As demonstrated consistently in other clinical trials (Hughes, Stead, & Lancaster, 2004
), including the larger trial population from which these participants were drawn (Brown et al., 2007
), bupropion was effective for smoking cessation across all follow-up time points post quit date, with attenuation by 12 months. However, our primary line of enquiry was the examination of potential gene × bupropion interactions on sustained abstinence following a quit attempt. As noted, the differences in outcomes across all time points between genotype dichotomies were small in both the bupropion and placebo conditions for all three candidate gene variants (DRD2
-Taq1A, SLC6A3 3′ VNTR, CYP2B6
1459 C→T). However, outcomes diverged at 6 months according to DRD2
genotype. Specifically, participants who received bupropion and possessed the DRD2
-Taq1A2/A2 genotype were nearly three times as likely to be abstinent from smoking at the 6-month follow-up, compared with their A2/A2 counterparts who received placebo. In contrast, bupropion did not appear to be effective among participants with at least one A1 allele.
It is quite conventional to analyze outcomes at the end of treatment and more distant posttreatment follow-ups separately, as seen in other pharmacogenetic trials (Lerman et al., 2003
; Lerman, Shields et al., 2002
; Swan et al., 2005
; Yudkin et al., 2004
). This is the case because after cessation of treatment, genes would not, by definition be interacting with treatment per se, but rather would be interacting with persistent neuropsychological adaptations resulting from treatment. Therefore, it would be biologically plausible for divergence of gene × “treatment” effects, between the end of treatment and subsequent follow-up. Even so, the precise mechanisms underlying the DRD
2 × bupropion interaction on 6-month point prevalence from smoking cannot be determined from this study.
In the present study, participants with two A2 alleles treated with bupropion demonstrated lower self-reported cigarette craving than those with one or more A1 alleles who were treated with bupropion. These results are consistent with a nested, laboratory-based substudy from this clinical trial indicating that the DRD2
-Taq1A2/A2 genotype predicted a reduction in cessation-related craving (David et al., 2003
) and cue-elicited craving among smokers attempting to quit with bupropion (David et al., 2001
). However, the gene × treatment × craving interaction demonstrated early in the treatment phase, during the period of heightened nicotine withdrawal, does not explain why this interaction would affect 6-month smoking cessation outcomes. It is conceivable that, combined with the intensive CBT, persistent biobehavioral neuroadaptations translated into less persistent cigarette craving beyond the treatment phase combined with better coping skills for resisting and preventing relapse triggers. However, this possibility is purely speculative given that we do not have self-reported craving or coping data at the 6-month follow up. That said, Hays and colleagues (2001)
demonstrated that bupropion was effective in preventing relapse as long as 6 months beyond the end of treatment. Examination of the neurobehavioral mechanisms underlying the observed DRD2
× bupropion × craving interaction, if robust, will require collection of additional cognitive and affective data well beyond the end of treatment and might be augmented by application of functional neuroimaging.
The mechanisms underlying the observed DRD2
interaction may not be intuitively apparent. As noted in the introduction, the hepatic P450 isoenzyme CYP2B6 contributes to nicotine metabolism, particularly during states of high serum nicotine, such as the period immediately following cigarette smoking (Yamanaka et al., 2005
). Individuals with one or more CYP2B6
1459 T alleles have been shown to express CYP2B6
at substantially lower levels than C/C counterparts in liver (Lang et al., 2001
) and brain (Miksys et al., 2003
). Furthermore, individuals with slow nicotine metabolism—resulting from specific variants in the CYP2A6
gene—tend to smoke fewer cigarettes per day and are more likely to quit smoking than normal or rapid metabolizers (Munafò et al., 2004
; Schoedel, Hoffmann, Rao, Sellers, & Tyndale, 2004
). Given the lower expression of the CYP2B6 enzyme in CYP2B6
1459 T allele carriers, it is conceivable that these individuals would be slower metabolizers of nicotine and subsequently smoke fewer cigarettes than their C/C counterparts. Therefore, it is not surprising that the individuals demonstrating the highest long-term abstinence rates would be those with DRD2
-Taq1A A2/A2 genotypes (conferring relatively “normal” striatal dopamine set points) and CYP2B6
1459 C→T alleles, the latter perhaps resulting in lower nicotine tolerance and cigarette consumption. Although we do not report the analysis here, significant differences were found in total FTND score or number of cigarettes per day according to CYP2B6
1459 C→T genotype (p
Another plausible explanation would be that altered nicotine metabolism resulting from the CYP2B6 1459 C→T polymorphism would affect accumbal dopamine release, potentially influencing the neuroadaptations resulting in incentive sensitization to nicotine. If this were the case, the DRD2-Taq1A genotype would potentially interact with the CYP2B6 1459 C→T genotype at the level of the nucleus accumbens. Again, such a mechanism cannot be concluded based on the data in the present study but would require the combination of pharmacogenetic and functional neuroimaging for resolution in future work.
The present results differ somewhat from those of existing pharmacogenetic studies of bupropion. In particular, Lerman and colleagues (2003)
examined the role of the SLC6A3 3′ VNTR and DRD2-
Taq1A polymorphisms in a clinical trial of bupropion with a design and sample similar to those in the present study. No significant genotype × treatment effects on prolonged abstinence or 7-day point prevalence were observed at the end of treatment (10–12 weeks) or 6-month follow-up. Instead, a SLC6A3
interaction predicted prolonged abstinence at the end of treatment (independent of treatment condition), an effect not seen in the present study. In addition, as described above, bupropion is metabolized by cytochrome P450 2B6 (CYP2B6) enzyme. In another examination from the same trial (Lerman, Shields et al., 2002
), the CYP2B6
1459 C allele increased the likelihood of smoking relapse; however, the effect was attenuated by bupropion among women. In the present study, the CYP2B6
C→T substitution did not predict prolonged abstinence as a main effect or interaction with bupropion treatment. Thus, although consistent with one prior study suggesting a gene × bupropion effect on craving, the results of the present study differ from the Lerman, Shields et al. (2002)
study with regard to the effects of the CYP2B6 1459 C→T polymorphism on smoking cessation.
Certain limitations to the present study should be highlighted. The present sample size was modest, which will have reduced the power of our study and may have limited the generalizability of our results, particularly given the somewhat specific demographics of our participant sample. Approximately 60% of the subjects in the original clinical trial agreed to blood collection for genotyping. Although the subject characteristics appeared to be quite similar between those who agreed to genotyping and those who did not, other differences between these groups may not have been ascertainable. However, compared with other pharmacogenetic studies using retrospective genotyping, our study had a blood collection rate within the norm (David et al., 2002
; Johnstone et al., 2004
; Yudkin et al., 2004
Finally, the intervention used was more intensive than might be typical, including guided group therapy. A usual consequence of such intensive therapy is an increase in quit rates, and if a more typical treatment model were used, the attenuation in global quit rates might also attenuate the genotype × treatment interaction effect reported here. This issue should be addressed in future studies that attempt to replicate our findings.
The precise mechanism of action of bupropion remains unclear (Ascher et al., 1995
), but it is widely accepted that it acts (although not exclusively) on the dopaminergic pathway, serving to inhibit reuptake. For this reason, dopaminergic genes represent good candidates for the pharmacogenetic study of potential genetic moderators of bupropion treatment efficacy.
Our data support the view that likely genetic moderators will consist, at least in part, of genes that influence dopaminergic biosynthesis and receptor systems. It is somewhat surprising, however, given this theoretical perspective, that we did not demonstrate an effect of the dopamine transporter on treatment response. However, multiple candidate polymorphisms would ideally be included in a comprehensive pharmacogenetic evaluation of bupropion and dopamine system genes.
This point brings up one of the weaknesses in the present study’s design, namely that the functional significance of the DRD2
-Taq1A RFLP and the SLC6A3 3′ VNTR remains speculative. As noted previously, the Taq1A RFLP is present in the ANKK1
gene (approximately 10 kb 3′ from the end of the DRD2
gene) rather than in the DRD2
gene itself (Neville et al., 2004
). Jonsson and colleagues (1999)
demonstrated that the DRD2
-Taq1A polymorphism influences striatal DRD2
receptor density, but the mechanisms underlying how a polymorphism in an adjacent tyrosine kinase gene affects striatal D2 receptor expression are not known. Indeed, two functional SNPs shown to influence transcriptional efficiency—DRD2
−141 ins/del (Arinami et al., 1997
) and DRD2
C957T (Duan et al., 2003
)—may also affect bupropion efficacy and, ideally, should be considered in future exploration of DRD2
× bupropion interactions on smoking cessation.
Furthermore, even though the SLC6A3
3′ VNTR does not appear to affect expression of the transporter in vitro
(Greenwood & Kelsoe, 2003
; Mill, Asherson, Craig, & D’Souza, 2005
), other SNPs and mutations do appear to influence dopamine transporter expression (Horschitz, Hummerich, Lau, Rietschel, & Schloss, 2005
; Kelada et al., 2005
; Lin & Uhl, 2003
). This observation may account in part for the lack of replication in studies of associations between smoking phenotypes and the SLC6A3
3′ VNTR polymorphism (Bierut et al., 2000
; Jorm et al., 2000
; Lerman et al., 1999
; Sabol et al., 1999
; Vandenbergh et al., 2002
). Also, other reasonable SNP candidates in the CYP2B6
gene should be considered for study. Thus, until future studies are designed with sufficient statistical power to include all known functional SNPs in genes throughout the dopamine system, conclusions on the presence or absence of these gene × gene interactions are premature.
Interest is growing in other potential moderators of treatment efficacy, most notably possible sex differences in treatment response to bupropion (Scharf & Shiffman, 2004
) and nicotine replacement therapy (Munafò et al., 2004a
; Shiffman, Sweeney, & Dresler, 2005
). However, the investigation of multiple potential treatment moderators brings with it the usual dangers associated with multiple statistical testing and should not proceed without firm theoretical grounds for investigating specific variables (Munafò, Johnstone, Murphy, & Walton, 2001
). In this respect, dopaminergic genes represent strong candidates, but if multiple candidates are to be investigated simultaneously this will require explicitly designed pharmacogenetic studies adequately powered to detect effect sizes likely to be modest.
The increased understanding of the mechanisms of nicotine addiction and smoking behavior that has developed from the study of genetic influences on these phenotypes has led to a growing interest in the possibility of offering personalized smoking cessation therapy to patients based on their genotype (Audrain et al., 1997
; Lerman & Niaura, 2002
). Our data reinforce this possibility, in particular because the patients in the present study who possessed at least one copy of the A1 allele did not appear to gain any benefit from bupropion relative to placebo. Any strong claims regarding translational implications of our research would be premature, however, and these data should be regarded as preliminary until replicated. Even assuming that such replication does occur, the potential implication of these data—that there might be grounds for not offering bupropion treatment to those statistically unlikely to benefit on the basis of their genotype—brings with it profound ethical, social, and policy implications. Not least, the acceptability of such an approach to those seeking treatment for smoking cessation remains to be determined.