Nicotine is known to be metabolised to its major metabolite cotinine by members of the cytochrome P450 monooxygenase superfamily. Although CYP2A6 has now been identified as the principal enzyme which catalyses this biotransformation, CYP2D6 is also an active nicotine C-oxidase. Some 8% of the Caucasian population have reduced or absent CYP2A6 activity; CYP2D6 may play a significant role in nicotine metabolism in these individuals. CYP2D6 is highly polymorphic - a number of studies linking CYP2D6 genotype to smoking behaviour have now been published. CYP2D6 may have an important constitutive function in neurotransmitter metabolism and CYP2D6 genotype is thought to be a critical determinant in the success of antidepressant drug treatment.
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INTRODUCTION FOR NICOTINE ADDICTION WORKSHOP BY MARCUS MUNAFO AND MIKE MURPHY: The ICRF General Practice Research Group in Oxford recently hosted a workshop on the theme of "Neuroscience, molecular genetics and nicotine addiction". It was chaired by Professor Neal Benowitz from the University of California at San Francisco, a specialist in the field of nicotine pharmacology spending an invited week in the University to work with the Group, and included about 30 other experts from groups around the UK and Sweden, including the ICRF Molecular Pharmacology Unit in Dundee and the ICRF Health Behaviour unit in London. Their backgrounds included neuroscience, molecular genetics, genetic epidemiology, pharmacology, psychology and clinical medicine, but they were united in a belief about the importance of tobacco control.
It is now recognised that addiction to nicotine lies at the heart of the tobacco control problem. Without the marketing of tobacco its use would dwindle. But while it is so widely promoted smoking research focuses heavily on factors affecting why people continue smoking after trying it and how to get them to stop with psychological support and the use of effective drugs. Although tobacco use is rightly viewed as a social and psychological problem we also need to see it as a pharmacological one with a potential genetic contribution to the addictive process. The need to integrate knowledge about the brain chemistry underlying addiction, the molecular genetics of these cellular processes, and how the drugs which are effective work, was the workshop's subject.
Each of the meeting's 4 sessions kicked off with a brief introduction by speakers who had precirculated draft papers. The sessions considered in turn: (1)how nicotine is metabolised and cleared from the body (2)the brain receptors in which nicotine "docks" to exert its effects (3)the brain pathways involving the neurotransmitter,dopamine,which are thought to contribute to the rewarding properties of nicotine which reinforce a smoker's desire to smoke (4)other non-dopamine pathways that are activated by nicotine and may underly such effects as nicotine withdrawal symptoms.
Understanding how the drugs which are effective (Nicotine Replacement Therapy and some antidepressants) interact with the expression of individual genotypes in the brain and elsewhere to predict success in quitting smoking will help to shed light on which bits of the brain are important in nicotine addiction at both the anatomical and cellular level. The workshop participants emerged pleased to have caught up with advances in fields which were not their speciality and with a strong sense that more enduring closer contact and cooperation will be possible in the future.
1. Nicotine, the major addictive component of tobacco, is a tertiary amine which binds nicotinic cholinergic and acetylcholine receptors in the brain. Nicotine binding leads to the release of a variety of neuro- transmitters including acetylcholine, noradrenaline, dopamine and serotonin (Benowitz 1996 and references therein).
2. The major metabolite (70-80%) of nicotine is cotinine (Figure 1), the formation of which is catalysed by a cytochrome P450 enzyme, CYP2A6 (Messina et al. 1997). Although CYP2A6 is now thought to be the principal nicotine C-oxidase, certain other P450 isozymes including CYP2B6 and CYP2D6 have also shown to be active in catalysing this reaction in recombinant systems (McCracken et al. 1992; Yamazaki et al. 1999, Table 1).
FIGURE 1: CYP2A6 catalyses the rate-limiting step in nicotine metabolism
TABLE 1: Relative nicotine C-oxidase activities of recombinant human P450s expressed in baculovirus (Yamazaki et al. 1999)
P450 Km Vmax
CYP2A6 11 11 CYP2B6 105 8.2 CYP2D6 132 8.6
3. The relative activities of CYP2A6 and CYP2D6 towards nicotine have also been compared by analysis of individual P450 isozyme expression in a panel of human liver microsomes by Western blotting and correlation of the level of expression of individual isozymes with nicotine C-oxidation rates (Yamazaki et al. 1999). The results of these experiments demonstrated that the strongest correlation was observed between CYP2A6 expression and enzyme activity. In addition, no inhibition of enzyme activity was observed with the CYP2D6 inhibitor quinidine, suggesting that CYP2D6 does not make a significant contribution to nicotine metabolism in human liver. This was further confirmed by the observation that the CYP2D6 phenotype of an individual known to be deficient in nicotine C-oxidation was that of an extensive metaboliser (Benowitz et al. 1995).
4. Considerable inter-individual variation has been observed in nicotine metabolism, and individuals with severely compromised nicotine C-oxidation activities identified (Benowitz et al. 1995). Recent evidence suggests that the principal defect in nicotine oxidation arises from genetic polymorphism at the CYP2A6 gene locus. Approximately 8% of the Caucasian population and 27% of Chinese appear to have diminished or absent CYP2A6 activity due to heterozygous or homozygous CYP2A6 polymorphisms (Oscarson et al. 1999). This ethnic variation in CYP2A6 allele frequencies is likely to go some way to rationalise the ethnic differences observed in nicotine and cotinine metabolism (Benowitz et al. 1999). It may be hypothesised that while the catalytic activity of CYP2D6 towards nicotine is negligible in the presence of CYP2A6, CYP2D6 may contribute to nicotine metabolism in the absence of CYP2A6.
5. Unlike CYP2A6, which is thought to play a relatively minor role in drug metabolism, CYP2D6 is known to be responsible for the metabolism of more than 25% of commonly prescribed drugs. The expression of CYP2D6 is also subject to genetic polymorphism, with an ever increasing number of allelic variants with differing catalytic properties described (Daly et al. 1996, Table 2). Approximately 7% of the Caucasian population are homozygous for a combination of several inactive CYP2D6 alleles (Sachse et al. 1997, Table 2). These individuals, "poor metabolisers (PMs)", express no functional CYP2D6 protein and therefore have a severely compromised ability to metabolise compounds which are CYP2D6 substrates. In addition, 1-3% of Caucasians are so-called "ultra-rapid metabolisers", carrying multiple copies of CYP2D6 arranged in tandem. This gene amplification can involve as many as 13 copies of CYP2D6 and results in extremely rapid substrate metabolism (Johansson et al. 1993). Like CYP2A6, the frequency of both CYP2D6 poor and ultra-rapid metabolisers varies in different ethnic groups (Table 3), with obvious consequences for prescribing regimens for the affected drugs.
TABLE 2: Compilation of CYP2D6 alleles
Allele name Nucleotide Protein Enzyme Allele New Old sequence sequence activity frequency changes changes [%]
*1 wt "Wild-type" None normal 36.4
*2 L 1661 G>C, 2850 C>T, R296C, down 32.4 4180 G>C S486T
*1x2 CYP2D6 duplicated 2 active UP 0.51 genes
*2xN L2 CYP2D6 duplicated N active UP 1.34 genes
*3 A 2549 A deleted Frameshift 0 2.04
*4 B 100 C>T, 1846 G>A, Frameshift 0 20.7 4180 G>C of splicing signal
*5 D CYP2D6 deleted None 0 1.95 expressed
*6 T 1707 T deleted Frameshift 0 0.93
*9 C 2613-2615 AGA K281 down 1.78 deleted deleted
*10 J/Ch 100 C>T, 1661 G>C, P34S, down 1.53 4180 G>C S486T
Alleles of CYP2D6 (updated at http://www.imm.ki.se/CYPalleles/cyp2d6.htm), found to be most frequent among white Caucasians (frequencies from Sachse et al. 1997). Consensus CYP2D6 gene nomenclature is according to Daly et al. 1996.
TABLE 3: Ethnic differences in CYP2D6 allele frequencies
Population Subjects Poor metaboli- Allele frequency Reference sample [n] sers (PMs)[%] of *MxN [%]
Koreans 152 0.0 0.3 Roh et al. 1996 Chinese 113 0.0 1.3 Johansson et al. 1994 Swedes 270 8.0 1.0 Dahl et al. 1995 Germans 589 7.0 2.0 Sachse et al. 1997 French 265 8.4 1.9 Legrand et al. 1998 S.Spaniards 217 2.8 3.5 Agundez et al. 1995 Anatolians 404 1.5 5.8 Aynacioglu et al. 1997 Saudi Arabs 101 2.0 10 McLellan et al. 1997 Blk Ethiopians 122 1.8 16 Aklillu et al. 1995 Blk Tanzanians 113 7.0 4.1 Wennerholm et al. 1999 Blk Americans 246 3.3 2.4 London et al. 1997 Nicaraguans 137 3.6 1.1 Agundez et al. 1997 Wht Americans 464 5.8 2.2 London et al. 1997
Population frequencies of CYP2D6 PM alleles and the ultrarapid CYP2D6 gene duplication in populations of different ethnic origin. a CYP2D6*MxN is a general designation of this allele; it includes duplications as well as higher amplifications (known from the literature: N = 2, 3, 4, ..., 13) of different alleles (known from the literature: M = *1, *2, *4).
6. A number of studies have addressed the issue of whether CYP2D6 genotype is associated with desire to smoke (Turgeon et al. 1995; Cholerton et al. 1996) or whether genotype may reinforce smoking behaviour in committed smokers (Boustead et al. 1997). The majority of these studies do not report a strong association between CYP2D6 genotype and smoking behaviour but were, however, mostly performed before CYP2A6 was identified as the major nicotine C-oxidase.
7. Much of the data on CYP2D6 genotype in smokers are found contained within larger studies investigating the CYP2D6 polymorphism as a susceptibility factor in lung cancer. The findings of many of these studies are contradictory, however, with meta-analysis of the available data suggesting no overall association between CYP2D6 genotype and susceptibility to lung cancer, irrespective of smoking status (Christensen et al. 1997; London et al. 1997). Many early studies were additionally complicated by the use of phenotyping rather than genotyping methods to assess CYP2D6 activity.
8. Although some positive findings have been reported relating CYP2D6 genotype to smoking history, the results of these studies have, in general, been based on the analysis of relatively small populations and on qualitative rather than quantitative assessment of smoking behaviour. It is therefore difficult to confidently correlate these variables with a quantitative assessment of CYP2D6 genotype.
9. A recent study (Saarikoski et al. 2000), however, reports a significant over-representation of the CYP2D6 ultra-rapid genotype in smokers, with a two-fold increase observed in heavy smokers compared to occasional smokers (OR=2.3, 95%CI=1.2-4.4) and a four-fold increase in heavy compared to never smokers (OR=4.2, 95%CI=1.8-9.8). Although the population tested was a mixture of cancer patients and healthy controls, when all possible confounding factors were included a significant trend was still observed suggesting increased tobacco use correlated with increased CYP2D6 metabolic activity.
10. Several independent epidemiological studies have suggested that smoking is protective against Parkinson's disease (e.g. Tzourio et al. 1997, OR=0.4). Nicotine is known to stimulate dopamine release in the substantia nigra, the area of the brain which is progressively destroyed in Parkinson's disease. CYP2D6 expression has been demonstrated in this area of the brain and a possible role for the enzyme in dopamine transport proposed (Niznik et al. 1990). CYP2D6 in vitro activity is competitively inhibited by serotonin, tryptamine and dopamine, and noncompetitively inhibited by adrenaline and noradrenaline (Martinez et al. 1997). More importantly, CYP2D6 has been shown to be active in the metabolic conversion of tyramine to dopamine (Hiroi et al. 1998), and in the metabolism of tryptamine (Martinez et al. 1997). Lack of active CYP2D6 protein in poor metabolisers may therefore result in reduced dopamine formation, a hypothesis which is supported by several epidemiological studies comparing CYP2D6 PM frequencies in PD patients and healthy controls (Smith et al. 1992; Lucotte et al. 1996). More over, CYP2D6 is known to metabolise MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Gilham et al. 1997), a contaminant of synthetic narcotics, exposure to which results in the development of Parkinson's-like symptoms - studies in animal models have demonstrated a protective effect of nicotine on neuronal loss in the substantia nigra following exposure to MPTP (Janson & Moller 1993).
11. Further evidence that CYP2D6 may have a constitutive role in neurotransmission comes from Sweden (Bertilsson et al. 1989), where CYP2D6 PMs were shown to demonstrate increased vitality, efficiency and alertness, and from a Spanish study where PMs were found to be more anxiety-prone and less successfully socialized (Llerena et al. 1993).
12. Links between smoking and depression are well documented - depressive and schizophrenic illnesses are often exacerbated by smoking cessation (Covey et al. 1990; Borrelli et al. 1996), and the higher frequency of smokers among psychiatric patients is believed to represent a self-medication mechanism (Stefanis & Kokkevi 1986). Antidepressant drugs are routinely prescribed as adjutant therapy in therapeutic intervention to aid smoking cessation. Interestingly, many of these antidepressants including nortriptyline and fluoxetine are CYP2D6 substrates (Table 4), and in vivo concentrations of many of these drugs are significantly dependent on CYP2D6 genotype (e.g. clomipramine, Bertilsson et al. 1997; desipramine, Dahl et al. 1993; nortriptyline, Dalen et al. 1998). Therefore, the CYP2D6 polymorphism is likely to be a critical determinant of the success of antidepressant drug therapy.
TABLE 4: Drugs used in the treatment of psychiatric diseases which are CYP2D6 substrates
Class of drugs Examples
Antidepressants: Amitriptyline, Clomipramine, Citalopram, Desipramine,Fluoxetine, Fluvoxamine, Imipramine, Nortriptyline,Paroxetine, Trimipramine, Venlafaxine
Antipsychotics: Chlorpromazine, Clozapine, Haloperidol, Olanzapine, Perazine, Perphenazine, Remoxipride, Risperidone, Thioridazine, Zuclopenthixol
(Not all of these drugs are metabolised exclusively by CYP2D6)
13. CYP2A6, not CYP2D6 appears to play a significant role in nicotine metabolism, and should therefore be the primary target for treating nicotine addiction. However, some studies have associated smoking behaviour with CYP2D6 genotype - the potential impact of CYP2D6 in neurotransmitter metabolism suggests a further link with smoking addiction. In addition, CYP2D6 is known to metabolise the majority of commonly prescribed antidepressant drugs. Where these drugs are prescribed to facilitate smoking cessation, CYP2D6 genotype may influence individual metabolic capabilities and therefore be a critical determinant of therapeutic efficacy.
The authors acknowledge the financial support of the Ministry of Agriculture, Fisheries and Food (MAFF).
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