang, 1985). In terms of sex differences, females have higher coumarin
7-hydroxyase activities (specific to CYP2A5) in some strains examined (e.g. DBA/2, 129,
CBA/Ca) but not in others (e.g. C3H/He, BALB/c, A/J) (van Iersel et al., 1994).
The 494-amino acid protein encoded by the Cyp2a5 gene is located on
chromosome 7 (6.5 cM; 9 exons) (Mouse Genome Informatics 4.01) and shares 86%
amino acid sequence identity with human CYP2A6. Mouse coumarin hydroxylase
activity is genetically variable between mouse strains (Wood and Taylor, 1979). Thus far
there are 38 identified single nucleotide polymorphisms (SNPs) that exist in Cyp2a5.
Two of the identified SNPs are in the coding region and result in amino acid changes
(non-synonymous): Valine117Alanine (V117A) and Threonine486Asparagine (T486N).
Twenty-nine of the SNPs are located in the introns; one is 1853-bp downstream of the
coding sequence, three are in the 3’-UTR of the mRNA, two are located in coding
sequence but are synonymous changes and one is located in the 5’-flanking region. The
full updated list of SNPs is found at Mouse Genome Informatics (MGI 4.01).
Polymorphisms in Cyp2a5 can have functional consequences; for example, the DBA/2J
strain expresses the high coumarin hydroxylase activity variant (valine at position 117)
whereas the C57BL/6J strain expresses the low activity variant (alanine at position 117).
The reported Km for coumarin 7-hydroxylase activities of the fractional purified CYP2A5
from the high (DBA/2) and low (C57Bl/6) activity variants are 0.35 µM and 4.0 µM,
respectively (Kaipainen et al., 1984).
6
Section 1.3.2 Mouse Flavin-containing Monoxidase 1 (FMO1)
In general, FMOs are endoplasmic reticulum enzymes responsible for the oxidation of
nucleophilic oxygen, nitrogen, sulphur, phosphorus, or selenium atoms from a wide
range of substrates such as amines, amides, thiols, and sulfides (Eswaramoorthy et al.,
2006). In mice, the FMO1 enzyme appears to be responsible for the conversion of
nicotine to nicotine N-oxide (Itoh et al., 1997), although the contributions of
polymorphisms and other FMO isoforms to this process have not been determined. In
vitro, the metabolism of nicotine in mouse hepatocytes (DBA/Jax) showed that nicotine
N-oxide constitutes ~26% of all metabolites formed from nicotine (Kyerematen et al.,
1990). It is unclear if nicotine N-oxide mediates any pharmacological actions in mice.
In mice (129/Sv), the Fmo1 mRNA is found in similar levels in the liver, lung, and
kidney, while the brain has over 10-fold lower levels of the transcript (Janmohamed et al.,
2004). The relative levels of this protein in various tissues have not been examined. In
terms of sex, the mRNA is found at 10-20% higher in male liver, lung, and brain
compared to females. In contrast, the Fmo1 mRNA is two times higher in the kidneys of
female compared to male mice (Janmohamed et al., 2004).
The mouse Fmo1 gene is located on chromosome 1 (92.6 cM) and encodes a
532-amino acid protein (Mouse Genome Informatics 4.01). Bioinformatic searches
indicated there are 152 polymorphisms in this gene: eight upstream, nine downstream,
two synonymous, 25 in the 3’-UTR of mRNA, and the remaining SNPs are found in the
intron (MGI 4.01). Between DBA/2J and C57BL/6J mice, there are 50 SNPs: four
upstream, two non-synonymous, nine 3’-UTR, four downstream, and the remaining
SNPs are found in introns (MGI 4.01). Despite the large number of polymorphisms
present in Fmo1, there is little difference in the mRNA transcript levels and catalytic
7
activities between mouse strains (Falls et al., 1995; Siddens et al., 2007), suggesting a
small impact of these SNPs on enzyme function.
Section 1.3.3 Mouse Aldehyde Oxidase 1 (AOX1)
Aldehyde oxidase is a member of the molybdo-flavoenzymes that require both flavin
adenine dinucleotide and molybdenum cofactor for its catalytic activity (Garattini et al.,
2003). The substrate specificity of mouse AOX1 is not well studied (Vila et al., 2004).
Aldehyde oxidase is responsible for the conversion of the nicotine-∆5’(1’)-iminium ion to
cotinine (Brandange and Lindblom, 1979). In mouse hepatic microsomes (plus cytosol)
nicotine-∆5’(1’)-iminium ion is metabolized to cotinine, although the enzyme responsible
for this reaction was not identified (Gorrod and Hibberd, 1982). However, it is likely that
mouse AOX1 is involved in this process due to its amino acid sequence similarity with
the human counterpart (83% identity, NCBI Blast). It is thought that, at least in humans,
the conversion of the nicotine-∆5’(1’)-iminium ion to cotinine is not rate limiting in this two-
step process of cotinine formation since the Km of aldehyde oxidase for the iminium ion
(Km = 0.85 µM, Vmax = 5300 pmol/min/mg; step 2) is much lower than the Km of nicotine
C-oxidation (step 1) (Fig. 1) (Murphy et al., 2005b; Obach, 2004). In addition, in our in
vitro studies we used sufficient amount of cytosol to ensure that aldehyde oxidase is not
rate limiting (Fig. 10d).
In mice, the expression of AOX1 is tissue- and sex-dependent. In CD-1 mice,
Aox1 mRNA is found in the liver, heart, lung, and esophagus; in contrast, the protein is
found in liver, lung, brain, and spinal cord, suggesting transcriptional or post-translational
control of enzyme expression (Kurosaki et al., 1999). With regards to sex, the AOX1
protein levels are higher in the liver but lower in the lung of males compared to females
(Kurosaki et al., 1999). Such sex difference may be determined by levels of sex
8
hormones as hepatic AOX1 protein increases in both sexes upon treatment with
testosterone (Vila et al., 2004).
The mouse Aox1 gene is located on chromosome 1 (23.2 cM) and encodes a
300-kDa homo-dimer (Kurosaki et al., 1999). Based on mouse genome informatic
searches, there are 81 polymorphisms in the mouse Aox1 gene: 14 insertion-deletion
and/or SNPs in the upstream locus; three synonymous; one non-synonymous; 55 intron
polymorphisms; and eight in the 3’-UTR region (MGI 4.01). Between DBA/2J and
C57BL/6J mice, Aox1 has 56 SNPs: two intronic and one upstream deletion with the
remaining found in the introns. The functional consequences of these SNPs have not
been determined; however, DBA/2 mice have significantly less hepatic AOX1 protein
compared to C57Bl/6 (Vila et al., 2004), suggesting possible influence of these
polymorphisms on protein levels.
Section 1.4 Nicotine Metabolism in Humans
The in vivo metabolism and disposition of nicotine and its metabolites in humans have
been examined in detail. A summary of the nicotine metabolic pathway and major
nicotine metabolites are shown in figure 1A (a comparison of known enzymes
responsible for nicotine metabolism between human and mice is shown in figure 1B). In
addition to these metabolites, there are other minor metabolites that are present at
extremely low levels (Hecht et al., 1999).
Section 1.5 Nicotine Pharmacokinetics in Humans
Nicotine is distributed widely but is found mainly in blood, brain, lung, liver, kidney,
spleen, muscle, but are very low in adipose tissues (Urakawa et al., 1994). During
9
Nic
oti
ne (
8-1
0%
)
Co
tin
ine
(10-1
5%
)
Trans-3
’-h
yd
rox
yc
oti
nin
e (
33-4
0%
)
Tra
ns-3
’-h
ydro
xyco
tin
ine O
’-glu
cu
ronid
e(7
-9%
)
Nic
otine
N’-o
xid
e (
4-7
%)
Nic
otine
N’-glu
cu
ron
ide
(3-5
%)
Cotinin
eN
’-o
xid
e (
2-5
%)
Co
tin
ine
N’-glu
curo
nid
e(1
2-1
7%
)
No
rco
tin
ine
(1-2
%)
No
rnic
otine
(0.4
-0.8
%)
Nic
otine iso
me
thon
ium
ion
(0
.4-1
.0%
)
5’-h
ydro
xyco
tin
ine
(1
.2-1
.6%
)5’-h
ydro
xyn
orc
otinin
e (
~0
.1%
)
[Nic
otine-∆
1’(
5’)-i
min
ium
ion]
4-o
xo
-4-(
3-p
yrid
yl)-
bu
tanoic
acid
(1-2
%)
4-h
ydro
xy-4
-(3
-pyrid
yl)-
bu
tano
ica
cid
(7
-9%
)
CY
P2A
6
CY
P2A
6
AO
X1
CY
P2A
6
CY
P2A
6FM
O3
UG
T2B
10
UG
T2B
10
UG
T2B
7
Fig
ure
1a. M
eta
bolis
m a
nd
in
viv
od
isp
ositio
n o
f n
icotine
and
meta
bolit
es in h
um
ans.
Appro
xim
ate
ly 8
0%
of
nic
otin
e is m
eta
boliz
ed (
5’
C-o
xid
ation)
to the n
ico
tine ∆
5’(1
’)-im
iniu
m ion b
y C
YP
2A
6, th
is is f
ollo
wed
by the c
onvers
ion
of th
eim
iniu
mio
n t
o c
otin
ine
by a
ldehyde
oxid
ase.
A s
ign
ific
ant
port
ion (
~60%
) of
cotin
ine
is f
urt
her
meta
bo
lized
to tra
ns-3
’-h
ydro
xyco
tin
ine b
y C
YP
2A
6. N
um
be
rs in the b
rackets
repre
se
nt th
e a
ppro
xim
ate
% r
ecovery
of
each c
om
po
und in
urine
fro
m a
syste
mic
dose o
f nic
otine.
Str
uctu
res o
f n
icotin
e, nic
otine
-
∆1
’(5’)-i
min
ium
ion
, cotinin
e, a
nd tra
ns-3
’-hydro
xycotinin
e a
re s
how
n in insets
. R
efe
ren
ce
s (
Be
now
itz
et a
l 1
994,
Beno
witz
and J
acob
2001
, C
hen e
t al. 2
007, H
ukkan
en
et
al 200
5, K
aiv
ors
aari
et a
l 2
007, K
yere
ma
ten
et al 19
90,
Murp
hy e
t al 19
99, Y
am
anaka e
t al
2005
a, Y
am
ana
ka e
t al 2005
b).
CY
P2A
6/
CY
P2B
6
CY
P2A
6
N
N
H
CH
3
N+
N
H
CH
3
N
N
H
CH
3
O
N
N
H
CH
3
OH
Nic
oti
ne (
8-1
0%
)
Co
tin
ine
(10-1
5%
)
Trans-3
’-h
yd
rox
yc
oti
nin
e (
33-4
0%
)
Tra
ns-3
’-h
ydro
xyco
tin
ine O
’-glu
cu
ronid
e(7
-9%
)
Nic
otine
N’-o
xid
e (
4-7
%)
Nic
otine
N’-glu
cu
ron
ide
(3-5
%)
Cotinin
eN
’-o
xid
e (
2-5
%)
Co
tin
ine
N’-glu
curo
nid
e(1
2-1
7%
)
No
rco
tin
ine
(1-2
%)
No
rnic
otine
(0.4
-0.8
%)
Nic
otine iso
me
thon
ium
ion
(0
.4-1
.0%
)
5’-h
ydro
xyco
tin
ine
(1
.2-1
.6%
)5’-h
ydro
xyn
orc
otinin
e (
~0
.1%
)
[Nic
otine-∆
1’(
5’)-i
min
ium
ion]
4-o
xo
-4-(
3-p
yrid
yl)-
bu
tanoic
acid
(1-2
%)
4-h
ydro
xy-4
-(3
-pyrid
yl)-
bu
tano
ica
cid
(7
-9%
)
CY
P2A
6
CY
P2A
6
AO
X1
CY
P2A
6
CY
P2A
6FM
O3
UG
T2B
10
UG
T2B
10
UG
T2B
7
Fig
ure
1a. M
eta
bolis
m a
nd
in
viv
od
isp
ositio
n o
f n
icotine
and
meta
bolit
es in h
um
ans.
Appro
xim
ate
ly 8
0%
of
nic
otin
e is m
eta
boliz
ed (
5’
C-o
xid
ation)
to the n
ico
tine ∆
5’(1
’)-im
iniu
m ion b
y C
YP
2A
6, th
is is f
ollo
wed
by the c
onvers
ion
of th
eim
iniu
mio
n t
o c
otin
ine
by a
ldehyde
oxid
ase.
A s
ign
ific
ant
port
ion (
~60%
) of
cotin
ine
is f
urt
her
meta
bo
lized
to tra
ns-3
’-h
ydro
xyco
tin
ine b
y C
YP
2A
6. N
um
be
rs in the b
rackets
repre
se
nt th
e a
ppro
xim
ate
% r
ecovery
of
each c
om
po
und in
urine
fro
m a
syste
mic
dose o
f nic
otine.
Str
uctu
res o
f n
icotin
e, nic
otine
-
∆1
’(5’)-i
min
ium
ion
, cotinin
e, a
nd tra
ns-3
’-hydro
xycotinin
e a
re s
how
n in insets
. R
efe
ren
ce
s (
Be
now
itz
et a
l 1
994,
Beno
witz
and J
acob
2001
, C
hen e
t al. 2
007, H
ukkan
en
et
al 200
5, K
aiv
ors
aari
et a
l 2
007, K
yere
ma
ten
et al 19
90,
Murp
hy e
t al 19
99, Y
am
anaka e
t al
2005
a, Y
am
ana
ka e
t al 2005
b).
CY
P2A
6/
CY
P2B
6
CY
P2A
6
N
N
H
CH
3
N+
N
H
CH
3
N
N
H
CH
3
O
N
N
H
CH
3
OH
10
Nic
oti
ne
Co
tin
ine
Trans-3
’-h
yd
rox
yc
oti
nin
e
[Nic
otine
-∆1’(
5’)-i
min
ium
ion
]
CY
P2
A6
CY
P2
A6
AO
X1
FM
O3
Fig
ure
1b. C
om
pari
so
n o
f nic
otine m
eta
bolis
m in h
um
an
s a
nd m
ice b
y know
n e
nzym
es.
Re
fere
nces (
Benow
itz
et
al 1994,
Beno
witz
and J
acob 2
001, C
hen
et al. 2
007, G
orr
od
and H
ibbert
1982,
Hu
kkanen
et al 2005, Itoh
et
al 1
997, K
aiv
ors
aari
et al 2007, K
yere
mate
n
et al 1990, M
urp
hy e
t a
l 1
999, Y
am
anaka e
t al 2005a, Y
am
anaka e
t al 20
05b).
Nic
oti
ne
Co
tin
ine
Trans-3
’-h
yd
rox
yc
oti
nin
e
[Nic
otin
e-∆
1’(5
’)-i
min
ium
io
n]
CY
P2
A5
CY
P2
A5
AO
X1
Nic
otine
N’-o
xid
eF
MO
1
Hu
ma
ns
Mic
e
Nic
oti
ne
Co
tin
ine
Trans-3
’-h
yd
rox
yc
oti
nin
e
[Nic
otine
-∆1’(
5’)-i
min
ium
ion
]
CY
P2
A6
CY
P2
A6
AO
X1
FM
O3
Fig
ure
1b. C
om
pari
so
n o
f nic
otine m
eta
bolis
m in h
um
an
s a
nd m
ice b
y know
n e
nzym
es.
Re
fere
nces (
Benow
itz
et
al 1994,
Beno
witz
and J
acob 2
001, C
hen
et al. 2
007, G
orr
od
and H
ibbert
1982,
Hu
kkanen
et al 2005, Itoh
et
al 1
997, K
aiv
ors
aari
et al 2007, K
yere
mate
n
et al 1990, M
urp
hy e
t a
l 1
999, Y
am
anaka e
t al 2005a, Y
am
anaka e
t al 20
05b).
Nic
oti
ne
Co
tin
ine
Trans-3
’-h
yd
rox
yc
oti
nin
e
[Nic
otin
e-∆
1’(5
’)-i
min
ium
io
n]
CY
P2
A5
CY
P2
A5
AO
X1
Nic
otine
N’-o
xid
eF
MO
1
Hu
ma
ns
Mic
e
11
smoking, nicotine is rapidly absorbed via the lung and reaches the brain within 20
seconds (Benowitz, 1990) and its plasma levels peak within five minutes (Armitage et al.,
1975). However, unlike mice, the elimination of nicotine follows a two compartmental
model with an initial rapid distribution (half-life 8.1±6.4 min) followed by elimination (half-
life 140±16 min) (Benowitz and Jacob, 1993; Benowitz et al., 1991b). In vivo, ~80% of
nicotine is metabolized to cotinine and 98% of a given nicotine dose can be recovered in
the urine as parent and metabolites within 24 hours (Benowitz et al., 1994).
Cotinine is the major metabolite of nicotine and it represents ~26% (free and
conjugated) of an intravenously infused dose of nicotine (Benowitz and Jacob, 1994).
Cotinine has a half-life of ~17.5 hr, although there is considerable variation between
subjects (range 8.1 to 29.3 hr) (Benowitz et al., 1983). The primary metabolite of cotinine
is trans 3’-hydroxycotinine (3-HC), and it represents ~40% (free and conjugated) of an
intravenously infused dose of nicotine (Benowitz and Jacob, 2001). The plasma half-life
of a single dose of 3-HC is ~6.6 hr (range 4.6 to 8.3 hr) (Benowitz and Jacob, 2001), but
the elimination of 3-HC is generation-limited (Dempsey et al., 2004). The primary
metabolite of 3-HC is trans 3’-hydroxycotinine glucuronide (Neurath and Pein, 1987).
Section 1.6 Human Nicotine Metabolizing Enzymes
Section 1.6.1 Human Cytochrome P450 2A6 (CYP2A6; Coumarin 7-
hydroxylase)
Similar to mouse CYP2A5, human CYP2A6 was originally named coumarin 7-
hydroxylase due to its exclusive ability to catalyze the formation of 7’-hydroxycoumarin
from coumarin (Pelkonen et al., 2000; Yamano et al., 1990). CYP2A6 is the primary
enzyme responsible for the C-oxidation of nicotine (Messina et al., 1997; Nakajima et al.,
1996b). It also contributes to the metabolism of a variety of compounds such as the
12
anesthetic halothane (Spracklin et al., 1996), the platelet activator factor inhibitor SM-
12502 (Nunoya et al., 1996), the anti-convulsants losigamone (Torchin et al., 1996) and
valproic acid (Sadeque et al., 1997), the anti-neoplastic agent tegafur (Ikeda et al., 2000),
and it also activates the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone (NNK), which is further described in section 2.3.5 (Tiano et al., 1993).
CYP2A6 also metabolizes several endogenous substrates such as trans-retinoic acid,
testosterone, estradiol, and progesterone althought its contribution to their metabolism is
minor (Di et al., 2009).
In humans, the two-step inactivation pathway of nicotine to cotinine is the same
as mice: nicotine is C-oxidized by CYP2A6 to form the ∆5’(1’)-iminium ion, which is
subsequently converted to cotinine by aldehyde oxidase (Gorrod and Hibberd, 1982). In
the cDNA-expressed system, CYP2A6 has an apparent Km of 11 to 58 µM for nicotine
(Ho et al., 2008; Messina et al., 1997; Nakajima et al., 1996b; Yamazaki et al., 1999). In
human liver microsomes, the Km of cotinine formation also varies greatly (range 13-131
µM) (Messina et al., 1997; Nakajima et al., 1996b). Similarly, in microsomes the Vmax of
cotinine formation from nicotine ranges from 4.2 to 120 nmol/mg protein/min (Messina et
al., 1997). These variations may be due to the genetic (e.g. CYP2A6 or transcription
factor variants) and/or environmental (e.g. dietary) influences. Chemical inhibitors and
antibodies were used to determine that the contribution of hepatic microsomal CYP2A6
to the metabolism of nicotine to cotinine to be ~90% (Messina et al., 1997; Nakajima et
al., 1996b). Consistent with this, individuals genetically lacking CYP2A6 produce little
cotinine (Kitagawa et al., 1999; Mwenifumbo and Tyndale, 2007; Yamanaka et al., 2004).
In addition to nicotine, CYP2A6 also metabolizes cotinine to 3-HC (Nakajima et
al., 1996a). In vitro, hepatic microsomes produce 3-HC from cotinine with a Km of ~234.5
µM (range 201.9 to 288 µM). This activity is highly correlated with coumarin 7-
13
hydroxylase activity and is inhibited by both coumarin and a CYP2A6 inhibitory antibody
(Nakajima et al., 1996a). In vivo, individuals lacking CYP2A6 do not produce 3-HC
(Dempsey et al., 2004; Yamanaka et al., 2004). Finally, CYP2A6 can also metabolize
nicotine to nornicotine, and cotinine to 5’-hydroxycotinine and norcotinine (Fig. 1)
(Murphy et al., 1999; Yamanaka et al., 2005a).
CYP2A6 mRNA is found predominantly in the liver, but is also found in lung,
trachea, nasal mucosa, and skin (Ding and Kaminsky, 2003; Janmohamed et al., 2001).
The transcript is not found in the kidney, duodenum, peripheral lymphocytes, or uterine
endometrium (Koskela et al., 1999). CYP2A6 protein is expressed primarily in the liver
(Ding and Kaminsky, 2003; Yamano et al., 1990) and is also found in the lungs (Zhang
et al., 2007). Its protein levels in other tissues have not been described. CYP2A6 protein
is found at higher levels in females than males (Al Koudsi et al., 2006b). This finding is
consistent with pharmacokinetic studies showing females have significantly faster
nicotine to cotinine metabolism (~20%) (Benowitz et al., 2006a).
CYP2A6 is 494 amino acids in length and is found on chromosome 19 (19q13.2;
9 exons) (Ensembl Genome Browser). CYP2A6 is highly polymorphic – currently there
are 34 named alleles (*1 to *34). Variant alleles contain non-synonymous SNP including
deletion, duplication, frameshift, and gene conversion, and promoter region alleles. The
full updated list of CYP2A6 polymorphisms can be found at
http://www.cypalleles.ki.se/cyp2a6.htm. The functional aspects of the alleles vary
ranging from a significant drop in nicotine metabolic activity (*2, *4, *7, *10, *17, *19, *20,
*23, *26, and *27) (Benowitz et al., 2001; Fukami et al., 2005a; Fukami et al., 2005b;
Fukami et al., 2004; Ho et al., 2008; Mwenifumbo et al., 2008a; Xu et al., 2002), a
moderate drop in activity (*9 and *12) (Benowitz et al., 2006b; Oscarson et al., 2002;
Yoshida et al., 2003), no change in activity (*8, *14, *16, *18, *21, *24, *25, and *28) (Al
Koudsi et al., 2006a; Fukami et al., 2005b; Ho et al., 2008; Mwenifumbo et al., 2008a;
14
Nakajima et al., 2006a; Xu et al., 2002), to increased activity (*1B, *1X2A, and *1X2B)
(Fukami et al., 2007; Mwenifumbo et al., 2008b; Rao et al., 2000; Wang et al., 2006a).
The functional impact of several other SNPs (*5, *6, *11, *13, *15, and *22) are predicted
to have reduced activities (Daigo et al., 2002; Haberl et al., 2005; Kitagawa et al., 2001;
Kiyotani et al., 2002; Oscarson et al., 1999) while others (*29, *30, *32, *33, and *34)
remain to be published. The impact of reduced nicotine metabolism on smoking
behaviour is briefly discussed in section 2.3.4.
Section 1.6.2 Human Cytochrome P450 2A13 (CYP2A13)
CYP2A13 is 93.5% identical in amino acid sequence with CYP2A6 (NCBI Pubmed).
Despite this homology, CYP2A13 has only approximately one-tenth the coumarin 7-
hydroxylase activity (Vmax/Km) of CYP2A6 (He et al., 2004b). CYP2A13 also metabolizes
tobacco alkaloids such as nicotine, cotinine (Bao et al., 2005), and NNK (Su et al., 2000).
CYP2A13 exhibits a similar affinity (Km) for cotinine formation from nicotine as
CYP2A6 (20 vs. 26 µM, respectively) in a heterologous expression system (Bao et al.,
2005). However, the enzyme is much more efficient in metabolizing cotinine to 3HC
compared to CYP2A6 (45 vs. 265 µM, respectively) (Bao et al., 2005). However, due to
the absence of CYP2A13 protein in the liver (Zhu et al., 2006), CYP2A6 remains the
main hepatic nicotine and cotinine metabolizing enzyme.
CYP2A13 mRNA is primarily found in the respiratory tract (Su et al., 2000). The
transcript is also found in liver, brain, mammary gland, prostate, testis, and uterus but
not in kidney, heart, small intestine, spleen, and stomach (Koskela et al., 1999; Su et al.,
2000). Using a CYP2A13-specific antibody the protein was found to be expressed in the
15
lung but not the liver (Zhu et al., 2006). Thus far, sex differences in CYP2A13 expression
have not been examined.
CYP2A13 is a CYP2A6 homologue located in the same gene cluster on
chromosome 19 (19q13.2) with the same number (nine) of exons encoding the same
number (464) of amino acids. As with CYP2A6, CYP2A13 is polymorphic. Currently
there are nine named alleles (*1 to *9) which include one wild-type allele, seven alleles
that contain at least one non-synonymous SNPs, and one frame-shift allele. The full
updated list of CYP2A13 polymorphisms can be found at
http://www.cypalleles.ki.se/cyp2a13.htm. The functional consequences of the variation
range from decrease in function (*2, *5, *6, *8, and *9) (Schlicht et al., 2007; Zhang et al.,
2002) to loss-of-function (*4 and *7) (Cauffiez et al., 2004; Wang et al., 2006b). The
contribution of CYP2A13 to systemic nicotine and cotinine levels is likely to be minor
considering that the protein is not expressed in the liver (Zhu et al., 2006) and individuals
who are poor metabolizers of CYP2A6 do not produce 3’-hydroxycotinine (Yamanaka et
al., 2004). However, it may contribute to local metabolism of xenobiotics such as nicotine
and NNK, particularly in the lung where the protein is predominantly expressed (Bao et
al., 2005; Su et al., 2000). There is no identified structural/functional homologue of this
enzyme in mice.
Section 1.6.3 Human Cytochrome P450 2B6 (CYP2B6)
CYP2B6 was originally named as the phenobarbital-inducible cytochrome P450IIB;
CYP2B6 metabolizes a wide range of important compounds including nicotine
(Yamazaki et al., 1999), the smoking cessation drug/anti-depressant bupropion
(Faucette et al., 2000), the HIV treatment drug efavirenz (Ward et al., 2003), and various
16
endogenous substrates such as testosterone and estrone (Imaoka et al., 1996; Shou et
al., 1997).
In an expression system, CYP2B6 has a low affinity for nicotine C-oxidation (Km
of 105±25 µM) (Yamazaki et al., 1999). Therefore, the contribution of CYP2B6 in the
metabolism of nicotine to cotinine is expected to be negligible at low concentrations, but
it may increase significantly when the nicotine concentration is high (Nakajima et al.,
1996b). The in vivo involvement of CYP2B6 in cotinine formation is debatable. For
instance, in a study of individuals deficient in CYP2A6 (CYP2A6*4), the metabolism of
nicotine was faster in those who had a CYP2B6 functional genetic variant (CYP2B6*6)
(Jinno et al., 2003), while this variant had no impact in the individuals with wild-type
CYP2A6 (Ring et al., 2007). In contrast, in another study, the same CYP2B6*6 variant
had no impact on in vivo nicotine plasma concentrations (obtained from nicotine patch)
regardless of the activity of CYP2A6 (Lee et al., 2007). Considering that a recent study
found CYP2B6*6 to be transcriptionally unstable resulting in a significant decrease in
protein production (Hofmann et al., 2008), additional studies will be required to reconcile
the observed differences. Nonetheless, a role for CYP2B6 may underlie the observation
that individuals lacking CYP2A6 still produce cotinine (~5% of normal level) following
nicotine administration, but the contribution of other enzymes has not been ruled out
(Yamanaka et al., 2004).
The contribution of CYP2B6 to nicotine metabolism may also be important under
situations of CYP2B6 enzyme induction. For example, in non-human primates
phenobarbital treatment led to greater than 6-fold increase in CYP2B6agm protein and
doubled its contribution to nicotine metabolism (Schoedel et al., 2003). CYP2B6 also
takes part in the N-demethylation of nicotine to form nornicotine although its involvement
is secondary to CYP2A6 (Yamanaka et al., 2005a).
17
CYP2B6 mRNA and protein are found at low levels in the liver and are also found
in the brain, lung, and kidney, and intestine (Gervot et al., 1999; Miksys et al., 2003).
Sex difference exists for CYP2B6 as females tend to have higher levels of mRNA,
protein, and activity (S-mephenytoin N-demethylation) (Lamba et al., 2003; Stresser and
Kupfer, 1999).
CYP2B6 is located in the same gene cluster as CYP2A6 and CYP2A13 on
chromosome 19 (19q13.3; 9 exons) and the protein is 491 amino acids in length
(Ensembl Genome Browser). CYP2B6 is also polymorphic and currently there are 29
named alleles for CYP2B6 (*1 to *29). There are at least 25 alleles; the effects of many
of these variants on nicotine metabolism have not been studied. Mouse Cyp2b10 is the
most similar to human CYP2B6 in nucleic acid sequence similarity (~75%; NCBI Blast);
however, whether CYP2B10 metabolizes nicotine or its metabolites in mice has not been
studied.
Section 1.6.4 Human Aldehyde Oxidase 1 (AOX1)
AOX1 is responsible for the metabolism of a large number of nitrogen-containing
xenobiotics including the antiviral famcyclovir, the antineoplastics methotrexate and 6-
mercaptopurine, and the ethanol metabolite acetaldehyde (Garattini et al., 2003). And as
discussed in the previous section (1.3.3), AOX1 metabolizes the nicotine-∆5’(1’)-iminium
ion to cotinine (Brandange and Lindblom, 1979).
In humans, AOX1 mRNA is found in highest level in the liver followed by the
kidney and adrenal gland, and it is found in trace levels in various peripheral tissues
(Nishimura and Naito, 2006). There are no apparent sex differences in aldehyde oxidase
activity (Al-Salmy, 2001).
18
AOX1 is located on chromosome 2 (2q33; 35 exons) and encodes a 1338 amino-
acid protein (Ensemble Genome Browser). The human AOX1 is highly polymorphic with
125 polymorphisms identified thus far, but only one non-synonymous SNP is predicted
to have any functional consequence (Steinberg et al., 2007). Despite the lack of the
impact of genetic variation, there is considerable variation in the activity of hepatic AOX1
activity (up to 50-fold depending on substrate tested) (Al-Salmy, 2001; Sugihara et al.,
1997). AOX1 variation is not expected to affect the formation of cotinine because up to
90% of variability in cotinine formation can be explained by CYP2A6 immunoreactivity
(Messina et al., 1997; Mwenifumbo and Tyndale, 2009)
Section 1.6.5 Human Flavin-containing Monooxygenase 3 (FMO3)
FMO3 metabolizes various compounds such as the pesticide fenthion (Furnes and
Schlenk, 2004), the anti-thyroid drug methimazole (Krueger and Williams, 2005),
triethylamine, as well as the conversion of nicotine to nicotine N-oxide (Cashman et al.,
1992). In humans, ~4-7% of administered nicotine is excreted in the urine as nicotine N-
oxide (Fig. 1) (Benowitz et al., 1994; Meger et al., 2002) and this percentage is three- to
four-fold higher in individuals lacking CYP2A6 (Yamanaka et al., 2004).
Like many other drug metabolizing enzymes the FMO3 transcript is mainly found
in the liver with minor transcript levels in both kidney and the lung (Zhang and Cashman,
2006), consistent with its protein expression (Krause et al., 2003; Overby et al., 1997).
There is marked inter-individual variation (over 9-fold) in the protein’s expression
(Krause et al., 2003; Overby et al., 1997). On the other hand, FMO3 expression and
nicotine N-oxide activity is uninfluenced by gender (Cashman et al., 1992; Krause et al.,
2003).
19
The FMO3 gene is located on chromosome 1 (1q23; 8 coding exons and 1 non-
coding exon) and encodes a 532 amino-acid protein (Ensemble Genome Browser).
There are currently 40 identified FMO3 alleles with the majority being non-synonymous
SNPs. In addition, there are two deletion alleles and one truncation allele. The list of
alleles can be found at http://human-
fmo3.biochem.ucl.ac.uk/Human_FMO3/Tables/tableall.html. Thus far there have been
no studies examining the direct impact of FMO3 polymorphism on nicotine disposition
kinetics; however, in rare cases (<1%) individuals with loss-of-function FMO variants
there may be minor re-routing of nicotine metabolism.
Section 1.6.6 Human Uridine Diphosphate (UDP)-Glucuronosyltransferase
(UGT)
UGTs are phase II enzymes responsible for the transfer of glucuronic acid from UDP-
glucuronic acid to various endogenous and exogenous substrates which increases their
water-solubility and subsequent excretion (Kiang et al., 2005). In humans, nicotine N-
glucuronide represents approximately 3-5% of total urinary metabolite of a dose of
nicotine (Benowitz et al., 1994). Individuals deficient for CYP2A6 may excrete up to
~45% of nicotine as the glucuronidated product over 24 hours due to metabolic re-
routing (Yamanaka et al., 2004). Cotinine and trans 3’-hydroxycotinine are also found as
glucuronidated products in the urine (12-17% and 7-9%, respectively) (Benowitz et al.,
1994).
The enzymes responsible for the N-glucuronidation of nicotine was thought to be
UGT1A4 with some contribution by UGT1A9 (Kuehl and Murphy, 2003; Nakajima et al.,
2002). Similarly, cotinine was thought to be N-glucuronidated by UGT1A4 (Kuehl and
Murphy, 2003; Nakajima et al., 2002). However, with improved detection method, it was
20
later identified that UGT2B10 to be the primary UGT enzyme that catalyzes the N-
glucuronidation of nicotine and cotinine (Kaivosaari et al., 2007). This was confirmed by
pharmacogenetic studies showing that hepatic microsomes from individuals with the
loss-of-function UGT2B10 enzyme (UGT2B10*2) showed a 5-fold reduction in nicotine
N-glucuronide production (Chen et al., 2007). This isoform also plays a predominant role
in the N-glucuronidation of cotinine (Kaivosaari et al., 2007) and UGT2B10*2 individuals
showed a 16-fold reduction in cotinine N-glucuronide production (Chen et al., 2007).
Trans 3’-hydroxycotinine is mainly O-glucuronidated and the primary enzyme involved in
this reaction is UGT2B7 (Yamanaka et al., 2005b).
Both UGT2B10 and UGT2B7 transcripts are found at high levels in the liver and
kidney (Turgeon et al., 2001). However, the variation in the expression of these two
genes between males and females has not been examined. Numerous UGT2B variants
have been identified in recent years; a current list of the variants can be found at
http://galien.pha.ulaval.ca. Both the UGT2B10 and UGT2B7 genes are located on
chromosome 4 (4q13; both are encoded by 6 exons) and are 528 and 529 amino acids
in length, respectively (Ensemble Genome Browser). Currently there are two identified
UGT2B10 and four UGT2B7 alleles with the variants coding for non-synonymous SNPs.
In mice neither nicotine and cotinine are glucuronidated (3-HC was not examined)
(Ghosheh and Hawes, 2002), suggesting a lack of involvement of UGT enzymes in
these metabolic pathways.
21
Section 2 Pharmacological Treatments for Smoking Cessation
Section 2.1 Primary Treatments
Section 2.1.1 Nicotine Replacement Therapies (NRTs)
Nicotine replacement products are the first line treatments for nicotine dependence (Siu
and Tyndale, 2007b). Since nicotine is the primary addictive component of cigarettes
(Russel, 1987), NRTs promote smoking cessation by delivering nicotine to replace
nicotine from cigarettes. The potential use of nicotine to alleviate withdrawal symptoms
was recognized in the 1970s (Schneider et al., 1977). Currently there are several
nicotine delivery devices available that include gum (2 and 4 mg), transdermal patch (7,
14, and 21 mg), vapor inhaler, nasal spray, sublingual tablet (2 mg), and lozenge (2 and
4 mg).
The general feature of NRTs is that the nicotine is absorbed through the skin or
the nasal/buccal mucosal membrane in order to avoid the extensive hepatic first-pass
metabolism (~80%; unclear in mice) associated with oral ingestion (Benowitz et al.,
1991a; Benowitz et al., 1991b; Zins et al., 1997). However, in practice, a large portion of
the nicotine delivered via the gum (Benowitz et al., 1987), tablets (Molander and Lunell,
2001), and lozenges (Choi et al., 2003) is swallowed and metabolized before reaching
systemic circulation. After a single cigarette puff, nicotine reaches peak plasma
concentration in 5 to 8 min (Hukkanen et al., 2005b). On the other hand, NRTs in
general have slower nicotine delivery kinetics with slower peak times after administration
(gum – 30 min; patch – 3 to12 hr; inhaler – 30 min; nasal spray – 11 to 18 min; tablet
and lozenges – 1 hr) (Hukkanen et al., 2005b). The slower delivery and absorption
kinetics of nicotine from NRTs significantly reduces their abuse liabilities (Henningfield
and Keenan, 1993; West et al., 2000) but may also reduce their long term success as
smoking cessation treatments.
22
A systemic review of 111 studies examining the efficacies of various NRT
products found that the relative risk (RR) of smoking abstinence after six months of
follow up with NRTs was 1.58 (95% CI 1.50 to 1.66) (Stead et al., 2008). Between NRTs,
nasal spray appeared to be the most effective [RR 2.02 (95% CI 1.49 to 3.73)] while the
gum appeared to be the least effective [RR 1.43 (95% CI 1.33 to 1.53)]. The RRs for
patch, inhaler, and lozenges are 1.66 (95% CI 1.53 to 1.81), 1.90 (95% CI 1.36 to 2.67),
and 2.00 (95% CI 1.63 to 2.45), respectively. It should be noted that not all studies in
each of the NRT forms showed definitive benefits over smoking cessation. There
appeared to be no overall effect of the type of NRTs on success of treatment outcome
(Stead et al., 2008). Behavioural support (e.g. advice, counseling) plus NRT
pharmacotherapy provided only minor advantage compared to pharmacotherapy alone
(Stead et al., 2008). There are conflicting evidence as to whether higher dose of nicotine
provided better outcome. For instance, 4 mg gum has been found to be provide better
cessation rates compared to 2 mg gum in some studies but not others (Garvey et al.,
2000; Herrera et al., 1995; Kornitzer et al., 1987). Nicotine patch studies provided less
evidence of dose-dependent efficacies (Killen et al., 1999; Tonnesen et al., 1999). The
primary adverse affects of nicotine replacement therapies depend on the type of
products used (e.g. skin irritation for patches), but common side effects include dizziness,
nausea, and headache (Stead et al., 2008).
Section 2.1.2 Bupropion
Smoking and depression are strongly associated perhaps because both are influenced
by dopamine levels (Paperwalla et al., 2004; Quattrocki et al., 2000). Bupropion is an
atypical antidepressant and was the first non-nicotine product approved as a first-line
therapy for smoking cessation. At therapeutic doses, bupropion binds to striatal
23
dopamine transporters (~20% occupancy). This potentially prevents dopamine reuptake
(Argyelan et al., 2005; Learned-Coughlin et al., 2003) which contribute to the mechanism
for reducing withdrawal symptoms. The noradrenergic system is also implicated in the
pharmacological effects of bupropion, possibly via noradrenaline reuptake inhibition
(Foley et al., 2006). The above mechanisms may explain, in part, bupropion’s ability to
reduce cravings in abstinent smokers and alleviate certain withdrawal symptoms which
may explain its usefulness in smoking cessation (Durcan et al., 2002; Jorenby et al.,
1999; Shiffman et al., 2000; Tashkin et al., 2001). Finally, bupropion can also act as a
nicotinic receptor antagonist via a non-competitive inhibition mechanism, suggesting that
it may attenuate the rewarding effect of nicotine (Alkondon and Albuquerque, 2005;
Fryer and Lukas, 1999; Slemmer et al., 2000).
A Cochrane systemic review of 31 studies found that the odds ratio of smoking
abstinence after six months of follow up with bupropion alone was 1.94 (95% CI 1.72 to
2.19) (Hughes et al., 2007). As with NRT, not all studies showed significant benefit of
bupropion over placebo. The intensity of behavioural support did not alter the cessation
rate although it is not known if counseling plus bupropion had significant advantages
over bupropion alone (Hughes et al., 2007). The analysis also found no effect of dose on
cessation rate from two studies (150 mg vs 300 mg at 12 months after target quit date).
However, a study directly comparing different dose of bupropion (100, 150 and 300
mg/day) found that bupropion was effective in smoking cessation in a dose-dependent
manner, although statistical comparisons between dosing groups were not made (Hurt et
al., 1997). Bupropion was not advantageous over NRTs for relapse prevention (Hughes
et al., 2007). The major adverse reactions associated with bupropion are insomnia,
nausea/vomiting, and dizziness (Boshier et al., 2003).
Section 2.1.3 Varenicline
24
Varenicline is a partial agonist of the α4β2 nicotinic cholinergic receptor (Coe et al.,
2005). In mouse studies, the β2 receptor subunit is required for nicotine reward, as
measured by conditioned place preference, and the α4 receptor subunit is important for
reward and development of tolerance to nicotine (McCallum et al., 2006; Tapper et al.,
2004). Varenicline is hypothesized to reduce activation of the dopaminergic system via
partial blockade of nicotinic receptors stimulated by high levels of nicotine during
smoking, thus lowering the rewarding effects of nicotine. In addition, as a weak agonist,
varenicline may also stimulate dopamine release by the mesolimbic neurons and reduce
craving as well as withdrawal during abstinence (Coe et al., 2005; Papke and
Heinemann, 1994).
Four studies that examined the 52-week continuous abstinence with varenicline
compared to placebo have all found varenicline to provide significantly better quit rate
compared to placebo (Gonzales et al., 2006; Jorenby et al., 2006; Nides et al., 2006;
Oncken et al., 2006). When analyzed together the odds of quitting were 3.22 (95% CI
2.43 to 4.27) favouring varenicline (Cahill et al., 2007). Two out of three studies that
compared the efficacy of varenicline to bupropion have found varenicline to be superior
(statistically significant) in maintaining 52-week continuous abstinence rate compared to
bupropion (Gonzales et al., 2006; Jorenby et al., 2006; Nides et al., 2006). Furthermore,
varenicline was more effective in reducing withdrawal symptoms compared to bupropion
(West et al., 2008). Varenicline was significantly more effective in preventing relapse
compared to placebo [198 (95% CI 159-260) days vs 87 (95% CI 58-143) days,
respectively; log-rank P<0.001] (Tonstad et al., 2006). The main adverse events
associated with varenicline are relatively mild and includes nausea, abnormal dreams,
and insomnia (Oncken et al., 2005). However, recent evidence indicated that varenicline
25
may induce depression and suicidal ideation (Kuehn, 2008) and a public health advisory
had been placed on this drug by the FDA
(http://www.fda.gov/bbs/topics/NEWS/2008/NEW01788.html).
Section 2.2 Secondary and Investigative (Non-approved) Smoking
Cessation Therapies
Section 2.2.1 Nortriptyline
Nortriptyline is a second-line therapy for smoking cessation but has not yet been
approved for this indication by the US Federal Drug Administration. Nortriptyline is a
tricyclic antidepressant that inhibits the reuptake of serotonin and noradrenalin. A meta-
analysis comparing the efficacy of nortriptyline vs. placebo found the pooled odds ratio
to be 2.34 (95% CI 1.61 to 3.41) (Hughes et al., 2007). The smoking cessation rate
achieved with nortriptyline was comparable to those achieved with bupropion [pooled
ORs: 2.1 (1.5 – 3.1) vs. 2.0 (1.7 – 3.4) for nortriptyline and bupropion, respectively]
(Hughes et al., 2005). However, nortriptyline appears to cause greater adverse events
compared to bupropion (Hall et al., 2002; Wagena et al., 2005) and may increase
abnormal cardiac rhythms (QTc interval) and increased adverse cardiac events in
ischemic heart disease patients (Roose et al., 1998; van Noord et al., 2009), thus
preventing its wide use.
Section 2.2.2 Clonidine
The α2-adrenergic receptor agonist clonidine is primarily used for the treatment of
hypertension; however, it has also been used for treatment of opiate and alcohol
withdrawal (Gossop, 1988; Manhem et al., 1985). Clonidine can alleviate smoking
26
withdrawal symptoms (Glassman et al., 1984; Ornish et al., 1988). A Cochrane analysis
of six studies found that clonidine treatment was associated with increased smoking
cessation [pooled OR: 1.89 (1.30 – 2.74)]; however, only one study showed significant
improvement in smoking cessation when the studies were analyzed separately (Gourlay
et al., 2004). Finally, adverse effects such as dry mouth, sedation, and postural
hypotension limit its general use.
Section 2.2.3 Serotonergic Agents
Section 2.2.3.1 Buspirone
The serotonergic system is responsible for the regulation of mood and appetite.
Buspirone is serotonin receptor (5-HT1a) partial agonist used for the treatment of
generalized anxiety disorder. The drug appears to function via the inhibition of the
release of serotonin and may potentially increase noradrenergic and dopaminergic
activity (Eison and Temple, 1986). Buspirone appears to benefit smokers with high level
of anxiety. For instance, in an eight-week treatment with maximum dose of 60 mg of
buspirone, the one-month abstinence in the high-anxiety group was 88% (treatment)
compared to 61% (placebo) but in the low-anxiety group the abstinence was 60%
(treatment) compared to 89% (placebo). At 12 months, there was no difference between
treatment and placebo in both anxiety group (Cinciripini et al., 1995). However, a
subsequent study revealed that buspione (six-week treatment; maximum dose of 60 mg)
was ineffective in smoking cessation regardless of degree of anxiety (Schneider et al.,
1996). In another study, buspirone (up to 40 mg for four weeks) was found to be equally
as effective as nicotine patch (fixed: 21/22 mg for six weeks; tapered: 21/22 mg for four
weeks, 14 mg for 4 weeks, and 7 mg for 4 weeks) (Hilleman et al., 1994). While the drug
27
appears to be safe, with little adverse effects, the overall usefulness of buspirone in
smoking cessation is inconclusive.
Section 2.2.3.2 Selective Serotonin Reuptake Inhibitors (SSRIs)
SSRIs are used for the treatment of depression and anxiety (Nutt et al., 1999).
Fluoxetine has not been demonstrated to be effective for smoking cessation but may
decrease the negative and increase the positive affects after quitting (Blondal et al.,
1999; Cook et al., 2004; Niaura et al., 2002; Saules et al., 2004). Paroxetine was not
effective beyond four weeks (during a 9-week treatment protocol) (Killen et al., 2000)
and sertraline did not increase abstinence compared to placebo (Covey et al., 2002).
SSRIs do not appear to be effective smoking cessation aides.
Section 2.2.4 Naltrexone
Endogenous opioids (endorphins, enkephalins, dynorphins), together with their receptors
(mu, kappa, delta) are responsible for various biological processes which include
autonomic, neuroendocrine, hedonic, and emotional responses (Kieffer and Evans,
2009). There are several lines of evidence that suggested the involvement of the opioid
system in nicotine addiction. First, animals exhibit similar somatic withdrawal signs from
both nicotine and opiates (e.g. tremors, scratches, chewing, etc.) (Malin and Goyarzu,
2009). Second, in rats nicotine administration led to increased turnover of Tyr-Gly-Gly, a
marker of enkephalin release (Houdi et al., 1991), and smoking cigarettes containing
more nicotine led to greater plasma levels of β-endorphin compared to cigarettes with
low nicotine (Gilbert et al., 1992). Third, mice with the preproenkephalin or the mu-opioid
receptor genes knocked-out do not respond to the rewarding properties of nicotine as
28
demonstrated by lack of nicotine-induced place preference (Berrendero et al., 2002;
Berrendero et al., 2005). Finally, in rats the opioid antagonist naloxone precipitated
nicotine withdrawal as well as prevented nicotine alleviation of withdrawal (Malin et al.,
1993; Malin et al., 1996) further suggesting the involvement of the opioid system in
nicotine dependence.
Naltrexone is a non-specific antagonist of the opioid receptors used for the
treatment of opioid-dependence (Modesto-Lowe and Van Kirk, 2002). Due to the
connection between nicotine dependence and opioid system, naltrexone has been
investigated as a potential therapy for smoking cessation. However, it should be noted
that several studies have found that naltrexone, when administered alone, led to
significant adverse effects due to precipitation of nicotine withdrawal (Covey et al., 1999;
Wong et al., 1999). On the contrary, studies of naltrexone with nicotine replacement
have found the drug combination to be more effective. For example, in a preliminary
study naltrexone (50 mg) plus nicotine patch (21 mg) resulted in higher 2-week
continuous abstinence rate (weeks 3-4 in a 4 week study) compared to placebo plus
patch (56.3% vs. 31.3%) (Krishnan-Sarin et al., 2003). In a larger study examining
naltrexone (0, 25, 50, and 100 mg/d) plus nicotine patch (21 mg) for smoking cessation,
it was found that naltrexone only at 100 mg/d (but not 25 and 50 mg/day) plus nicotine
patch led to significantly better 6-week continuous abstinence rate compared to placebo
plus patch. However, subsequent exploratory analzysis of 7-day point-prevalence
abstinence at 3-, 6-, and 12-months did not find any difference in cessation between
naltrexone vs placebo (O'Malley et al., 2006). As mentioned previously, naltrexone
causes withdrawal symptoms such as nausea, vomiting, diahrea, depression, and
insomnia which may limit its utility (O'Malley et al., 2006).
Section 2.2.5 Rimonabant (SR141716)
29
The involvement of the endocannabinoid system with nicotine dependence was
established when it was observed that chronic nicotine administration increased the level
of the endogenous cannabinoid receptor agonists anandamide (AEA) and 2-
arachidonoly-glycerol (2-AG) (for CB1 and CB2 receptors, respectively). The increases in
these agonists are localized to the brainstem, a region involved in drug withdrawal
(Gonzalez et al., 2002; Shoemaker et al., 2005; Steffens et al., 2005). In rats,
administration of the CB1 receptor antagonist SR141716 (rimonabant) blocked nicotine-
induced dopamine release in the nucleus accumbens (Cohen et al., 2002). Rimonabant
also prevented IV nicotine self-administration, conditioned stimuli-induced nicotine-
seeking behaviors and nicotine-conditioned place preference in rats (Cohen et al., 2005;
Cohen et al., 2002; Le Foll and Goldberg, 2004). The precise mechanism of actions of
CB1 receptor antagonism on nicotine reward/reinforcement remains unclear.
A recent Cochrane review found that the pooled odds ratio (from STRATUS-US
and STRATUS-EUROPE) of quitting smoking at one year with 20 mg rimonabant to be
1.61 (95% CI 1.12 to 2.30) with the 5 mg showing no benefit over placebo (Cahill and
Ussher, 2007). When analyzed separately, only the STRATUA-US study showed a
significant benefit in cessation. Rimonabant showed potential benefit in relapse
prevention (STRATUS-Worldwide); both 5 mg and 20 mg treatment provided
approximately 1.5-fold increase in remaining abstinent (Cahill and Ussher, 2007). The
most common side effects were nausea and upper respiratory tract infection (Anthenelli,
2005). Due to the inconclusive evidence showing rimonabant to be effective in smoking
cessation, the drug is currently not approved for the indication in the United States. As of
2008, the European Medicines Agency has suspended the use of rimonabant in the
treatment of obesity due to increased risk of psychiatry disorders in overweight or obese
patients (Allchurch, 2008).
30
Section 2.2.6 Nicotine Vaccines
The nicotine vaccine is another potential treatment for smoking. The mechanism behind
this therapeutic strategy is to prevent nicotine from entering the brain via binding to
nicotine-specific antibodies (Hieda et al., 2000; Pentel et al., 2000). An advantage of
nicotine vaccines is that only occasional booster shots, rather than daily administration,
should be needed for therapeutic effect. In a 38-week study it was found that during ad
libitum smoking, nicotine vaccine, at all doses tested (50, 100, and 200 µg; 1 primary
challenge shot and 3 booster shots at each dose), did not cause compensatory smoking
behaviors or precipitate withdrawal (Hatsukami et al., 2005). In addition, the 30-day
continuous abstinence rates were higher in patients receiving the highest vaccine dose
(~38%) compared with placebo (~10%) (Hatsukami et al., 2005). Careful interpretation of
these results is needed as the sample size was small and the study was primarily
designed to evaluate the safety and immunogenicity of the vaccine. In a subsequent
study with larger sample size (N=341) and a different vaccine, it was found that the four-
month continuous abstinent rate was significantly higher in individuals with the highest
antibody titer compared to placebo (56.6% [N=30 out of 53] vs. 31.3% [N=25 out of 80];
OR = 2.9; p<0.01) (Cornuz et al., 2008). The vaccine is associated with greater adverse
events such as flu-like symptoms and pyrexia (Cornuz et al., 2008). Currently nicotine
vaccine is still being investigated for efficacy in Phase III clinical trials (Clinicaltrial.gov).
Section 2.2.7 Monoamine Oxidase Inhibitors (MAOIs)
MAO enzymes are responsible for the metabolism of various endogenous substrates
(e.g. adrenaline, noradrenaline, dopamine, 5-hydroxytryptamine, 2-phenylethylamine,
31
tryptamine, tyramine) and thus they can play an important role in smoking behaviours
(Youdim et al., 2006). The relationship between MAO activities and smoking was first
observed almost three decades ago (Oreland et al., 1981). In smokers, the brain MAO-A
and MAO-B activities are ~30 and ~40% lower compared to non-smokers, respectively
(Fowler et al., 1996a; Fowler et al., 1996b). MAO-B is the major isoform of the
monoamine oxidase in the brain (~75-80%) (Saura Marti et al., 1990; Squires, 1972).
The inhibition of MAO-B is reversed after long-term abstinence from smoking, likely due
to the synthesis of new enzymes (Gilbert et al., 2003). These findings suggest MAO-B
inhibitor(s) may be present in tobacco smoke (Khalil et al., 2000) that may inhibit MAO-
mediated dopamine metabolism, thereby enhancing the rewarding effects of nicotine
during smoking (Lewis et al., 2007). However, the specific tobacco smoke compounds
that lead to long term inhibition of MAO-B have not been identified.
Through inhibition of MAO-A and MAO-B, the metabolism of dopamine can
potentially be reduced and this may enhance the rewarding effects of nicotine during
smoking. In a preliminary study, the reversible MAO-A inhibitor moclobemide
significantly reduced self-reported smoking rates, although at one year there was no
difference in abstinence compared with placebo (Berlin et al., 1995). The reversible
inhibitor of MAO-B lazabemide has also been evaluated for smoking cessation, but the
study was terminated due to concerns of hepatotoxicity (Berlin et al., 2002).
The irreversible MAO-B inhibitor selegiline has also demonstrated some efficacy
in several small studies. In one study, oral selegiline (10 mg) decreased physical
symptoms (e.g. hand shakes, increased heart rate) and craving during abstinence as
well as reducing smoking satisfaction during smoking (Houtsmuller et al., 2002). In
another study, 5 mg b.i.d. oral selegiline increased the trial end point (8-week) 7-day
point prevalent abstinence compared with placebo [OR 4.64 (95% CI: 1.02-21.00
p<0.05)]; however at 6 months the 7-day point prevalent abstinence was not significant
32
even though the OR was 4.75. A limitation of the study was the small sample size (N=40)
(George et al., 2003). In a third study that used a combination of oral selegiline and
nicotine patch, selegiline plus nicotine patch doubled the 52-week continuous abstinence
rate compared to nicotine patch alone (25% vs. 11%), although the difference was not
significant possibly due to small number of subjects (N=38) (Biberman et al., 2003).
Common adverse effects associated with selegiline monotherapy (5 mg b.i.d.)
are nausea and dizziness (Heinonen and Myllyla, 1998). Despite the dopamine
potentiating effect of selegiline, as well as the production of L-methamphetamine and L-
amphetamine metabolites (instead of the more potent D-isomers) from selegiline, there
is no indication that selegiline is addictive (Schneider et al., 1994).
Section 2.2.7.1 Selegiline (l-deprenyl; Eldepryl®)
Section 2.2.7.1.1 Selegiline Pharmacodynamics
Selegiline is used clinically in conjunction with levodopa for the treatment of Parkinson’s
disease (Gerlach et al., 1996). The recommended dose of selegiline for Parkinson’s
disease is 10 mg (5 mg p.o. b.i.d.) (Gerlach et al., 1996). This is the same dose of
selegiline that was used in several smoking cessation trials. At these clinical doses,
selegiline is a selective and irreversible inhibitor of MAO-B (Gerlach et al., 1996). The
drug inhibits MAO-B by covalently binding to its flavine-adenine dinucleotide (FAD) co-
factor (Youdim, 1978), effectively decreasing the amount of enzyme available to
metabolize dopamine. At higher doses the selectivity of selegiline decreases and it also
inhibits MAO-A (Gerlach et al., 1992).
In addition to enhancing dopamine availability by inhibiting MAO-B, selegiline
may have another mechanism of action that modulates dopaminergic activities that may
be important to smoking cessation. Phenylethylamine (β-PEA), an endogenous
33
substrate of MAO-B, exists in trace concentrations in the brain and was found to be
significantly higher (up to 10-fold) in selegiline-treated Parkinson’s patients compared to
non-treated patients (Reynolds et al., 1978). In monkeys, selegiline increased brain β-
PEA levels (Paterson et al., 1995). In rats, selegiline increased β-PEA levels in the
striatum and enhanced the neuronal response to dopamine stimulation (Berry et al.,
1994; Paterson et al., 1991). In addition, direct administration of β-PEA into the rat
striatum caused the release of dopamine (Bailey et al., 1987; Kuroki et al., 1990). β-PEA
has been proposed to be a neuromodulator of dopaminergic transmission although the
mechanism by which this occurs has not been determined (Paterson et al., 1990).
Therefore, the selegiline-mediated increase in β-PEA may enhance neuronal stimulation
by dopamine. The selegiline metabolite desmethylselegiline can also inhibit MAO-B
activity in vivo in humans although its contribution may be minor (Heinonen et al., 1997).
Section 2.2.7.1.2 Selegiline Metabolism
In humans, selegiline is extensively metabolized; less than 1% of an oral dose is
excreted unchanged in the urine (Shin, 1997). In correlation analyses, CYP2B6 accounts
for the largest variation in the metabolism of selegiline to its primary metabolites
desmethylselegiline (77%) and L-methamphetamine (79%) (Benetton et al., 2007). Both
CYP2A6 and CYP3A4 contribute to these metabolic pathways to some extent (14-15%
for both desmethylselegiline and L-methamphetamine by CYP2A6 and 33-35% for both
desmethylselegiline and L-methamphetamine by CYP3A4) (Benetton et al., 2007). The
plasma half-life of 10 mg of oral selegiline is 1.0 ± 0.9 hr; however, this increased to 2.7
± 1.8 hr following chronic (8 days) administration, indicating the metabolism slows with
chronic selegiline treatment. In addition, the observed accumulation ratio of the drug is
34
higher at 2.7 compared to the estimated accumulation ratio of 1.0 based on elimination
half-life (although not statistically significant), indicating possibly that selegiline, or a
metabolite, down-regulates the enzyme(s) responsible for its metabolism or inhibits
selegiline’s clearance (Laine et al., 2000). Age and sex were not found to contribute to
differences in selegiline pharmacokinetics (Barrett et al., 1996a; Barrett et al., 1996b).
The plasma elimination half-lives of desmethylselegiline and L-
methamphetamine are 31 ± 8 and 161 ± 38 hr, respectively. L-amphetamine, the major
metabolite of desmethylselegiline, has a half-life of 40 ± 7 hr (Laine et al., 2000). Similar
to selegiline, the three metabolites showed accumulation over eight days to some extent.
L-methamphetamine and L-amphetamine are further metabolized to various p-
hydroxylation and β-hydroxylation products whose pharmacokinetic profiles have not
been examined in detail (Shin, 1997).
In mice, an inhibitor study showed that CYP2E1 is responsible for more than
50% of the metabolism of selegiline to all three of its metabolites (desmethylselegiline,
methamphetamine, and amphetamine) with a minor contribution from mouse CYP3A
enzyme(s) (Valoti et al., 2000). The contribution of CYP2A5 to the metabolism of
selegiline was not examined (Valoti et al., 2000). Similar to humans, the plasma half-life
of selegiline in mice is between 0.9 to 1.8 hr (Magyar et al., 2007).
The observation that CYP2A6 is involved in selegiline metabolism and chronic
administration of this drug decreases its own metabolism suggests that selegiline may
inhibit CYP2A6. These findings have implications for selegiline in the smoking cessation
and will be further described in sections 2.3.3.2 and 5.3.2.
Section 2.3 The Inhibition of Nicotine Metabolism and its Application to
Smoking Cessation
Section 2.3.1 Inhibitors of Human CYP2A6
35
CYP2A6 has a compact, narrow, hydrophobic active site (Yano et al., 2005); therefore,
compounds that are good inhibitors of CYP2A6 are small and planar (e.g. structurally
related to nicotine) (Denton et al., 2005; Rahnasto et al., 2005; Yano et al., 2006). A
variety of compounds can inhibit CYP2A6 in vitro including pharmaceuticals [e.g.
methoxsalen (Maenpaa et al., 1993), pilocarpine (Kimonen et al., 1995), clotrimazole,
miconazole (Draper et al., 1997), tranylcypromine, tryptamine (Zhang et al., 2001),
amphetamine (Rahnasto et al., 2005)], naturally occurring substances [furanocoumarins
(Maenpaa et al., 1993), various nicotine-related alkaloids (in vitro) (Denton et al., 2004;
Denton et al., 2005), soy isoflavones (in vivo) (Nakajima et al., 2006b), grapefruit juice
(in vivo) (Hukkanen et al., 2006), star fruit juice (in vitro) (Yang, 2007)], and endogenous
substances (in vitro) [e.g. β-PEA (Rahnasto et al., 2003), dopamine (Higashi et al.,
2007b)]. Of the above, methoxsalen can also inhibit nicotine metabolism in vivo and may
be clinically significant in its application in smoking cessation (further discussed in
sections 2.3.3.1 and 5.3.1).
Section 2.3.2 Inhibitor of Mouse CYP2A5
Some CYP2A6 inhibitors also appear to be inhibitors of CYP2A5. Examples are
furanocoumarins-derivatives such methoxsalen (Maenpaa et al., 1993), pilocarpine
(Kimonen et al., 1995), and amphetamine (Rahnasto et al., 2005). Other sample
compounds that inhibit CYP2A5 include, various lactone (coumarin-like) compounds
(Juvonen et al., 2000), as well as etomidate, metomidate and metyrapone (Kojo et al.,
1989). These results suggest a similar inhibitor profile between the human CYP2A6 and
mouse CYP2A5 and the mouse may be useful in the evaluation of novel CYP2A6
inhibitors.
36
Section 2.3.3 Mechanism-Based Inhibition
Mechanism-based (suicide) inhibitors (MBIs) are compounds that inhibit enzyme
activities by covalent modification. As a result, the enzyme is no longer functional, and
its activity can only be recovered via synthesis of new enzyme. There are several ways
in which mechanism-based inhibition can occur (Osawa and Pohl, 1989). First is the
covalent modification of the prosthetic heme in which the reactive metabolite binds to the
heme and together they dissociate from the enzyme. Another is the covalent binding of a
reactive metabolite to the holoenzyme. Third is the covalent binding of the heme to the
protein mediated by the reactive metabolite. In addition, the reactive metabolite can also
covalently bind to the enzyme co-factor [e.g. binding of selegiline reactive metabolite to
the flavin adenine dinucleotide (FAD) of MAO-B (Youdim, 1978)].
There are seven experimental criteria that can be used to establish whether a
compound is a MBI (Silverman, 1995). First, there is time-dependent loss of activity due
to reduction in active enzyme. Second, the inhibition is saturable – the rate of
inactivation of enzyme is proportional to the number of inactivator molecules added until
all enzyme molecules are saturated. Third, a substrate or a competitive (reversible)
inhibitor should slow down inactivation by the potential MBI (substrate protection). Fourth,
the inhibition is irreversibe such that dialysis or filtration (gel or membrane) does not
restore enzyme activity. Fifth, there should be inactivator stoichiometry in which only one
inactivator molecule is attached to each enzymatic site. Sixth, the involvement of a
catalytic step – the enzyme needs to convert the inhibitor to an active species that
actually carries out the inactivation step and therefore an enzymatic reaction is required.
Finally, the active species formed from enzymatic activity must inactivate the enzyme
prior to its release from the active site.
37
Section 2.3.3.1 Methoxsalen (8-methoxypsoralen; 8-MOP): a CYP2A5 and
CYP2A6 MBI
Methoxsalen is a naturally occurring psoralen compound that is used for the treatment of
psoriasis and cutaneous T-cell lymphoma in conjunction with UV exposure (Bond et al.,
1981; Young, 1993). Methoxsalen is a competitive (Ki), non-competitive (Kiu), and a
mechanism-based (KI and kinact) inhibitor of CYP2A6 inhibiting both coumarin (Ki =1.5 µM
[human liver microsomes]; Kiu =0.5 µM; KI =0.3-0.8 µM [kinact =0.5 min-1]) and nicotine (Ki
=0.8 µM) metabolism (Draper et al., 1997; Koenigs et al., 1997; Maenpaa et al., 1994;
Zhang et al., 2001). In mice, methoxsalen is a competitive (Ki not determined), non-
competitive (Kiu =1.7 µM), and mechanism-based (kinact =0.17 min-1) inhibitor of
microsomal CYP2A5-mediated coumarin metabolism (Visoni et al., 2008). Methoxsalen
also inhibits human CYP1A2, CYP2B6, CYP2A13, and to some extent several other
P450 enzymes (von Weymarn et al., 2005; Zhang et al., 2001) in addition to human
CYP2A6 and mouse CYP2A5.
Section 2.3.3.2 Selegiline as a MBI
As mentioned in section 2.2.7.1, selegiline is an irreversible MBI of MAO-B. Selegiline
belongs to the acetylene group of chemicals that contain a carbon-carbon triple bond,
which are known to be potent MBIs (Correia and Ortiz de Montellano, 2005). Selegiline
is a MBI of CYP2B1, the rat nicotine metabolizing enzyme, although the precise
mechanism of inactivation is unclear (Sharma et al., 1996). It is unknown what other
additional rodent P450 enzymes selegiline may inhibit; however, in humans selegiline
38
has little to no inhibitory effects on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A
in vitro (Polasek et al., 2006). Considering that CYP2A6 is involved in the metabolism of
selegiline in vitro (Benetton et al., 2007) and chronic selegiline treatment reduces its own
metabolism (Laine et al., 2000), it is possible that selegiline is a MBI of CYP2A6.
Section 2.3.4 Improving Smoking Cessation through CYP2A6 Inhibition
The impact of CYP2A6 genotype on nicotine metabolism and smoking behaviors has
been examined in detail (Ho and Tyndale, 2007; Malaiyandi et al., 2005; Mwenifumbo
and Tyndale, 2007). The most striking observation of the influence of CYP2A6 genetics
on smoking behaviours comes from slow metabolizers of nicotine (50% or less predicted
CYP2A6 activity). For instance, slow metabolizers are less likely to be current smokers
(OR 0.52 [95% CI: 0.29-0.95], p=0.03) (Schoedel et al., 2004), smoke significantly fewer
(20-25%) cigarettes (Malaiyandi et al., 2006; Schoedel et al., 2004), and have
significantly reduced (~30%) total inhalation volume (Strasser et al., 2007). In addition,
when smokers are ranked according to rates of nicotine metabolism (as measured by
the 3-hydroxycotinine to cotinine ratio), individuals in slowest quartile are more likely to
quit smoking after counseling (32%) compared to those in the fastest quartile (10%)
(Patterson et al., 2008). Furthermore, with nicotine patch treatment, individuals in the
upper quartiles (faster nicotine metabolism) have lower chance of quitting smoking
compared to those in the lowest quartile at the end of treatment and 6 months’ follow-up
(OR 0.72 [95% CI: 0.83-1.33], P=0.005) (Lerman et al., 2006). Similar observation is
also seen in light-smokers treated with nicotine gum (Ho et al., 2009).
Based on the above findings, it was hypothesized that one could
pharmacologically mimic impairments in the CYP2A6 enzyme (phenocopying) to
increase nicotine levels and decrease the number of cigarettes smoked (Sellers et al.,
39
2000; Tyndale and Sellers, 2002). By inhibiting nicotine metabolism, the systemic
availability of nicotine is increased; therefore, the concomitant use of CYP2A6 inhibitors
with NRT products should enhance smoking cessation. For example, some of the
nicotine released from nicotine gum is absorbed by the buccal cavity, while a significant
portion is swallowed (Benowitz et al., 1987). Of the nicotine swallowed (from nicotine
gum, lozenges, or pills), approximately 80% is metabolized via first-pass metabolism
before entering systemic circulation (Benowitz et al., 1991b). Inhibition of CYP2A6 would
decrease first-pass metabolism and increase the bioavailability of nicotine as well as
other CYP2A6 substrates (see Section 1.6.1). In a preliminary study, after three days of
treatment with methoxsalen (10 mg t.i.d.) plus nicotine gum (4 mg), the mean plasma
nicotine levels were significantly higher compared to placebo plus nicotine gum (15.3 vs.
10.1 ng/ml; p<0.01) (Sellers et al., 2002). Similarly, the pill form of nicotine for smoking
cessation may also benefit from the co-administration of a CYP2A6 inhibitor. This is due
to the fact that higher doses of oral nicotine that are required to overcome first-pass
metabolism can cause gastrointestinal irritation (Benowitz et al., 1991b). Compared with
oral nicotine alone, the addition of methoxsalen (10 and 30 mg) to 4 mg of oral nicotine
significantly increased mean plasma nicotine levels (>70%) and decreased the desire to
smoke (p<0.05) (Tyndale et al., 1999). In another study, methoxsalen (30 mg) plus oral
nicotine (4 mg) also significantly altered smoking behaviours. Participants on this
treatment smoked fewer cigarettes (3.1 vs. 4.1; p<0.01) during a 90 minute free smoking
session, had lower levels of breath carbon monoxide (4.6 vs 8.7 ppm; p<0.01), lower
numbers of puffs (~28 vs. ~38; p<0.01) and inhalation intensity compared with placebo.
The subjects also had increased latency to lighting the second cigarette (~43 vs. ~32
mins; p<0.01) on methoxsalen plus nicotine compared to placebo plus nicotine (Sellers
et al., 2000). Finally, CYP2A6 inhibitor alone may be able to reduce smoking and
40
increase quitting, similar to the pharmacogenetic findings in slow metabolizers
(Malaiyandi et al., 2006; Patterson et al., 2008; Rao et al., 2000; Schoedel et al., 2004);
however, this remains to be tested. An advantage of this strategy is that the long-term
behavioral effects of mimicking a defect in nicotine metabolism can be predicted based
on available pharmacogenetic and behavioral studies.
Section 2.3.5 Reduction of Tobacco-Specific Nitrosamine Activation
through Human CYP2A6 Inhibition
One goal of smoking cessation is to reduce smoking-associated morbidity and mortality.
The tobacco-specific nitrosamine NNK is a potent procarcinogen found in tobacco
smoke (Hecht and Hoffmann, 1988). NNK is primarily detoxified by conversion to 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), followed by conjugation to NNAL-
glucuronide (Hecht et al., 1993; Maser et al., 1996). Alternatively, NNK can be
metabolically activated to a reactive metabolite capable of forming DNA adducts via α-
hydroxylation (Hecht et al., 1988; Peterson et al., 1990); one enzyme responsible for this
process is CYP2A6 (Nishikawa et al., 2004). The other enzyme that exhits the highest in
vitro activity towards NNK activity is CYP2A13 (with at least 100-fold greater activity than
CYP2A6) and it is primarily expressed in the lung and may be one of the enzyme
responsible for NNK activation in situ (Jalas et al., 2005). However, CYP2A13 level in
lung tissues is variable between individuals and thus CYP2A6, as well as other enzymes
with lower in vitro NNK activation activities, such as CYP1A1/2, CYP2B6, CYP2D6,
CYP2E1, and CYP3A4, may contribute to NNK activation in those lacking CYP2A13
(Jalas et al., 2005; Zhang et al., 2007). The involvement of CYP2A6 in lung
carcinogenesis is supported in a recent epidemiology study: individuals that were
CYP2A6-poor metabolizers had significantly less risk for developing lung cancer, even
41
after adjusting for the number of cigarettes smoked (Fujieda et al., 2004). Furthermore,
the expression of CYP2A6 is higher in large lung adenocarcinoma tissues and tumors
that have metastasized compared to smaller tumor and non-metastasized tissues,
respectively, suggesting the involvement of CYP2A6 in lung carcinogenesis (Matsuda et
al., 2007).
In mice, NNK exposure causes the formation of lung tumors (Hecht et al., 1978;
Hecht et al., 1994; Okubo et al., 2005), and administration of the mouse CYP2A5
inhibitor, methoxsalen, reduced lung malignancies (Miyazaki et al., 2005; Takeuchi et al.,
2003). The finding is corroborated with findings in humans in which methoxsalen
administration in smokers re-routed excretion of NNK to NNAL and NNAL-glucuronide,
suggesting the α-hydroxylation of NNK was inhibited (Sellers et al., 2003a).
CYP2A13 also contributes to the α-hydroxylation of NNK (He et al., 2004a). Due
to the high level of CYP2A13 in the respiratory tract (Su et al., 2000), it is thought that
CYP2A13 plays a significant role in the bioactivation of NNK in the lung. This is
supported by the finding that NNK activation is significantly correlated with CYP2A13
levels in lung microsomes (Zhang et al., 2007). In contrast, the association between
CYP2A13 levels/activities with NNK metabolism was only found in a subgroup of lung
cancer biopsy samples (Brown et al., 2007). It is possible that inhibition of CYP2A
enzymes (CYP2A6 and CYP2A13) may decrease the risk of development of NNK-
induced lung cancer.
42
Section 3 Nicotine Pharmacological Behavioural Models
Section 3.1 Nicotine Self-Administration
Nicotine has reinforcing properties in mice as demonstrated through the use of self-
administration paradigms. Intravenous nicotine self-administration (NSA) is a widely
used method of testing nicotine’s reinforcing properties in rats (Corrigall and Coen, 1991;
Corrigall et al., 1994) but is more technically demanding in mice due to their small size
(Sparks and Pauly, 1999). Oral NSA, whilst not modeling smoking behaviours directly,
has been studied extensively in mice (Adriani et al., 2002; Aschhoff et al., 1999; Meliska
et al., 1995; Robinson et al., 1996; Stolerman et al., 1999) and has some similar features
to smoking, which may make it a suitable method of studying nicotine pharmacology. For
instance, oral NSA occurs intermittently primarily during the wake period. There exhibits
a wide spectrum of preference for oral nicotine between mouse strains (Aschhoff et al.,
1999; Robinson et al., 1996), suggesting a role of genetic influences in NSA, similar to
smoking behaviours in humans (Ho and Tyndale, 2007). Chronic oral NSA produces
pharmacologically relevant levels of nicotine in the plasma of mice. When nicotine is
provided in a free-access two-bottle choice paradigm, mice consume an amount of
nicotine that results in a plasma concentration (~35 ng/ml) similar to those seen in
humans during ad libitum smoking (10 to 50 ng/ml) (Hukkanen et al., 2005b; Rowell et
al., 1983). Chronic oral NSA can also lead to physical dependence as measured by
somatic signs (paw tremors, backing, and head shakes) although this occurs at high
nicotine levels (200 µg/ml) (Grabus et al., 2005). In addition, oral NSA up-regulates both
α4 and α7 nicotinic acetylcholine receptors in mouse brain (Sparks and Pauly, 1999);
this is consistent with findings that chronic nicotine and smoking increase nicotinic
receptors in baboons and smokers, respectively (Kassiou et al., 2001; Perry et al., 1999).
43
Oral nicotine also alters the activities of enzymes (e.g. CREB, pERK) in pathways
thought to mediate nicotine reward (Brunzell et al., 2003). Behaviourally, in mice oral
nicotine is rewarding at an optimal range: too low a dose and nicotine is not rewarding
and at high doses it is aversive (Robinson et al., 1996). This finding is in line with human
and other animal studies (Rose and Corrigall, 1997). Together, these data suggest that
oral NSA in mice may be a suitable method of studying some aspects of the
pharmacology of nicotine.
Section 3.2 Analgesic Effect of Nicotine and Application in Nicotine
Studies
Nicotine’s analgesic properties have long been documented (Meyer et al., 2000). The
mechanism of action is thought to be mediated indirectly by the activation of the opioid
system. In a study examining the anti-nociceptive effect of nicotine on tail-pinch in mice,
nicotine induced the release of the endogenous opioid peptide, Met-enkephalin (agonist
at both µ- and δ-opioid receptors), but not dynorphins and endorphins, in the spinal cord.
The analgesic action was mediated in part by the nicotinic acetylcholine receptors as
mecamylamine ameliorated the effect (Kiguchi et al., 2008). In addition to analgesia,
nicotine also has both hypothermic and ataxic effects (Kita et al., 1988; Marks et al.,
1984).
The tail-flick and hot-plate tests are used to study nicotine’s analgesic effects
(Marubio et al., 1999; Rogers and Iwamoto, 1993). The tail-flick response is a spinal
reflex (Caggiula et al., 1995); therefore, the ataxic-properties of nicotine are not thought
to be confounding variables in this behavioural test (Rogers and Iwamoto, 1993). On the
other hand, the hot-plate response is thought to be mediated by supra-spinal (i.e. brain)
processes, thus it is conceivable that ataxia induced by nicotine may confound
44
interpretation of results from this method (Caggiula et al., 1995). A recent study has
found that the tail-flick reflex requires α4β2 receptors whereas response to hot-plate
requires α4β2 and possibly α7 or other receptor subsets (Damaj et al., 2007a), both
indicating the direct involvement of nicotinic receptors in nicotine-induced analgesia.
45
Section 4 Outline of Chapters
Section 4.1 Chapter 2: Characterization and Comparison of Nicotine and
Cotinine Metabolism In Vitro and In Vivo in DBA/2 and
C57BL/6 Mice
Rationale
In order to determine whether the mouse is a suitable model of nicotine metabolism, the
disposition kinetics of nicotine and its metabolites, as well as the enzyme responsible for
the primary metabolism of nicotine, need to be assessed. Second, to examine whether
interstrain differences in nicotine pharmacology could be contributed to by differences in
nicotine metabolism, nicotine pharmacokinetics between mouse strains also need to be
studied. We hypothesized that the mouse CYP2A5 is responsible for the primary
metabolism of nicotine to cotinine and that strain differences (in CYP2A5) could result in
differences in nicotine pharmacokinetics.
Outline
This chapter describes the studies undertaken to examine the pharmacokinetics of
nicotine and cotinine in mice. Immuno-inhibition studies indicated that, similar to human
CYP2A6, the mouse CYP2A5 is responsible for the primary metabolism of nicotine to
cotinine and cotinine to 3-hydroxycotinine in vitro. The metabolism of nicotine and
cotinine, both in vitro and in vivo, between two commonly studied mouse strains (DBA/2
and C57BL/6) were also described. Pharmacokinetic studies and in vitro assays
indicated that the metabolism of cotinine, but not nicotine, is different between these two
mouse strains.
Contribution to Thesis
46
This chapter contributes to the overall thesis by first describing the pharmacokinetics of
nicotine and cotinine in mice, and second, by defining the contribution of a specific
enzyme (i.e. CYP2A5) responsible for the (in vitro) metabolism of these compounds. The
latter observation provided a line of evidence that the mouse could be a suitable model
for studying inhibitors of human nicotine metabolism. The chapter also suggests that
interstrain genetic variations may affect xenobiotic metabolism in a substrate-dependent
manner.
Section 4.2 Chapter 3: Nicotine Self-Administration in Mice is Associated
with Rates of Nicotine Inactivation by CYP2A5
Rationale
In humans, smoking (nicotine self-administration) is significantly affected by CYP2A6
variations (structure/function). We have previously demonstrated that CYP2A5 is
responsible for the primary metabolism of nicotine to cotinine in mice. However, the
association of nicotine self-administration behaviours with CYP2A5 variations has not
been examined. We hypothesized that oral nicotine self-administration in mice is
associated with CYP2A5 levels and rates of nicotine inactivation.
Outline
This chapter describes the study carried out to determine the amount of CYP2A5 protein
as well as the rates of nicotine metabolism in a group of F2 mice. The F2 mice were
previously generated by breeding a high nicotine consumption strain with a low nicotine
consumption strain. The F2 mice were also tested for nicotine self-administration in a 2-
bottle choice paradigm. In male mice, the amount of nicotine self-administered was
47
significantly associated with the amount of CYP2A5 protein as well as rates of in vitro
nicotine metabolism.
Contribution to Thesis
The above findings further demonstrated that: 1) nicotine metabolism is associated with
CYP2A5; 2) genetic variation in CYP2A5 between DBA/2 and C57Bl/6 mice can alter
nicotine metabolism; and 3) CYP2A5 (and nicotine metabolism) is associated with, and
potentially influencing, nicotine pharmacology including, importantly, nicotine self-
administration. This chapter contributes to the overall thesis by demonstrating that
CYP2A5 influences nicotine metabolism through its expression/activity. In addition,
CYP2A5 can potentially alter the behavioural pharmacology of nicotine. These
observations strengthen the application of the mouse model of nicotine metabolism for
both inhibitor studies as well as genetic studies.
Section 4.3 Chapter 4: Inhibition of Nicotine Metabolism by Methoxysalen:
Pharmacokinetic and Pharmacological Studies in Mice
Rationale
We have previously demonstrated that CYP2A5 levels/activities are significantly
associated with nicotine behavioural pharmacology (i.e. oral nicotine self-administration
behaviour) in mice. This finding is analogous to human studies in which CYP2A6
function is significantly correlated with levels of smoking. We are interested in
determining whether direct manipulation (i.e. inhibition) of CYP2A5, can alter nicotine
metabolism as well as the resulting behavioural response to nicotine. We hypothesized
that inhibition of CYP2A5 would significantly reduce nicotine metabolism and enhance
the pharmacological effects of nicotine.
48
Outline
This chapter describes the use of the CYP2A6 inhibitor, methoxsalen, to determine
whether it can inhibit nicotine metabolism in mice. Initially the examination of the
inhibition of nicotine metabolism in mouse hepatic microsomes was performed to
determine whether this inhibitor is effective in mice (specifically in inhibiting CYP2A5-
mediated nicotine metabolism). This was followed by the in vivo administration of
methoxsalen and the characterization on its impact on nicotine pharmacokinetics and
resulting behavioural pharmacology. The anti-nociception effect of nicotine on tail-flick
and hot-plate procedures was measured as the indicator of the pharmacological effects
of nicotine. Methoxsalen significantly inhibited nicotine metabolism both in vitro and in
vivo. More importantly, the reduction in clearance of nicotine was associated with a
dramatic prolongation in its anti-nociception effect as measured by tail-flick and hot-plate
procedures.
Contribution to Thesis
This chapter contributes to the thesis by demonstrating that: 1) Mice may be suitable for
studying CYP2A6 inhibitors; and 2) inhibition of nicotine metabolism can directly alter the
behavioural pharmacological effects of nicotine. These observations further support the
use of the mouse for the in vivo screenings of novel inhibitors of nicotine metabolism,
especially for the purpose of smoking cessation.
Section 4.4 Chapter 5: Selegiline (L-Deprenyl) is a Mechanism-based
Inactivator of CYP2A6 Inhibiting Nicotine Metabolism in
Humans and Mice
49
Rationale
Selegiline is an anti-Parkinsonian drug currently being investigated as a potential
therapy for smoking cessation due to its ability to decrease dopamine metabolism
through inhibition of MAO-B. In humans chronic treatment with selegiline decreases its
own metabolism. Selegiline belongs to the acetylene group of compounds that are
known mechanism-based inhibitors. Since CYP2A6 has been demonstrated to
metabolize selegiline, we investigated whether selegiline could also inhibit mouse and
human nicotine metabolism. We hypothesized that selegiline can inhibit nicotine
metabolism by mouse CYP2A5 and human CYP2A6. This may be an additional
mechanism by which selegiline mediates smoking cessation.
Outline
This chapter described the various studies performed to characterize the inhibitory effect
of selegiline on nicotine metabolism in mouse and human hepatic microsomes, in cDNA-
expressed CYP2A5 and CYP2A6, and in mouse in vivo. Inhibition of nicotine metabolism
in vitro with major selegiline metabolites (desmethylselegline, L-methamphetamine, and
L-amphetamine) was also performed. Selegiline significantly inhibited nicotine
metabolism in all in vitro enzyme systems studied. Selegiline also inhibited nicotine
metabolism in mice in vivo. Most importantly, selegiline was found to be a potent
mechanism-based inhibitor of CYP2A6-mediated nicotine metabolism.
Contribution to Thesis
This chapter contributes to the thesis by demonstrating that in vitro, and especially in
vivo, the mouse model of nicotine metabolism is a potentially useful for the screening of
novel inhibitors of CYP2A6.
50
CHAPTER 2: CHARACTERIZATION AND COMPARISON OF NICOTINE AND COTININE METABOLISM IN VITRO AND IN VIVO IN DBA/2 AND C57Bl/6 MICE
Eric C. K. Siu and Rachel F. Tyndale As published in: Molecular Pharmacology, 2007, Mar; 71(3):826-834.
All of the experiments, data analyses, and writing of the manuscript were performed by
ECK Siu except for the mass-spectrometry analysis which was performed at the
Proteomic and Mass Spectrometry Centre at the University of Toronto. The study was
designed by ECK Siu and RF Tyndale.
Reprinted from Molecular Pharmacology, Volume 71, Siu et al., “CHARACTERIZATION AND COMPARISON OF NICOTINE AND COTININE METABOLISM IN VITRO AND IN VIVO IN DBA/2 AND C57Bl/6 MICE”, pp 826-834, Copyright (2007) with permission from The American Society for Pharmacology and Experimental Therapeutics. © 2007 The American Society for Pharmacology and Experimental Therapeutics
51
Abstract
DBA/2 and C57Bl/6 are two commonly used mouse strains that differ in response to
nicotine. Previous studies have shown that the nicotine metabolizing enzyme CYP2A5
differs in coumarin metabolism between these two strains suggesting differences in
nicotine metabolism. Nicotine was metabolized to cotinine in vitro by two enzymatic
sites. The high-affinity sites exhibited similar parameters (Km: 10.7 ± 4.8 vs. 11.4 ± 3.6
µM; Vmax: 0.58 ± 0.18 vs. 0.50 ± 0.07 nmol/min/mg, for DBA/2 and C57Bl/6,
respectively). In vivo, the elimination half-lives of nicotine (1 mg/kg, s.c.) were also
similar between DBA/2 and C57Bl/6 mice (8.6 ± 0.4 vs. 9.2 ± 1.6 min, respectively);
however, cotinine levels were much higher in DBA/2 mice. The production and identity of
the putative cotinine metabolite 3’-hydroxycotinine in mice was confirmed by LC/MS/MS.
The in vivo half-life of cotinine (1 mg/kg, s.c.) was significantly longer in the DBA/2 mice
compared to the C57Bl/6 mice (50.2 ± 4.7 vs. 37.5 ± 9.6 min, respectively, p<0.05). The
in vitro metabolism of cotinine to 3’-hydroxycotinine was also less efficient in DBA/2 than
C57Bl/6 mice (Km: 51.0 ± 15.6 vs. 9.5 ± 2.1 µM, p<0.05; Vmax: 0.10 ± 0.01 vs. 0.04 ± 0.01
nmol/min/mg, p<0.05, respectively). Inhibitory antibody studies demonstrated that the
metabolism of both nicotine and cotinine was mediated by CYP2A5. Genetic differences
in Cyp2a5 potentially contributed to similar nicotine but different cotinine metabolism,
which may confound interpretation of nicotine pharmacological studies and studies
utilizing cotinine as a biomarker.
52
Introduction
Nicotine is the primary component of cigarettes that is responsible for the addictive
properties of smoking, which include feelings of pleasure and reward (Henningfield and
Keenan, 1993). Rodents, in particular mice, have been widely used for studying the
pharmacological effects of nicotine (Aschhoff et al., 1999; Stolerman et al., 1999). Two
of the most commonly used mouse strains for studying nicotine behavioral effects are
the DBA/2 and C57Bl/6. A large number of studies have examined various aspects of
nicotine-mediated behaviors such as discrimination, self-administration, tolerance, and
withdrawal, and the majority of these studies have found some differences in nicotine
effects between these strains (Aschhoff et al., 1999; Stolerman et al., 1999).
The amino acid sequence of CYP2A5 is 84% identical to the human CYP2A6,
the main enzyme responsible for the metabolic inactivation of nicotine (Messina et al.,
1997; Nakajima et al., 1996b). The mouse Cyp2a5 gene is genetically polymorphic
(Lindberg et al., 1992). Specifically, the DBA/2 mice express the amino acid Val117 in
hepatic CYP2A5 and metabolized coumarin, a selective probe substrate for mouse
CYP2A5 and human CYP2A6, much more efficiently than C57Bl/6 mice, which express
the amino acid Ala117 (Lindberg et al., 1992). Similarly, mutagenesis of CYP2A6,
substituting the valine with alanine at the same position, also significantly reduced its
catalytic efficiency for coumarin (He et al., 2004b).
Genetic variation in human CYP2A6 can alter nicotine metabolism resulting in
altered smoking behaviors (Malaiyandi et al., 2006; Schoedel et al., 2004). For instance,
individuals who are homozygous for the CYP2A6 deletion variant (CYP2A6*4) produce
minimal cotinine (Yamanaka et al., 2004). These individuals smoke fewer cigarettes and
are less likely to be dependent on tobacco (Malaiyandi et al., 2006; Schoedel et al.,
2004). Likewise, in male mice we have previously shown that lower nicotine self-
53
administration behaviors were associated with lower CYP2A5 protein levels and rates of
nicotine metabolism (Siu et al., 2006). Furthermore, inhibition of CYP2A5-mediated
nicotine metabolism significantly enhanced the pharmacological (i.e. anti-nociceptive)
effects of nicotine in mice (Damaj et al., 2006). These data together suggest that, as in
humans, nicotine metabolism can significantly affect nicotine-mediated behaviors in mice.
Therefore, the main objective of the study was to characterize nicotine and cotinine
metabolism (both in vitro and in vivo) in both the DBA/2 and C57Bl/6 mouse strains.
Such differences may account for the variations observed in the pharmacological effects
of nicotine in these mice.
54
Materials and Methods
Animals
Adult male C57Bl/6 and DBA/2 mice (22-24g) were obtained from Charles River
Laboratories Inc. (Saint-Constant, PQ). Animals were housed in groups of three to four
on a 12-hour light cycle and had free access to food and water. We restricted the study
to male mice as we have previously found large variation in CYP2A levels and nicotine
metabolism among female mice (Siu et al., 2006). This may be due to hormonal
influences (mouse estrous cycle is approximately 3-7 days) as estrogen-only oral
contraceptives increased nicotine metabolism in human females (Benowitz et al., 2006a).
In addition a second CYP2A enzyme, CYP2A4, is present in female mice that may
metabolize nicotine (Murphy et al., 2005b) complicating the interpretation of our CYP2A5
studies.
Reagents
(-)-Nicotine hydrogen tartrate and (-)-cotinine were purchased from Sigma-Aldrich (St.
Louis, MO). Both nicotine and cotinine were dissolved in physiological saline (0.9%
sodium chloride) for use in in vivo studies. Trans-3’-hydroxycotinine was custom-made
by Toronto Research Chemicals Inc. (Toronto, ON). The internal standard 5-
methylcotinine was a generous gift from Dr. Peyton Jacob III at UCSF. All doses are
expressed as the free base of the drug. Inhibitory antibodies against human CYP2A6,
CYP2B6, and CYP2D6 were purchased from BD Biosciences (Mississauga, ON).
Membrane Preparations
Microsomal membranes were prepared from mouse livers for in vitro nicotine
metabolism assays as previously described (Messina et al., 1997; Siu et al., 2006) and
55
stored at -80°C in 1.15% KCl. The cytosolic fractions were acquired during membrane
preparation and were used as a source of aldehyde oxidase. All livers were collected
and frozen prior to 3 pm to avoid circadian effects on CYP2A5 expression.
Nicotine C-Oxidation Assay
Prior to determining the in vitro kinetic parameters (Km and Vmax) for nicotine metabolism
in C57Bl/6 and DBA mice, assay conditions were optimized as previously described (Siu
et al., 2006). Linear formation of cotinine from nicotine was obtained under assay
conditions of 0.5 mg/ml protein concentration with an incubation time of 15 min.
Incubation mixtures contained 1 mM NADPH and 1 mg/ml of mouse liver cytosol in 50
mM Tris-HCl buffer, pH 7.4 and were performed at 37oC in a final volume of 0.5 ml. The
reaction was stopped with a final concentration of 4% v/v Na2CO3. After incubation 5-
methylcotinine (70 µg) was added as the internal standard and the samples were
prepared and analyzed for nicotine and metabolites by HPLC system I as described
previously (Siu et al., 2006). The limits of quantification were 5 ng/ml for nicotine, 12.5
ng/ml for cotinine, and 10 ng/ml for 3’-hydroxycotinine.
Cotinine Hydroxylation Assay
Prior to determining the in vitro kinetic parameters (Km and Vmax) of cotinine metabolism
in C57Bl/6 and DBA mice, assay conditions were optimized. Linear formation of 3’-
hydroxycotinine from cotinine was obtained under assay conditions of 1 mg/ml protein
with an incubation time of 20 min. The incubation mixture was the same as above with
the exception that aldehyde oxidase was not added as cotinine metabolism to 3’-
hydroxycotinine does not require this cytosolic enzyme. Samples were then analyzed by
HPLC system I.
56
In Vivo Nicotine and Cotinine Treatments and Plasma Nicotine, Cotinine, and 3’-
Hydroxycotinine Measurements
To determine the in vivo kinetic parameters of nicotine and cotinine in C57Bl/6 and
DBA/2 mice, animals were injected with nicotine (1 mg/kg, s.c.) or cotinine (1 mg/kg,
s.c.). Blood samples were drawn by cardiac puncture at baseline from untreated animals
and from treated animals at various times after the injections. Immediately after
collection plasmas were prepared by centrifugation at 3000 x g for 10 min and frozen at
–20˚C until analysis. Sample collection took place prior to 3 pm. Total nicotine, cotinine,
and 3’-hydroxycotinine levels (free and glucuronides) were measured following
deconjugation by β-glucuronidase at a final concentration of 5 mg/ml in 0.2 M acetate
buffer, pH 5.0, at 37oC overnight. Samples were then analyzed by HPLC system I.
LC/MS/MS Analysis of Cotinine Metabolite
An alternative HPLC system (system II) suitable for separation of eluate for mass-
spectrometry was used for the characterization of the cotinine metabolite. This system
was similar to that previously described with minor modifications (Murphy et al., 1999).
Briefly, using the same column as HPLC system I, cotinine and its metabolites were
eluted with a linear gradient from 100% A’ (10 mM ammonium acetate buffer, pH 6.5) to
70% A’ and 30% acetonitrile over the course of 30 minutes at a flow rate of 1 ml/min.
Mass-spectrometry analysis was performed at the Proteomic and Mass
Spectrometry Centre at the University of Toronto (Toronto, ON). Data were acquired with
the Q TRAP LC/MS/MS System (Applied Biosystems/MDS Sciex, Toronto, ON). The
sample was injected into the sample loop and delivered to the mass spectrometer by
65% acetonitrile and 0.1% formic acid in water at 20 µl/min. Liquid chromatography
conditions were as described above (system II) except a flow rate of 0.8 ml/min was
57
used. The liquid was introduced to the mass spectrometer directly after 40:1 splitting.
Electrospray ionization was performed in enhanced mass scan (EMS) mode with
positive ionization. Nitrogen was used as curtain gas (25 psi), nebulizer gas (25 psi), and
heater gas (0 psi). The spray needle voltage was set at 5.5KV and collision-induced
dissociation gas was set at high. The decluster potential was 20V, collision energy was
30eV, and entrance potential was 10V. Enhanced product ion (EPI) was performed at a
collision energy of 30eV, all other parameters were same as described for EMS.
Antibody Inhibition of Nicotine and Cotinine Metabolism
We have previously demonstrated that the anti-CYP2A6 antibody was able to cross-
react with mouse CYP2A5 (Siu et al., 2006). Microsomes were preincubated with anti-
human selective P450 antibodies (anti-CYP2A6, anti-CYP2B6, and anti-CYP2D6), at
concentrations of 0, 2, 40, and 80 µl antibodies per mg microsomal protein, for 15
minutes on ice according to manufacturer's instruction. Substrate concentrations used
represented the high-affinity Km value concentrations for nicotine and cotinine
metabolism, specifically 11 µM for nicotine and 51 µM for cotinine for DBA/2 mice
microsomes and 11 µM for nicotine and 9.5 µM for cotinine for C57Bl/6 mice
microsomes.
In Vitro Kinetic and Pharmacokinetic Parameters Analyses
The Michaelis-Menten kinetic parameters Km and Vmax from in vitro metabolism studies
were calculated using Graphpad Prism (Graphpad Software Inc., San Diego, CA) and
were verified by the Eadee-Hofstee method. The equation used to determine Km and
Vmax for one and two enzymatic sites were v = Vmax [S] / (Km + [S]) and v = [Vmax1 [S] /
58
(Km1 + [S])] + [Vmax2 [S] / (Km2 + [S])], respectively, where [S] denotes substrate
concentration.
The in vivo pharmacokinetic parameters were determined using non-
compartmental analysis: AUC0-480, peak plasma concentration (Cmax), maximum plasma
concentration (Tmax). AUC0-480 was calculated using the trapezoidal rule. Elimination half-
life (T1/2) was estimated by the terminal slope. Since the bioavailabilities (F) of nicotine
and cotinine were unknown following subcutaneous injection in mice, CL (clearance)
was determined as a hybrid parameter CL/F and was calculated as Dose/AUC0-480. The
average weights of the animals of the strains were similar (24.8 ± 1.7 vs. 25.5 ± 1.1 g for
DBA/2 and C57Bl/6, respectively, n=50 for each strain), therefore the dose of 25 µg (1
mg/kg) was used for the calculation of CL/F for nicotine.
Statistical Analyses
Statistical analyses of in vitro kinetic parameters were tested by Mann-Whitney U test.
Assessment of in vivo nicotine, cotinine and 3’-hydroxycotinine plasma levels for the
entire time course was not possible from individual animals due to limited blood volume,
therefore each time point represented data from multiple mice. Due to this experimental
design pharmacokinetic parameters (e.g. half life) were estimated by resampling
methods using the PKRandTest software (H. L. Kaplan, Toronto, ON) (Damaj et al.,
2006).
59
Results
In Vitro Nicotine C-Oxidation in DBA/2 and C57Bl/6 Mice
We first assessed the in vitro kinetic parameters of nicotine C-oxidation in hepatic
microsomes prepared from DBA/2 and C57Bl/6 mice. Nicotine metabolism to cotinine,
demonstrated with Michaelis-Menten kinetics (Fig. 2A) and Eadee-Hofstee plotting (Fig.
2B), revealed two enzymatic sites in both strains. The high-affinity sites for hepatic
microsomes from both DBA/2 and C57Bl/6 showed similar Km, Vmax, and Vmax/Km values
for nicotine (Table 1). In contrast, the low-affinity enzymatic sites exhibited differing, and
much lower nicotine metabolic activities with a slight non-significantly higher Km for
C57Bl/6 and Vmax for DBA/2. The Vmax/Km value was significantly higher for the DBA/2
microsomes (Table 1).
Characterization of In Vivo Nicotine Metabolism in DBA/2 and C57Bl/6 Mice
Since the rate of drug metabolism in vitro does not necessarily reflect drug clearance in
vivo (e.g. presence of non-hepatic elimination processes), we determined whether the in
vivo clearance of nicotine was similar between the two mouse strains. Adult male mice
from both strains were treated with 1 mg/kg subcutaneous nicotine, a dose used
previously in nicotine behavioral studies (Zarrindast et al., 2003). In both mouse strains,
nicotine concentrations peaked at 10 minutes with DBA/2 mice having a significantly
greater maximum concentration compared to C57Bl/6 mice (Fig. 3A; Table 2). The
overall AUC0-480 of nicotine was also modestly higher for DBA/2 than for C57Bl/6 mice.
Both strains had similar elimination half-lives for nicotine but the clearance of nicotine
was slower in the DBA/2 mice compared to C57Bl/6 mice.
60
Figure 2. In vitro metabolism of nicotine to cotinine in DBA/2 and C57Bl/6 mice. In vitro kinetic parameters of nicotine metabolism were investigated using hepatic microsomes. A) Rate of formation of cotinine from nicotine (NIC) (mean ± SD of 4 to 5 mice). B) Eadee-Hofstee plots of the kinetic data for DBA and C57Bl/6 mice. See Table 1 for values of kinetics parameters. V: velocity; S: substrate
61
Table 1. In vitro nicotine metabolism (C-oxidation) parameters.
DBA/2 (n=5 mice) mean ± S.D.
C57Bl/6 (n=4 mice) mean ± S.D.
High Affinity Km (µM) 10.7 ± 4.8 11.4 ± 3.6
Vmax (nmol/min/mg) 0.58 ± 0.18 0.50 ± 0.07 Vmax/Km 0.05 ± 0.03 0.04 ± 0.02 Low Affinity Km (µM) 234 ± 77 306 ±126
Vmax (nmol/min/mg) 1.60 ± 0.63 1.18 ± 0.36 Vmax/Km 0.007 ± 0.001* 0.004 ± 0.001 *p < 0.05 compared to C57Bl/6 mice
62
Figure 3. Plasma nicotine and cotinine concentrations following nicotine treatment. Adult male DBA/2 and C57Bl/6 mice were injected with subcutaneous nicotine (1 mg/kg). A) The AUC0-480 of nicotine (NIC) was 15% higher while the elimination half-life was similar in the DBA/2 mice compared to C57Bl/6 mice. B) The AUC0-480 of cotinine (COT) in the DBA/2 mice was twice that of the C57Bl/6 mice. Each time point represents mean (± SD) of 5 to 7 animals for each strain. Values below the limit of quantification are not shown.
63
Table 2. Pharmacokinetic parameters of plasma nicotine and cotinine in mice treated with nicotine (1 mg/kg, s.c.). Results are derived using data from 5 to 7 animals at each time point.
Nicotine Cotinine DBA/2 C57Bl/6 DBA/2 C57Bl/6
AUC0-480 (ng•hr/ml) 92.9 ± 2.9* 80.8 ± 3.2 245 ± 10* 120 ± 7 T1/2 (min) 8.6 ± 0.4 9.2 ± 1.6 51.0 ± 4.1* 23.7 ± 2.0 CL/F (ml/min) 4.5 ± 0.1* 5.2 ± 0.2 ND ND Tmax (min) 10.0 ± 2.2 10.0 ± 4.7 45.0 ± 13.3* 20.0 ± 13.3 Cmax (ng/min) 201 ± 15* 160 ± 15 141 ± 7* 112 ± 12 *p < 0.05 compared to C57Bl/6 mice, ND – not determined
64
When examining the disposition kinetics of cotinine formed from injected nicotine, we
observed that the appearance of the cotinine metabolite was rapid and similar between
the two mouse strains and achieving peak concentrations around 15 minutes (Fig 3B). In
contrast, compared to C57Bl/6 mice, DBA/2 mice showed a significantly larger AUC0-480
and longer elimination half-life (Table 2).
LC/MS/MS Characterization of the Putative Cotinine Metabolite 3’-Hydroxycotinine
In humans cotinine is metabolized exclusively to trans-3’-hydroxycotinine by CYP2A6
(Dempsey et al., 2004; Nakajima et al., 1996a; Yamanaka et al., 2004). To our
knowledge no prior studies have examined or confirmed the production of trans-3’-
hydroxycotinine from cotinine in mice, therefore our immediate goal was to determine
whether mice metabolize cotinine to 3’-hydroxycotinine. In a preliminary in vitro study we
identified a cotinine metabolite that displayed the same retention time as the trans-3’-
hydroxycotinine standard (Fig. 4A). To confirm the identity of the putative trans-3’-
hydroxcotinine compound, a second HPLC system compatible with MS/MS analysis was
used. Both the trans-3’-hydroxycotinine standard and the cotinine metabolite eluted with
the same retention time (10.4 min) using the new HPLC system (Fig. 4B).
In the LC/MS/MS analyses, EMS indicated that the trans 3’-hydroxycotinine
standard had a retention time of 11.8 minutes with a m/z of 193 (Fig. 4C) and when the
metabolite of in vitro cotinine metabolism was monitored at m/z 193, a major peak was
present at 11.1 minutes (Fig. 4D). Fragmentation of the trans-3’-hydroxycotinine
standard ion (m/z 193) gave two fragments of m/z 80 and m/z 134 (Fig. 4E).
Fragmentation of the cotinine metabolite at m/z 193 also gave two major ions of m/z 80
and m/z 134 (Fig. 4F). The peak area ratios of m/z 80/134 for the trans-3’-
hydroxycotinine and the cotinine metabolite were 3.44 and 3.41, respectively. The m/z
65
Figure 4. Characterization of 3’-hydroxycotinine in mice by HPLC and LC/MS/MS. A) Cotinine and its metabolite (COT metabolite) from a pilot in vitro cotinine metabolism study using mouse liver microsomes (DBA/2) were separated with HPLC system I in the absence (left) or presence (right) of the trans-3’-hydroxycotinine standard (3-HC). B) Cotinine and trans-3’-hydroxycotinine standards (left) as well as products from a pilot in vitro cotinine metabolism study (right) were separated with HPLC system II. C) Enhanced mass scan at m/z 193 showed that the trans-3’-hydroxycotinine standard had a retention time of 11.8 minute. D) Liquid chromatography of products from in vitro cotinine metabolism monitored at m/z 193 identified a putative 3’-hydroxycotinine compound with a retention time of 11.1 minutes. E) Fragmentation of the m/z 193 ion (from C) at 30eV produced m/z 80 and m/z 134 ions. F) Fragmentation of the m/z 193 ion (from D) at 11.1 minutes at 30eV produced m/z 80 and m/z 134 ions.
66
80 and the m/z 134 fragments corresponded to (C5H5N)H+ and pyridyl-C3H4O+,
respectively (Murphy et al., 1999).
Characterization of In Vivo Cotinine Metabolism in DBA/2 and C57Bl/6 Mice
Having confirmed that 3’-hydroxycotinine is produced from cotinine in mice, we
proceeded with in vivo injections of cotinine (1 mg/kg, s.c.). Plasma cotinine
concentrations were maximal between 5 to 15 minutes and were similar for both DBA/2
and C57Bl/6 mice (Fig. 5A, Table 3). Similar to cotinine derived from nicotine injection,
following cotinine injection the cotinine AUC0-180 was much higher in the DBA/2 mice
compared to the C57Bl/6 mice. The clearance of cotinine was slower in DBA/2 mice,
which resulted in longer elimination half-life of cotinine compared to C57Bl/6 mice.
The plasma levels of 3’-hydroxycotinine formed from cotinine injections were also
monitored. The plasma AUC0-180 of 3’-hydroxycotinine was higher in the DBA/2 mice
compared to the C57Bl/6 mice (Fig. 5B, Table 3).
In Vitro Cotinine Metabolism in DBA/2 and C57Bl/6 Mice
To determine whether cotinine was metabolized to 3’-hydroxycotinine differently
between the two mouse strains, accounting for the differences in cotinine plasma
concentrations seen in vivo, we performed in vitro cotinine metabolism studies. We
found that cotinine metabolism to 3’-hydroxycotinine was characterized by Michaelis-
Menten kinetics (Fig. 6A), mediated by a single enzymatic site in both strains (Fig. 6B).
The DBA/2 mice had a significantly higher Km compared to the C57Bl/6 mice (Table 4),
while the Vmax for cotinine was much greater for DBA/2 than for C57Bl/6 mice. This
resulted in an overall lower catalytic efficiency (Vmax/Km) for DBA/2 compared to C57Bl/6.
67
Figure 5. Plasma cotinine and 3’-hydroxycotinine concentrations following cotinine injections. Adult male DBA/2 and C57Bl/6 mice were injected with subcutaneous cotinine (1 mg/kg). A) The AUC0-180 of cotinine (COT) was 37% higher and its elimination half-life was 34% longer in DBA/2 mice compared C57Bl/6 mice. B) The AUC0-180 of 3’-hydroxycotinine (3-HC) was 94% higher in the DBA/2 mice compared to C57Bl/6 mice. Each time point represents mean ± SD of 3 to 8 animals for each strain.
68
Table 3. Pharmacokinetic parameters of plasma cotinine and 3’-hydroxycotinine in mice treated with cotinine (1 mg/kg, s.c.). Results are derived using data from 3 to 8 animals at each time point.
Cotinine 3’-hydroxycotinine DBA/2 C57Bl/6 DBA/2 C57Bl/6
AUC0-180 (ng•hr/ml) 1087 ± 33* 796 ± 33 518 ± 30* 268 ± 19 T1/2 (min) 50.2 ± 4.7* 37.5 ± 9.6 ND ND CL/F (ml/min) 0.38 ± 0.01* 0.52 ± 0.02 ND ND Tmax (min) 15.0 ± 7.5 15.0 ± 0.0 60.0 ± 0.0 60.0 ± 23.6 Cmax (ng/ml) 748 ± 35 759 ± 39 255 ± 23* 110 ± 10 *p < 0.05 compared to C57Bl/6, ND – not determined
69
Figure 6. In vitro metabolism of cotinine to 3’-hydroxycotinine in DBA/2 and C57Bl/6 mice. In vitro kinetic parameters of cotinine metabolism were investigated using hepatic microsomes. A) Rate of formation of 3’-hydroxycotinine from cotinine (mean ± SD; n=4 samples per strain). DBA/2 and C57Bl/6 mice. Regression lines and intercepts for both DBA/2 and C57Bl/6 were calculated from Km and Vmax values derived from Michaelis-Menten fitting and no data points were censored. See Table 4 for values of kinetic parameters. V: velocity; S: substrate
70
Table 4. In vitro cotinine hydroxylation parameters.
DBA/2 (n=4) mean ± S.D.
C57Bl/6 (n=4) mean ± S.D.
Km (µM) 51.0 ± 15.6* 9.5 ±2.1
Vmax (nmol/min/mg) 0.10 ± 0.01* 0.04 ± 0.01 Vmax/Km 0.002 ± 0.000* 0.005 ± 0.002 *p < 0.05 compared to C57Bl/6 mice
71
Inhibition of In Vitro Nicotine and Cotinine Metabolism
Previously the mouse CYP2A5 was identified as the enzyme responsible for the high-
affinity metabolism of nicotine using cDNA-expressed CYP2A5 (Murphy et al., 2005b).
To extend these studies characterizing the enzyme involved, we tested the effect of
inhibitory antibodies on in vitro nicotine metabolism. Anti-CYP2A6 inhibitory antibodies
dose-dependently inhibited the formation of cotinine from nicotine in DBA/2 microsomes
with maximal inhibition of 70% at 40 µl antibody/mg microsomal protein (Fig. 7A, filled
symbols). Similar results were seen in hepatic microsomes from C57Bl/6 mice, tested at
80 µl antibody / mg protein (Fig. 7A, open symbols). Inhibitory antibodies against
CYP2B6 and CYP2D6, enzymes postulated to be involved in the remaining small
percentage of metabolism of nicotine in humans (Messina et al., 1997; Nakajima et al.,
1996b; Yamazaki et al., 1999), did not inhibit nicotine metabolism in either mouse strain
(Fig. 7A).
In humans, CYP2A6 is exclusively responsible for the metabolism of cotinine to
trans-3’-hydroxycotinine (Dempsey et al., 2004; Nakajima et al., 1996a). To determine
whether mouse CYP2A5 was also responsible for this metabolic pathway we performed
inhibition studies on cotinine metabolism. Anti-CYP2A6 inhibitory antibodies dose-
dependently inhibited the formation of 3’-hydroxycotinine from cotinine in DBA/2 mouse
liver microsomes (Fig. 7B, filled symbols), with a maximal inhibition of 90% at 40 µl
antibody / mg microsomal protein. Anti-CYP2A6 inhibitory antibodies also inhibited
cotinine metabolism (no detectable metabolite peak) in C57Bl/6 hepatic microsomes (Fig.
7B, open symbols). Inhibitory antibodies against CYP2B6 and CYP2D6 had no effect on
cotinine metabolism (Fig. 7B).
72
Figure 7. Antibody inhibits in vitro nicotine and cotinine metabolism in DBA/2 and
C57Bl/6 mice. A) At Km (11 µM) nicotine, anti-CYP2A6 inhibitory antibodies inhibited cotinine formation maximally by 70% in hepatic microsomes in DBA/2 mice (filled
symbols). At Km (11 µM) nicotine, anti-CYP2A6 inhibitory antibodies (80 µl antibodies / mg protein) inhibited cotinine formation by 70% in C57Bl/6 hepatic microsomes (open
symbols). B) At Km (51 µM) cotinine, anti-CYP2A6 inhibitory antibodies inhibited 3’-hydroxycotinine formation by >90% in hepatic microsomes DBA/2 mice (filled symbols).
At Km (9.5 µM) cotinine, anti-CYP2A6 inhibitory antibodies inhibited 3’-hydroxycotinine formation in C57Bl/6 hepatic microsomes (open symbols). *Activity of cotinine metabolism was below limit of quantification. Anti-CYP2B6 and anti-CYP2D6 antibodies had no effect on nicotine or cotinine metabolism. Values are expressed as a percent of activity in the absence of antibodies and were determined from pooled sample of 2 animals.
73
Discussion
In the present study we examined both in vitro metabolism and in vivo pharmacokinetics
of nicotine and cotinine in two commonly used inbred mouse strains, DBA/2 and C57Bl/6.
Mice have been used extensively for the study of nicotine behaviors; however, the
effects of nicotine vary widely between strains (Aschhoff et al., 1999; Stolerman et al.,
1999). One potential contributing factor may be differences in nicotine pharmacokinetics
which can significantly alter its pharmacology. CYP2A5 metabolizes nicotine (Murphy et
al., 2005b) and previous studies found that DBA/2 and C57Bl/6 mice differed in CYP2A5
structure and function (Lindberg et al., 1992). Therefore the primary goal of this study
was to determine whether nicotine and cotinine metabolism differed between these two
strains, which may be contributed by the Cyp2a5 polymorphism between these mice
(Lindberg et al., 1992).
The in vitro metabolism of nicotine to cotinine was mediated by a high- and a
low-affinity enzyme site in both mouse strains. The Km values for the high-affinity sites
reported here are consistent with those seen using cDNA-expressed CYP2A5 (7.7 ± 0.8
µM; 129/J mouse strain) (Murphy et al., 2005b) but are modestly higher affinity relative
to hepatic microsomes from ICR mice (18.6 ± 5.9 µM) (Damaj et al., 2006). The identity
of the high-affinity site was confirmed, as CYP2A5 inhibitory antibodies inhibited up to
70% of nicotine metabolism at Km for nicotine in both strains. The low-affinity sites in our
mice could potentially belong to the 2B family. In humans, cDNA-expressed CYP2B6
metabolizes nicotine but with much lower affinity and activity compared to CYP2A6
(Yamazaki et al., 1999). In monkeys, CYP2B6agm is a minor enzyme compared to
CYP2A6agm for the metabolism of nicotine to cotinine (Schoedel et al., 2003). In
contrast, rat CYP2B1/2 is the primary enzyme responsible for this process (Nakayama et
al., 1993). In our experiments, however, no indication of inhibition of nicotine metabolism
74
was seen at the highest CYP2B antibody concentration tested. Considering that at the
highest plasma nicotine concentrations observed (~200 ng/ml ≅ 1.2 µM) following
nicotine injection the estimated contribution of the low-affinity enzymes to nicotine
metabolism was only ≤10%, the identity of this enzyme was not pursued.
To determine if nicotine was metabolized similarly between DBA/2 and C57Bl/6
mice in vivo we administered nicotine subcutaneously as this route kinetically mimics,
somewhat, the route of nicotine intake from smoking in that it bypasses first-pass
metabolism. The in vivo kinetics of nicotine in these two strains differed though not
dramatically. The higher nicotine Cmax in DBA/2 mice may be due to a smaller volume of
distribution of nicotine: male DBA/2 mice have, on average, 34-40% more body fat and
10% lower lean mass compared to C57Bl/6 mice (Mouse Phenome Database, Jackson
Labs) which could result in higher levels of nicotine in the plasma (and other highly
perfused organs such as liver, kidneys, and the lung) (Urakawa et al., 1994). Despite
similar nicotine clearance, however, we found that cotinine was removed more slowly in
DBA/2 mice as demonstrated by the two-fold longer elimination half-life and higher
cotinine AUC0-480.
In humans, the main metabolites of cotinine recovered in urine are trans-3’-
hydroxycotinine and its glucuronide which account for 40-60% of the total administered
dose of nicotine (Hukkanen et al., 2005b). Initially we demonstrated that mice produced
3’-hydroxycotinine from cotinine with LC/MS/MS. We then confirmed that the metabolism
of cotinine to 3’-hydroxycotinine was mediated by CYP2A5 – up to 90% of cotinine
metabolism to 3’-hydroxycotinine was inhibited by anti-CYP2A6 inhibitory antibodies.
This is consistent with the metabolism of cotinine to 3’-hydroxycotinine being mediated
exclusively by human CYP2A6 in vitro and in vivo (Dempsey et al., 2004; Nakajima et al.,
1996a). Following cotinine injections, DBA/2 mice showed slower clearance of cotinine
75
compared to C57Bl/6 mice, which was consistent with the pharmacokinetics of cotinine
formed from nicotine injections. It is likely that DBA/2 mice have a slower hepatic
(intrinsic) clearance of cotinine to 3’-hydroxycotinine compared to C57Bl/6; this is
supported by our in vitro findings that cotinine was metabolized to 3’-hydroxycotinine
significantly slower in the DBA/2 compared to C57Bl/6 mice. Even at the maximum
plasma cotinine concentrations observed (~760 ng/ml ≅ 4.3 µM) our in vitro data
indicated that the DBA/2 mice metabolized cotinine slower than the C57Bl/6 mice (v =
0.008 vs. 0.0012 nmol/min/mg, respectively).
When examining the plasma concentrations of 3’-hydroxycotinine formed from
cotinine, we found that DBA/2 mice had a larger AUC0-180 compared to C57Bl/6 mice.
The higher level of 3’-hydroxycotinine in DBA/2 mice was likely to be due to reduced
elimination of 3’-hydroxycotinine. This could occur through slower rates of conjugation to
O-glucuronide and/or slower renal excretion of 3’-hydroxycotinine and its glucuronidated
metabolite. In mice, N-glucuronides of nicotine and its proximal metabolites have not
been detected nor identified (although O-glucuronides were not measured) (Ghosheh
and Hawes, 2002), while in humans, ~80% of trans-3’-hydroxycotinine is excreted
unchanged (Hukkanen et al., 2005b). Thus we believe the higher level of 3’-
hydroxycotinine was most likely due to slower renal excretion in the DBA/2 mice.
This study showed the in vitro and in vivo metabolism of nicotine was similar
between DBA/2 and C57Bl/6 mice. In contrast to nicotine, DBA/2 mice metabolized
cotinine to 3’-hydroxycotinine with lower efficiency compared to C57Bl/6 mice both in
vitro and in vivo. These data indicate that genetic differences in the structure of CYP2A5
between the two strains can potentially alter the rate of metabolism depending on the
specific substrate. The DBA/2 CYP2A5, which has valine at position 117, is more
efficient at metabolizing coumarin compared to C57Bl/6, which has an alanine in this
position (Lindberg et al., 1992; van Iersel et al., 1994). In contrast, this genetic variant
76
does not appear to alter the metabolism of nicotine, but rather, it reduces the catalytic
activity for cotinine. Mouse CYP2A5 oxidizes nicotine to ∆5’(1’)-iminium ion followed by
conversion to cotinine by aldehyde oxidase (Murphy et al., 2005b); however, no
functional polymorphisms for the mouse aldehyde oxidase genes have been reported
(Mouse Genome Informatics), and our in vivo data was consistent with our in vitro data
suggesting the variations in aldehyde oxidases were not contributing substantively to our
observations. Substrate-selective metabolisms by genetic variants of human CYP2A6
have been observed. For example, the CYP2A6*7 variant has reduced nicotine
metabolic activity, but coumarin metabolism was minimally affected (Ariyoshi et al.,
2001). In addition, different levels of cotinine following similar nicotine intake have been
observed in smokers (Benowitz et al., 1999), and this may be partly related to genetic
variations in CYP2A6 that have minor impacts on the metabolism of nicotine relative to
the impact on cotinine metabolism. Finally, these observations warrant further studies on
the metabolic activation of CYP2A5/6 substrates such as NNK, a tobacco-specific
nitrosamine known to cause lung cancer (Miyazaki et al., 2005), and the consequence of
these genetic variants on NNK activation. Future studies will focus on the expression of
variants V117A (found in CYP2A5 [Lindberg et al., 1992] and CYP2A13 [NCBI]) as well
as F118L (found in CYP2A6 [NCBI]), in all three enzymes and their impact on multiple
substrates including NNK.
The finding that cotinine is differentially metabolized relative to nicotine, between
strains, has implications with respect to interpreting nicotine pharmacological studies.
Cotinine can pass through the blood brain barrier (Lockman et al., 2005). In rats, cotinine
can bind to epibatidine-sensitive nicotinic receptors in frontal cortex and hippocampus
tissues, although with lower affinity than nicotine (Vainio and Tuominen, 2001).
Furthermore, administration of cotinine in rat striatal tissues evoked dopamine overflow
in a dose-dependent manner (Dwoskin et al., 1999). Thus, differing levels of cotinine
77
may result in altered pharmacological effects despite similar nicotine levels between the
two mouse strains, and the effects may be erroneously attributed to nicotine
pharmacology. On the other hand, the effects of cotinine in humans are less clear
(Buccafusco and Terry, 2003; Crooks and Dwoskin, 1997). Cotinine alone did not show
pharmacological effects, but it did interfere with the ability of nicotine patch to reduce
withdrawal symptoms (Hatsukami et al., 1998b). Cotinine also increased plasma nicotine
levels in smokers, possibly through increased smoking to compensate for the
interference of nicotine action by cotinine (Hatsukami et al., 1998a). In other studies
cotinine appeared to have some effects in reducing withdrawal symptoms (Benowitz et
al., 1983; Keenan et al., 1994).
Consideration should also be taken when using cotinine as a biomarker for
environmental tobacco smoke exposure (Benowitz, 1999) and cigarette intake (de Leon
et al., 2002) in humans, or nicotine consumption in mice (Sparks and Pauly, 1999), as
cotinine can be metabolized at different rates by human CYP2A6 and mouse CYP2A5.
Differing levels of cotinine could be erroneously interpreted as different levels of
exposure rather than differing rates of removal.
In conclusion, we have characterized nicotine and cotinine metabolism in two
mouse strains which differed in CYP2A5 enzyme structure (Lindberg et al., 1992). While
coumarin metabolism differed (Lindberg et al., 1992) we observed no substantial
difference in nicotine metabolism. In contrast, CYP2A5-mediated cotinine metabolism to
3’-hydroxycotinine was different between the mouse strains, which may confound
interpretation of pharmacological and biomarker studies.
78
Acknowledgments
We would like to thank Linda Liu for assistance in HPLC and Dr. Sharon Miksys and Jill
Mwenifumbo for critical review of this manuscript. RFT is a shareholder and chief
scientific officer of Nicogen Inc., a company focused on the development of novel
smoking cessation therapies; no funds were received from Nicogen for these studies
and this manuscript was not reviewed by other people associated with Nicogen prior to
submission or revision.
Footnote
This study was supported by CAMH and CIHR grant # MOP 53248. CIHR-Special
Training Program in Tobacco Control and OGS to ECKS and Canada Research Chair in
Pharmacogenetics to RFT.
79
SIGNIFICANCE OF CHAPTER
This study described the in vitro and in vivo metabolism of both the nicotine and cotinine
in mice. Similar to human CYP2A6, the mouse CYP2A5 is responsible for the majority of
metabolism of nicotine to cotinine, and it appears to be exclusively responsible for the
metabolism of cotinine to 3-HC. This provided a line of evidence that the mouse may be
a suitable model for studying inhibitors of nicotine metabolism. In addition, the study also
showed that polymorphism in CYP2A5 can affect cotinine metabolism differentially
compared with nicotine. This suggested that careful interpretations are required in
nicotine pharmacology studies as well as in studies where cotinine are used as
biomarkers.
80
CHAPTER 3: NICOTINE SELF-ADMINISTRATION IN MICE IS ASSOCIATED WITH RATES OF NICOTINE INACTIVATION BY CYP2A5
Eric C. K. Siu, Dieter B. Wildenauer, and Rachel F. Tyndale As published in: Psychopharmacology, 2006, Mar; 184(3-4):401-408.
The immunoblotting and in vitro enzyme activities assays, data analyses, and writing of
the manuscript were performed by ECKS. The nicotine self-administration study was
performed by DB Wildenauer. The study was designed by ECK Siu and RF Tyndale.
Reprinted from Pyschopharmacology, Volume 184, Siu et al., “NICOTINE SELF-ADMINISTRATION IN MICE IS ASSOCIATED WITH RATES OF NICOTINE INACTIVATION BY CYP2A5”, pp 401-408, Copyright (2006) with permission from Springer-Verlag. © 2006 Springer-Verlag
81
Abstract
Rationale: Cyp2a5, the mouse homologue of human CYP2A6, encodes for the enzyme
responsible for the primary metabolism of nicotine. Variation in human CYP2A6 activity
can alter the amount smoked such as numbers of cigarettes smoked per day and
smoking intensity. Different mouse strains self-administer different amounts of oral
nicotine and quantitative trait loci analyses in mice suggested that Cyp2a5 may be
involved in differential nicotine consumption behaviours. Objectives: The goal of this
study was to examine whether in vivo nicotine consumption levels were associated with
CYP2A5 protein levels and in vitro nicotine metabolism in mice. Methods: F2 mice
propagated from high (C57Bl/6) and low (St/bJ) nicotine consuming mice were analyzed
for CYP2A5 hepatic protein levels and in vitro nicotine metabolizing activity. Results:
We found that F2 male high nicotine (n=8; 25.1±1.2 µg nicotine/day) consumers had
more CYP2A5 protein compared to low (n=11; 3.8±1.4 µg nicotine/day) consumers
(10.2±1.0 vs. 6.5±1.3 CYP2A5 units). High consumers also metabolized nicotine faster
than the low consumers (6 µM: 0.18±0.04 vs. 0.14±0.07; 30 µM: 0.36±0.06 vs.
0.26±0.13; 60 µM: 0.49±0.05 vs. 0.32±0.17 nmol/min/mg). In contrast, female high
(25.1±2.1 µg nicotine/day) and low (4.7±1.4 µg nicotine/day) nicotine consumers did not
show pronounced differences in nicotine metabolism or CYP2A4/5 protein levels; this is
consistent with other studies of sex differences in response to nicotine. Conclusions:
These data suggested that among male F2 mice, increased nicotine self-administration
is associated with increased rates of nicotine metabolism, most likely as a result of
greater CYP2A5 protein levels.
82
Introduction
Nicotine is the main constituent of tobacco responsible for the addictive property
of cigarettes (Stolerman and Jarvis, 1995). Once dependent, smokers adjust their
smoking behaviours (e.g. number of cigarettes smoked, puff intensity) to maintain
constant nicotine plasma levels (McMorrow and Foxx, 1983; Russel, 1987). As a
consequence, factors affecting nicotine clearance alter an individual’s smoking
behaviours. In humans approximately 80% of nicotine is metabolically inactivated to
cotinine by the liver and approximately 90% of this reaction is carried out by CYP2A6
(Benowitz et al., 1994; Messina et al., 1997; Nakajima et al., 1996a). Individuals with
different reduced activity variants of CYP2A6 smoke lower numbers of cigarettes; for
instance, smokers who are slow metabolic inactivators of nicotine smoke fewer
cigarettes compared to those who are normal or faster metabolizers (Rao et al., 2000;
Schoedel et al., 2004). To further investigate the impact of nicotine metabolism on
nicotine consumption, a genetic and kinetic study in mice was initiated.
The mouse is a better suited model for the study of nicotine metabolism and
behaviours compared to rats for several reasons. First, the predominant enzymes
responsible for the metabolism of nicotine in rats belong to the CYP2B family
(Nakayama et al., 1993; Schoedel et al., 2001). Second, CYP2A5 is approximately 84%
identical to CYP2A6 in amino acid sequence, and cDNA-expressed CYP2A5
metabolizes nicotine with similar efficiency to human CYP2A6 (Murphy et al., 2005a).
Finally, the close structural similarity between CYP2A5 and CYP2A6 allows for the
development of inhibitors and in vivo screening of these compounds in mice. In addition,
the behavioural and physiological effects of nicotine in mice have been well documented
(Collins et al., 1988; Faraday et al., 2005; Grabus et al., 2005; Marks et al., 1985).
83
With respect to nicotine intake, it has been shown that different mouse strains self-
administer different amounts of oral nicotine (Aschhoff et al., 1999; Robinson et al.,
1996), and a variety of mechanisms may explain the differential preferences. These
include differences in the rewarding properties of nicotine and taste preference for bitter
compounds (i.e. nicotine) (Lush and Holland, 1988; Merlo Pich et al., 1999).
Acetylcholine receptor density and receptor affinity for nicotine could also potentially
alter nicotine preference (Marks et al., 1989; Marks et al., 1986). However, there are no
studies examining the effect of differing rates of nicotine metabolism on oral nicotine
self-administration in mice. Recently, a quantitative trait loci study revealed that the
Cyp2a5 gene is situated close to a marker on Chromosome 7 associated with differential
nicotine self-administration behaviours, suggesting a possible involvement of Cyp2a5 in
nicotine consumption behaviours in mice (Aschhoff et al., 1999; Wildenauer et al., 2005).
The goal of this study was to determine whether CYP2A5 protein levels, and resulting
nicotine metabolism, differed between the high and low nicotine consuming mice
examined in the QTL study. The mice were selected from the F2 generation and would
allow us to address the relationship between gene function and behaviour.
84
Materials and Methods
Animals
The F2 mice were established by crossing the F1 mice generated from a high
nicotine-consuming strain (C57Bl/6) and a low-nicotine consuming strain (St/bJ)
(Aschhoff et al., 1999). The mice were housed individually in polycarbonate cages with
wood-shaving bedding. Each cage contained two graduated bottles with one on the left
and one on the right side of each cage cover. The bottles were filled with tap water and
total consumption from each bottle was estimated from the decrease of the fluid cylinder
along the graduation of the tube every day during a 10 day acclimation period. On day
11 one bottle was filled with tap water, containing 5 µg/ml nicotine tartrate, while the
other one contained tap water only. Subsequently, nicotine concentrations were raised
to 10, 25, 50, 75 and 100 µg/ml at 5-day intervals. To minimize problems associated with
side preference, the position of the bottles at the right or left side of the cage was
changed every day after measuring consumption. During the experiment, animals were
fed ad libitum (24-hour free-access). There were a total of 485 F2 mice, of which the 46
lowest nicotine-consuming mice and the 47 highest nicotine-consuming mice were
selected for genotyping and QTL analyses (Wildenauer et al., 2005). From these we
analyzed the highest and lowest nicotine-consuming mice (males and females). High
nicotine consumers (HNC, n=22) and low nicotine consumers (LNC, n=23) were
selected based on the highest and lowest average amount of nicotine consumed over
the testing period, respectively. Animals were sacrificed after the last exposure at the
beginning of the light cycle at the end of the test. Following sacrifice the livers were
removed and frozen in liquid nitrogen and transferred to -80oC for storage.
85
Membrane Preparations Microsomal membranes were prepared from mouse livers for the in vitro nicotine
metabolism assay and stored at 80°C in 1.15% KCl as previously described (Messina
et al., 1997). Briefly, the livers were homogenized in 1.15% KCl buffer and centrifuged at
9,000 g for 20 minutes. The supernatant fractions were collected and centrifuged again
at 100,000 g for 90 minutes. The resultant microsomal pellets were resuspended in
1.15% KCl buffer for in vitro NCO assay. The cytosolic fractions (supernatant) were also
collected from each of the membrane preparations and were pooled for used as the
common source of aldehyde oxidase. For immunoblotting, microsomal membranes were
prepared as above with the use of 100 mM Tris, pH 7.4, 0.1 mM EDTA, 0.1 mM
dithiothreitol, 1.15% (w/v) KCl, and 20% (v/v) glycerol. Protein concentrations were
determined with the Bradford reagent according to manufacturer's protocol (Bio-Rad
Labs Ltd., Mississauga, ON).
Immunoblotting Immunoblotting was performed essentially as previously described (Schoedel et al.,
2003). To determine the linear range of CYP2A5 detection for the immunoblotting assay
mouse liver microsomes were serially diluted and used to construct standard curves
(0.05 to 12.8 µg protein). Membrane proteins from livers (4.5 µg) were separated by
SDS-polyacrylamide gel electrophoresis (4% stacking and 8% separating) and
transferred overnight onto nitrocellulose membranes. Human lymphoblastoid cDNA-
expressed CYP2A6 (BD Biosciences, San Jose, CA) was used as a positive control. For
the detection of CYP2A proteins, nitrocellulose membranes were preincubated for 1 h in
a blocking solution containing 1% skim milk powder (w/v), and 0.1% bovine serum
albumin (w/v) in Tris-buffered saline-Triton X-100 [50 mM Tris, pH 7.4, 150 mM NaCl,
86
0.1% (v/v) Triton X-100]. Membranes were probed with a monoclonal antibody to human
CYP2A6 (1:3000 dilution) (BD Biosciences), a horseradish peroxidase-conjugated anti-
mouse secondary antibody (1:20000 dilution) (Pierce Biotechnology, Rockfort, IL),
followed by detection using enhanced chemiluminescence (Pierce Biotechnology).
Controls included immunoblots that were incubated without primary antibody as well as
immunoblots loaded with several other cDNA-expressed CYPs for the assessment of
cross-reactivity. Nitrocellulose membranes were exposed to Progene autoradiographic
film (Ultident Scientific, St. Laurent, P.Q.) for 0.5 to 2 min. Immunoblots were analyzed
using the MCID imaging system (Imaging Research Inc., St. Catherines, ON, Canada).
Nicotine C-Oxidation (NCO) Assay To establish assay linearity, increasing concentrations of hepatic microsomal proteins (0,
0.25, 0.5, 1, 1.5, 2, 2.5, 3 mg/ml) were incubated with 65 and 650 µM of nicotine for 30
minutes; microsomal protein (0.5 mg/ml) were incubated for various durations (0, 5, 10,
15, 30, 45, 60 mins) with 65 and 650 µM of nicotine. These concentrations of nicotine
were originally examined based on human pharmacokinetic variables (Messina et al.,
1997), and then refined as data on the mice was generated. Varying concentrations of
cytosolic protein (0.3, 0.6, 0.75, 0.9, 1.1, 1.2 mg/ml) were incubated with 0.5 mg/ml of
microsomal protein and with 30 and 300 µM of nicotine for 15 minutes to ensure that
aldehyde oxidase was not rate limiting. This is important since the conversion of nicotine
to cotinine follows two steps: the first of which is the conversion of nicotine to nicotine-
∆1’(5’)-iminium ion by CYP2A5. The imimium ion is then converted to cotinine by aldehyde
oxidase which is present in the cytosolic fraction (Brandange and Lindblom, 1979). From
these studies a protein concentration of 0.5 mg/ml and an incubation time of 15 min was
selected for all subsequent assays. Incubation mixtures also contained 1 mM NADPH
and 1 mg/ml of mouse liver cytosol in 50 mM Tris-HCl buffer, pH 7.4 and were
87
performed at 37oC in a final volume of 0.5 ml. The reaction was stopped with 4%
Na2CO3, and 5-methylcotinine (70 µg) was added as the internal standard. Nicotine
metabolizing activity was analyzed as previously described (Messina et al., 1997) with
the modification of the solid phase extraction procedure for assessment of 3-
hydroxycotinine. Briefly, samples were extracted using ISOLUTE HM-N columns
(Argonaut Technologies, Redwood City, CA) with 13 ml of dichloromethane and were
dried under nitrogen. Samples were reconstituted with 105 µl of 0.01 M HCl and 90 µl of
each sample was analyzed by HPLC with UV detection (260 nm). Separation of nicotine,
cotinine, and 3-hydroxycotinine was achieved using a ZORBAX Bonus-RP column (5 µm,
150 × 4.6 mm; Agilent Technologies Inc., Mississauga, ON) and a mobile phase
consisting of acetonitrile/potassium phosphate buffer (10:90 v/v, pH 5.07) containing
3.3 mM heptane sulfonic acid and 0.5% triethylamine. The separation was performed
with isocratic elution at a flow rate of 0.9 ml/min. Nicotine, cotinine, and 3-
hydroxycotinine sample concentrations were determined from standard curves. The
quantitation limits were 5 ng/ml for nicotine, 12.5 ng/ml for cotinine, and 10 ng/ml for 3-
hydroxycotinine.
Statistical Analyses Data were analyzed by Graphpad Prism (version 4.00, San Diego, CA). Statistical
significance was calculated using Mann-Whitney U test and the significance level was
set at p=0.05. Correlation analyses were performed using Pearson’s method.
88
Results
CYP2A5 detection in mouse liver An immunoblotting assay was developed in order to detect CYP2A5 in male mouse liver
microsomes. The CYP2A5 signal was linear between 2.5 to 10 µg of microsomal protein
(Fig. 8a & b). Selectivity was confirmed by incubating the immunoblots without the
primary antibody (Fig. 8c). The primary antibody did not react with several alternative
cDNA-expressed CYPs (Fig. 8d).
Increased oral nicotine self-administration in male mice is associated with higher CYP2A5 protein levels Male mice in the high nicotine consumption (HNC) group self-administered significantly
higher amounts of oral nicotine compared to the low nicotine consumption (LNC) group
(Table 5; Fig. 9a). We first determined if the increased consumption of nicotine in the
male mice was associated with elevated levels of hepatic CYP2A5 protein. HNC mice
had ~60% more CYP2A5 protein compared to the LNC mice (10.2 ± 1.0 vs. 6.5 ± 1.3
units, respectively, p=0.03, Fig. 9a). Figure 9b shows a representative immunoblot of
CYP2A5 expression in both the high and the low consumption mouse groups.
In vitro NCO rate is faster in high nicotine consuming male mice In addition to affecting levels of protein expression, genetic variation in CYP2A5 may
also affect protein structure altering the interaction with nicotine (Km). We compared the
kinetic parameters of in vitro NCO activities between the HNC and LNC mice. We first
optimized assay conditions and linearity for mouse NCO reactions. We established good
separation of peaks (Fig. 10a) and identified appropriate concentration of cytosol (1
89
Figure 8. Selectivity and linearity of CYP2A5 immunoblotting for mouse liver microsomes. a) A dilution curve of mouse liver microsomal proteins detected by immunoblotting (± SEM, 3 experiments). Representative immunoblots incubated with (b) and without (c) primary antibody. d) cDNA-expressed CYP proteins as well as mouse liver microsomal protein detected (lanes 1:CYP1A1, 2:CYP1A2, 3:CYP2A2, 4:CYP3A2, 5:CYP1B1, 6:CYP2C11, 7:CYP2D, 8:CYP2E1, 9:CYP2A6; 0.2 pmol each; lane 10:mouse liver microsomal protein, 2 µg).
1047kDa
1 2 3 4 5 6 7 8 9
b)
c)
d)
2.4 3.2 4.8 6.4 9.6 µg0 12.8
a)
0.0 2.5 5.0 7.5 10.00
1
2
3
4
5
6
7
8
Protein (µµµµg)C
YP
2A
5 D
en
sit
y U
nit
s (
±± ±±S
EM
)
1047kDa
1 2 3 4 5 6 7 8 9
b)
c)
d)
2.4 3.2 4.8 6.4 9.6 µg0 12.8
a)
0.0 2.5 5.0 7.5 10.00
1
2
3
4
5
6
7
8
Protein (µµµµg)C
YP
2A
5 D
en
sit
y U
nit
s (
±± ±±S
EM
)
90
Table 5. Average nicotine consumption (µg/day; mean ± SD) in F2 male and female mice in a two-bottle choice paradigm.
LNC Group HNC Group Male 3.8 ± 1.4 (n=11) 25.1 ± 1.2* (n=8)
Female 4.7 ± 1.4 (n=12) 25.1 ± 2.1* (n=14) *p<0.05 compared to the LNC group
91
Figure 9. CYP2A5 levels are higher in male high nicotine consuming mice. a) Microsomal proteins from both male LNC (n=11) and HNC (n=8) mice were analyzed for CYP2A5 protein levels. For each animal, CYP2A5 protein levels were plotted against nicotine (NIC) consumption. Protein data are from 4 independent immunoblots for each animal. b) A representative immunoblot of CYP2A5 in LNC and HNC mice. *p=0.03
47 kDa
LNC HNCb)
a)
0 10 20 300
5
10
15
20
25LNC
HNC
NIC Consumption (µµµµg/day)
CY
P2
A5
De
ns
ity
Un
its
*
47 kDa
LNC HNCb)
a)
0 10 20 300
5
10
15
20
25LNC
HNC
NIC Consumption (µµµµg/day)
CY
P2
A5
De
ns
ity
Un
its
*
0 10 20 300
5
10
15
20
25LNC
HNC
NIC Consumption (µµµµg/day)
CY
P2
A5
De
ns
ity
Un
its
*
92
Figure 10. Optimization of nicotine-C-oxidation assay. a) HPLC separation of a blank sample containing the standards nicotine (NIC), cotinine (COT), 3-hydroxycotinine (3HC), and the internal standard 5-methylcotinine (5MC). Assay conditions were optimized for: b) hepatic microsomal protein concentration; c) incubation time and; d) I cytosolic protein concentration. Arrows at the X-axis indicate conditions used for subsequent experiments.
0 15 30 45 600
5
10
15
20
25
30
35
40
4565 µM
650 µM
Incubation time (mins)
Nic
oti
ne C
-oxid
ati
on
(nm
ol co
tin
ine f
orm
ed
/mg
pro
tein
)
0.0 0.3 0.6 0.9 1.2 1.50.0
0.1
0.2
0.3
0.4
0.530 µM
300 µM
Cytosolic protein (mg/ml)
Nic
oti
ne C
-oxid
ati
on
(nm
ol c
oti
nin
e f
orm
ed
/min
/mg
pro
tein
)
0 1 2 30.0
0.1
0.2
0.3
0.4
0.565 µM
650 µM
Microsomal protein (mg/ml)
Nic
oti
ne
C-o
xid
ati
on
(ng
co
tin
ine
fo
rmed
/min
)
c)
b)
d)
a)
COT
NIC
5 10 15
Retention time (min)
3HC
Ab
so
rban
ce (
mA
U)
5MC
0 15 30 45 600
5
10
15
20
25
30
35
40
4565 µM
650 µM
Incubation time (mins)
Nic
oti
ne C
-oxid
ati
on
(nm
ol co
tin
ine f
orm
ed
/mg
pro
tein
)
0.0 0.3 0.6 0.9 1.2 1.50.0
0.1
0.2
0.3
0.4
0.530 µM
300 µM
Cytosolic protein (mg/ml)
Nic
oti
ne C
-oxid
ati
on
(nm
ol c
oti
nin
e f
orm
ed
/min
/mg
pro
tein
)
0 1 2 30.0
0.1
0.2
0.3
0.4
0.565 µM
650 µM
Microsomal protein (mg/ml)
Nic
oti
ne
C-o
xid
ati
on
(ng
co
tin
ine
fo
rmed
/min
)
c)
b)
d)
a)
COT
NIC
5 10 15
Retention time (min)
3HC
Ab
so
rban
ce (
mA
U)
5MC
COT
NIC
5 10 15
Retention time (min)
3HC
Ab
so
rban
ce (
mA
U)
5MC
93
mg/ml, Fig. 10b) and microsomes (0.5 mg/ml, Fig. 10c), as well as the incubation time
(15 mins, Fig. 10d) for the assay.
Next, based on the recent publication of the mouse nicotine kinetics using
expressed cDNAs, the Km value (7.7 µM) from the cDNA-expressed CYP2A5 (Murphy et
al., 2005a) we examined nicotine at 6, 30, and 60 µM - concentrations predicted to allow
for the separation of structural (Km) and expression (Vmax) differences between the HNC
and LNC groups. We found that the NCO activities were significantly higher in the male
HNC group compared to the LNC group at all three nicotine concentrations (6 µM:
0.18±0.04 vs. 0.14±0.07 nmol/min/mg, p=0.02; 30 µM: 0.36±0.06 vs. 0.26±0.13
nmol/min/mg, p=0.01; 60 µM: 0.49±0.05 vs. 0.32±0.17 nmol/min/mg, p=0.01; Figs. 11a &
b), consistent with the higher levels of CYP2A5 protein. There was also a relatively good
association (R2=0.28; p=0.02; Fig. 11c) between NCO activities (60 µM of nicotine) and
CYP2A5 protein levels. We found that while the predicted Vmax was significantly higher in
the HNC mice, the apparent Km was not different between the two groups (Table 6). The
concentrations of nicotine, cotinine, and 3-hydroxycotinine, as measured by HPLC at 30
µM nicotine, are listed in Table 7.
Nicotine self-administration in female mice was not associated with CYP2A5 protein levels or in vitro NCO activity Similar to males, the female mice in the HNC group consumed significantly more
nicotine compared to the LNC group (Table 5; Fig. 12a & b). When we measured
CYP2A protein, no differences in CYP2A levels between the female HNC and LNC mice
were observed (Fig. 12a). When we assessed NCO activities, we found that the female
HNC (n=14) mice tended to metabolize nicotine (30 µM) at higher rates compared to the
LNC (n=12) but this did not reach significant difference (0.43 ± 0.18 vs. 0.39 ± 0.30
nmol/min/mg; p=0.12; Fig. 12b). There were also no differences in activities at 60 µM
94
Figure 11. Nicotine-C-oxidation activity in male high nicotine consuming mice is higher compared to the low nicotine consuming mice. a) Liver microsomes from male LNC and HNC mice were tested for velocity (V) of cotinine formation (mean ± SD). b) Velocities at 30 µM nicotine were plotted against averaged daily nicotine consumption. c) Correlation between CYP2A5 protein levels and velocity at 60 µM nicotine. *p=0.02 and **p=0.01 compared to the LNC group.
6 uM 30 uM 60 uM0.0
0.1
0.2
0.3
0.4
0.5
0.6 LNC
HNC
*
**
**
6 µµµµM 30 µµµµM
NIC
V (
nm
ol/
min
/mg
)
60 µµµµM
a) b)
c)
0 10 200.00
0.25
0.50
0.75
1.00LNC
HNC
R2=0.28p=0.02
CYP2A5 Density Units
V (
nm
ol/m
in/m
g)
0 10 20 300.00
0.25
0.50
0.75
1.00LNC
HNC
NIC Consumption (µµµµg/day)
V (
nm
ol/m
in/m
g) *
6 uM 30 uM 60 uM0.0
0.1
0.2
0.3
0.4
0.5
0.6 LNC
HNC
*
**
**
6 µµµµM 30 µµµµM
NIC
V (
nm
ol/
min
/mg
)
60 µµµµM
a) b)
c)
0 10 200.00
0.25
0.50
0.75
1.00LNC
HNC
R2=0.28p=0.02
CYP2A5 Density Units
V (
nm
ol/m
in/m
g)
0 10 20 300.00
0.25
0.50
0.75
1.00LNC
HNC
NIC Consumption (µµµµg/day)
V (
nm
ol/m
in/m
g) *
0 10 20 300.00
0.25
0.50
0.75
1.00LNC
HNC
NIC Consumption (µµµµg/day)
V (
nm
ol/m
in/m
g) **
95
Table 6. Estimated in vitro NCO kinetic parameters of male LNC and HNC mice (mean ± SD)
Km (µM) Vmax (nmol/min/mg) LNC (n=10) 7.8 ± 2.0 0.30 ± 0.16 HNC (n=8) 10.7 ± 4.7 0.48 ± 0.05* *p=0.003 compared to the LNC group
96
Table 7. Concentrations (mean ± SD) of nicotine, cotinine, and 3-hydroxycotinine (ng/mL) from the in vitro NCO activity assays (at 30 µM nicotine)
Nicotine (µg/mL)
Cotinine (µg/mL)
3-hydroxycotinine (µg/mL)
LNC (n=11) 4.63 ± 0.42 0.17 ± 0.09 ND Male
HNC (n=8) 4.58 ± 0.41 0.24 ± 0.04 ND LNC (n=12) 4.41 ± 0.36 0.26 ± 0.20 ND
Female HNC (n=14) 4.37 ± 0.34 0.28 ± 0.12 ND
ND: not detected
97
Figure 12. CYP2A4/5 levels and in vitro NCO activities do not differ between female LNC and HNC mice. Microsomal proteins from female LNC and HNC mice were analyzed for a) CYP2A4/5 protein levels and b) NCO activities at 30 µM of nicotine. Protein data are from 4 independent immunoblots for each animal.
0 10 20 30
0
5
10
15
20
25LNC
HNC
NIC Consumption (µµµµg/day)
CY
P2
A4
/5
a) b)
0 10 20 30
0.00
0.25
0.50
0.75
1.00LNC
HNC
NIC Consumption (µµµµg/day)
V (
nm
ol/m
in/m
g)
98
nicotine between LNC (n=7) and HNC (n=8) mice (0.61 ± 0.48 vs. 0.59 ± 0.17
nmol/min/mg; p=0.40).
99
Discussion
One determinant of the amount of smoking is the rate of nicotine metabolic inactivation,
whereby the systemic availability of nicotine influences the number of cigarettes smoked
and the intensity of inhalation (McMorrow and Foxx, 1983; Russel, 1987; Schoedel et al.,
2004). Here we showed that nicotine self-administration in male mice is associated with
the amount of nicotine metabolizing enzyme CYP2A5 as well as the rate at which
nicotine is metabolized in vitro. CYP2A5 protein levels were correlated with NCO
activities. Results from the current study are consistent with human data in which cyp2a6
genotype and/or phenotype can have a significant impact on cigarette consumption
behaviours (Rao et al., 2000; Schoedel et al., 2004) where reducing or increasing
nicotine clearance can decrease smoking (Sellers et al., 2000; Sellers et al., 2003b) or
increase smoking (Benowitz and Jacob, 1985), respectively.
There are several interpretations for the presence of higher CYP2A5 levels and
NCO activities in the HNC mice. Quantitive trait loci analyses have implicated the
potential involvement of CYP2A5 in differential nicotine consumption levels (Wildenauer
et al., 2005); therefore, genetic variation in CYP2A5 may contribute to the observed
nicotine intake behaviours. For instance, CYP2A5 levels may be related to
polymorphisms in cis- and/or trans-regulatory factors that influence transcriptional
efficiency, translational efficiency, as well as message stability. This is plausible since
the human CYP2A6 contains multiple polymorphisms that affect its expression and
activity (Nakajima and Yokoi, 2005). For example, the CYP2A6*9 polymorphism
encodes a TATA box mutation which reduces its mRNA production by 40-70% (Kiyotani
et al., 2003; Pitarque et al., 2001; Yoshida et al., 2003). Smokers who are homozygous
for CYP2A6*9 (slow metabolizers) have reduced nicotine metabolism by approximately
50% (Schoedel et al., 2004; Yoshida et al., 2003); and slow metabolizers smoke ~20%
100
fewer cigarettes compared to individuals who are normal metabolizers (Schoedel et al.,
2004). Polymorphism in CYP2A6 may also affect the structural stability of the enzyme.
The CYP2A6*2 variant codes for a protein with a single amino-acid substitution (L160H)
that results in an unstable and inactive enzyme (Yamano et al., 1990). In mice a single
nucleotide polymorphism causing a change in amino acid (V117A) in CYP2A5, and the
resultant coumarin-hydroxylase activity, has also been described (Lindberg et al., 1992).
Another potential explanation for the greater CYP2A5 levels in male HNC mice is
that mice that consume more nicotine may have induced CYP2A5, thus further
increasing nicotine consumption. However, studies have shown that chronic nicotine
administration decreases both the plasma clearance of nicotine in humans and CYP2A6
protein expression in monkeys (Benowitz and Jacob, 1993; Lee et al., 1987; Schoedel et
al., 2003). Therefore it is unlikely that the higher CYP2A5 levels in the male HLC mice
are the result of induction by nicotine consumption.
In addition to the nicotine metabolic pathway, other components (and their
respective genetic variations) involved in nicotine sensitivity and reward properties could
also affect nicotine intake, and many of these candidate genes have been investigated in
humans (Tyndale, 2003). In inbred mice there are marked differences in response to
nicotine across strains (Collins et al., 1988; Crawley et al., 1997; Marks et al., 1983). It is
therefore possible that nicotine self-administration in mice may also be related to factors
such as fluid intake and taste preference. Water consumption between the high (C57Bl/6)
and the low (St/bJ) nicotine consumption parental strains do not differ, indicating that the
difference in nicotine consumption is not related to fluid intake (Aschhoff et al., 1999).
Lush and Holland (1998) found that the C57Bl/6 mice have a greater preference for the
bitter compound copper glycinate compared to the St/bJ mice. However, this may be
difficult to interpret with regards to nicotine since the ranking of copper glycinate
101
preference does not match that of nicotine preference in several other strains (Aschhoff
et al., 1999; Lush and Holland, 1988).
In females, the higher overall CYP2A protein levels compared to males and lack
of difference in CYP2A protein between consumption groups is likely due to the
presence of the female specific CYP2A4 protein (Burkhart et al., 1985; Harada and
Negishi, 1984). The CYP2A4 protein is approximately 98% identical to CYP2A5 in amino
acid sequence and they are indistinguishable from one another using available
antibodies (Su et al., 1998). CYP2A4 metabolizes nicotine only at very high
concentrations (more than 15-fold lower in affinity) and should not contribute to
metabolism at the nicotine concentrations consumed (Murphy et al., 2005a). We
therefore investigated the in vitro NCO activities between the two female groups. We
observed no significant difference between the female high and low consumers in in vitro
nicotine metabolism; however, the distribution of nicotine metabolism within each group
is such that the majority of NCO activities were higher in the high consumption group
compared to the low consumption group.
It appears that components in addition to nicotine metabolism have a greater
contribution to nicotine consumption in females compared to males. The apparent lack of
association between nicotine consumption levels and nicotine metabolism in females
may be related to effects of steroids (Dempsey et al., 2002). For example, in humans,
pregnancy increases plasma nicotine and cotinine clearances, but nicotine intake from
cigarettes are similar between pregnant and post-partum females (Dempsey et al., 2002).
On the other hand, in ICR mice, administration of exogenous progesterone or 17β-
estradiol to female mice dose-dependently decreased the antinociceptive effect of
nicotine, but administration of testosterone to male mice did not alter pain sensitivity
(Damaj, 2001). The differential effects of these hormones were attributed to the ability of
progesterone and 17β-estradiol to antagonize the binding of nicotine to the α4β2
102
nicotinic-receptors (Damaj, 2001). Therefore, female sex hormones may modulate
nicotine-mediated behaviours (e.g. consumption) reducing the relative impact of nicotine
metabolism in these mice.
In conclusion, we found that varying levels of nicotine self-administration
behaviour in male mice were associated with different rates of nicotine metabolism, most
likely mediated by genetic controls over the differing expression levels of the CYP2A5
protein. This observation is similar to that observed in smokers in which the amount
smoked is related to the functional levels of CYP2A6 (Rao et al., 2000; Schoedel et al.,
2004). Using inducers and inhibitors, as well as various mouse strains, we can model
the influence of nicotine metabolism on various stages of smoking seen in humans such
as initiation, acquisition, maintenance, and cessation.
103
Acknowledgements
We would like to thank Amir Boutrous for his assistance in protein analysis; Sharon
Miksys for guidance and critical review of the manuscript; Helma Nolte and Raj Sharma
for their assistance with HPLC assessment.
Support
This study was supported by CAMH, CIHR 14173 & 53248. CIHR-Special Training
Program in Tobacco Use in Special Populations and OGS to ECKS and Canada
Research Chair in Pharmacogenetics.
104
SIGNIFICANCE OF CHAPTER
This is the first study to demonstrate the association between CYP2A5 and the
pharmacology of nicotine in mice. Specifically, we found that nicotine metabolism is
associated with the amount of CYP2A5 (expression/activity) as contributed by genetic
variations. More importantly, CYP2A5-mediated nicotine metabolism is associated with
NSA in which mice exhibiting faster in vitro nicotine metabolism self-administered more
nicotine. These findings are in line with humans in which CYP2A6 phenotype can have a
significant impact on cigarette consumption behaviours.
105
CHAPTER 4: INHIBITION OF NICOTINE METABOLISM BY METHOXSALEN: PHARMACOKINETIC AND PHARMACOLOGICAL STUDIES IN MICE
M. Imad Damaj, Eric C. K. Siu, Edward M. Sellers, Rachel F. Tyndale, and Billy R. Martin
As published in: Journal of Pharmacology and Experimental Therapeutics, 2007, Jan; 320(1):250-257
The in vitro enzyme activities assays, analyses of the pharmacokinetic data, writing of
the enzyme kinetics and pharmacokinetics section of the manuscript were performed by
ECKS. The behavioural work were performed by MI Damaj. The study was designed by
MI Damaj and RF Tyndale.
Reprinted from Molecular Pharmacology, Volume 320, Damaj et al., “INHIBITION OF NICOTINE METABOLISM BY METHOXSALEN: PHARMACOKINETIC AND PHARMACOLOGICAL STUDIES IN MICE”, pp 250-257, Copyright (2007) with permission from The American Society for Pharmacology and Experimental Therapeutics. © 2007 The American Society for Pharmacology and Experimental Therapeutics
106
Abstract
Studies were undertaken to examine whether methoxsalen, a specific and relatively
selective inhibitor of human CYP2A6, inhibited CYP2A5-mediated nicotine metabolism in
vitro. Furthermore, studies were performed in vivo to determine whether methoxsalen
would modulate acute nicotine pharmacokinetics and pharmacological effects
(antinociception and hypothermia) in ICR mouse. Our results demonstrated that
methoxsalen competitively inhibits in vitro nicotine metabolism in mice. The inhibition
was potent, as seen in human inhibition studies, with a Ki of 0.32 µM. In addition, we
found that administration of methoxsalen significantly increased the plasma half-life of
nicotine (approximately doubled) and increased its AUC compared to saline treatment.
There was a dose-dependent enhancement in the pharmacological effects of nicotine
(body temperature and analgesia) after methoxsalen treatment. Methoxsalen prolonged
the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg) for periods
up to 180 min post-nicotine administration. Furthermore, this prolongation in nicotine’s
effects after methoxsalen was associated with a parallel prolongation of nicotine plasma
levels in mice. These data strongly suggest that variation in the rates of nicotine
metabolic inactivation substantially alter nicotine’s pharmacological effects. In
conclusion, these results confirmed that methoxsalen did indeed inhibit the conversion of
nicotine to cotinine both in vitro and in vivo. They also suggest that mice may represent
a suitable model for studying variation in nicotine metabolism and its impact on
mechanisms of nicotine dependence, including the use of inhibitors to reduce nicotine
metabolism.
107
INTRODUCTION
Nicotine is known to induce several physiological and pharmacological effects
and to produce subjective feelings of reward and pleasure in humans and animals.
Several well-characterized behavioral models and tests are currently used for
investigating the biological and pharmacological mechanisms underlying nicotine
dependence. These models reveal a high level of variability among nicotine behavioral
responses due to factors such as differing genetic background, species, sex, age, initial
basal and behavioral state, route and regimen of administration. In addition, the narrow
effective dose range for many pharmacological and behavioral effects of nicotine further
compounds the variability (see review by (Picciotto, 2003)). Species differences also
represent an important consideration regarding in vivo responses to either acute or
chronic nicotine exposure, particularly mice versus rats. In general, mice are less
sensitive to the acute pharmacological effects of nicotine than rats. For example,
nicotine’s potency in the tail-flick and hot-plate tests after systemic administration in mice
is 5-10 fold lower than that reported for rats (Tripathi et al., 1982). One of the most
striking differences concerns nicotine’s locomotor effects. Although nicotine induces
locomotor hyperactivity in rats, it generally fails to induce locomotor hyperactivity in mice
at any dose (Castane et al., 2002; Damaj and Martin, 1993; Freeman et al., 1987;
Gaddnas et al., 2002; Itzhak and Martin, 1999; Kita et al., 1992; Marks et al., 1983;
Smolen et al., 1994). In addition, even though i.v. nicotine self-administration can be
accomplished in mice, a model using drug-naïve mice not restricted in their movement
and a limited access schedule similar to that frequently used in rats is not reported. This
rat-mouse variability can be attributed to various factors, such as pharmacokinetics,
stress-induced by behavioral manipulations, physiological status, and/or psychological
context. Given the very rapid metabolism of nicotine in mice (plasma and brain t1/2 = 5.9-
6.9 min) (Petersen et al., 1984) compared to that of the rat (45 min) (Hwa Jung et al.,
108
2001), distribution and kinetic factors appear to play an important role in the behavioral
response to nicotine in mice. A recent study reported that nicotine oral self-
administration in male mice is associated with the amount of nicotine metabolizing
enzyme CYP2A5 as well as the rate at which nicotine is metabolized in vitro (Siu et al.,
2006).
The main purpose of this study was to elucidate the impact of nicotine
metabolism on nicotine’s acute pharmacological effects in mice. The mouse is a better-
suited model for the study of nicotine metabolism and behaviors, compared to rats, since
the predominant enzymes responsible for the metabolism of nicotine in rats belong to
the CYP2B family (Nakayama et al., 1993; Schoedel et al., 2001; Siu et al., 2006). In
contrast to rats, human CYP2A6 and mouse CYP2A5 (mouse orthologous form of
human CYP2A6) are the main enzymes involved in nicotine metabolism (Messina et al.,
1997; Murphy et al., 2005b). There is close structural (84% amino acid sequence
similarity) and functional (CYP2A5 metabolizes nicotine with similar efficiency to human
CYP2A6) similarity between CYP2A5 and CYP2A6 (Murphy et al., 2005b; Siu et al.,
2006). In addition a similarly large portion of nicotine is metabolized to cotinine in
humans and mice, relative to the rat, making the mouse a good model for the
pharmacological impacts of manipulating nicotine metabolism in vivo. Accordingly,
studies were undertaken to examine whether methoxsalen [a specific and relatively
selective inhibitor of human CYP2A6, (Zhang et al., 2001)] inhibited CYP2A5-mediated
nicotine metabolism in vitro. Subsequently studies were performed in vivo to determine
whether methoxsalen would modulate acute nicotine pharmacokinetics and
pharmacological effects (antinociception and hypothermia) in the mouse.
109
Materials and Methods
Animals
Male ICR mice (20-25g) obtained from Harlan Laboratories (Indianapolis, IN) were used
throughout the study. Animals were housed in groups of six and had free access to food
and water. Animals were housed in an AAALAC approved facility and the study was
approved by the Institutional Animal Care and Use Committee of Virginia
Commonwealth University.
Drugs
Mecamylamine hydrochloride was supplied as a gift from Merck, Sharp and Dohme &
Co. (West Point, PA). (-)-Nicotine hydrogen tartrate was obtained from Aldrich Chemical
Company, Inc. (Milwaukee, WI) and converted to the ditartrate salt as described by
(Aceto et al., 1979) (1979). Methoxsalen was purchased from Sigma-Aldrich (St. Louis,
MO). All drugs were dissolved in physiological saline (0.9% sodium chloride). All doses
are expressed as the free base of the drug.
Membrane Preparations
Microsomal membranes were prepared from mouse livers for the in vitro nicotine
metabolism assay as previously described (Messina et al., 1997; Siu et al., 2006) and
stored at 80°C in 1.15% KCl. The cytosolic fractions were also acquired during the
membrane preparation and were used as a source of aldehyde oxidase.
Nicotine C-Oxidation Assay
Assay conditions were optimized for ICR mouse microsomes as previously described
(Siu et al., 2006). Linear formation of cotinine from nicotine was obtained under assay
110
conditions of 0.5 mg/ml protein concentration with an incubation time of 15 min.
Incubation mixtures contained 0.5 mg/ml microsomal protein, 1 mM NADPH and 1
mg/ml of mouse liver cytosol in 50 mM Tris-HCl buffer, pH 7.4 and were performed at
37oC in a final volume of 0.5 ml. The reaction was stopped with 4% Na2CO3, and 5-
methylcotinine (70 µg) was added as the internal standard. In vitro kinetic parameters
(Km and Vmax) of nicotine metabolism in ICR mice were characterized using substrate
concentrations of 0, 3.0 to 1000 µM nicotine. Nicotine metabolizing activity was analyzed
by HPLC as previously described (Messina et al., 1997) with the modification of the solid
phase extraction procedure for assessment of 3-hydroxycotinine (Siu et al., 2006).
Separation of nicotine and cotinine was achieved using a ZORBAX Bonus-RP column (5
mm, 150 x 4.6 mm; Agilent Technologies Inc., Mississauga, ON) and a mobile phase
consisting of acetonitrile/potassium phosphate buffer (10:90 v/v, pH 5.07) containing 3.3
mM heptane sulfonic acid and 0.5% triethylamine. The separation was performed with
isocratic elution at a flow rate of 0.9 ml/min. Nicotine, cotinine, and 3-hydroxycotinine
sample concentrations were determined from standard curves. The limits of
quantification were 5 ng/ml for nicotine and 12.5 ng/ml for cotinine.
Methoxsalen Inhibition of Cotinine Formation from Nicotine
To determine the apparent Ki value of inhibition of cotinine formation by methoxsalen in
the ICR mouse microsomes, 0, 0.04, 0.1, 0.2, and 0.4 µM methoxsalen were used. The
substrate (nicotine) concentrations were 10, 20 and 40 µM, which were approximately
equal to 1/2Km, Km, and 2Km.
Plasma nicotine and cotinine levels measurement
To determine plasma nicotine and cotinine levels, blood samples were drawn by cardiac
puncture at 5, 15, 30, 45, 60, 120, and 180 minutes after nicotine administration (2.5
111
mg/kg, s.c.). Animals were pretreated with saline or methoxsalen for a variety of times
before nicotine administration. Immediately afterwards the plasma samples were
prepared by centrifugation at 3000 x g for 10 min and frozen at –20˚C until analysis. To
measure total nicotine and cotinine levels (free and glucuronides) the samples were
incubated with β-glucuronidase at a final concentration of 5 mg/ml in 0.2 M acetate
buffer, pH 5.0, at 37oC overnight. After incubation the samples were processed and
analyzed for nicotine and metabolite levels by HPLC as described above.
Behavioral tests
1. Tail-flick test. Antinociception was assessed by the tail-flick method of D'Amour and
Smith (d'Amour and Smith, 1941) as modified by Dewey et al. (Dewey et al., 1970). A
control response (2-4 sec) was determined for each mouse before treatment, and a test
latency was determined after drug administration. In order to minimize tissue damage, a
maximum latency of 10 sec was imposed. Antinociceptive response was calculated as
percent maximum possible effect (% MPE), where % MPE = [(test-control)/(10-control)] x
100.
2. Hot-plate Test. Mice were placed into a 10 cm wide glass cylinder on a hot plate
(Thermojust Apparatus) maintained at 55.0oC. Two control latencies at least ten min
apart were determined for each mouse. The normal latency (reaction time) was 8 to 12
seconds. Antinociceptive response was calculated as percent maximum possible effect
(% MPE), where % MPE = [(test-control)/(40-control) x 100]. The reaction time was
scored when the animal jumped or licked its paws. In order to minimize tissue damage,
a maximum latency of 40 sec was imposed.
Groups of eight to twelve animals were used for each dose and for each
treatment. Studies were carried out by pretreating the mice with either saline or
112
methoxsalen 30 min before nicotine. The animals were tested 5 min after administration
of nicotine.
3. Body temperature. Rectal temperature was measured by a thermistor probe
(inserted 24 mm) and digital thermometer (Yellow Springs Instrument Co., Yellow
Springs, OH). Readings were taken just before and at 30 min after the s.c. injection of
either saline or nicotine. The difference in rectal temperature before and after treatment
was calculated for each mouse. The ambient temperature of the laboratory varied from
21-24oC from day to day.
Statistical analysis
Kinetic (i.e. apparent Km, Vmax, Ki) parameters were calculated using Graphpad Prism
(Graphpad Software Inc., San Diego, CA) and were verified by the Eadee-Hofstee plots.
The type of inhibition by methoxsalen was further assessed by the Dixon method.
Assessment of in vivo nicotine and cotinine plasma levels for the entire time course from
individual animals was not possible due to limited blood volume; therefore, each time
point represents data from individual mice. Due to this restriction to the experimental
design, statistical parameters (i.e. half life) were estimated by resampling methods using
the PKR and Test software (H. L. Kaplan, Toronto, ON). Statistical analysis of all
analgesic and behavioral studies was performed using either t-test or analysis of
variance (ANOVA) with Tukey’s test post hoc test when appropriate. All differences
were considered significant if at p < 0.05. ED50 values with 95% CL for behavioral data
were calculated by unweighted least-squares linear regression as described by Tallarida
and Murray (Tallarida and Murray, 1987).
113
Results
In vitro nicotine C-oxidation activity of mice
We first established the kinetic parameters apparent Km and Vmax for in vitro nicotine
metabolism in ICR mice. Nicotine metabolism followed two-site Michaelis-Menten
kinetics as illustrated by the Eadee-Hofstee plot (Fig. 13 & inset). The estimated kinetic
parameters for the in vitro nicotine metabolism, derived from five animals, for both sites
can be found in Table 8. One site exhibited high affinity for nicotine with an apparent Km
of 18.6 ± 5.9 µM. The second site had a much lower affinity for nicotine with an
apparent Km of 215.2 ± 60.0 µM. The overall catalytic efficiency (Vmax/Km) was greater
for the high-affinity site compared to the low-affinity site (0.013 ± 0.007 vs. 0.003 ±
0.001).
Inhibition of in vitro nicotine C-oxidation in mouse microsomes by methoxsalen
Previous studies indicated that CYP2A5 is the primary enzyme responsible for nicotine
metabolism in mice (Murphy et al., 2005b). We tested whether the human CYP2A6
inhibitor methoxsalen (Zhang et al., 2001) was also an inhibitor of nicotine metabolism in
ICR mouse microsomes. Methoxsalen inhibited nicotine metabolism with a Ki of 0.32 ±
0.03 µM (Fig. 14 & Table 8). Methoxsalen was a competitive inhibitor with some
indication of a mixed-type of inhibition consistent with it also being a mechanism-based
inhibitor in vitro.
Effects of methoxsalen on nicotine and cotinie plasma levels after in vivo treatment
Having established that methoxsalen can inhibit in vitro nicotine metabolism in the ICR
mice, we tested the effect of the inhibitor on in vivo plasma nicotine and cotinine levels.
114
Figure 13. In vitro nicotine C-oxidation activity of ICR mice. Microsomal proteins (0.5 mg/ml) from adult male ICR mice were incubated with increasing concentrations of nicotine for 15 minutes. The plot shows the average cotinine formation velocity ± SD values for 5 individual animals. Inset: a representative Eadee-Hofstee plot indicating the presence of two enzymatic sites responsible for the metabolism of nicotine.
0 250 500 750 10000.0
0.2
0.4
0.6
0.8
1.0
1.2
NIC (µµµµM)
Nic
oti
ne C
-oxid
ati
on
V (
nm
ol co
tin
ine f
orm
ed
/min
/mg
pro
tein
)
0.00 0.01 0.02 0.030.00
0.25
0.50
0.75
1.00
1.25
V/S
V (
nm
ol/
min
/mg
)
0 250 500 750 10000.0
0.2
0.4
0.6
0.8
1.0
1.2
NIC (µµµµM)
Nic
oti
ne C
-oxid
ati
on
V (
nm
ol co
tin
ine f
orm
ed
/min
/mg
pro
tein
)
0.00 0.01 0.02 0.030.00
0.25
0.50
0.75
1.00
1.25
V/S
V (
nm
ol/
min
/mg
)
115
Table 8. Kinetic parameters of in vitro nicotine C-oxidation activities of adult male ICR mouse liver microsomes. Km, Vmax and Ki values were determined independently for each animal and the average ± SD values are shown.
ICR (n=5)
High Affinity Km (µM) 18.6 ± 5.9
Vmax (nmol/min/mg) 0.22 ± 0.05
Vmax/Km 0.013 ± 0.007
Low Affinity Km (µM) 215.2 ± 60.0
Vmax (nmol/min/mg) 0.65 ± 0.08
Vmax/Km 0.003 ± 0.001
Methoxsalen Inhibition Ki (µM) 0.32 ± 0.03
116
Figure 14. A representative Dixon plot of inhibition of cotinine formation by ICR mouse microsomes in presence of varying concentrations of methoxsalen. In such plots, the abscissa of the point where the regression lines intersect is the negative of the estimated value of Ki. Three animals were used to establish Ki value. Velocity is expressed as nmol/min/mg.
-0.4 -0.2 0.0 0.2 0.4 0.6
20
40
60
8010 µM
20 µM
40 µM
8-MOP (µµµµM)
1/V
Ki = 0.3 µM
-0.4 -0.2 0.0 0.2 0.4 0.6
20
40
60
8010 µM
20 µM
40 µM
8-MOP (µµµµM)
1/V
Ki = 0.3 µM
117
Administration of methoxsalen significantly increased the plasma half-life of nicotine and
increased its AUC compared to saline treatment (Fig. 15A & Table 9). Methoxsalen had
minimal effect on the maximum nicotine concentration (Table 9) consistent with the
inhibition of nicotine given by the subcutaneous route. The plasma levels of the primary
CYP2A5-mediated metabolite of nicotine, cotinine, were also reduced as a result of
inhibition of this nicotine metabolic pathway by methoxsalen (Fig 15B).
Effects of methoxsalen on nicotine acute pharmacological effects (antinociception and
hypothermia): Time-course, potency and selectivity studies
Methoxsalen given s.c. was evaluated for its ability to enhance a 2.5 mg/kg dose (an
ED84 dose) of nicotine-induced antinociception (tail-flick and hot-plate procedures) and
hypothermia after nicotine s.c. injection of the drug. We first carried out a time-course
study of methoxsalen (10 mg/kg) enhancement of nicotine’s effects to determine an
optimal pretreatment time. Mice were given methoxsalen at different times and then
received nicotine (2.5 mg/kg s.c.). The mice were tested 45 min after nicotine
administration. As illustrated in Fig. 16A, the potentiation of nicotine’s antinociceptive
effects by methoxsalen pretreatment in the tail-flick and hot-plate tests was time-
dependent with maximum enhancement occurring between 30 and 60 min. A similar
time-course was seen with nicotine-induced hypothermia (Fig. 16B) with maximum
enhancement occurring between 30 and 120 min. Based on the time-course results,
subsequent studies were carried out by pretreating the mice with methoxsalen 30 min
before nicotine. We then determined its potency of enhancing nicotine’s effects at this
pretreatment time. As showed in Fig. 17, methoxsalen dose-dependently enhanced
nicotine-induced antinociception in the tail-flick (Fig. 17A), hot-plate (Fig. 17B) assays
and hypothermia (Fig. 17C).
118
Figure 15. Time course of nicotine and cotinine plasma concentration in mice pretreated with methoxsalen. Nicotine was administered s.c. (2.5 mg/kg) 30 min after pretreatment with ( ) saline or ( ) methoxsalen. Each time point represents the mean ± SD of 4 to 6 animals. For the saline pretreatment nicotine plasma levels, five of seven values at 120 min, and all values at 180 min, were below the limits of detection. They were included in the plot as 1 ng/ml for visualization of the data.
(A) (B)(A) (B)
119
Table 9. Pharmacokinetic parameters of nicotine in adult male ICR mouse after pretreatment with methoxsalen. Values are shown as mean ± standard deviation.
Cmax (ng/ml)
AUC
(ng*hr/ml) T1/2
(min)
Saline/Nicotine 314 ± 170 132 ± 14 14.3 ± 8.1
Methoxsalen/Nicotine 354 ± 26 334 ± 24* 30.1 ± 13.5*
* p < 0.05 compared to Saline/Nicotine
120
Figure 16. Time-course of methoxsalen effects on nicotine-induced (A) antinociception and (B) hypothermia after s.c. administration of 10 mg/kg in mice. Nicotine was given at the dose of (2.5 mg/kg, s.c.) and animals were tested 45 min after injection. Each point represents the mean ± SE of 8 to 12 mice. *P <0.05 compared to correspondent zero time point.
0
20
40
60
80
100
0 5 15 30 60 120 180
Tail-FlickHot-Plate
% M
PE
Time (min)
*
* **
**
*
*
-7
-6
-5
-4
-3
-2
-1
0
0 5 15 30 60 120 180
²ÞC
Time (min)
** *
*
(A) (B)
∆o
% m
axim
um
po
ssib
le e
ffec
t
121
Figure 17. Dose-response enhancement of nicotine by methoxsalen after s.c. administration in mice. Methoxsalen at different doses was administered s.c. 15 min before nicotine (2.5 mg/kg, s.c.) and mice were tested 45 min later in (A) the tail-flick and (B) the hot-plate tests. Each point represents the mean ± SE of 8 to 12 mice. *P <0.05 compared to saline (zero dose) treatment.
0
25
50
75
100
% M
PE
0 1 5 10 15
8-MOP (mg/kg)
0
25
50
75
100
% M
PE
0 1 5 10 15
8-MOP (mg/kg)
(A) (B)
*
* *
*
* *
% m
ax
imu
m p
oss
ible
eff
ect
% m
ax
imu
m p
oss
ible
eff
ect
122
By itself, methoxsalen did not significantly change tail-flick or hot-plate basal latencies,
or body temperature at the indicated doses and times. Furthermore, the antinociceptive
and hypothermic effects of methoxsalen/nicotine combination were totally blocked by a
pretreatment with mecamylamine, a non-competitive nicotinic antagonist, at 2 mg/kg
(Fig. 18 & Table 10).
To determine whether methoxsalen produced similar effects on other analgesic
substances, it was given at a dose of 10 mg/kg before the administration of an inactive
dose of morphine. Pretreatment with methoxsalem failed to significantly enhance the
effect of morphine in the tail-flick test [Saline/Saline = 7 ± 5 % MPE; Saline/Morphine
(0.5 mg/kg, s.c.) = 20 ± 7 % MPE; Methoxsalen (10 mg/kg)/Morphine (0.5 mg/kg, s.c.) =
25 ± 8 % MPE]. Thus, the effect of methoxsalen appears not to be generalized to other
analgesic substances since it did not enhance the effects of morphine after systemic
administration.
Effects of methoxsalen on the time-course of nicotine’s pharmacological effects
The onset of action for nicotine (2.5 mg/kg, s.c.) in the tail-flick test was rapid with
maximum antinociception occurring between 0 and 5 min. The duration of
antinociception was relatively brief in that the effect had disappeared completely within
45 min after nicotine administration in mice (Fig. 19A). A similar time-course pattern was
observed in the hot-plate test (Fig. 19B). The duration of nicotine-induced hypothermia
was however longer compared to that of antinociception. Indeed, nicotine’s effect
disappeared completely within 180 min after s.c. administration in mice (Fig. 19C).
When animals were pretreated with methoxsalen (10 mg/kg, s.c.), the effects of nicotine
in both analgesic tests were significantly prolonged. Nicotine-induced antinociception
did not disappear completely till 180 min after nicotine administration in mice (Fig. 19A &
123
Figure 18. Blockade of methoxsalen’s effects on nicotine-induced antinociception in the tail-flick test after s.c. administration in mice. Animals were pretreated with either saline or mecamylamine (2 mg/kg, s.c.) followed by methoxsalen (10 mg/kg, s.c). Mice received 30 min later nicotine at a dose of 2.5 mg/kg and were tested 45 min after injection. Each point represents the mean ± SE of 8 to 12 mice. MOP (10) = methoxsalen at 10 mg/kg; Sal: Saline; Nic (2.5): Nicotine at 2.5 mg/kg; Meca (2): Mecamylamine at 2 mg/kg. *P <0.05 compared to control.
0
25
50
75
100
% M
PE
Control MOP (10) Sal/Nic (2.5) MOP (10)/Nic (2.5) Meca (2)/MOP (10)/Nicc (2.5)
*%
max
imu
m p
oss
ible
eff
ect
124
Table 10. Blockade of methoxsalen’s effects on nicotine acute pharmacological responses after s.c. administration in mice. Animals were pretreated with either saline or mecamylamine (2 mg/kg, s.c.) followed by methoxsalen (10 mg/kg, s.c). Mice received 30 min later nicotine at a dose of 2.5 mg/kg and were tested 45 min after injection. Methoxsalen (10) = methoxsalen at 10 mg/kg; Meca (2): Mecamylamine at 2 mg/kg. Each point represents the mean ± SE of 8 to 12 mice.
Treatment
(mg/kg) Tail-flick Test
% MPE (Mean ± SEM)
Hot-plate Test % MPE
(Mean ± SEM)
Body temperature ∆
oC (Mean ± SEM)
Saline/Saline/Saline 7 ± 2 8 ± 2 -0.1 ± 0.2
Saline/Methoxsalen (10)/Saline 10 ± 3 12 ± 5 -1.5 ± 0.2
Meca (2)/Methoxsalen (10)/Saline 15 ± 7 10 ± 5 -1.8 ± 0.5
Meca (2)/Saline/Saline 8 ± 3 6 ± 4 -0.5 ± 0.3
Meca (2)/Saline/Nicotine (2.5) 5 ± 2 6 ± 2 -1.9 ± 0.6*
Saline/Saline/Nicotine (2.5) 10 ± 5 15 ± 5 -4.1 ± 0.6
Saline/Methoxsalen (10)/Nicotine (2.5) 80 ± 10* 83 ± 8* -6.8 ± 0.4*
Meca (2)/Methoxsalen (10)/Nicotine (2.5) 15 ± 8 23 ± 10 -2.2 ± 0.5*
*P <0.05 compared to control.
125
Figure 19. Effects of methoxsalen on the time-course of nicotine’s effects in (A) the tail-flick test, (B) the hot-plate assay and (C) body temperature in mice. Animals were pretreated with either ( ) saline or ( ) methoxsalen (10 mg/kg, s.c) and 30 min later received nicotine at a dose of 2.5 mg/kg. Mice were tested at different times after injection. Each point represents the mean ± SE of 8 to 12 mice. *P <0.05 compared to control.
0
25
50
75
100
% M
PE
0 30 60 90 120 150 180 0
25
50
75
100
% M
PE
0 30 60 90 120 150 180
Time (min)
-8
-6
-4
-2
0
2
²ÞC
0 30 60 90 120 150 180
Time (min)
(A) (B)
(C)
*
*
*
* * *
**
*
*
** * **
**
* *
*
*
**
* *
*
*
% m
ax
imu
m p
oss
ible
eff
ect
% m
ax
imu
m p
oss
ible
eff
ect
126
B). On the other hand, the effects of nicotine on body temperature were still significant
beyond the 180 min time point (Fig. 19C). Further times were not evaluated.
The prolongation of nicotine’s acute pharmacological effects correlated well with
the prolongation of nicotine plasma concentrations after methoxsalen administration in
mice. Indeed, as shown in Figure 20, there was a corresponding increase in nicotine’s
sensitivity in the tail-flick test to the increased plasma concentration of nicotine after
methoxsalen pretreatment as measured by the time-course curves. A comparable
profile can also be seen in the hot-plate test and body temperature changes (data not
shown).
127
Figure 20. Relationship between nicotine-induced antinociception in the tail-flick test and plasma levels of nicotine in the ICR mice after methoxsalen treatment. MOP = methoxsalen; Sal: Saline; TF = Tail-flick effect; Conc: plasma concentration of nicotine.
0
50
100
150
200
250
300
350
400
0
20
40
60
80
100
5 15 30 45 60 120 180
Sal/Conc
MOP/Conc
Sal/TF
MOP/TF
Co
ncen
trati
on
(n
g/m
l)
%M
PE
Time (min)
% m
ax
imu
m p
oss
ible
effe
ct
128
Discussion
A previous study examining nicotine metabolism in C57Bl/6 and DBA/2 mice found that
nicotine is metabolized by two enzymatic sites with the high affinity sites having Km’s of
~10 µM (Siu et al., unpublished) consistent with the nicotine metabolism mediated by
cDNA-expressed CYP2A5 (Km ~ 7.7 ± 0.8 µM; 129/J mouse strain) (Murphy et al.,
2005b). This current study indicates that ICR mice microsomes also contain two
enzymatic sites; the high affinity site had a modestly lower affinity for nicotine (~18 µM)
compared to that observed in C57Bl/6 and DBA/2 mice. Such small differences may be
attributed to unidentified polymorphisms in the ICR mouse strain altering the structure of
the enzyme to a minor extent (Lindberg et al., 1992). The low affinity enzyme site in mice
may be a member of the CYP2B family consistent with the low affinity site in human and
monkey hepatic nicotine metabolism studies (Messina et al., 1997; Schoedel et al.,
2003); the rat CYP2B can also metabolize nicotine (Schoedel et al., 2001).
The present study was the first to demonstrate that the human CYP2A6 inhibitor
methoxsalen (Zhang et al., 2001) can inhibit in vitro nicotine metabolism in mice. The
inhibition was competitive with some indication of mixed inhibition. The inhibition was
potent, as seen in human inhibition studies, with a Ki of 0.32 µM, indicating a 60-fold
higher affinity for the mouse enzyme compared to nicotine (Km = 19 µM, Table 8). These
pharmacokinetic values are similar to human where the Km is estimated to be 65 µM in
human liver microsomes (Messina et al., 1997) and the Ki was found to be 0.01 µM
(Zhang et al., 2001). As methoxsalen may be able to inhibit both CYP2A5 and CYP2B
enzymes in mice (Maenpaa et al., 1994), it is possible that methoxsalen reduces nicotine
metabolism by inhibiting both the high affinity CYP2A5 enzyme site (Murphy et al.,
2005b; Siu et al., 2006) as well as the second low affinity site. The in vitro mouse
pharmacokinetic data (Table 8) suggest that even at the very high concentrations initially
129
achieved following 2.5 mg/kg s.c. nicotine injections (100-300 ng/ml) that approximately
80% of the metabolism is mediated by the high affinity site kinetic site (Table 8).
Several studies have suggested that methoxsalen acts as a competitive, non-
competitive and/or mechanism-based inhibitor of human CYP2A6 in vitro (Draper et al.,
1997; Koenigs et al., 1997; Maenpaa et al., 1994; Zhang et al., 2001). Methoxsalen
potently inhibited CYP2A6-mediated nicotine metabolism in vivo in humans however a
clear indication of mechanism-based inhibition was not seen using either coumarin
(Kharasch et al., 2000) or nicotine (Sellers et al., 2000) as a substrate.
This is the first study to examine the pharmacokinetic impact of methoxsalen in
vivo in mice. We found that the half-life of plasma nicotine in mice treated with
methoxsalen was significantly longer (approximately doubled) compared to the saline
inhibition group. In addition, as evident in Fig. 16, methoxsalen’s effects increased with
pretreatment times from zero to 60 minutes suggesting that this pretreatment time
enhanced methoxsalen’s ability to inhibit nicotine metabolism. Together with the in vitro
data this suggests that methoxsalen may be acting in vivo in mice as a competitive
inhibitor and through an additional mechanism, possibly mechanism-based inhibition. It
has been shown in vitro that mouse CYP2A5 can metabolize methoxsalen (Maenpaa et
al., 1994). Mechanism-based inhibitors, also known as suicide substrates, are
compounds which are metabolically activated by the enzyme and the metabolite binds
irreversibly to the enzyme inactivating it. Therefore, it is possible that metabolites of
methoxsalen, even if produced by other enzymes such as CYP2B, may inhibit the
CYP2A5 through a variety of mechanisms including mechanism-based inhibition,
competitive inhibition and/or binding to the heme moiety of the enzyme. Together this
suggests that preinhibition with methoxsalen, prior to administration of a substrate such
as nicotine, may lead to enhanced metabolic inhibition via one of these mechanisms.
The similar maximal plasma nicotine levels seen with and without pretreatment with
130
methoxsalen are consistent with the subcutaneous route of nicotine’s administration, but
predicts a large increase in Cmax if nicotine is given by an oral route where the inhibition
would also block a first pass metabolism.
Our results confirmed that methoxsalen did indeed inhibit the conversion of
nicotine to cotinine both in vitro and in vivo. Pretreatment with methoxsalen increased
the AUC 2.5 fold and prolonged the nicotine plasma levels; it was also associated with a
2-fold increase in the half-life of the drug (Table 9). The nicotine plasma levels remained
above 10 ng/ml in the saline pretreatment for approximately 90 minutes but with
pretreatment with methoxsalen the nicotine plasma levels were still above 10 ng/ml at
180 min. Similar to the marked effects of CYP2A5 inhibition on nicotine metabolism,
there were dramatic enhancement in the pharmacological effects of nicotine (body
temperature and analgesia) after methoxsalen treatment. The effects of methoxsalen on
nicotine were dose-dependent and reached maximal enhancement at a dose of 15
mg/kg. Furthermore, the duration of methoxsalen-induced potentiation of nicotine’s
antinociceptive and hypothermic lasted close to 180 min after drug pretreatment.
Pharmacogenetic studies in humans have showed that smoking behaviours are
affected by polymorphisms in CYP2A6 that alter nicotine metabolism (Ariyoshi et al.,
2002; Malaiyandi et al., 2006; Minematsu et al., 2006; Rao et al., 2000; Schoedel et al.,
2004). For instance, individuals who are slow metabolizers of nicotine (e.g. those with
50% of less activity due to having inactive and/or decreased function alleles) smoked
significantly fewer cigarettes compared to individuals that are normal nicotine
metabolizers (those without variants) (Ariyoshi et al., 2002; Malaiyandi et al., 2006; Rao
et al., 2000; Schoedel et al., 2004). Furthermore, inhibition of orally delivered nicotine in
smokers using methoxsalen (to phenocopy slower metabolism found in those with
defects in CYP2A6) led to increases in plasma nicotine levels, reduction of cravings for
nicotine, and fewer numbers of cigarettes smoked relative to nicotine alone;
131
methoxsalen alone, in smokers, also increased the latency between lighting the first and
second cigarette (Sellers et al., 2000). Consistent with human data, we have shown that
CYP2A5 levels and in vitro nicotine metabolism rates are associated with nicotine oral
self-administration in male mice (Siu et al., 2006). These findings suggest that mice may
represent a suitable model for studying variation in nicotine metabolism, including the
use of inhibitors to reduce nicotine metabolism.
The main purpose of the current study was to determine the impact of nicotine
metabolism (i.e. inhibition) on nicotine’s acute pharmacological effects in mice.
Methoxsalen prolonged the duration of nicotine-induced antinociception and
hypothermia (2.5 mg/kg) for periods up to 180 min post-nicotine administration.
Although this increase in nicotine’s time course was seen in all responses measured, it
was more dramatic in antinociceptive tests (an additional 150 min compared to the
control group). This differential effect is probably due to the fact that nicotine-induced
antinociception time-course is much shorter than the effects on body temperature.
Furthermore, this prolongation in nicotine’s effects after methoxsalen was associated
with a parallel prolongation of nicotine plasma levels in mice (Figure 20). Consistent
with these present results, we have shown that CYP2A5 levels and in vitro nicotine
metabolism rates are associated with nicotine oral self-administration in male mice (Siu
et al., 2006). These findings suggest that mice may represent a suitable model for
studying variation in nicotine metabolism and its impact on mechanisms of nicotine
dependence, including the use of inhibitors to reduce nicotine metabolism. Although the
study of nicotine’s acute effects is important to the understanding of nicotine
dependence, one must also consider responses after chronic exposure of the drug in
animals. Animal models studying the different aspects of dependence such as
tolerance, withdrawal and reward involve a chronic/repeated exposure of the animals to
nicotine. Assessing the impact of nicotine metabolism in these behavioral models will
132
enhance our understanding of the molecular mechanisms involved in nicotine
dependence and will facilitate the development of new strategies for smoking cessation
therapies.
These data strongly suggest that variation in the rates of nicotine metabolic
inactivation substantially alter nicotine’s pharmacological effects. These data suggest
that in mice, with very rapid rates of metabolism, small differences in levels or function of
CYP2A5 between different mouse strains may result in substantially differing nicotine
pharmacological effects. Thus pharmacokinetic differences likely contribute to
differences in effects seen between studies employing the use of different mouse
strains.
In conclusion we have shown that the human CYP2A6 inhibitor methoxsalen is
also a potent inhibitor of mouse CYP2A5, both in vitro and in vivo. We have shown that
the pharmacological effects of inhibiting nicotine’s metabolism are profound, illustrating
the differences in resulting effects of nicotine that will occur between individuals and also
between animal strains that differ in levels of CYP2A-mediated nicotine metabolism.
133
Acknowledgments
The authors greatly appreciate the technical assistance of Tie Han and W Zhang.
Support
This work was supported by National Institute on Drug Abuse grant # DA-05274,
the Centre for Addiction and Mental Health, and the Canadian Institutes for
Health Research grants # MOP53248 and #MOP14173. E Siu acknowledges
the support of the CIHR STPTR scholarship and RFT is grateful for the support
of the Canada Research Chairs program.
Disclosures
EMS and RFT are shareholders in Nicogen, a company focused on novel
treatment approaches involving inhibition of hepatic CYP2A6. No support from
Nicogen was used and no benefit to the company was obtained.
134
SIGNIFICANCE OF CHAPTER
This chapter demonstrated the application of the inhibition of nicotine metabolism on
nicotine pharmacology in a mouse model. Specifically, the human CYP2A6 inhibitor
methoxsalen significantly inhibited the CYP2A5-mediated nicotine metabolism in mice
both in vitro and in vivo. The inhibition of nicotine metabolism in mice led to a substantial
increase in nicotine’s pharmacological effect (i.e. tail-flick and hot-plate tests).
135
CHAPTER 5: SELEGILINE (L-DEPRENYL) IS A MECHANISM-BASED INACTIVATOR OF CYP2A6 INHIBITING NICOTINE METABOLISM IN HUMANS AND MICE
Eric C. K. Siu and Rachel F. Tyndale As published in: Journal of Pharmacology and Experimental Therapeutics, 2008, Mar;
324(3):992-999
All of the experiments, data analyses, and writing of the manuscript were performed by
ECK Siu. The study was designed by ECK Siu and RF Tyndale.
Reprinted from Journal of Pharmacology and Experimental Therapeutics, Volume 324, Siu et al., “SELEGILINE (L-DEPRENYL) IS A MECHANISM-BASED INACTIVATOR OF CYP2A6 INHIBITING NICOTINE METABOLISM IN HUMANS AND MICE”, pp 992-999, Copyright (2008) with permission from The American Society for Pharmacology and Experimental Therapeutics. © 2008 The American Society for Pharmacology and Experimental Therapeutics
136
Abstract
Selegiline (L-deprenyl) is in clinical treatment trials as a potential smoking cessation drug.
We investigated the impact of selegiline and its metabolites on nicotine metabolism. In
mice, selegiline was a potent inhibitor of nicotine metabolism in hepatic microsomes and
cDNA-expressed CYP2A5; the selegiline metabolites desmethylselegiline, L-
methamphetamine, and L-amphetamine, also inhibited nicotine metabolism. Pre-
treatment with selegiline and desmethylselegiline increased inhibition (IC50) in
microsomes by 3.3- and 6.1-fold, respectively. In mice in vivo, selegiline increased AUC
(90.7±5.8 vs. 57.4±5.3 ng • hr/ml, p < 0.05), decreased clearance (4.6±0.4 vs. 7.3±0.3
ml/min, p < 0.05), and increased elimination half-life (12.5±6.3 vs. 6.6±1.4 min, p < 0.05)
of nicotine. In vitro, selegiline was a potent inhibitor of human nicotine metabolism in
hepatic microsomes and cDNA-expressed CYP2A6; desmethylselegiline and L-
amphetamine also inhibited nicotine metabolism. Selegiline pre-incubation increased
inhibition in microsomes (3.7-fold) and CYP2A6 (14.8-fold); the Ki for CYP2A6 was 4.2
µM. Selegiline dose- and time-dependently inhibited nicotine metabolism by CYP2A6 (KI
= 15.6±2.7 µM; kinact = 0.34±0.04 min-1) and the inhibition was irreversible in the
presence of NADPH, indicating that it is a mechanism-based inhibitor of CYP2A6. Thus,
inhibition of mouse nicotine metabolism by selegiline was competitive in vitro, and
significantly increased plasma nicotine in vivo. In humans where selegiline is both a
competitive and mechanism-based inhibitor, it is likely to have even greater effects on in
vivo nicotine metabolism. Our findings suggest that an additional potential mechanism of
selegiline in smoking cessation is through inhibition of nicotine metabolism.
137
Introduction
Nicotine is the primary psychoactive component in tobacco responsible for the addictive
properties of cigarettes (Henningfield and Keenan, 1993). One action of nicotine is the
binding to nicotinic receptors stimulating dopamine release in the nucleus accumbens,
an area of the brain responsible for reward (Balfour, 2004). In the brain, dopamine is
metabolized by monoamine oxidases A and B (MAO-A and MAO-B) (Youdim et al., 2006)
although MAO-B appears to be the major form (Fowler et al., 1987). The amount of
MAO-B activity in the brain of smokers is ~40% lower compared to non-smokers (Fowler
et al., 1996a) and this inhibition of MAO-B is reversed during long-term smoking
abstinence (Gilbert et al., 2003), suggesting the presence of a MAO-B inhibitor in
tobacco smoke (Khalil et al., 2000). These findings suggest that the reduced metabolism
of dopamine, as mediated by the inhibition of MAO, may enhance the rewarding effects
of nicotine during smoking (Lewis et al., 2007).
Selegiline (L-deprenyl) is a selective and irreversible MAO-B inhibitor used in
conjunction with L-dopa to alleviate symptoms associated with Parkinson’s disease
(Gerlach et al., 1996). Due to its ability to reduce dopamine metabolism, selegiline has
been investigated as a potential therapy for smoking cessation. Several small-scale
studies have shown that selegiline is effective in reducing withdrawal symptoms and
increasing abstinence compared to placebo. For instance, in one study 10 mg of oral
selegiline decreased craving during abstinence and reduced smoking satisfaction during
smoking (Houtsmuller et al., 2002). In another study, 5 mg b.i.d. oral selegiline increased
the trial end point (8-week) 7-day point prevalent abstinence compared with placebo by
three-fold (George et al., 2003). In a third study that used a combination of oral selegiline
and nicotine patch, selegiline plus nicotine patch doubled the 52-week continuous
abstinence rate compared to nicotine patch alone although the difference was not
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significant due to small subject numbers (Biberman et al., 2003). Other MAO inhibitors
have also been investigated for their effects on smoking cessation, but due to poor
efficacy (moclobemide) or toxicity (lazabemide) they are no longer studied (McRobbie et
al., 2005).
Selegiline is metabolized to desmethylselegiline and L-methamphetamine, both
of which can be further metabolized to L-amphetamine as well as other minor
metabolites (Fig. 21) (Shin, 1997; Valoti et al., 2000); despite this, there is no evidence
indicating that selegiline is addictive (Schneider et al., 1994). In humans, chronic
treatment with selegiline reduces the metabolism of selegiline and its metabolites,
suggesting that selegiline or its metabolites may inhibit or down-regulate its own
metabolic enzymes (Laine et al., 2000). Selegiline belongs to the acetylene group of
compounds that contain a carbon-carbon triple bond, which are known to be potent
mechanism-based inhibitors (Correia and Ortiz de Montellano, 2005). Since the main
human nicotine metabolizing enzyme CYP2A6 appears to play a role in the metabolism
of selegiline in vitro (Benetton et al., 2007), we examined whether selegiline and its
metabolites (desmethylselegiline, L-methamphetamine, and L-amphetamine) (Shin,
1997) could inhibit nicotine metabolism in vitro in human and mouse hepatic microsomes,
as well by both cDNA-expressed major human nicotine metabolizing enzyme CYP2A6
and mouse CYP2A5 (Murphy et al., 2005b; Siu and Tyndale, 2007a). Genetically slow
CYP2A6 metabolizers have a greater likelihood (1.75-fold) of quitting smoking (Gu et al.,
2000) suggesting that if selegiline inhibits nicotine metabolism, this may be an additional
mechanism through which it reduces smoking. In addition, selegiline could potentially be
used to enhance the efficacies of current nicotine
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Figure 21. Selegiline and its metabolites. Selegiline is metabolized to desmethyselegiline and L-methamphetamine, both of which can be metabolized to L-amphetamine (Shin, 1997; Valoti et al., 2000).
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replacement therapies, or it could be combined with nicotine as an oral combination
therapy with nicotine for smoking cessation.
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Materials and Methods
Animals
Adult male DBA/2 mice, previously characterized in nicotine metabolism studies (Siu and
Tyndale, 2007a), were obtained from Charles River Laboratories Inc. (Saint-Constant,
PQ). Animals were housed in groups of three to four on a 12-hour light cycle and had
free access to food and water.
Reagents
(-)-Nicotine hydrogen tartrate, (-)-cotinine, selegiline (R-(-)-deprenyl hydrochloride), R-(-
)-desmethylselegiline, thiamine hydrochloride, and 5-aminolevulinic acid (ALA) were
purchased from Sigma-Aldrich (St. Louis, MO). L-methamphetamine and L-
amphetamine were purchased from Research Biochemicals Inc. (Natwick, MA). The
internal standard 5-methylcotinine was custom-made by Toronto Research Chemicals
(Toronto, ON). All dosed drugs are expressed as the free base of the drug. Ampicillin
and lysozyme were purchased from BioShop Canada (Burlington, ON). IPTG (Isopropyl-
beta-D-thiogalactopyranoside) was purchased from MBI Fermentas (Burlington, ON).
The monoclonal antibody to human CYP2A6 was purchased from BD Biosciences
(Mississauga, ON). Horseradish peroxidase-conjugated anti-mouse secondary antibody
and enhanced chemiluminescence were purchased from Pierce Biotechnology (Rockfort,
IL). Nitrocellulose membrane was purchased from Bio-Rad Labs (Mississauga, ON).
Autoradiographic film was purchased from Ultident Scientific (St. Laurent, PQ).
Membrane Preparations
Microsomal membranes were prepared from mouse and human livers for in vitro nicotine
metabolism assays as previously described (Siu et al., 2006) and stored at -80°C in
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1.15% KCl. The human liver is from the previously characterized K-series liver bank
(Messina et al., 1997) and was a generous gift from Dr. T. Inaba at the University of
Toronto. The cytosolic fractions were acquired during membrane preparation and were
used as a source of aldehyde oxidase. All mouse livers and plasma samples were
collected and frozen prior to 3 pm to minimize circadian effects on CYP2A5 expression.
Membrane protein concentrations were determined with Bradford reagent according to
manufacturer's protocol (Bio-Rad Labs Ltd., Mississauga, ON).
Expression of CYP2A5
Cyp2a5 cDNA vector in Escherichia coli was a generous gift from Dr. Xinxin Ding
(Wadsworth Center, New York State Department of Health, NY) and prepared as
previously described with modifications (Gu et al., 1998). Briefly, E. coli colonies from
ampicillin plates were inoculated in 2 ml of LB broth with 100 µg/ml of ampicillin and
incubated overnight (no more than 16 hours) at 37oC shaking at 200 rpm. The culture
was then diluted (1:100) in TB broth, with final concentrations of 100 µg/ml of ampicillin,
1 mM thiamine, 0.5 ml of ALA, and 1 mM IPTG, and incubated for 48 hours at 25oC
shaking at 150 rpm. Following incubation the culture was centrifuged at 2800xg at 4oC
for 20 min and the pellet was resuspended in 1/20th culture volume of ice-cold TSE
buffer (100 mM Tris, pH 7.4, 0.1 mM EDTA, 0.1 mM dithiothreitol, 1.15% KCl, 110 g/L
sucrose) and 1/20th culture volume of ice-cold water. Lysozyme was then added to a
final concentration of 0.25 mg/ml and this was shaken gently at 4oC for 1 hr followed by
centrifugation at 2800xg at 4oC for 20 min. After centrifugation the pellet was
resuspended in 1/25th culture volume of ice-cold TE buffer. The resuspension was
placed on ice and sonicated in 3 x 30 sec bursts with a Branson Digital Sonifier (Model
S-450D; Branson Ultrasonics, Markham, ON) set at 30% output. The suspension was
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spun at 12,000xg at 4oC for 12 min and the supernatant was re-spun at 180,000xg at
4oC for 60 min. The pellet containing the bacterial membrane fraction was resuspended
in 1.15% KCl and stored at -80oC.
Quantification by Immunoblotting of CYP2A5
Immunoblotting was performed for CYP2A5 and the reference lymphoblastoid cDNA-
expressed human CYP2A6 (BD Biosciences, San Jose, CA) essentially as previously
described (Siu et al., 2006). To determine the linear ranges of detection of CYP2A5 and
CYP2A6, in order to quantify the amount of CYP2A5 in the bacterial membrane, the
bacterial membrane fraction and CYP2A6 were serially diluted from 0.25 to 2 µg protein
and from 0.06 to 1.5 pmol, respectively.
In Vitro Nicotine C-Oxidation Assay
The linear conditions for nicotine C-oxidation to cotinine (nicotine metabolism) in DBA
mouse liver microsomes were previously described (Siu et al., 2006) (0.5 mg/ml protein;
15 min). In human liver microsomes, the linear conditions of nicotine metabolism were
obtained under assay conditions of 0.5 mg/ml protein with an incubation time of 20 min.
For CYP2A5, the linear conditions of nicotine metabolism were obtained under assay
conditions of 60 pmol P450/ml, 60 pmol of reductase and cytochrome b5 (Invitrogen,
Carlsbad, CA) with an incubation time of 15 min. For expressed CYP2A6 (containing
P450 reductase and cytochrome b5) (BD Gentest, Woburn, MA), the linear conditions of
nicotine metabolism were obtained under assay conditions of 20 pmol P450/ml with an
incubation time of 15 min. All incubation mixtures contained 1 mM NADPH and 1 mg/ml
of liver cytosol in 50 mM Tris-HCl buffer, pH 7.4 and were performed at 37oC in a final
volume of 0.5 ml. Unless specified, nicotine concentrations used were 10 µM (mouse
liver microsomes), 60 µM (CYP2A5), 20 µM (human liver microsomes), and 100 µM
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(CYP2A6). The reactions were stopped with a final concentration of 4% v/v Na2CO3.
After incubation, 5-methylcotinine (70 µg) was added as the internal standard and the
samples were prepared and analyzed for nicotine and metabolites by HPLC system as
described previously (Siu et al., 2006). The limits of quantification were 5 ng/ml for
nicotine and 12.5 ng/ml for cotinine.
In Vitro Inhibition of Nicotine Metabolism
In all in vitro inhibition experiments assays were conducted as above with the addition of
the inhibitors at the same time as nicotine. In experiments with the pre-incubation step,
reactions containing increasing concentrations of the inhibitor were initiated by pre-
warming the mixture for two minutes prior to the addition of NADPH and pre-incubation
of 15 minutes at 37oC prior to addition of nicotine and incubation.
For the NADPH-dependent inactivation experiment, 0 or 100 µM of selegiline
was added to the mixture in the presence or absence of NADPH. Following pre-
incubation, samples were loaded into the Microcon YM-30 centrifuge membrane filters
(Dyck and Davis, 2001) (Millipore, Etobicoke, ON) and centrifuged for at least 30 min at
4oC. Retentates (typically 40-50 µl) were added to fresh reaction mixtures containing
nicotine and the reactions were allowed to proceed.
In Vivo Inhibition of Nicotine Metabolism
Nicotine and selegiline were dissolved in physiological saline (0.9% sodium chloride)
and adjusted to pH 7.4 for use in in vivo studies. All animals were injected
intraperitoneally with nicotine or nicotine plus selegiline (both at 1 mg/kg). The nicotine
dose was chosen based on previous pharmacokinetic studies (Siu and Tyndale, 2007a)
and relevance in behavioural models for nicotine in mice (Marks et al., 1985). The
selegiline dose was based on its relevance in the mouse MPTP-model of Parkinson’s
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disease (Fredriksson and Archer, 1995). Blood samples were drawn by cardiac puncture
at various times after the injection. Immediately after collection plasma was prepared by
centrifugation at 3000xg for 10 min and kept at –20˚C until analysis. Total plasma
nicotine levels (free and glucuronides) were measured following deconjugation by β-
glucuronidase at a final concentration of 5 mg/ml in 0.2 M acetate buffer, pH 5.0, at 37oC
overnight. Samples were then analyzed by HPLC.
In Vitro Kinetic and Pharmacokinetic Parameters Analyses and Statistical Analyses
The Michaelis-Menten kinetic parameters Km and Vmax were calculated using Graphpad
Prism (Graphpad Software Inc., San Diego, CA) and were verified by the Eadie-Hofstee
method. The equation used to determine Km and Vmax was v = Vmax [S] / (Km + [S]) (Eqn.
1) where [S] denotes substrate concentration. These kinetic parameters were used to
determine nicotine concentrations used. Statistical analyses of in vitro kinetic parameters
were tested by student’s t-test.
The in vivo pharmacokinetic parameters were determined using non-
compartmental analysis: AUC0-40 was calculated using the trapezoidal rule. Elimination
half-life (t1/2) was estimated by the terminal slope. Since the bioavailability (F) of nicotine
was unknown following intraperitoneal injection in mice, CL (clearance) was determined
as a hybrid parameter CL/F and was calculated as Dose/AUC0-40. The average weights
of the animals were similar (24.4 ± 0.7 g, n=24), therefore an estimated dose of 25 µg
was used for the calculation of CL/F for nicotine. Assessment of in vivo nicotine levels
for the entire time-course was not possible from individual animals due to limited blood
volume; therefore each time point represented data from multiple mice. Due to this
experimental design, pharmacokinetic parameters (e.g. half-life) were estimated by
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resampling methods using the PKRandTest software (H. L. Kaplan, Toronto, ON) (Siu
and Tyndale, 2007a).
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Results
Inhibition of Nicotine Metabolism by Selegiline and Metabolites
Selegiline and desmethylselegiline had the highest inhibitory activities on nicotine
metabolism in mouse liver microsomes (MLMs) while L-methamphetamine and L-
amphetamine had smaller effects (Fig. 22a). Selegiline also inhibited CYP2A5 to a great
extent while desmethylselegiline, L-methamphetamine, and L-amphetamine had similar
inhibitory effects (Fig. 22b). In human liver microsomes (HLMs), selegiline and L-
amphetamine appeared to show the greatest inhibitory activities on nicotine metabolism
closely followed by desmethylselegiline (Fig. 22c), while L-methamphetamine did not
inhibit cotinine formation (Fig. 22c). As with the three previous enzyme sources,
selegiline caused the greatest inhibition on nicotine metabolism in CYP2A6 (Fig. 22d).
Desmethylselegiline and L-amphetamine showed almost similar inhibition whereas L-
methamphetamine did not alter nicotine metabolism (Fig. 22d), as was seen with HLMs
(Fig. 22c).
The above findings suggested that selegiline had the greatest inhibitory effect on
CYP2A5-mediated nicotine metabolism in vitro in mice followed by selegiline’s three
metabolites. Likewise, selegiline, desmethylselegiline, and L-amphetamine could inhibit
CYP2A6-mediated human nicotine metabolism in vitro whereas L-methamphetamine did
not.
Effects of Selegiline and Desmethylselegiline Pre-incubation on Nicotine Metabolism in
Mouse Liver Microsomes and CYP2A5
As selegiline and desmethylselegiline contain a carbon-carbon triple bond found in
mechanism-based inhibitors (MBI), the effects of pre-incubation of these compounds on
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Figure 22. Selegiline and its metabolites inhibited nicotine metabolism. a) MLMs, b) CYP2A5, c) HLMs, and d) CYP2A6 were incubated with 0 (control), 10, and 100 µM of selegiline (SEL), desmethylselegiline (DES), L-methamphetamine (L-MAMP), or L-amphetamine (L-AMP) in the presence of nicotine and analyzed for cotinine formation. Activity remaining for each compound was compared to the corresponding control treatment. Each data point represented the average of 2 to 5 independent experiments. * p < 0.05 compared to control.
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nicotine metabolism in MLMs and CYP2A5 were investigated. If pre-treatment increased
inhibition, this could indicate that the inhibitor is either acting as a MBI or is metabolized
to a more potent inhibitor during the pre-incubation. Selegiline dose-dependently
inhibited cotinine formation in both MLMs and CYP2A5; pre-incubation with selegiline
decreased the IC50 in MLMs (3.3-fold) but not in CYP2A5 (Figs. 23a & b).
Desmethylselegiline also dose-dependently inhibited nicotine metabolism in MLMs but
its effect was much weaker in expressed CYP2A5 (Figs. 23c & d). As with selegiline,
pre-incubation with desmethylselegiline enhanced inhibition in MLMs (6.1-fold change in
IC50) but not in CYP2A5 (Figs. 23c & d). This indicated that selegiline and
desmethylselegiline were competitive inhibitors, but not MBIs, of CYP2A5 in vitro; and
other enzymes in MLMs can metabolize selegiline and desmethylselegiline to inhibitors
of greater potency.
Effect of Selegiline on Nicotine Metabolism in vivo in DBA/2 Mice
To determine if selegiline could also inhibit nicotine metabolism in vivo, we treated
DBA/2 mice with selegiline and nicotine. Selegiline co-administration decreased the
clearance of nicotine by ~40%, resulting in 58% greater AUC and almost doubling the
elimination half-life (Fig. 24, Table 11). These results demonstrated that selegiline is an
effective inhibitor of nicotine metabolism in vivo in mice.
Effects of Selegiline and Desmethylselegiline Pre-incubation on Nicotine Metabolism in
Human Liver Microsomes and CYP2A6
To further investigate the effect of selegiline and desmethylselegiline on nicotine
metabolism in humans, HLMs and CYP2A6 were pre-incubated with the inhibitors.
Selegiline dose-dependently inhibited cotinine formation in both HLMs and expressed
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Figure 23. Pre-incubation of MLMs, but not CYP2A5, with selegiline increased inhibition of nicotine metabolism. MLMs (a & c) and CYP2A5 (b & d) were pre-incubated (+PRE) for 15 minutes with increasing concentrations (0, 0.3, 1, 3, 10, 30, and 100 µM) of selegiline (a & b) or desmethylselegiline (c & d) prior to addition of nicotine and an additional incubation of 15 minutes. Samples were also incubated with the inhibitors and nicotine without pre-treatment (-PRE). Activity remaining was calculated as % control (no inhibitor, same incubation conditions) within each treatment group. IC50 was determined as the concentration of inhibitor required to decrease nicotine metabolism by 50%. Each data point represented the average of 2 to 5 independent experiments. * p < 0.05 compared to no pre-incubation.
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Figure 24. Selegiline decreased nicotine metabolism in vivo in DBA/2 mice. DBA/2 mice were injected with nicotine (1 mg/kg, i.p.) (-SEL) or with nicotine plus selegiline (1 mg/kg, i.p.) (+SEL). Following nicotine injection plasma samples were collected at the indicated time points and analyzed for nicotine levels. Each time point represents mean (± S.D.) of 3 to 6 animals for each treatment; statistical comparisons are listed in Table 1.
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Table 11. Pharmacokinetic parameters of plasma nicotine in mice treated intraperitoneally with nicotine (1 mg/kg) or nicotine plus selegiline (1 mg/kg). Results were derived using data (Fig. 3) from three to six animals at each time point.
-SEL (mean ± SD)
+SEL (mean ± SD)
AUC0-40 (ng • hr/ml) 57.4 ± 5.3 90.7 ± 5.8* t1/2 (min) 6.6 ± 1.4 12.5 ± 6.3* CL/F (ml/min) 7.3 ± 0.3 4.6 ± 0.4*
*p < 0.05 compared to –SEL
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CYP2A6 (Figs. 25a & b). In contrast to the mouse enzymes, pre-incubation of both
HLMs and CYP2A6 with selegiline decreased the IC50 by 3.7- and 14.8-fold, respectively
(Figs. 25a & b). Desmethylselegiline also dose-dependently inhibited nicotine
metabolism in HLMs (Fig. 25c) but seemed to be a weaker inhibitor of CYP2A6 (Fig.
25d). Pre-incubation with desmethylselegiline did not enhance inhibition in HLMs but
may modestly increase inhibition in CYP2A6 (Figs. 25c & d). The above findings
suggested that selegiline is acting as a MBI of CYP2A6 or that selegiline could be
metabolized by HLMs and CYP2A6 to metabolites that are even more potent inhibitors
than selegiline. Following preincubation, a Ki of 4.2 µM for the competitive inhibition of
CYP2A6 was observed (Fig 26a).
Time-, Concentration-, and NADPH-Dependent Irreversible Inhibition of CYP2A6-
Mediated Nicotine Metabolism
In addition, to determine if the inhibition of CYP2A6-mediated nicotine metabolism by
selegiline is mechanism-based, we first carried out time- and concentration-dependent
inactivation assays. Figure 26b indicates that the cotinine formation decreases with
increasing pre-incubation time and the decreases were dose-dependent. Using a
double-reciprocal plot, the KI and kinact were estimated to be 15.6 ± 2.7 µM and 0.34 ±
0.04 min-1, respectively (Fig. 26c).
To determine if the decrease in nicotine metabolism was due to covalent
modification of CYP2A6 by the metabolically activated selegiline, we used a centrifuge
filtering system that would allow for the removal of the unbound inhibitors from the pre-
incubation mixture (Dyck and Davis, 2001). In the absence of NADPH in the pre-
incubation mixture with selegiline, filter removal of unbound selegiline (-N+I) did not lead
to a significant decrease in enzyme activity compared to control (-N-I) (Fig. 26d),
indicating a requirement for the cofactor NADPH. In contrast, in the presence of NADPH
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Figure 25. Pre-incubation of human hepatic microsomes and CYP2A6 with selegiline increased inhibition of nicotine metabolism. HLMs (a & c) and CYP2A6 (b & d) were pre-incubated (+PRE) for 15 minutes with increasing concentrations (0, 1, 3, 10, 30, and 100 µM) of selegiline (a & b) or desmethylselegiline (c & d) prior to addition of nicotine and an additional incubation of 15 or 20 (HLMs) minutes. Samples were also incubated with the inhibitors and nicotine without pre-treatment (-PRE). Activity remaining was calculated as % control (no inhibitor, same incubation conditions) within each treatment group. IC50 was determined as the concentration of inhibitor required to decrease nicotine metabolism by 50%. Each data point represented the average of 2 to 5 independent experiments. * p < 0.05 compared to no pre-incubation.
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Figure 26. Selegiline inhibited CYP2A6-mediated nicotine metabolism in a time-, concentration-, and NADPH-dependent manner. a) A Dixon plot of competitive inhibition: CYP2A6 were pre-incubated with increasing concentrations of selegiline (0, 0.5, 1, 2, and 4 µM) for 15 minutes. Following pre-incubation 50, 100, or 200 µM of nicotine was added to each of the samples and reactions were carried out for an additional 15 min. b) CYP2A6 was pre-incubated with increasing concentrations of selegiline (0, 1, 2.5, 10, 20, 40 µM) for 0, 1, 3, and 5 minutes. Following pre-incubation nicotine was added to each sample and the reactions were carried out for an additional 15 minutes. Data was plotted as the natural log (Ln) of % activity remaining against the pre-incubation time. Regression lines represent the initial slope of the inhibition curve (Ghanbari et al., 2006). c) Double reciprocal plot of the rate of inactivation (kobs) of cotinine formation as a function of the concentration of selegiline. Data represents duplicate experiments. d) CYP2A6 was pre-incubated without NADPH or inhibitors (-N-I), without NADPH but with 100 µM of selegiline or desmethylselegiline (-N+I), or with NADPH and 100 µM of selegiline or desmethylselegiline (+N+I) for 15 minutes in the absence of nicotine. This was followed by removal of the unbound inhibitors through centrifugal filtration. Retentates were added to fresh reaction mixture containing nicotine and incubated for an additional 15 minutes. Data were averaged from 2 to 3 experiments. * p < 0.05 compared to the -N+I treatment group.
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during pre-incubation (+N+I), filter removal of unbound selegiline did not lead to
significant recovery of cotinine formation activity, suggesting that CYP2A6 was
inactivated by metabolic activation of selegiline. Similar findings were also seen with
desmethylselegiline (Fig. 26d). The above findings demonstrated that the inhibition of
CYP2A6-mediated nicotine metabolism by selegiline is irreversible in the presence of
NADPH and that selegiline is a MBI of CYP2A6.
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Discussion
It is estimated that 20% of adults in the US are current smokers (National, 2007).
Previous studies demonstrated that selegiline was an effective aide in smoking
cessation (Biberman et al., 2003; George et al., 2003). At least four clinical trials are
currently underway to evaluate the effectiveness of selegiline as a potential therapy in
smoking cessation (www.clinicaltrial.gov). Selegiline can prevent dopamine metabolism
by irreversibly inhibiting MAO-B (Youdim, 1978). Selegiline also appeared to be able to
inhibit its own metabolism in vivo (Laine et al., 2000), possibly via inhibition of CYP2A6.
In the present study we showed that nicotine metabolism in both mice and human liver
microsomes and expressed enzymes could be inhibited by selegiline,
desmethylselegiline, L-methamphetamine (only in mice), and L-amphetamine. In
addition, we also demonstrated that selegiline inhibited nicotine metabolism in vivo in
mice. More importantly, we showed that selegiline is a mechanism-based inhibitor of
human CYP2A6.
In all in vitro systems tested in our study, selegiline (and desmethylselegiline in
MLMs) inhibited nicotine metabolism with the greatest potency compared to its
metabolites. In mice, a greater level of inhibition was seen in MLMs compared to the
expressed CYP2A5. However, the enhanced inhibition of nicotine metabolism following
selegiline pre-incubation in MLMs, not observed with CYP2A5, suggested that selegiline
is unlikely to be a MBI of CYP2A5. The greater inhibition in MLMs (compared to
expressed CYP2A5; and with pre-incubation in MLMs) may be due to the formation of
more potent inhibitory metabolite(s) of selegiline. None of the tested selegiline
metabolites inhibited CYP2A5 with greater potency compared to selegiline. However,
another metabolite that is produced in mice is selegiline-N-oxide (Levai et al., 2004). It is
possible that in addition to the compounds tested here, that selegiline-N-oxide, produced
by other enzymes in MLMs, can also inhibit CYP2A5-mediated nicotine metabolism.
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For both HLMs and CYP2A6, pre-incubation with selegiline led to an increase in
inhibition potency. This enhancement in inhibition was more dramatic in CYP2A6
compared to HLMs, which might reflect the metabolism of selegiline in HLMs by other
CYPs such as CYP2B6 and CYP2C8 (Benetton et al., 2007), thus reducing its
availability to inhibit CYP2A6. More importantly, the inhibition of CYP2A6, in addition to
being competitive, was likely mechanism-based. This was supported by the findings that
in the time- and concentration-dependent inhibition study, the decrease in nicotine
metabolism was enhanced with increasing inhibitor pre-incubation time and with
increasing inhibitor concentration. Furthermore, the inhibition of CYP2A6 by selegiline
was irreversible in the presence of NADPH, suggesting that selegiline was metabolically
activated by CYP2A6 and the reactive intermediate formed a covalent linkage with the
holoenzyme. The general mechanism of acetylene-mediated destruction of P450s
appears to be the direct N-alkylation of the porphyrin (Correia and Ortiz de Montellano,
2005). However the precise mechanism by which selegiline irreversibly inhibited
CYP2A6 activity was not determined. The minor inhibition seen in the presence of
selegiline without NADPH was likely due to residual inhibitor left in the retentate
transferred to the fresh nicotine-containing reaction mixture.
Since desmethylselegiline also contains the carbon-carbon triple bond and
irreversibly inhibited MAO-B and decreased its activity by up to 65% in vivo (Heinonen et
al., 1997), we also characterized its effects on nicotine metabolism in both species. In
mice in the absence of pre-incubation, desmethylselegiline inhibited nicotine metabolism
in MLMs to the same extent as selegiline. The greater inhibition by desmethylselegiline
in MLMs (compared to CYP2A5), and the potentiation of inhibition with pre-incubation, is
likely due to the production of more potent metabolite(s) of desmethylselegiline by other
enzymes in MLMs. The only known metabolites of desmethylselegiline are L-
amphetamine and its hydroxylated products (Shin, 1997); however, L-amphetamine had
159
weaker inhibitory activities. Thus the specific desmethylselegiline metabolite (e.g.
potentially N-oxides) that may be contributing to the inhibition in MLMs, but not in
CYP2A5, is unknown.
Interestingly in HLMs pre-incubation with desmethylselegiline did not increase
the inhibition potency but it appeared to show minor effects on CYP2A6. One possibility
is that desmethylselegiline had weaker affinity for CYP2A6 compared to other enzymes
present in HLMs (i.e. inhibiting or metabolized by other enzymes) and thus was less
available to inhibit nicotine metabolism by CYP2A6. The inhibition of CYP2A6 by
desmethylselegiline could potentially be mechanism-based since in the presence of
NADPH the inhibition of nicotine metabolism was irreversible.
In our study L-methamphetamine inhibited nicotine metabolism in mice but not in
humans. L-amphetamine inhibited nicotine metabolism almost to the same extent as
selegiline in CYP2A5 and HLMs but had the weakest effect in CYP2A6. Again, it is
possible that L-amphetamine was metabolized to more potent inhibitors of nicotine
metabolism by other enzymes in HLMs. The effects of the D-isomers of
methamphetamine and amphetamine on CYP2A5 or CYP2A6 activities are unknown;
however, one study found that racemic (D,L-) amphetamine was a weak inhibitor of
CYP2A6 and a much weaker inhibitor of CYP2A5 (Rahnasto et al., 2003).
We and others have previously demonstrated that nicotine, at relevant
pharmacological concentrations, is metabolized essentially exclusively by CYP2A5 in
mice (Murphy et al., 2005b; Siu and Tyndale, 2007a). We have also shown that
subcutaneous administration of the CYP2A5/6 inhibitor methoxsalen significantly
inhibited nicotine metabolism when nicotine was given subcutaneously and this increase
in nicotine plasma levels subsequently increased the pharmacological actions of nicotine
(Damaj et al., 2007b), demonstrating the value of mice in studying nicotine metabolism
and pharmacology. Since selegiline inhibited CYP2A5-mediated nicotine metabolism in
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MLMs and CYP2A5, we determined its effect on nicotine clearance in mice in vivo. We
treated mice with nicotine and selegiline intraperitoneally as this route mimics oral
delivery of both drugs in animals, the route of choice for a novel smoking cessation
product. Administration of selegiline together with nicotine caused almost a 40%
decrease in clearance of nicotine and doubled its elimination half-life. These data
suggested that selegiline and its metabolites can act as competitive inhibitors of nicotine
in vivo even though selegiline is not a MBI of CYP2A5. It is likely that in humans,
selegiline can decrease the first-pass metabolism and elimination half-life of nicotine via
mechanism-based inactivation as well as competitive inhibition of CYP2A6. Genetically
slow CYP2A6 metabolizers have an increased likelihood of quitting smoking (Gu et al.,
2000) suggesting that inhibition of CYP2A6 by selegiline may contribute to selegiline’s
ability as a smoking cessation therapeutic agent.
Our findings suggest that oral selegiline may also be combined with oral nicotine
to create an orally bioavailable and clinically effective form of nicotine due to the high
hepatic extraction of selegiline (Heinonen et al., 1994) (suggesting that selegiline will be
able to rapidly inhibit CYP2A6 prior to reaching the systemic circulation) and the
relatively smaller Ki and KI of selegiline towards nicotine by CYP2A6 (4.2 and 15.6 µM
respectively) compared to the Km for nicotine (~65 µM) (Messina et al., 1997). Currently
there is no pill form of nicotine replacement therapy as the higher doses required to
overcome first-pass metabolism may cause nausea and diarrhea due to gastrointestinal
irritation (Benowitz et al., 1991b). Oral administration of even low doses (4 mg) of
nicotine to those genetically deficient in CYP2A6 increased nicotine AUC by over 3-fold
(Xu et al., 2002), suggesting that inhibition of nicotine metabolism can make nicotine
orally bioavailable. We have also shown that the combination of the CYP2A6 inhibitor
methoxsalen and nicotine significantly increased the mean plasma level of nicotine in
humans as well as decreased the number of cigarettes smoked, reduced craving, the
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number of puffs, the inhalation intensity, and breath carbon monoxide levels, a
biomarker of smoke inhalation (Sellers et al., 2000). Furthermore, inhibition of CYP2A6
by methoxsalen also re-routed the tobacco-specific pro-carcinogen NNK to NNAL and
glucuronidation pathways (Sellers et al., 2003a), indicating the additional potential
benefit for harm-reduction.
Here we have shown that selegiline is a mechanism-based inhibitor of human
CYP2A6, the major enzyme responsible for nicotine metabolism. In mice, where
selegiline and its metabolites are competitive inhibitors, selegiline substantially reduced
nicotine metabolism in vivo resulting in prolonged elevated nicotine plasma levels. These
findings suggest that in humans, where selegiline is a competitive and MBI of CYP2A6-
mediated nicotine metabolism, selegiline can potentially be an effective inhibitor of
nicotine metabolism.
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Acknowledgments
We would like to thank Dr. Sharon Miksys for the assistance in the development of the in
vivo protocol and Dr. Bin Zhao for assistance with HPLC. We would also like to thank
members of the Tyndale laboratory for critical review of this manuscript. RFT is a
shareholder and chief scientific officer of Nicogen Inc., a company focused on the
development of novel smoking cessation therapies; no funds were received from
Nicogen for these studies nor was this manuscript reviewed by other people associated
with Nicogen.
163
SIGNIFICANCE OF CHAPTER
In this study we found selegiline to be an inhibitor of CYP2A5-mediated in vitro nicotine
metabolism. Selegiline also inhibited in vivo nicotine metabolism in mice. More
importantly, in humans the pre-incubation of hepatic microsomes and cDNA-expressed
CYP2A6 with selegiline enhanced the inhibition nicotine metabolism. This with additional
studies showing that selegiline irreversibly inhibited CYP2A6 under condition of
metabolic activation demonstrated that selegiline to be a MBI of CYP2A6-mediated
nicotine metabolism. The latter may represent an additional mechanism by which
selegiline mediates smoking cessation.
164
CHAPTER 6 GENERAL DISCUSSION
Prior to the approval of a candidate drug for use in humans, the compound must first be
tested for its pharmacological and toxicological effects in vivo in animal models. In this
thesis I examined the mouse as a potential model of nicotine metabolism and its
application in studying inhibitors and the consequence of genetic variation in nicotine
metabolism and pharmacology. In order to determine the suitability of the mouse as an
experimental model, a fundamental requirement is that the metabolism of nicotine to
cotinine occurs similarly to that seen in humans (e.g. the reaction is primarily catalyzed
by a homologous enzyme with similar activities). We were interested in the mouse as an
experimental model to study nicotine metabolism based on several reasons. First, a
preliminary quantitative trait loci study in mice suggested the possible involvement of
Cyp2a5 in nicotine self-administration (Wildenauer et al., 2005). Second, mouse
CYP2A5 shares high amino acid sequence similarity to human CYP2A6. Third, the
pharmacodynamics of nicotine in several inbred mouse strains have been well
established (Collins et al., 1988; Faraday et al., 2005; Grabus et al., 2005; Marks et al.,
1985) and this may be combined with nicotine metabolism data to study the contribution
of genetic variation to nicotine pharmacology. Fourth, mice can be genetically modified
(e.g. introducing human cytochrome P450 genes). Finally, mice are small, easily
maintained, and are economical compared to other animal species.
Section 5 Metabolism Topicss
Section 5.1 In Vitro Metabolism
Section 5.1.1 In Vitro Nicotine Metabolism
165
The in vitro nicotine metabolism data from our studies are in accordance with similar
studies published during this period. The apparent Kms of cotinine formation in the
hepatic microsomes from DBA/2, C57BL/6 (Siu and Tyndale, 2007a), and the F2 hybrid
(C57BL/6 x ST/bJ) mice (Siu et al., 2006) are similar to the Km of the cDNA-expressed
CYP2A5 (from the 129/J strain) (range 8 to 11 µM) (Murphy et al., 2005b). The Kms
observed here are lower compared to the outbred ICR mice (~19 µM) (Damaj et al.,
2007b). Another study found the Km for cotinine formation to be much higher in the
CD2F1 mice (~68 µM) (Raunio et al., 2008). It is likely that polymorphisms in CYP2A5
account for the variation in the observed Kms. For example, substitution of amino acid
207 from glycine (polar, uncharged) to proline (non-polar, hydrophobic) substantially
increased the Km (~100-fold) of CYP2A5 for coumarin 7-hydroxylation, possibly through
structural alteration increasing the size of the substrate binding pocket (Juvonen et al.,
1993). The Cyp2a5 nucleic acid sequences of ICR and CD2F1 mice and further kinetic
studies are required to confirm this hypothesis. Also, one cannot rule out the contribution
of differences in experimental procedure. Nevertheless, compared to humans, the Kms of
cotinine formation in the examined mouse strains (cDNA-expressed CYP2A5 and
hepatic microsomes; range 8 to 68 µM) largely overlap with those seen in humans
(cDNA-expressed CYP2A6 and hepatic microsomes; range 13 to 162 µM) (Messina et
al., 1997; Nakajima et al., 1996b; Yamazaki et al., 1999), suggesting similar enzymatic
activities, at least for cotinine formation.
The DBA/2 and C57BL/6 mice also have higher Vmaxs for nicotine metabolism
compared to the CD2F1 mice (Raunio et al., 2008) and this could be due to several
reasons. First, between-strain variation in the cis-acting regulatory elements and/or
trans-acting regulatory factors can affect both Cyp2a5 transcriptional and post-
transcriptional processes, thereby altering protein levels. For example, the human
166
CYP2A6*9 contains a polymorphism in the TATA box resulting in decreased
transcription (Kiyotani et al., 2003; Pitarque et al., 2001). Hepatic nuclear factor-4α
(HNF-4α) is a major transcriptional factor for CYP2A6 and Cyp2a5 (Kamiyama et al.,
2007; Ulvila et al., 2004) and bioinformatic database showed that both human and
mouse HNF-4α are highly polymorphic (MGI 4.12, Ensembl). Other regulatory factors
that can influence CYP2A6 expression include PXR, PPAR-γ, ER, C/EBPα, and hnRNP-
A1 (Christian et al., 2004; Di et al., 2009; Higashi et al., 2007a; Pitarque et al., 2005).
Genetic variations in HNF-4α or other regulatory elements could potentially alter the
expression of Cyp2a5. Second, polymorphisms affecting various steps in the P450
catalytic cycle and the interaction of the P450 oxidase with heme or oxidoreductase may
also affect the rate of substrate metabolism. However, such a change would likely alter
the Km to some extent as well since Km is defined as 1
21
k
kk +− where k2 is the turnover
number (i.e. the catalytic step) that is dependent on the overall efficiency of the catalytic
cycle (after taking into consideration the total number of catalytic sites). For instance,
CYP2A6*6 contains an amino acid substitution at position 128 where arginine (polar,
basic) is replaced with glutamine (polar, uncharged). The substitution resulted in a 10-
fold decrease in Vmax and 5-fold increase in Km for coumarin metabolism, and this was
due to decreased heme binding and disruption of the holoprotein structure (Kitagawa et
al., 2001). Random mutagenesis in CYP2A6 found that substitution of residue 476 from
lysine (polar, basic) to glutamine acid (polar, acidic) caused a 20-fold decrease in kcat
(=k2, and by extension Vmax) but only a 25% decrease in Km for coumarin hydroxylation,
a result thought to be due to reduced electron transfer efficiency (Kim et al., 2005).
Our in vitro studies demonstrated that, as in humans, there exists a second
nicotine metabolizing enzyme in mice. However, the catalytic efficiency of this enzyme
for cotinine formation is at least 10-fold lower compared to CYP2A5, suggesting that this
167
enzyme does not contribute significantly to the overall metabolism of nicotine under
normal pharmacological levels/exposures. At the moment it is difficult to speculate which
cytochrome P450 is contributing to the minor formation of cotinine as inhibitory
antibodies against human CYP2B6 and CYP2D6 did not alter nicotine metabolism in
mice hepatic microsomes (Fig. 7A).
Section 5.1.2 In Vitro Cotinine Metabolism
We have shown, from in vitro kinetic and inhibitory antibody studies that cotinine is
metabolized to 3-HC and this process appeared to be mediated almost entirely by
CYP2A5 (Figs. 6 & 7B). This finding parallels those in humans in which CYP2A6 is
solely responsible for the formation of 3-HC (Dempsey et al., 2004; Nakajima et al.,
1996a). The affinity for 3-hydroxylation of cotinine is much higher in mice (~10 to ~50 µM)
hepatic microsomes compared to humans (~235 µM) (Nakajima et al., 1996a;
Yamanaka et al., 2004). Genetic variation may contribute to the difference in the affinity
between mouse strains. Overall, our findings show that mice exhibit similar
characteristics in the metabolism of nicotine and its primary metabolite cotinine, as
observed in humans.
Section 5.1.3 Effect of a CYP2A5 Polymorphism on Substrate Metabolism
It is interesting to note that despite the reported amino acid and coumarin hydroxylase
activity differences in CYP2A5 between DBA/2 (Val117; high COH activity) and C57BL/6
(Ala117; low COH activity) (both amino acids are non-polar and hydrophobic) (Lindberg et
al., 1992), there is no difference in their cotinine formation capacity as measured by Km
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in our study. In contrast, the Km for 3-HC formation is five-fold higher in DBA/2 mice.
Previous studies have also demonstrated that amino acid 117 determines whether
CYP2A5 possesses 7β- or 2α-hydroxylase activity for dehydroepiandrosterone (Uno et
al., 1997). In CYP2A6, mutagenesis of residue 117 from valine to alanine in the cDNA-
expressed protein did not affect its Km but resulted in a substantial drop in Vmax for
coumarin hydroxylation (He et al., 2004b; Yano et al., 2005). Although crystallography
studies of the CYP2A6 crystal structure coupled with coumarin showed that amino acid
117 is in close proximity with coumarin (He et al., 2004b; Yano et al., 2005), the
aforementioned effect was hypothesized to be an indirect role of residue 117 in
coumarin hydroxylation through modulation of the function of surrounding amino acids
potentially involved in proton/electron transfer and reaction stabilization (He et al.,
2004b). It may be that these observations are restricted only to the effect of valine and
alanine on coumarin. As yet this particular mouse polymorphism has not been found in
the general human population and the impact of this alteration on nicotine and cotinine
metabolism in CYP2A6 has not been studied. The high sequence similarity of CYP2A5
with CYP2A6 suggests that residue 117 of CYP2A5 should also be located in the
substrate binding pocket and potentially be in contact with the substrate. It is possible
that the changes in amino acid 117 in mouse CYP2A5 may affect binding and catalytic
activity depending on the substrate.
Section 5.2 In Vivo Metabolism
Our data demonstrated that the in vivo clearance of nicotine in mice following
subcutaneous administration is rapid (t1/2 ~8 to 9 min) consistent with findings in these
mice following intraperitoneal injection (~6 to 7 min) (Petersen et al., 1984). The half-life
of plasma nicotine is longer at 14 minutes in the ICR strain. This is consistent with in
169
vitro data showing lower nicotine metabolic efficiency (Vmax/Km) in the ICR (0.01) mice
compared to the DBA/2 (0.05) and C57BL/6 (0.04) mice. The elimination of plasma
nicotine in mice is monophasic compared to the biphasic elimination in humans; this
could be due to the much more rapid rate of metabolism compared to distribution of
nicotine following absorption in mice. The percentage recoveries of nicotine and its
metabolites in the urine of these mice were not measured. However, urinary analysis in
the CD2F1 strain indicated that nicotine represented <1% of the systemic dose 24 hours
following its oral administration (Raunio et al., 2008), whereas in humans (normal
CYP2A6 activity), the recovery is higher (~8 to 10%) (Benowitz and Jacob, 1994; Meger
et al., 2002). The difference could be due to the high activity of CYP2A5 as well as the
rapid hepatic blood flow in mice (four-times faster) compared to humans (Bischoff et al.,
1971; Boxenbaum, 1980), resulting in the rapid metabolic clearance of nicotine.
The results from our study, and from others, indicate that in mice cotinine is the
primary metabolite of nicotine in plasma and has a significantly longer half-life (e.g. 24 to
51 min) compared to nicotine (DBA/2; C57BL/6; C3H) (Figs. 3 & 5A) (Petersen et al.,
1984). In the urine of CD2F1 mice, cotinine represented ~3-4% of the systemic nicotine
dose (Raunio et al., 2008). The above findings are compared with humans in which the
half-life of cotinine is much longer (>10 hours) (Benowitz and Jacob, 1994) and its
urinary recovery is ~10-15% of an oral dose of nicotine (Meger et al., 2002).
In mice (and in humans) unconjugated 3-HC is the predominate metabolite of
nicotine that is accumulated in the urine (77% in CD2F1 mice; 33-40% in humans)
(Benowitz and Jacob, 1994; Meger et al., 2002; Raunio et al., 2008). We found that in
the DBA/2 and C57BL/6 mice the clearance of 3-HC is slower compared to cotinine
whereas in humans, 3-HC clearance is formation dependent (Dempsey et al., 2004).
This may be due to differences in the speed of formation (e.g. slower in mice) and renal
clearance of 3-HC considering metabolic clearance (i.e. glucuronidation) does not
170
contribute to the removal 3-HC in mice (Ghosheh and Hawes, 2002) and only plays a
small role (up to ~25%) in the clearance of 3-HC in humans (Nakajima and Yokoi, 2005).
One possibility is that the renal clearance of 3-HC is influenced by genetic differences.
For instance, a human twin study found the genetic component accounted for up to 61%
of the variance in the renal clearance of nicotine and cotinine (clearance of 3-HC not
examined) (Benowitz et al., 2008). The genetic variation was suggested to be
contributed by the variation in the active secretory or reabsorptive components for both
nicotine and cotinine, although this remains to be determined for 3-HC. Another
possibility is that CYP2A5 and CYP2A6 produce different isomers of 3-HC which have
different affinities for renal excretion, but this remains to be verified.
In our studies we did not examine the kinetics of nicotine N-oxide and cotinine N-
oxide formation. Raunio et al (Raunio et al., 2008) found that cotinine N-oxide was the
second most abundant mouse urinary metabolite (~16%) followed by nicotine N-oxide,
which was found at lower levels (~3-5%). In humans, the urinary recovery of nicotine N-
oxide and cotinine N-oxide are 4-7% and 2-5%, respectively.
Section 5.2.1 Biomarker of Nicotine Metabolism
Many studies in humans have used ratios of nicotine metabolites as indicators of
CYP2A6 activity as majority of nicotine is metabolized by CYP2A6 (Nakajima et al.,
1996a), and it is the only enzyme that metabolizes cotinine to 3-HC (Nakajima et al.,
1996a). For example, salivary and plasma 3-HC/cotinine ratios have been found to be
highly correlated with oral nicotine clearance (first-pass metabolism and systemic
clearance) (Dempsey et al., 2004). The 3-HC/cotinine ratio following systemic
(intravenous) administration of nicotine is also indicative of CYP2A6 genotype as
grouped by activity (Benowitz et al., 2006b). In addition, the urinary and plasma 3-
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HC/cotinine ratios are highly correlated within subjects and also with the clearance of
nicotine by the cotinine pathway, suggesting that urinary 3-HC/cotinine ratio may be
used as a marker of CYP2A6 activity (Benowitz et al., 2006b). In addition, those without
CYP2A6 have no 3-HC production making the ratio both selective and sensitive.
Levels of nicotine metabolites (particularly in urine) in mice can also potentially
be used as indicators of CYP2A5 activity in inhibition studies; this may minimize
between-animal variations when studying multiple inhibitors and reduce the number of
animals needed. We showed that administration of methoxsalen to the ICR mice
significantly increased nicotine and decreased cotinine plasma levels (Fig. 15) although
metabolite ratios were not measured in either plasma or urine. In the CD2F1 mice
(Raunio et al., 2008), urinary metabolite ratios were examined as a measure of CYP2A5
activity. Following in vivo administration of methoxsalen, 24-hour total urinary 3-HC and
cotinine N-oxide levels (the two most abundant metabolites) decreased and nicotine
increased leading to significant decreases in 3-HC/nicotine and cotinine N-oxide/nicotine
ratios (Raunio et al., 2008). However, since the production of both 3-HC and cotinine N-
oxide requires another metabolic step after cotinine formation, the use of nicotine as the
denominator in the ratio may increase variability. Furthermore, due to the high variability
in the level of urinary metabolites (Raunio et al., 2008), the metabolite ratio may be less
sensitive to detecting the effect of weaker inhibitors.
Section 5.3 Mice (CYP2A5) as a Model to Study CYP2A6 Inhibition
Apart from nicotine metabolism, with respect to the structural/functional differences
between CYP2A5 and CYP2A6, inhibitor and modeling studies showed that CYP2A5
possess a larger active site compared to CYP2A6 (Juvonen et al., 2000; Poso et al.,
2001) and this has been suggested to be important for the relative potency of inhibitors
172
(Juvonen et al., 2000). For instance, compounds with the bulkier lactone ring structure
were demonstrated to be better inhibitors of CYP2A5 than CYP2A6 (Juvonen et al.,
2000). However, subsequent experiments showed substantial differences in the
inhibition efficacies between compounds of similar sizes, suggesting that the size of the
active site may play a secondary role to the chemical properties of the inhibitors and
their interactions with the binding site (Rahnasto et al., 2003).
Section 5.3.1 Methoxsalen
Methoxsalen is one of the most thoroughly studied inhibitors of CYP2A5 and CYP2A6. In
mice, methoxsalen decreased NNK-induced lung tumor formation (Section 2.3.5)
(Miyazaki et al., 2005; Takeuchi et al., 2003). In humans, methoxsalen increased plasma
nicotine level and decreased smoking behaviours (Section 2.3.3.1) (Sellers et al., 2002;
Sellers et al., 2000; Tyndale et al., 1999) as well as decreased NNK activation (Section
2.3.5) (Sellers et al., 2003a).
Our data showed that in vitro methoxsalen acted as a competitive and potentially
a mixed-type inhibitor of nicotine metabolism (Fig. 14). This is consistent with the
compound acting as a mixed-type (competitive and non-competitive) inhibitor of
CYP2A5-mediated coumarin metabolism (Visoni et al., 2008). Methoxsalen is also a MBI
of CYP2A5 but the mechanism through which this occurs has not been studied (Visoni et
al., 2008). Results from another study suggested that the active metabolite(s) of
methoxsalen covalently modifies the apo-protein of CYP2A5 (Mays et al., 1990). During
in vivo behavioural tests, pre-treatment of methoxsalen 30 to 60 minutes prior to
subcutaneous nicotine injection in the ICR mice led to the greatest change in the anti-
nociceptive and hypothermic effects of nicotine (tested 45 minutes after nicotine injection;
Fig. 16). This also led to a substantial change in nicotine pharmacokinetics as
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demonstrated by a ~30% decrease in its half-life (Fig. 15). However, since we only
examined nicotine pharmacokinetics following 30 minutes (but not 60 minutes) pre-
treatment with methoxsalen, one cannot be definitive regarding the direct correlation
between behavioural changes and plasma nicotine levels across time. The
pharmacokinetics of methoxsalen in the ICR mice has not been studied and therefore it
is uncertain which inhibition mechanism (i.e. competitive and/or mechanism-based
inhibition) is responsible for the in vivo inhibition (Visoni et al., 2008). It is also unknown
if metabolites of methoxsalen in mice are capable of inhibiting CYP2A5.
In humans, methoxsalen acts as both a competitive and a MBI of CYP2A6 in
vitro (Section 2.3.3.1) (Draper et al., 1997; Koenigs et al., 1997; Maenpaa et al., 1994;
Zhang et al., 2001). The mechanism through which methoxsalen inactivates CYP2A6 is
unclear but it may be due to denaturation of the apo-protein, heme modification, or
covalent binding of methoxsalen to the active site of CYP2A6 (Koenigs et al., 1997). In
vivo, pre-administration of methoxsalen at clinical doses resulted in a statistically
significant decrease in the Cmax of the coumarin metabolite 7-hydroxycoumarin and its
AUC (Kharasch et al., 2000) suggesting inhibition of coumarin metabolism; however, the
pharmacokinetics of coumarin was not determined.
Section 5.3.2 Selegiline
We showed for the first time that selegiline, and to some extent, its metabolites, are
capable of inhibiting CYP2A6- and CYP2A5-mediated nicotine metabolism. The
inhibition seemed to be slightly more potent for CYP2A6 than CYP2A5. However, the
mechanisms through which selegiline inhibits these enzymes differ as selegiline is an in
vitro MBI of CYP2A6 but not of CYP2A5. In vivo in mice, co-administration of selegiline
with nicotine led to a significant increase in the plasma half-life of nicotine but had no
174
effect on Cmax (Fig. 24). It is likely that both selegiline and its metabolites contribute to
the in vivo inhibition observed in mice. A recent study of the disposition kinetics of
selegiline and its metabolites in NMRI mice showed that following intraperitoneal
injection of selegiline, selegiline was the primary compound found in the plasma (Magyar
et al., 2007). In contrast, when the drug was administered orally, the bioavailability of
selegiline was substantially lowered (by more than three-fold) (Magyar et al., 2007). The
reduced selegiline bioavailability was not thought to be due to incomplete oral absorption,
suggesting instead that the majority of selegiline has reached hepatic circulation and the
liver (i.e. inhibiting CYP2A5) prior to reaching the systemic circulation, in contrast to
intraperitoneal administration. Thus, these data suggests that intraperitoneal
administration (in our study) may be underestimating the impact of selegiline on the in
vivo inhibition of CYP2A5-mediated nicotine metabolism in mice compared to oral
administration. Nevertheless, confirmation is needed as our study was done in the
DBA/2 mice and strain differences may alter the pharmacokinetics of selegiline. Ideally
in clinical setting both the nicotine and inhibitor would be given orally and these data
suggest that an even greater inhibition of nicotine metabolism may be seen with oral
selegiline administration alone.
In humans, the effect of selegiline on nicotine metabolism in vivo has not been
examined. The fold-reduction in intrinsic clearance by the MBI can be roughly estimated
by the equation: fold reduction in deg
deg
int
][
][
k
KI
kIk
CLI
inact
+
×+
= where kdeg is the degradation
rate constant of the enzyme (CYP2A6) and [I] is the concentration of the inhibitor at the
active site, the latter of which can be determined from total and free concentrations in
the plasma (Rostami-Hodjegan and Tucker, 2004). The KI value needs to be corrected
for non-specific binding of the inhibitor to microsomes. The effect of the inhibitor in vivo
175
also depends on several factors. These include the properties of the substrate co-
administered, the relative fraction of the substrate metabolized by the enzyme being
inhibited (Ghanbari et al., 2006), and the potential inhibition caused by different
metabolites (e.g. desmethylselegiline, L-amphetamine, and L-methamphetamine). For
example, the plasma AUCs’ of metabolites following oral selegiline administration are
substantially greater than selegiline (demethylselegiline: 31-fold; L-methamphetamine:
161-fold; L-amphetamine: 50-fold) as well as having longer elimination half-lives (Laine
et al., 2000). Since we showed that these metabolites are capable of inhibiting CYP2A6
in vitro (Fig. 22), they may potentially contribute to the inhibition of in vivo nicotine
metabolism. By determining the effect of the inhibitor on nicotine clearance in vivo, the
relative contribution of the inhibitory effect of selegiline on CYP2A6 vs. MAO-B inhibition
in smoking cessation can potentially be estimated. For instance, by comparing the
nicotine pharmacokinetics resulting from selegiline-mediated CYP2A6 inhibition to
individuals with a particular CYP2A6 genotype that produces similar nicotine
pharmacokinetics, the extent of smoking cessation contributed by CYP2A6 and MAO-B
inhibition can be inferred from smoking behaviours of individuals with the CYP2A6
genotype being compared. Alternatively, if a compound exhibits similar MAO-B inhibition
characteristics but does not interact with CYP2A6 or other nicotine metabolizing
enzymes, its effect on smoking cessation may be compared with and estimate the
contribution of inhibition of MAO-B or CYP2A6 by selegiline. Practically speaking, the
concomitant inhibition of nicotine and dopamine metabolism provides selegiline an
advantage by targeting two distinct aspects important in nicotine dependence. As
mentioned in Section 2.3.4., individuals with normal CYP2A6 activity are less likely to
quit smoking with nicotine replacement therapy (transdermal and gum) or with
counseling compared to individuals with impaired CYP2A6 activity (Ho et al., 2009;
Lerman et al., 2006; Patterson et al., 2008); therefore, normal metabolizers may obtain
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the greatest benefit from inhibition of CYP2A6 by selegiline. Selegiline at 10 mg daily
dose plus oral nicotine may be particularly useful for smokers with Parkinson’s disease
although this remain to be tested.
Section 6 Factors Affecting Nicotine Intake
Section 6.1 Impact of Genetic Variations
Section 6.1.1 Variations in Nicotine Metabolism
We showed that oral nicotine consumption is associated with CYP2A5 levels and in vitro
nicotine metabolism in male mice. This is consistent with observations in humans where
CYP2A6 activity is associated with smoking behaviours (Section 2.3.4). In our study we
examined in vitro nicotine metabolism in mice from two extreme ends of the
consumption spectrum rather than from across the entire consumption range; therefore,
we were unable to assess the contribution of nicotine metabolism (and Cyp2a5 genotype)
to the variability in oral NSA. Since one of the parental strains (ST/bJ) self-administrated
significantly less amount of oral nicotine compared to the C57BL/6 mice, it would be
advantageous to have the Cyp2a5 sequence data and hepatic microsomes from the
ST/bJ mice to verify that they have a different Cyp2a5 sequence and slower nicotine
metabolism than the C57BL/6 mice. Similarly, the lower affinity of the ICR hepatic
microsomes for nicotine C-oxidation may be due to a polymorphism in Cyp2a5 which
may contribute to differences in oral NSA (and other nicotine behavioural responses),
but this remains to be verified. A recent genetic study in mice using the F2 offspring of
C57BL/6J (high nicotine consumption strain) and C3H/HeJ (low nicotine consumption
strain) has revealed that at least four QTLs in the mouse genome are associated with
oral NSA (LOD score of >2.0 [suggestive association]), and these QTLs contributed to
~62% of the variance in this behaviour (Li et al., 2007). One of the loci is located on
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chromosome 7 and the highest combined LOD score found on this loci is 5.9 (highly
significant association). Overall, the QTL on chromosome 7 was estimated to contribute
~13% of the variance in oral NSA. However, the study only reported data derived from
10 cM and onward, and Cyp2a5 is located at ~6.5 cM, thus it is unclear what the
contribution of this region is (i.e. Cyp2a5) to the overall variance in oral NSA (Li et al.,
2007). It is also possible that other genes influencing nicotine consumption co-segregate
with Cyp2a5 but this remains to be investigated. In order to directly test the contribution
of CYP2A5-mediated nicotine metabolism on NSA, inhibitors of CYP2A5 could be used.
Similarly in humans, genetics play a significant role in various aspects of smoking
behaviour which include initiation (Li et al., 2003; Vink et al., 2005), persistence to
regular smoking (Maes et al., 2004), number of cigarettes smoked (Koopmans et al.,
1999), degree of nicotine dependence (Vink et al., 2005), withdrawal severity (Xian et al.,
2003), and smoking cessation (Broms et al., 2006). The heritability estimates for the
amount of cigarettes smoked (once initiated) was shown to be up to 86% (Koopmans et
al., 1999), which is similar to the results from the above mouse study. Vast numbers of
pharmacogenetic studies have demonstrated that the CYP2A6 genotype to be highly
associated with many aspects of smoking (Section 2.3.4). The heritability estimates of
CYP2A6 to different dimensions of smoking behaviour have not been specifically defined
but a genome-wide linkage scan found this gene to be situated near a locus associated
with smoking frequency (Swan et al., 2006).
Section 6.1.2 Non-Nicotine Metabolism Variations
In addition to nicotine metabolism, other genetic factors may contribute to variations in
oral NSA among mouse strains. One particular striking feature is that a significant
association exists between the amount of nicotine self-administered and the threshold of
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nicotine-induced seizures observed between 19 strains of mice (r=0.89, P<0.01)
(Crawley et al., 1997); although this may be related to the sensitivity of mice to nicotine
which is in part controlled by the bioavailability of oral nicotine following hepatic first-pass
metabolism. An earlier study conducted by Hatchell and Collins showed a lack of clear-
cut association between brain nicotine levels or hepatic nicotine clearance and motor
depression between mouse strains, suggesting differential sensitivity to the
pharmacological effects of nicotine (Hatchell and Collins, 1980). Nicotine sensitivity may
be influenced by polymorphisms in genes of the neurotransmitter system involved in
nicotine reward, which include the dopaminergic (David et al., 2006), serotonergic
(Villegier et al., 2006), cholinergic (Levin et al., 2008), GABAnergic (Markou et al., 2004),
opiate (Berrendero et al., 2002), and cannabinoid (Merritt et al., 2008) systems. Although
currently there are no known studies demonstrating the association between
polymorphisms in these genes with nicotine sensitivity in mice, studies using inhibitors
and knock-out animals have revealed their direct role in nicotine pharmacology. For
instance, administration of the dopamine D1 receptor antagonist SCH 23390, or the
nicotinic receptor antagonist DHβE, disrupted intra-cranial self-administration of nicotine
to the ventral tegmental area, a brain region important for drug reward (David et al.,
2006). Mice with the β2 nicotinic acetylcholine receptors knocked out showed reduced
oral NSA in the first weeks of testing but nicotine consumption level returned to the wild-
type levels within the first month. In contrast, α7 nicotinic acetylcholine receptors
knockout mice showed normal oral NSA at the beginning of test but their nicotine
consumption level declined after several weeks (Levin et al., 2008). In humans, evidence
indicates that polymorphisms in the neurotransmitter systems of the nicotine reward
pathways potentially influence smoking behaviours. These include dopamine receptors
(e.g. DRD2) (Comings et al., 1996), transporter (DAT) (Ling et al., 2004), and
179
metabolism (e.g. TH) (Olsson et al., 2004); serotonin transport (5-HTT) (Gerra et al.,
2005) and metabolism (TPH1) (Sullivan et al., 2001); nicotinic acetylcholine receptors
(e.g. α4) (Li et al., 2005); GABA receptor (GABAB2) (Beuten et al., 2005a); opiate
receptor (OPRM1) (Swan et al., 2006); and brain-derived neurotrophic factor (Beuten et
al., 2005b).
Section 6.2 Non-Genetic Factors
Although in general oral nicotine self-administration appears to mimic nicotine
consumption via smoking (Section 3.1), the two behaviours may be influenced by
different factors. For instance, in addition to nicotine, cigarette smoke contains numerous
chemicals and additives that may affect smoking behaviour through their interactions
with various cranial nerves, such as the olfactory nerve (smell), trigeminal nerve
(irritation), as well as facial, glossopharyngeal, vagal nerves (taste) (Megerdichian et al.,
2007). Studies have shown that application of anesthetic to the airway significantly
reduced satisfaction and craving from smoking (Rose et al., 1985; Rose et al., 1984) and
blockade of olfactory/taste stimuli from smoking reduced self-administration of cigarette
puffs (Perkins et al., 2001). In addition, studies have also shown that neither
denicotinized cigarettes nor pure nicotine alone completely abolish cravings or provide
the subjective satisfaction of smoking regular cigarettes (Rose et al., 2003; Rose et al.,
2000). This evidence suggests the importance of non-nicotinic factors in contributing to
smoking behaviours and they may be different to oral NSA.
There appears to be another important distinction between nicotine self-
administration in mice and smoking in humans. Stress is considered to be an important
factor for the amount of cigarettes smoked in humans (Baker et al., 2004; Todd, 2004).
180
However, a genetic study using F2 mice comprised of low, average, and high stress
responders revealed that despite showing different response to stress, the mice did not
show different level of NSA. A caveat of this study was that the degree of NSA was not
measured against the degree of stress induced within the parental strain (or within the
stress-responder groups), thus the potential influence of genetic components (e.g.
nicotine metabolism) in NSA cannot be ruled out (Bilkei-Gorzo et al., 2008).
Section 7 Sex-Dependent Association between Nicotine Metabolism
and NSA
Even though our study chose male and female mice that were matched for their NSA
level, on average the female mice tend to metabolize nicotine slightly faster compared to
males. Similar observations are also seen in humans (Benowitz et al., 2006a). It has
been reported that on average, both men and women smoke a similar number of
cigarettes (Benowitz and Hatsukami, 1998; Benowitz et al., 2006a; Zeman et al., 2002).
However, women in general have a faster rate of nicotine metabolism (e.g. fractional
clearance of nicotine to cotinine [>15%] and 3-HC/cotinine ratios [>20%]) compared to
men (Benowitz et al., 2006a; Zeman et al., 2002). This is supported by our previous
findings that human females expressed a greater amount of CYP2A6 protein and have
faster in vitro nicotine metabolism compared to males (Al Koudsi et al., 2006b).
We also noted that female mice tend to have more variable rates of nicotine
metabolism compared to males. One potential explanation is the fluctuation in the level
of steroid hormones in females altering the level of CYP2A5 (as the mouse oestrous
cycle is less than one week). 17β-estradiol has been shown to induce a significant rise in
CYP2A6 mRNA in human hepatocytes (likely via the estrogen receptor-α by interacting
181
with the estrogen response element [AGGTCAnnnCAACCT]), although protein levels
were not measured (Higashi et al., 2007a). Immunostaining also revealed that CYP2A6
protein levels tend to be higher during the follicular phase compared to the luteal phase
in endometrial tissues (Higashi et al., 2007a). The effect of sex steroids on nicotine
metabolism in mice has not been examined; however, there also appears to be an
estrogen response element in mice as well [AGGGCAnnnTGACCT] although it is
unclear if it is involved in the regulation of mouse Cyp2a5 (Orimo et al., 1995). It is
possible that sex hormones may induce other nicotine-metabolizing hepatic enzymes in
mice.
In contrast to the above in vitro study, pharmacokinetic studies in humans have
revealed some discrepancies on the role of female sex hormones on nicotine
metabolism. One study found that the pharmacokinetics of nicotine or cotinine are not
affected by the menstrual cycle, as measured during the mid-follicular and mid-luteal
phases (Hukkanen et al., 2005a). In contrast, other studies have shown that pregnancy
and the use of estrogen-containing oral contraceptives significantly increases the
clearance of nicotine via the cotinine pathway (~25% and ~40%, respectively) (Benowitz
et al., 2006a; Dempsey et al., 2002). This inconsistency may be due to the varying
amount and the duration of exposure to estrogen during these two distinctive
physiological phases. Other available evidence indicates that smoking behaviour is
influenced by menstrual cycle (increased during late luteal phase) but does not change
during pregnancy (DeBon et al., 1995; Dempsey et al., 2002; Snively et al., 2000),
suggesting the role of other influences on nicotine intake in females. 17β-estradiol and
progesterone have been shown to decrease nicotine’s antinociceptive effect in female
mice, through antagonism of α4β2 nicotinic receptor, whereas testosterone had no effect
in males (Damaj, 2001). These findings suggest a balance exists between nicotine
182
metabolism and nicotine pharmacodynamics as influenced by female steroids (both
appeared to be, in part, affected by 17β-estradiol) in controlling nicotine intake,
especially in females. This may also underlie our observation that nicotine metabolism is
less correlated with NSA in female mice compared to the male mice. The complex
interactions between nicotine metabolism and sex may also contribute to the conflicting
evidence showing reduced efficacy of NRTs in females compared to males in some
studies but not others (Bjornson et al., 1995; Perkins and Scott, 2008; Schnoll et al.,
2009; Wetter et al., 1999).
Section 8 Effect of Chronic Nicotine Treatment on Nicotine Metabolism
In our studies we examined nicotine metabolism in mice in acute settings (i.e. following a
single nicotine administration); however, in clinical settings, nicotine metabolism and in
particular, nicotine-mediated behavioural effects, take place during chronic nicotine
exposure. Therefore, nicotine metabolism during chronic exposure is of particular
interest.
In monkeys, chronic nicotine treatment (0.3 mg/kg b.i.d.) for three weeks led to a
~60% decrease in CYP2A6agm protein levels and ~40% decrease in in vitro nicotine
metabolism (Schoedel et al., 2003). Cigarette smoke exposure in human led to only a
small reduction in the fractional clearance of nicotine to cotinine (Benowitz and Jacob,
2000). However, 7-day tobacco abstinence led to a 39% increase in non-renal nicotine
clearance (Lee et al., 1987) and a recent study showed that the clearance of oral
nicotine is reduced by up to 25% in smokers compared to non-smokers (Mwenifumbo et
al., 2007). Therefore, chronic nicotine exposure would likely decrease the metabolism of
CYP2A6 substrates, in particular nicotine. We have previously examined the effect of
nicotine metabolism in mice following chronic nicotine treatments. In a first study, DBA/2
183
mice were treated with a single injection of nicotine (1 mg/kg s.c.) once daily for seven
days. On the last day plasma concentrations of nicotine were measured but no
differences were found in its elimination kinetics compared to those of acute treatment
(unpublished observations). This may be due to the rapid clearance of nicotine in the
mice. In a subsequent study osmotic mini-pumps providing 1 mg/kg/hr or 5 mg/kg/hr of
nicotine for seven days were implanted in DBA/2 mice. On day seven the mice were
sacrificed and livers were removed to examine hepatic CYP2A5 protein levels.
Compared to saline, 1 mg/kg/hr nicotine did not caused significant changes in CYP2A5
levels. In contrast, 5 mg/kg/hr nicotine caused a drop in CYP2A5 levels although nicotine
at this level were toxic to mice, therefore we felt that nicotine at this level were of little
pharmacological relevance (unpublished observations). A recent study found that
chronic nicotine exposure in DBA/2 and C57Bl/6 mice, while leading to changes in
various behavioural measures, not all the changes were congruent in terms of direction
of change. For example, during withdrawal both strains showed decreases in somatic
withdrawal signs between day 1 and day 3; in contrast, in the plus-maze test, the DBA/2
mice increased time spent whereas C57Bl/6 mice decreased time spent in the opening.
Therefore, the contribution of chronic nicotine exposure to behavioural changes by
potential differences in nicotine metabolism could be secondary to and/or confounded by
the impact of other physiological/neurological changes (Jackson et al., 2009). The above
data suggested while in both humans and monkeys chronic nicotine exposure could lead
to reduction in CYP2A6-mediated nicotine metabolism, which may play a role in altered
nicotine pharmacology in chronic settings; its impact on mouse nicotine metabolism has
not yet been demonstrated or is minimal, possibly due to rapid elimination of the drug in
this species.
Section 9 Future Directions
184
Section 9.1 Selegiline
Previous study indicated that mouse deficient for MAO-B showed no changes in brain
dopamine levels, suggesting its lack of involvement in dopamine metabolism (Grimsby et
al., 1997). In contrast, dopamine metabolism in mice appeared to be mediated in part by
MAO-A (Shih et al., 1999). Thus, although selegiline is an irreversible inhibitor of MAO-B,
it would have no impact on dopamine metabolism in mice. Consequently, the effect of
selegiline on nicotine pharmacology can be tested and the changes in nicotine-mediated
behaviour can be attributed to the CYP2A5-inhibition (i.e. inhibition of nicotine
metabolism) aspect of selegiline. On the other hand, this also excludes the use of mice
for studying the impact of selegiline on dopamine metabolism and its subsequent effects
on nicotine pharmacodynamics.
Based on our observation that selegiline is an MBI of CYP2A6, it is reasonable to
assume that it will exert its greatest impact on the first-pass metabolism (i.e. inhibition) of
orally delivered nicotine. Considering existing clinical trials only examine selegiline either
alone (capsule or patch) or in combination (capsule plus nicotine patch) for smoking
cessation (Clinicaltrials.gov), it would also be important to examine the effect of oral
selegiline in combination with oral nicotine capsule/pill. It is likely that the smoking
cessation effect may be greater when oral selegiline is used in combination with oral
nicotine due to inhibition of dopamine metabolism and decreased nicotine metabolism.
Consider the involvement of various non-nicotine factors in nicotine-dependence, the
above therapeutic combination may be particularly useful as an initial treatment in
conjunction with behavioural therapies for smokers consider quitting.
Section 9.2 Mouse Model of Nicotine Metabolism
185
In this thesis we demonstrated the usefulness of the mouse model in studying nicotine
metabolism in the context of pharmacological inhibition. However, extrapolation of
pharmacokinetic data from animals to humans has limitations due to species differences
in physiology and conservation of drug metabolism enzymes. The former can be
addressed by allometric scaling which proposed that certain pharmacokinetic information
(e.g. plasma-concentration-time profile, clearance, and distribution) can be estimated
between species as a function of body weight (Boxenbaum, 1984; Martignoni et al.,
2006). It has also been suggested that, in general, small animals have a higher hepatic
P450 content per unit body weight due to a larger liver to body weight ratio as well as
faster hepatic blood flow (Davies and Morris, 1993). As a consequence, smaller animals
have a faster metabolic clearance of xenobiotics compared to humans (Davies and
Morris, 1993). However, species variations in P450 represents a major factor in drug
metabolism difference between species, particularly for compounds that are extensively
metabolized (Martignoni et al., 2006). Therefore in these circumstances pharmacokinetic
data from animals may not be reasonably applied to humans. For instance, while
existing evidence, and results from our study, indicate that both human CYP2A6 and
mouse CYP2A5 exhibit similar nicotine metabolizing properties, studies with selegiline
showed that this drug to be a MBI of CYP2A6 but not CYP2A5. The endogenous
compound 2-phenylethyamine is a far better inhibitor of CYP2A6 than CYP2A5 in vitro
(IC50 ratio of <0.04) (Rahnasto et al., 2003) and other experimental compounds also
exhibit different inhibition potencies for these enzymes (Juvonen et al., 2000). Thus,
evaluating the impact of CYP2A6 inhibitors in mice in vivo may yield different results.
Furthermore, potential pharmacokinetic differences in the metabolism of the inhibitors
between species can also alter their inhibitory properties. While the mouse model may
be imperfect, it represents the best small animal model we have to date and may prove
useful for the first line in vivo animal studies considering that the main nicotine
186
metabolizing enzymes in rats, the other well studied small animal species, belong to the
CYP2B family (Nakayama et al., 1993).
In the past few years humanized mice have been developed using several
strategies. One method is to introduce human P450 cDNA with tissue-specific promoter
or the entire gene (with regulatory elements) into the pronuclei of the mice (Cheung and
Gonzalez, 2008). With this method, however, the presence of endogenous counterparts
(homologous or orthologous genes) may interfere with, and confound the effects of, the
introduced genes. Therefore mice with the corresponding gene knocked out should be
used. Another method is to introduce cDNA into the site of the endogenous gene
thereby disrupting its function (Cheung and Gonzalez, 2008). An advantage of this
model is that once generated, the transgenic line can be maintained indefinitely.
Conceivably this model is ideal for studying the impact of various Cyp2a5 and CYP2A6
genetic polymorphisms on nicotine-mediated behaviours. In the case of inhibitor studies,
(human) enzymes involved in the metabolism of the inhibitor should ideally be
introduced to increase the predictability of the animal model; however, this may not be
feasible since different inhibitors will likely be metabolized by different enzymes. We are
aware that humanized CYP2A6 mice (Cyp2a5-null background) are currently being
developed and this model may be invaluable in studying CYP2A6 inhibitors in vivo.
Another method that has been developed is the transplantation of human
hepatocytes into mice (Katoh et al., 2008). This model employs a urokinase-type
plasminogen activator+/+/ severe combined immunodeficient mouse strain in which
human hepatocytes are directly injected into the spleen of the mice and the resultant
mice have between 80-90% of their hepatocytes replaced (Tateno et al., 2004). This
model may provide a much more accurate picture of in vivo metabolism (and inhibition)
as all human hepatic enzymes and their regulatory factors are present (Katoh et al.,
2008). This model may also indicate whether the target compound is inducing the
187
expression of other enzymes through human (rather than mice) xenobiotic receptors,
which is particularly important for drug-drug interactions (Katoh et al., 2008). A drawback
of this particular model is that the chimera mice have a shorter life-span due to organ
failure caused by graft vs. host disease and administration of protease inhibitor is also
required to prolong survival (Tateno et al., 2004). The mice also need to be constantly
generated and thus costly and time-consuming. Their short life-span and inability to
generate offspring carrying human hepatocytes can limit their use in behavioural studies.
Clearly, each of the above mouse models has its advantages and drawbacks in
studying nicotine metabolism. Further development of these models, either through
creating mice with multiple human transgenes, or refinement of the hepatocyte
transplant model, are needed to increase their applicability to humans; however, a
disadvantage associated with the humanized mouse model is that the generation of a
panel of mice each with different CYP2A6 genotype is both time and resource
consuming. Continual functional characterization of CYP2A5 (and in vivo nicotine
metabolism) from different mouse strains may also be useful in correlating their
metabolism phenotype with that of CYP2A6 and its functional variants. Generation of
mutations in mouse Cyp2a5 that mimic various CYP2A6 genotypes may also be useful
in understanding the contribution of these genotypes in altering nicotine pharmacology.
Finally, even though nicotine is largely metabolized by the liver in mice, the contribution
of other pharmacokinetic factors such as absorption, distribution, and extrahepatic
metabolism need to be considered.
Section 10 Conclusion
Based on our findings that: 1) CYP2A5, as with CYP2A6, is the primary enzyme
responsible for the metabolism of nicotine to cotinine and cotinine to 3-HC; 2) genetic
188
variations in CYP2A5 may alter nicotine metabolism; 3) inhibition of CYP2A5-mediated
nicotine metabolism led to change in nicotine pharmacokinetics and pharmacodynamics;
and 4) identification of an existing drug (selegiline) as an inhibitor of CYP2A5 and
CYP2A6 suggested the mouse may be a suitable model for studying novel CYP2A6
inhibitory compounds. Overall, the use of CYP2A5 inhibitors in the mouse model may be
suitable for studying the direct effect of nicotine metabolism on nicotine pharmacology.
On the other hand, genetic studies across strains may be useful for dissecting the
genetic architecture of nicotine-mediated behaviours, in this case oral NSA.
189
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