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CYP2B6 Genotype Alters Abstinence Rates in a Bupropion Smoking Cessation Trial
CYP2B6 Genotype Alters Abstinence Rates in a Bupropion Smoking Cessation Trial Anna M. Lee, Christopher Jepson, Ewa Hoffmann, Leonard Epstein, Larry W. Hawk, Caryn Lerman, and Rachel F. Tyndale Background: CYP2B6 is the primary enzyme involved in bupropion (Zyban; GlaxoSmithKline, Research Triangle Park, North Carolina) metabolism. Genetic polymorphisms in CYP2B6, such as CYP2B6*6, can alter bupropion metabolism and may affect bupropion treatment outcome. Methods: Subjects participated in a smoking cessation clinical trial of bupropion versus placebo. The main outcome was a 7-day point prevalence abstinence rate measured 10 weeks after the start of treatment (i.e., end of treatment) and at the 6-month follow-up; secondary outcomes were severity of adverse effects, withdrawal, and urge to smoke. Subjects were haplotyped for the CYP2B6*6 variants. Results: Among smokers in the CYP2B6 *6 group (CYP2B6 *1/ *6 or CYP2B6 *6/ *6 genotype, n ⫽ 147, 45% of the population), bupropion produced significantly higher abstinence rates than placebo at the end of treatment (32.5% vs. 14.3%, p ⫽ .01) and at the 6-month follow-up (31.2% vs. 12.9%, p ⫽ .008). In contrast, bupropion was no more effective than placebo for smokers in the CYP2B6 *1 group (CYP2B6 *1/ *1, n ⫽ 179) at the end of treatment (31.0% vs. 31.6%, p ⫽ .93) or at the 6-month follow-up (22.0% vs. 21.5%, p ⫽ .94). There was a significant genotype by treatment interaction at the end of treatment (odds ratio [OR] ⫽ 2.97, confidence interval [CI] ⫽ 1.05– 8.40, p ⫽ .04), which was similar at 6-month follow-up (OR ⫽ 2.98, CI ⫽ .98 –9.06, p ⫽ .05). Conclusions: These data suggest that smokers with the CYP2B6 *6 genotype have a higher liability to relapse on placebo and that they may be good candidates for bupropion treatment for smoking cessation. Key Words: Bupropion, CYP2A6, CYP2B6, genetic, nicotine, pharmacogenetic, smoking
B
upropion (Zyban; GlaxoSmithKline, Research Triangle Park, North Carolina) was the first Food and Drug Administration (FDA)-approved nonnicotine pharmacotherapy for smoking cessation and is also widely used to treat depression (Wellbutrin; GlaxoSmithKline). Although the biological mechanism of action for bupropion has yet to be fully elucidated, there is evidence that it can inhibit norepinephrine and dopamine reuptake (Stahl et al. 2004) and act as a nicotinic acetylcholine receptor antagonist (Slemmer et al. 2000). Although the efficacy of bupropion relative to placebo is firmly established (Hughes et al. 2004; Hurt et al. 1997; Jorenby et al. 1999), the majority of smokers relapse to smoking within 6 months after a quit attempt. Varenicline, an ␣42 nicotinic acetylcholine receptor partial agonist, was recently approved by the FDA for smoking cessation and may have superior efficacy to bupropion (Gonzalez et al. 2006). Pharmacogenetic analyses may be valuable for identifying those smokers most likely to benefit from bupropion therapy or other pharmacotherapies for the treatment of nicotine dependence. Bupropion is metabolized to hydroxybupropion, erythrohydrobupropion, and threohydrobupropion (Faucette et al. 2000; Hsyu et al. 1997). Carbonyl reductase metabolizes bupropion to erythrohydrobupropion and threohydrobupropion (Jefferson et al.
From the Centre for Addiction and Mental Health and the Department of Pharmacology (AML, EH, RFT), University of Toronto, Ontario, Canada; Department of Psychiatry (CJ, CL), Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania; and the Department of Pediatrics (LE), School of Medicine and Biomedical Sciences, and Department of Psychology (LWH), The University at Buffalo, State University of New York, Buffalo, New York. Address reprint requests to Rachel F. Tyndale, M.Sc., Ph.D., University of Toronto, Department of Pharmacology, 1 King’s College Circle, Room 4326, Toronto, Ontario M5S 1A8, Canada; E-mail: [email protected]. Received July 2, 2006; revised October 4, 2006; accepted October 5, 2006.
0006-3223/07/$32.00 doi:10.1016/j.biopsych.2006.10.005
2005). CYP2B6 is the main enzyme that metabolizes bupropion to hydroxybupropion (Faucette et al. 2000) and CYP2B6 does not appear to be involved in further metabolism of hydroxybupropion (Hsyu et al. 1997; Welch et al. 1987). Bupropion and hydroxybupropion are pharmacologically active, whereas erythrohydrobupropion and threohydrobupropion have little pharmacological activity (Damaj et al. 2004). CYP2B6 is genetically variable (http://www. cypalleles.ki.se/cyp2b6.htm); some alleles, such as CYP2B6 *4 (A785G), CYP2B6*5 (C1459T), and CYP2B6*6 (G516T and A785G), have been shown to alter CYP2B6 activity (Hesse et al. 2004; Kirchheiner et al. 2003; Lang et al. 2001; Lerman et al. 2002) and may thus alter the therapeutic effects of bupropion. We have previously found that the C1459T single nucleotide polymorphism in exon 9, which is associated with decreased protein and activity (Lang et al. 2001), is associated with an increase in craving and higher smoking relapse rates on placebo; this effect was reversed by bupropion among female subjects (Lerman et al. 2002). CYP2B6 can metabolize nicotine (Yamazaki et al. 1999) and is found in the brain (Miksys et al. 2003); CYP2B6 may contribute to the association of the C1459T variant with smoking relapse. Current single nucleotide polymorphism assays were unable to distinguish the CYP2B6*9 (G516T), CYP2B6*4 (A785G), and CYP2B6*6 (G516T and A785G) alleles. We developed a novel haplotyping assay and have investigated these alleles in a bupropion clinical trial. The CYP2B6*6 haplotype is of particular interest as it has allele frequencies of 25%, 30%, and 15% in Caucasians (Jacob et al. 2004; Kirchheiner et al. 2003; Klein et al. 2005; Zukunft et al. 2005), African Americans (Klein et al. 2005), and Asians (Cho et al. 2004; Klein et al. 2005; Tsuchiya et al. 2004), respectively; therefore, approximately 45%, 50%, and 25% of Caucasians, African Americans, and Asians, respectively, have at least one CYP2B6*6 allele. Thus, a clinical impact of this allele on cessation would affect a large portion of the smoking population. The CYP2B6 *6 allele results in a structurally altered enzyme that has been associated with both decreased (Haas et al. 2004; Lang et al. 2001; Rotger et al. 2005; Tsuchiya et al. 2004) and increased (Jinno et al. 2003; Xie et al. 2003) metabolism of BIOL PSYCHIATRY 2007;62:635– 641 © 2007 Society of Biological Psychiatry
636 BIOL PSYCHIATRY 2007;62:635– 641 various CYP2B6 substrates. CYP2B6 *6 has been associated with decreased bupropion metabolism in human liver samples in vitro (Hesse et al. 2004). A recent in vivo study demonstrated a reduction in bupropion metabolic activity for CYP2B6 *6 using the ratio of the areas under the curve for hydroxybupropion compared with bupropion; one subject with a CYP2B6 *6/ *6 genotype had a considerably smaller ratio than the subjects with CYP2B6 *1/ *1 genotypes (8.1 vs. 18.5) (Loboz et al. 2006). We postulated that smokers in the CYP2B6*6 group (CYP2B6*1/*6 or CYP2B6*6/*6 genotype) may have decreased bupropion metabolism leading to increased bupropion plasma levels but a similar rate of hydroxybupropion metabolism as the CYP2B6*1 group (CYP2B6*1/*1 genotype). Therefore, we hypothesized that there would be a larger therapeutic impact of bupropion in the CYP2B6*6 group compared with the CYP2B6*1 group.
Methods and Materials Participants Participants were enrolled from April 1999 to October 2001 at Georgetown University (Washington, DC) and State University of New York (SUNY) Buffalo (New York). Recruitment, demographics, and the protocol have been reported in detail elsewhere (Lerman et al. 2002, 2006). A total of 555 participants enrolled in the pharmacogenetic trial, of whom 423 were successfully genotyped; the rest had insufficient DNA remaining for this analysis. Of the 423 for whom CYP2B6 genotyping was performed, 342 were Caucasians; 16 had a CYP2B6 *4 genotype (Results) and were omitted, leaving 326 for the final analysis. Eligible participants smoked at least 10 cigarettes per day. Exclusion criteria included pregnancy, a history of DSM-IV psychiatric disorder, seizures, and current use of antidepressants and other psychotropic medications. Study Protocol The protocol number was 703463 (Biobehavioral Lung Cancer Prevention Program) and was originally approved on November 30, 1999. The clinical trial registration number is NCT00322205. The Institutional Review Board of the University of Toronto has also approved this study (#17520). At an initial visit to the smoking clinics, participants provided a 40 mL blood sample for genotyping and completed a set of standardized self-report questionnaires that collected information on demographics (e.g., age, education, marital status) and smoking (age at smoking initiation, cigarettes per day, and nicotine dependence as measured by the Fagerström Test for Nicotine Dependence). Participants then received 10 weeks of either placebo or bupropion treatment, plus seven sessions of behavioral group counseling. Bupropion treatment was given according to the standard therapeutic dose (150 mg/day for the first 3 days, followed by 300 mg/day). All participants were instructed to quit on a target quit date 2 weeks after initiating medication. Smoking status was assessed at the end of treatment (EOT; 8 weeks after target quit date), and at 6-month follow-up using a validated timeline follow-back method. To be categorized as abstinent, a participant had to report no smoking at all in the 7 days prior to the assessment and provide a breath sample containing ⱕ10 ppm of carbon monoxide. Participants were asked to refrain from use of nicotine replacement therapy or other smoking cessation therapies during the treatment and follow-up phase; however, usage during the follow-up phase was not monitored. For midtreatment assessments, a 17-item self-report index of adverse effects was administered at sessions 2 through 7; a www.sobp.org/journal
A.M. Lee et al. summary score was created by summing the responses to all items (Lerman et al. 2002, 2006). A 19-item self-report index of withdrawal symptoms and craving was administered at baseline and at sessions 2 through 6; a summary score was created as the sum of responses to items 1 through 18 and an urge subscale was created as the sum of responses to items 1 (cravings for cigarettes) and 19 (urges to smoke) (Lerman et al. 2002, 2006). The analysis was restricted to smokers of Caucasian European ancestry to reduce bias due to population stratification. Genotyping for CYP2B6 was performed as part of a secondary analysis; thus, posttreatment plasma samples for bupropion and hydroxybupropion levels were not collected. Statistical Analysis The analysis was conducted on a modified intent-to-treat basis that included all participants who attended the initial randomization session and the first treatment session, regardless of completion. Participants who could not be reached for the follow-up or were unavailable to biochemically confirm abstinence were considered to be smokers based on the recommendations of the SRNT Subcommittee on Biochemical Verification (2002). Two-tailed Student t tests and chi-square tests were used to examine associations of selected baseline variables (sex, educational level, marital status, body mass index [BMI], age, age at smoking initiation, cigarettes smoked per day, nicotine dependence) with CYP2B6 genotype and abstinence. Logistic regression models of abstinence at end of treatment and the 6-month follow-up were then estimated, using CYP2B6 genotype, treatment arm, and the interaction of CYP2B6 genotype and treatment arm as the predictors. (None of the abovementioned baseline variables were included in these models, as they did not display significant bivariate associations either with CYP2B6 genotype or with abstinence at either time point.) Repeated measures multivariate analyses of variance were used to model total scores on withdrawal symptoms and craving, urge to smoke scores, and total adverse effect scores over the course of treatment, with time as the within-subjects factor, and CYP2B6 genotype and treatment type as the between-subjects factors. Analyses of withdrawal symptoms, craving, and urge to smoke were restricted to participants who were abstinent (biochemically confirmed) at all treatment phase time points. CYP2B6 and CYP2A6 Genotyping The CYP2B6 gene is highly polymorphic, and the G516T and A785G single nucleotide polymorphisms occur together in minor alleles in addition to CYP2B6 *6, namely CYP2B6 *7, CYP2B6 *13, CYP2B6 *19, and CYP2B6 *20. Currently, the very rare CYP2B6 *13, CYP2B6 *19, and CYP2B6 *20 have not yet been found in Caucasians; lower activity is suggested by expression studies (Klein et al. 2005; Lang et al. 2004). The CYP2B6 *7 allele is infrequent in Caucasians (1.1–3.0%) (Hesse et al. 2004; Klein et al. 2005) and the in vivo functional impact is unknown; the expected impact of the CYP2B6 *7 allele in our population is minimal compared with that of the decreased function CYP2B6 *6 allele, which occurs with much higher frequency. A haplotyping assay was developed to detect the CYP2B6 polymorphisms G516T and A785G using a two-step allele-specific polymerase chain reaction (PCR). CYP2B6 primers were ordered from ACGT Corporation (Toronto, Ontario, Canada). Reagents used in the assays were purchased as a kit from Fermentas (Burlington, Ontario, Canada) with the Taq polymerase enzyme. The deoxyribonucleotide triphosphate (dNTP) set and 1-kb Gene Ruler DNA ladder were also purchased from Fermentas.
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Caucasians, CYP2A6 *2, CYP2A6 *4, CYP2A6 *9, and CYP2A6 *12, was performed as previously described (Malaiyandi et al. 2005; Schoedel et al. 2004).
Table 1. Primer Sequences for CYP2B6*6 (G516T and A785G) Haplotyping Assay First Amplification Primers 2B6ln3F-1 (intron 3 forward primer) 2B6ln5R-1 (intron 5 reverse primer) Second Amplification Primers 2B6G516-F (G516T forward primer wild-type) 2B6T516-F (G516T forward primer variant) 2B6A785-R (A785G reverse primer wild-type) 2B6G785-R (A785G reverse primer variant)
5’-GGTCTGCCCATCTATAAAC-3’ 5’-CTCTCCCTGTCTCAGTTCC-3’
Results
5’-CCCACCTTCCTCTTCCAG-3’ 5’-CCCACCTTCCTCTTCCAT-3’ 5’-AGGTGTCGATGAGGTCCT-3’ 5’-AGGTGTCGATGAGGTCCC-3’
Polymerase chain reaction amplifications were carried out on PTC-200 Peltier Thermal Cycler (BioRad, Toronto, Ontario, Canada). Agarose was purchased from ONBIO Inc. (Richmond Hill, Ontario, Canada). For the first gene-specific amplification, each sample reaction contained 2.5 L of 10 X Taq PCR buffer containing 100 mmol/L Tris-HCl (pH 8.8 at 25°C), 500 mmol/L potassium chloride (KCl), and .8% Nonidet P40, .2 L of a dNTP mixture containing 25 mmol/L of each nucleotide, 1.7 L of 25 mmol/L magnesium dichloride (MgCl2,) .125 L each of the forward and reverse primers at 12.5 m, .25 L of Taq, 1.0 L of the sample DNA (5 to 50 ng/L stock concentrations), and 19.1 L of water. The conditions were an initial denaturation at 95°C for 1 min, 31 cycles at 95°C for 15 sec, 52°C for 30 sec, 72°C for 2 min, followed by a final extension at 72°C for 7 min. For the second haplotype-specific amplification, each reaction contained 2.5 L of 10 X Taq PCR buffer, .1 L of the dNTP mixture, 1.2 L of 25 mmol/L MgCl2, .25 L each of the forward and reverse primers at 12.5 m, .15 L of Taq, .8 L of the sample DNA, and 19.75 L of water. The conditions were an initial denaturation at 95°C for 1 min, 16 cycles at 95°C for 15 sec, 60°C for 30 sec, 72°C for 1 min and 30 seconds, followed by a final extension at 72°C for 7 min. A list of primers for the haplotyping assay is given in Table 1. Genotyping for the common reduced function alleles in
In our sample, 423 participants were successfully genotyped, including 342 Caucasians. The CYP2B6*9 allele was not found. The Caucasian genotype frequencies were CYP2B6*1/*1 (52.3%), CYP2B6*1/*6 (36.3%), CYP2B6*6/*6 (6.7%), CYP2B6*1/*4 (4.1%), and CYP2B6*4/*6 (.6%). The Caucasian CYP2B6*6 and CYP2B6*4 allele frequencies were 25.2% and 2.3%, respectively (n ⫽ 342 total); both alleles were in Hardy-Weinberg equilibrium (p ⫽ .96 and p ⫽ .53, respectively). Smokers with a CYP2B6*1/*4 genotype, indicating increased rates of bupropion metabolism (Kirchheiner et al. 2003; Loboz et al. 2006), were omitted from subsequent analyses due to low power to assess the impact. Smokers with the CYP2B6*4/*6 genotype were omitted, as the effect of this genotype on bupropion metabolism is unclear (Loboz et al. 2006). The final sample of Caucasians (n ⫽ 326) was grouped into a CYP2B6*1 (n ⫽ 179) and a CYP2B6*6 group (n ⫽ 147). There were no significant differences across genotype groups in any sociodemographic variables, treatment group assignment, or Fagerström nicotine dependence scores (Table 2). At the end of treatment (8 weeks following the target quit date), the overall rate of biochemically verified smoking cessation was 27.9% (91 of 326 participants). In the bupropion arm, the abstinence rate was 31.6% (56 of 177) compared with 23.5% (35 of 149) in the placebo arm [2(1) ⫽ 2.67, p ⫽ .10]. At 6 months, the abstinence rate was 22.1% [26.0% in the bupropion arm compared with 17.4% in the placebo arm; 2(1) ⫽ 3.43, p ⫽ .064]. At both EOT and at 6 months, abstinence was not significantly associated with CYP2B6 genotype [at EOT, 2(1) ⫽ 2.24, p ⫽ .13; at 6 months, 2(1) ⫽ .02, p ⫽ .89], sex, educational level, marital status, BMI, age, age at smoking initiation, number of cigarettes smoked per day at baseline, or baseline nicotine dependence (all p ⱖ .10). In the logistic regression model of abstinence at EOT, there was a significant CYP2B6 genotype ⫻ treatment arm interaction (odds ratio [OR] ⫽ 2.97, confidence interval [CI] ⫽ 1.05– 8.40, p ⫽
Table 2. Demographic Characteristics by CYP2B6 Genotype Mean (SD) Among Variable Baseline Nicotine Dependencea Cigarettes Per Day at Baseline Age at Smoking Initiation (Years) Body Mass Index Age (Years)
CYP2B6*1
CYP2B6*6
t Value
p Value
5.31 (2.21) 22.3 (10.5) 16.7 (3.0) 27.3 (4.8) 44.9 (11.0)
5.25 (2.04) 21.1 (7.3) 17.2 (3.8) 26.5 (4.7) 43.7 (12.3)
.24 1.18 ⫺1.29 1.48 .91
.81 .24 .20 .14 .36
N (%) Among Variable
CYP2B6*1
CYP2B6*6
2
p Value
Female Subjects Marital Status Never Married Married Divorced/Separated/Widowed Education No College Some College College Graduate
94 (52%)
84 (57%)
.70 .52
.40 .77
40 (23%) 87 (49%) 50 (28%)
33 (23%) 76 (52%) 36 (25%) .25
.88
33 (19%) 66 (37%) 78 (44%)
29 (20%) 56 (39%) 60 (41%)
a
Fagerström Test for Nicotine Dependence score.
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Table 3. Logistic Regression Models of Abstinence at EOT and 6-Month Follow-Up Variable End of Treatment CYP2B6 Genotype (CYP2B6*1 ⫽ 0, CYP2B6*6 ⫽ 1) Treatment (Placebo ⫽ 0, Bupropion ⫽ 1) CYP2B6 Genotype by Treatment 6-Month Follow-Up CYP2B6 Genotype (CYP2B61 ⫽ 0, CYP2B66 ⫽ 1) Treatment (Placebo ⫽ 0, Bupropion ⫽ 1) CYP2B6 Genotype by Treatment
Odds Ratio
Confidence Interval
p Value
.36 .97 2.97
.16, .82 .51, 1.83 1.05, 8.40
.02 .93 .04
.54 1.03 2.98
.22, 1.30 .50, 2.10 .98, 9.06
.17 .94 .054
Each of these models includes a significant interaction between genotype and treatment; therefore, the p-values for the component main effect variables will not correspond to the results of bivariate analyses (2 tests) testing associations of those variables with abstinence. EOT, end of treatment.
.04); the interaction was similar in magnitude and close to significance at 6 months (OR ⫽ 2.98, CI ⫽ .98 –9.06, p ⫽ .054) (Table 3). Within the CYP2B6 *6 group, bupropion significantly increased abstinence rates compared with placebo at EOT [32.5% vs. 14.3%, 2(1) ⫽ 6.68, p ⫽ .01] and at the 6-month follow-up [31.2% vs. 12.9%, 2(1) ⫽ 7.06, p ⫽ .008] (Figure 1A and 1B). Thus, bupropion had a large benefit over placebo for 45% of the sample, the CYP2B6 *6 group. Within the CYP2B6 *1 group, bupropion did not increase abstinence rates compared with placebo at EOT [31.0% vs. 31.6%, 2(1) ⫽ .01, p ⫽ .93] or at the 6-month follow-up [22.0% vs. 21.5%, 2(1) ⫽ .01, p ⫽ .94]. Thus, for 55% of the sample, the CYP2B6 *1 group, bupropion did not provide a benefit over placebo. The CYP2B6 *6 group did not have a higher abstinence rate on bupropion compared with the CYP2B6 *1 group at EOT [32.5% vs. 31.0%, 2(1) ⫽ .04, p ⫽ .84] but had a nonsignificantly higher abstinence rate than the CYP2B6 *1 group at the 6-month follow-up [31.2% vs. 22.0%; 2(1) ⫽ 1.90, p ⫽ .17]. The abstinence rate in the CYP2B6 *6 group treated with bupropion did not decrease between EOT and the 6-month follow-up (32.5% vs. 31.2%; p ⫽ .99 by McNemar test); however, the abstinence rate decreased significantly from EOT to the 6-month follow-up in the CYP2B6 *1 group (31.0% vs. 22.0%; p ⫽ .04). The CYP2B6 *6 group had a smaller change in the abstinence rate between the EOT and the 6-month follow-up compared with the CYP2B6 *1 group (1.3% vs. 9%; p ⫽ .13 by longitudinal logistic regression) but this did not reach significance. These data suggest there may be an impact of the CYP2B6 *6 allele at the 6-month follow-up with respect to the maintenance of abstinence. The CYP2B6 *1 group achieved a significantly higher abstinence rate on placebo compared with the CYP2B6 *6 group at
EOT [31.6% vs. 14.3%, 2(1) ⫽ 6.22, p ⫽ .01] (Figure 1A). The results were similar at 6-month follow-up [21.5% vs. 12.9%, 2(1) ⫽ 1.93, p ⫽ .16], although not statistically significant (Figure 1B). There was a gene-dose effect on placebo abstinence rates at EOT. The placebo abstinence rates were 31.6% for homozygous CYP2B6 *1, 16.4% for heterozygous CYP2B6 *1/ *6, and 0% for homozygous CYP2B6 *6 (p ⫽ .03). The 6-month abstinence rates were 21.5%, 14.8%, and 0%, respectively (p ⫽ .21). Gender differences in abstinence rates have been observed in bupropion clinical trials (Lerman et al. 2002; Swan et al. 2003), including an earlier analysis indicating that in female subjects only, there was decreased abstinence on placebo with the rarer CYP2B6 C1459T variant (Lerman et al. 2002). In the current study, there was no main effect of gender on abstinence at EOT or at the 6-month follow-up. The CYP2B6 genotype distribution and the gene-specific effect of treatment arm were similar between male subjects and female subjects at the end of treatment and at the 6-month follow-up (the sex ⫻ CYP2B6, sex ⫻ treatment arm, and sex ⫻ CYP2B6 ⫻ treatment arm interactions were not significant at either time point (all p ⬎ .10). To investigate potential mediators of the difference in abstinence rates between genotype groups, we examined associations of CYP2B6 genotype with pretreatment and midtreatment smoking-related variables. There were no differences between CYP2B6 genotype groups in reported age of smoking initiation, cigarettes per day at baseline, and baseline scores on nicotine dependence in the placebo group or among bupropion-treated participants (all p ⬎ .15, results not shown). In addition, we examined the main and interacting effects of CYP2B6 genotype and treatment arm on midtreatment withdrawal symptoms, urge to smoke, and total adverse effects using multivariate analysis of Figure 1. Significant CYP2B6 genotype by drug treatment effects on abstinence rates. The CYP2B6*6 group (n ⫽ 147) had a significantly higher abstinence rate on bupropion compared with placebo at the end of treatment and at the 6-month follow-up. The CYP2B6*1 group (n ⫽ 179) did not benefit from bupropion compared with placebo at the end of treatment (A) or at the 6-month follow-up (B). (C) The sample was restricted to normal nicotine metabolizers with a CYP2A6*1/*1 genotype (n ⫽ 274). The pattern of abstinence between the CYP2B6*1 and the CYP2B6*6 groups is similar to that found in the whole group (B). Numbers within the bars indicate the percent of the subsample that was abstinent.
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A.M. Lee et al. variance. No significant effects of genotype or genotype by treatment arm interactions were found and adverse events did not appear to mediate the abstinence rates in either the CYP2B6 *6 or CYP2B6 *1 group. CYP2A6 metabolizes nicotine (Messina et al. 1997) and can contribute to altered hepatic nicotine metabolism (Xu et al. 2002) and smoking behaviors (Schoedel et al. 2004); therefore, we examined the impact of common CYP2A6 decreased activity alleles (*2, *4, *9, and *12) on abstinence. There was no significant main effect of CYP2A6 genotype on abstinence at EOT or 6-month follow-up (p ⱖ .15), nor was there a CYP2A6CYP2B6 gene-gene interaction at either time point (p ⱖ .14). However, there was a significant CYP2A6 gene ⫻ treatment arm interaction at EOT (OR ⫽ .20, 95% CI ⫽ .04 –.95; p ⫽ .04). Among cases with normal CYP2A6 metabolism (n ⫽ 274), similar EOT abstinence rates were seen compared with the general population on placebo (21.5%) and bupropion (34.6%) [2(1) ⫽ 5.70, p ⫽ .02]. In contrast, among the small proportion of cases with intermediate or slow CYP2A6 metabolism (16%, n ⫽ 51), EOT abstinence rates were high on placebo (32.1%) and low on bupropion (13.0%) [2(1) ⫽ 2.56, p ⫽ .11]. The CYP2A6 gene ⫻ treatment arm interaction was not significant at 6-month follow-up (OR ⫽ .38, 95% CI ⫽ .06 –2.46; p ⫽ .31). We repeated the analysis of abstinence by CYP2B6 among participants with CYP2A6 *1/ *1 genotype only (those with normal CYP2A6 metabolism, n ⫽ 274) and found that the abstinence rates within the CYP2B6 *1 and CYP2B6 *6 groups (Figure 1C) were similar to those seen in the total sample, which included CYP2A6 intermediate and slow metabolizers (Figure 1B).
Discussion This study illustrates the importance of investigating genetic variation in drug-metabolizing enzymes to predict smoking cessation and therapeutic response to bupropion. Without investigating CYP2B6 genetics, the overall effect on abstinence of bupropion is modest. However, when CYP2B6 genotype is incorporated, bupropion is significantly more efficacious than placebo for ⬃45% of the population (those in the CYP2B6 *6 group) but not in the remaining ⬃55% (those in the CYP2B6 *1 group). At EOT, the CYP2B6 *6 group had low abstinence rates on placebo but responded well with bupropion, whereas the CYP2B6 *1 group had high abstinence rates on placebo and these were not significantly enhanced by bupropion (Figure 1). The CYP2B6 *1 and CYP2B6 *6 groups achieved maximal abstinence rates of approximately 33% on bupropion at EOT, similar to other studies (Hurt et al. 1997; Tonnesen et al. 2003). Thus, the CYP2B6 *6 group does not have an advantage over the CYP2B6 *1 group when examining only the response to bupropion treatment, and the observed interaction is due to differences in placebo response. The change in abstinence rates from EOT to 6-month follow-up also appears to be modified by CYP2B6 genotype. Abstinence rates after the termination of treatment tend to drop as participants relapse (Hurt et al. 1997). Among participants treated with bupropion, the abstinence rate was maintained at 6 months by the CYP2B6 *6 group (Figure 1), suggesting that bupropion may be more efficacious for preventing relapse in the CYP2B6 *6 group. Among placebo-treated participants in the CYP2B6 *6 group, there was also no change in abstinence, but these rates were low at both time points. The CYP2B6 *1 group did not maintain abstinence at the 6-month follow-up compared with EOT on either bupropion or placebo, suggesting that the
BIOL PSYCHIATRY 2007;62:635– 641 639 maintenance of abstinence is specific to the CYP2B6 *6 group. The maintenance of abstinence at 6 months by the CYP2B6 *6 group may be due to an intermediate outcome measure that was not assessed with our current self-report questionnaires. Although bupropion has been shown to decrease withdrawal and craving (Brody et al. 2004; Shiffman et al. 2000; Teneggi et al. 2005), there were no significant differences by genotype in midtreatment withdrawal symptoms or adverse effects in either the placebo or bupropion groups. This suggests that the decreased abstinence on placebo in the CYP2B6 *6 group, and the enhanced response to bupropion in this group, is not mediated by these variables. Further, the data suggest that these effects may not be mediated by differences in bupropion metabolism, because we did not see genotype associations in adverse effect scores in the bupropion-treated group. The high placebo abstinence rates (Hurt et al. 1997; Tonnesen et al. 2003) observed in the CYP2B6 *1 group may be due to other factors. Firstly, the increased placebo abstinence rate may reflect a function of CYP2B6 that is currently unknown. CYP2B6 is expressed in extrahepatic locations, such as lung (Gervot et al. 1999) and brain (Miksys et al. 2003), and metabolizes endogenous substrates such as testosterone (Imaoka et al. 1996). The structurally altered CYP2B6 *6 has also been associated with increased nicotine metabolism (Ring et al, unpublished data) compared with CYP2B6 *1. CYP2B6 *1 may be involved in a mechanism of smoking that provides good short-term abstinence (occurring during the behavioral counseling treatment) compared with the CYP2B6 *6 in the placebo-treated groups, resulting in the higher abstinence rate. The advantage in quitting on placebo conferred by CYP2B6 *1, however, appears to be unaltered by bupropion treatment. Conversely, CYP2B6 *6 may be involved in a mechanism of smoking that contributes to decreased abstinence in the placebo-treated arm but which is responsive to bupropion. Secondly, CYP2B6 *1 may be in linkage disequilibrium with another gene conferring high abstinence rates. We investigated the CYP2A6 gene, as the CYP2A6 enzyme metabolizes nicotine (Messina et al. 1997), and can contribute to differences in hepatic nicotine metabolism (Xu et al. 2002) and smoking behavior (Schoedel et al. 2004). It is located in close proximity to CYP2B6 on chromosome 19 (Hoffman et al. 2001). For example, slow CYP2A6 metabolism has been previously associated with shorter smoking durations (Schoedel et al. 2004) and increased quit rates (Gu et al. 2000). Although there was a significant CYP2A6 gene ⫻ treatment arm interaction at EOT, there was no significant main effect of CYP2A6 genotype on abstinence and no detectable CYP2A6-CYP2B6 gene-gene interaction. In addition, when restricted to CYP2A6 normal metabolizers, the abstinence rates within the CYP2B6 *1 and CYP2B6 *6 groups were similar to those in the total sample (Figure 1C). CYP2B6 *1 may be in linkage disequilibrium with another unknown gene that confers an advantage in quitting on placebo compared with the CYP2B6 *6 group; however, on inspection of the chromosomal region, no other obvious candidates were identified. One limitation of the study was that the population was restricted to persons of European ancestry. Further studies will be required to confirm these effects in other ethnic groups. Another limitation was the lack of plasma bupropion and hydroxybupropion levels to confirm the effect of the CYP2B6 genotype. Subsequent prospective clinical trials to test the findings observed here should also include the measurement of bupropion and hydroxybupropion plasma levels. If the present CYP2B6 genotype results are replicated, genetic testing prior to www.sobp.org/journal
640 BIOL PSYCHIATRY 2007;62:635– 641 treatment would provide an opportunity to match smokers with a treatment that would maximize abstinence rates. We have shown that the CYP2B6 *1 group achieves high quit rates on placebo and does not benefit from bupropion therapy for smoking cessation. This subgroup of smokers may be better treated with an alternative therapy, such as behavioral counseling. In contrast, the CYP2B6 *6 group did poorly on placebo and exhibited a significant benefit of bupropion treatment at EOT, an effect that was maintained at 6-month follow-up. In the future, if these findings are validated and as genetic testing becomes more economical, it could be advantageous to genotype smokers for CYP2B6 and offer bupropion treatment for those with the CYP2B6 *6 genotype. This work was supported by a Canadian Tobacco Control Research Initiative student grant 16803 (AML), Canadian Institute of Health Research (CIHR) Tobacco Use in Special Populations Fellowship (AML), grants from the National Cancer Institute and National Institutes on Drug Abuse, P5084718, RO1 CA63562 (CL) and DA020830 (RFT, CL), CIHR grant MOP53248 (RFT), and a Canada Research Chair in Pharmacogenetics (RFT). Study medication was provided at no cost by GlaxoSmithKline. CL has served as a consultant to GlaxoSmithKline. RFT holds shares in Nicogen Inc., a company focused on creating novel smoking cessation treatments. No funding for this study was received from Nicogen and no benefit to the company was obtained. Brody AL, Mandelkern MA, Lee G, Smith E, Sadeghi M, Saxena S, et al. (2004): Attenuation of cue-induced cigarette craving and anterior cingulate cortex activation in bupropion-treated smokers: A preliminary study. Psychiatry Res 130:269 –281. Cho JY, Lim HS, Chung JY, Yu KS, Kim JR, Shin SG, et al. (2004): Haplotype structure and allele frequencies of CYP2B6 in a Korean population. Drug Metab Dispos 32:1341–1344. Damaj MI, Carroll FI, Eaton JB, Navarro HA, Blough BE, Mirza S, et al. (2004): Enantioselective effects of hydroxy metabolites of bupropion on behavior and on function of monoamine transporters and nicotinic receptors. Mol Pharmacol 66:675– 682. Faucette SR, Hawke RL, Lecluyse EL, Shord SS, Yan B, Laethem RM, et al. (2000): Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 28:1222–1230. Gervot L, Rochat B, Gautier JC, Bohnenstengel F, Kroemer H, de Berardinis V, et al. (1999): Human CYP2B6: Expression, inducibility and catalytic activities. Pharmacogenetics 9:295–306. Gonzales D, Rennard SI, Nides M, Oncken C, Azoulay S, Billing CB, et al.(2006): Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs. sustained-release bupropion and placebo for smoking cessation: A randomized controlled trial. JAMA 296:47–55. Gu DF, Hinks LJ, Morton NE, Day IN (2000): The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit. Ann Hum Genet 64:383–390. Haas DW, Ribaudo HJ, Kim RB, Tierney C, Wilkinson GR, Gulick RM, et al. (2004): Pharmacogenetics of efavirenz and central nervous system side effects: An Adult AIDS Clinical Trials Group study. AIDS 18:2391–2400. Hesse LM, He P, Krishnaswamy S, Hao Q, Hogan K, Moltke LL, et al. (2004): Pharmacogenetic determinants of interindividual variability in bupropion hydroxylation by cytochrome P450 2B6 in human liver microsomes. Pharmacogenetics 14:225–238. Hoffman SM, Nelson DR, Keeney DS (2001): Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 11:687– 698. Hsyu PH, Singh A, Giargiari TD, Dunn JA, Ascher JA, Johnston JA (1997): Pharmacokinetics of bupropion and its metabolites in cigarette smokers versus nonsmokers. J Clin Pharmacol 37:737–743. Hughes J, Stead L, Lancaster T (2004): Antidepressants for smoking cessation. Cochrane Database Syst Rev CD000031.
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A.M. Lee et al. Hurt RD, Sachs DP, Glover ED, Offord KP, Johnston JA, Dale LC, et al. (1997): A comparison of sustained-release bupropion and placebo for smoking cessation. N Engl J Med 337:1195–1202. Imaoka S, Yamada T, Hiroi T, Hayashi K, Sakaki T, Yabusaki Y, et al. (1996): Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. Biochem Pharmacol 51:1041–1050. Jacob RM, Johnstone EC, Neville MJ, Walton RT (2004): Identification of CYP2B6 sequence variants by use of multiplex PCR with allele-specific genotyping. Clin Chem 50:1372–1377. Jefferson JW, Pradko JF, Muir KT (2005): Bupropion for major depressive disorder: Pharmacokinetic and formulation considerations. Clin Ther 143:415– 426. Jinno H, Tanaka-Kagawa T, Ohno A, Makino Y, Matsushima E, Hanioka N, et al. (2003): Functional characterization of cytochrome P450 2B6 allelic variants. Drug Metab Dispos 31:398 – 403. Jorenby DE, Leischow SJ, Nides MA, Rennard SI, Johnston JA, Hughes AR, et al. (1999): A controlled trial of sustained-release bupropion, a nicotine patch, or both for smoking cessation. N Engl J Med 340:685– 691. Kirchheiner J, Klein C, Meineke I, Sasse J, Zanger UM, Murdter TE, et al. (2003): Bupropion and 4-OH-bupropion pharmacokinetics in relation to genetic polymorphisms in CYP2B6. Pharmacogenetics 13:619 – 626. Klein K, Lang T, Saussele T, Barbosa-Sicard E, Schunck WH, Eichelbaum M, et al. (2005): Genetic variability of CYP2B6 in populations of African and Asian origin: Allele frequencies, novel functional variants, and possible implications for anti-HIV therapy with efavirenz. Pharmacogenet Genomics 15:861– 873. Lang T, Klein K, Fischer J, Nussler AK, Neuhaus P, Hofmann U, et al. (2001): Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 11:399–415. Lang T, Klein K, Richter T, Zibat A, Kerb R, Eichelbaum M, et al. (2004): Multiple novel nonsynonymous CYP2B6 gene polymorphisms in Caucasians: Demonstration of phenotypic null alleles. J Pharmacol Exp Ther 311:34–43. Lerman C, Shields PG, Wileyto EP, Audrain J, Pinto A, Hawk L, et al. (2002): Pharmacogenetic investigation of smoking cessation treatment. Pharmacogenetics 12:627– 634. Lerman C, Jepson C, Wileyto EP, Epstein LH, Rukstalis M, Patterson F, et al. (2006): Role of functional genetic variation in the dopamine D2 receptor (DRD2) in response to bupropion and nicotine replacement therapy for tobacco dependence: Results of two randomized clinical trials. Neuropsychopharmacology 31:231–242. Loboz KK, Gross AS, Williams KM, Liauw WS, Day RO, Blievernicht JK, et al. (2006): Cytochrome P450 2B6 activity as measured by bupropion hydroxylation: Effect of induction by rifampin and ethnicity. Clin Pharmacol Ther 80:75– 84. Malaiyandi V, Sellers EM, Tyndale RF (2005): Implications of CYP2A6 genetic variation for smoking behaviors and nicotine dependence. Clin Pharmacol Ther 77:145–158. Messina ES, Tyndale RF, Sellers EM (1997): A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. J Pharmacol Exp Ther 282: 1608 –1614. Miksys S, Lerman C, Shields PG, Mash DC, Tyndale RF (2003): Smoking, alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain. Neuropharmacology 45:122–132. Rotger M, Colombo S, Furrer H, Bleiber G, Buclin T, Lee BL, et al. (2005): Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients. Pharmacogenet Genomics 15:1–5. Schoedel KA, Hoffmann EB, Rao Y, Sellers EM, Tyndale RF (2004): Ethnic variation in CYP2A6 and association of genetically slow nicotine metabolism and smoking in adult Caucasians. Pharmacogenetics 14: 615– 626. Shiffman S, Johnston JA, Khayrallah M, Elash CA, Gwaltney CJ, Paty JA, et al. (2000): The effect of bupropion on nicotine craving and withdrawal. Psychopharmacology (Berl) 148:33– 40. Slemmer JE, Martin BR, Damaj MI (2000): Bupropion is a nicotinic antagonist. J Pharmacol Exp Ther 295:321–327. SRNT Subcommittee on Biochemical Verification (2002): Biochemical verification of tobacco use and cessation. Nicotine Tob Res 4:149 –159. Stahl SM, Pradko JF, Haight BR, Modell JG, Rockett CB, Learned-Coughlin S (2004): A review of the neuropharmacology of bupropion, a dual norepinephrine and dopamine reuptake inhibitor. Prim Care Companion J Clin Psychiatry 6:159 –166.
A.M. Lee et al. Swan GE, Jack LM, Curry S, Chorost M, Javitz HM, McAfee T, et al. (2003): Bupropion SR and counseling for smoking cessation in actual practice: Predictors of outcome. Nicotine Tob Res 5:911–921. Teneggi V, Tiffany ST, Squassante L, Milleri S, Ziviani L, Bye A (2005): Effect of sustained-release (SR) bupropion on craving and withdrawal in smokers deprived of cigarettes for 72 h. Psychopharmacology (Berl) 183:1–12. Tonnesen P, Tonstad S, Hjalmarson A, Lebargy F, Van Spiegel PI, Hider A, et al. (2003): A multicentre, randomized, double-blind, placebo-controlled, 1-year study of bupropion SR for smoking cessation. J Intern Med 254:184 –192. Tsuchiya K, Gatanaga H, Tachikawa N, Teruya K, Kikuchi Y, Yoshino M, et al. (2004): Homozygous CYP2B6 *6 (Q172H and K262R) correlates with high plasma efavirenz concentrations in HIV-1 patients treated with standard efavirenz-containing regimens. Biochem Biophys Res Commun 319:1322–1326.
BIOL PSYCHIATRY 2007;62:635– 641 641 Welch RM, Lai AA, Schroeder DH (1987): Pharmacological significance of the species differences in bupropion metabolism. Xenobiotica 17: 287–298. Xie HJ, Yasar U, Lundgren S, Griskevicius L, Terelius Y, Hassan M, et al. (2003): Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation. Pharmacogenomics J 3:53– 61. Xu C, Rao YS, Xu B, Hoffmann E, Jones J, Sellers EM, et al. (2002): An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles. Biochem Biophys Res Commun 290:318 –324. Yamazaki H, Inoue K, Hashimoto M, Shimada T (1999): Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes. Arch Toxicol 73:65–70. Zukunft J, Lang T, Richter T, Hirsch-Ernst KI, Nussler AK, Klein K, et al. (2005): A natural CYP2B6 TATA box polymorphism (⫺82T¡ C) leading to enhanced transcription and relocation of the transcriptional start site. Mol Pharmacol 67:1772–1782.
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B
upropion (Zyban; GlaxoSmithKline, Research Triangle Park, North Carolina) was the first Food and Drug Administration (FDA)-approved nonnicotine pharmacotherapy for smoking cessation and is also widely used to treat depression (Wellbutrin; GlaxoSmithKline). Although the biological mechanism of action for bupropion has yet to be fully elucidated, there is evidence that it can inhibit norepinephrine and dopamine reuptake (Stahl et al. 2004) and act as a nicotinic acetylcholine receptor antagonist (Slemmer et al. 2000). Although the efficacy of bupropion relative to placebo is firmly established (Hughes et al. 2004; Hurt et al. 1997; Jorenby et al. 1999), the majority of smokers relapse to smoking within 6 months after a quit attempt. Varenicline, an ␣42 nicotinic acetylcholine receptor partial agonist, was recently approved by the FDA for smoking cessation and may have superior efficacy to bupropion (Gonzalez et al. 2006). Pharmacogenetic analyses may be valuable for identifying those smokers most likely to benefit from bupropion therapy or other pharmacotherapies for the treatment of nicotine dependence. Bupropion is metabolized to hydroxybupropion, erythrohydrobupropion, and threohydrobupropion (Faucette et al. 2000; Hsyu et al. 1997). Carbonyl reductase metabolizes bupropion to erythrohydrobupropion and threohydrobupropion (Jefferson et al.
From the Centre for Addiction and Mental Health and the Department of Pharmacology (AML, EH, RFT), University of Toronto, Ontario, Canada; Department of Psychiatry (CJ, CL), Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania; and the Department of Pediatrics (LE), School of Medicine and Biomedical Sciences, and Department of Psychology (LWH), The University at Buffalo, State University of New York, Buffalo, New York. Address reprint requests to Rachel F. Tyndale, M.Sc., Ph.D., University of Toronto, Department of Pharmacology, 1 King’s College Circle, Room 4326, Toronto, Ontario M5S 1A8, Canada; E-mail: [email protected]. Received July 2, 2006; revised October 4, 2006; accepted October 5, 2006.
0006-3223/07/$32.00 doi:10.1016/j.biopsych.2006.10.005
2005). CYP2B6 is the main enzyme that metabolizes bupropion to hydroxybupropion (Faucette et al. 2000) and CYP2B6 does not appear to be involved in further metabolism of hydroxybupropion (Hsyu et al. 1997; Welch et al. 1987). Bupropion and hydroxybupropion are pharmacologically active, whereas erythrohydrobupropion and threohydrobupropion have little pharmacological activity (Damaj et al. 2004). CYP2B6 is genetically variable (http://www. cypalleles.ki.se/cyp2b6.htm); some alleles, such as CYP2B6 *4 (A785G), CYP2B6*5 (C1459T), and CYP2B6*6 (G516T and A785G), have been shown to alter CYP2B6 activity (Hesse et al. 2004; Kirchheiner et al. 2003; Lang et al. 2001; Lerman et al. 2002) and may thus alter the therapeutic effects of bupropion. We have previously found that the C1459T single nucleotide polymorphism in exon 9, which is associated with decreased protein and activity (Lang et al. 2001), is associated with an increase in craving and higher smoking relapse rates on placebo; this effect was reversed by bupropion among female subjects (Lerman et al. 2002). CYP2B6 can metabolize nicotine (Yamazaki et al. 1999) and is found in the brain (Miksys et al. 2003); CYP2B6 may contribute to the association of the C1459T variant with smoking relapse. Current single nucleotide polymorphism assays were unable to distinguish the CYP2B6*9 (G516T), CYP2B6*4 (A785G), and CYP2B6*6 (G516T and A785G) alleles. We developed a novel haplotyping assay and have investigated these alleles in a bupropion clinical trial. The CYP2B6*6 haplotype is of particular interest as it has allele frequencies of 25%, 30%, and 15% in Caucasians (Jacob et al. 2004; Kirchheiner et al. 2003; Klein et al. 2005; Zukunft et al. 2005), African Americans (Klein et al. 2005), and Asians (Cho et al. 2004; Klein et al. 2005; Tsuchiya et al. 2004), respectively; therefore, approximately 45%, 50%, and 25% of Caucasians, African Americans, and Asians, respectively, have at least one CYP2B6*6 allele. Thus, a clinical impact of this allele on cessation would affect a large portion of the smoking population. The CYP2B6 *6 allele results in a structurally altered enzyme that has been associated with both decreased (Haas et al. 2004; Lang et al. 2001; Rotger et al. 2005; Tsuchiya et al. 2004) and increased (Jinno et al. 2003; Xie et al. 2003) metabolism of BIOL PSYCHIATRY 2007;62:635– 641 © 2007 Society of Biological Psychiatry
636 BIOL PSYCHIATRY 2007;62:635– 641 various CYP2B6 substrates. CYP2B6 *6 has been associated with decreased bupropion metabolism in human liver samples in vitro (Hesse et al. 2004). A recent in vivo study demonstrated a reduction in bupropion metabolic activity for CYP2B6 *6 using the ratio of the areas under the curve for hydroxybupropion compared with bupropion; one subject with a CYP2B6 *6/ *6 genotype had a considerably smaller ratio than the subjects with CYP2B6 *1/ *1 genotypes (8.1 vs. 18.5) (Loboz et al. 2006). We postulated that smokers in the CYP2B6*6 group (CYP2B6*1/*6 or CYP2B6*6/*6 genotype) may have decreased bupropion metabolism leading to increased bupropion plasma levels but a similar rate of hydroxybupropion metabolism as the CYP2B6*1 group (CYP2B6*1/*1 genotype). Therefore, we hypothesized that there would be a larger therapeutic impact of bupropion in the CYP2B6*6 group compared with the CYP2B6*1 group.
Methods and Materials Participants Participants were enrolled from April 1999 to October 2001 at Georgetown University (Washington, DC) and State University of New York (SUNY) Buffalo (New York). Recruitment, demographics, and the protocol have been reported in detail elsewhere (Lerman et al. 2002, 2006). A total of 555 participants enrolled in the pharmacogenetic trial, of whom 423 were successfully genotyped; the rest had insufficient DNA remaining for this analysis. Of the 423 for whom CYP2B6 genotyping was performed, 342 were Caucasians; 16 had a CYP2B6 *4 genotype (Results) and were omitted, leaving 326 for the final analysis. Eligible participants smoked at least 10 cigarettes per day. Exclusion criteria included pregnancy, a history of DSM-IV psychiatric disorder, seizures, and current use of antidepressants and other psychotropic medications. Study Protocol The protocol number was 703463 (Biobehavioral Lung Cancer Prevention Program) and was originally approved on November 30, 1999. The clinical trial registration number is NCT00322205. The Institutional Review Board of the University of Toronto has also approved this study (#17520). At an initial visit to the smoking clinics, participants provided a 40 mL blood sample for genotyping and completed a set of standardized self-report questionnaires that collected information on demographics (e.g., age, education, marital status) and smoking (age at smoking initiation, cigarettes per day, and nicotine dependence as measured by the Fagerström Test for Nicotine Dependence). Participants then received 10 weeks of either placebo or bupropion treatment, plus seven sessions of behavioral group counseling. Bupropion treatment was given according to the standard therapeutic dose (150 mg/day for the first 3 days, followed by 300 mg/day). All participants were instructed to quit on a target quit date 2 weeks after initiating medication. Smoking status was assessed at the end of treatment (EOT; 8 weeks after target quit date), and at 6-month follow-up using a validated timeline follow-back method. To be categorized as abstinent, a participant had to report no smoking at all in the 7 days prior to the assessment and provide a breath sample containing ⱕ10 ppm of carbon monoxide. Participants were asked to refrain from use of nicotine replacement therapy or other smoking cessation therapies during the treatment and follow-up phase; however, usage during the follow-up phase was not monitored. For midtreatment assessments, a 17-item self-report index of adverse effects was administered at sessions 2 through 7; a www.sobp.org/journal
A.M. Lee et al. summary score was created by summing the responses to all items (Lerman et al. 2002, 2006). A 19-item self-report index of withdrawal symptoms and craving was administered at baseline and at sessions 2 through 6; a summary score was created as the sum of responses to items 1 through 18 and an urge subscale was created as the sum of responses to items 1 (cravings for cigarettes) and 19 (urges to smoke) (Lerman et al. 2002, 2006). The analysis was restricted to smokers of Caucasian European ancestry to reduce bias due to population stratification. Genotyping for CYP2B6 was performed as part of a secondary analysis; thus, posttreatment plasma samples for bupropion and hydroxybupropion levels were not collected. Statistical Analysis The analysis was conducted on a modified intent-to-treat basis that included all participants who attended the initial randomization session and the first treatment session, regardless of completion. Participants who could not be reached for the follow-up or were unavailable to biochemically confirm abstinence were considered to be smokers based on the recommendations of the SRNT Subcommittee on Biochemical Verification (2002). Two-tailed Student t tests and chi-square tests were used to examine associations of selected baseline variables (sex, educational level, marital status, body mass index [BMI], age, age at smoking initiation, cigarettes smoked per day, nicotine dependence) with CYP2B6 genotype and abstinence. Logistic regression models of abstinence at end of treatment and the 6-month follow-up were then estimated, using CYP2B6 genotype, treatment arm, and the interaction of CYP2B6 genotype and treatment arm as the predictors. (None of the abovementioned baseline variables were included in these models, as they did not display significant bivariate associations either with CYP2B6 genotype or with abstinence at either time point.) Repeated measures multivariate analyses of variance were used to model total scores on withdrawal symptoms and craving, urge to smoke scores, and total adverse effect scores over the course of treatment, with time as the within-subjects factor, and CYP2B6 genotype and treatment type as the between-subjects factors. Analyses of withdrawal symptoms, craving, and urge to smoke were restricted to participants who were abstinent (biochemically confirmed) at all treatment phase time points. CYP2B6 and CYP2A6 Genotyping The CYP2B6 gene is highly polymorphic, and the G516T and A785G single nucleotide polymorphisms occur together in minor alleles in addition to CYP2B6 *6, namely CYP2B6 *7, CYP2B6 *13, CYP2B6 *19, and CYP2B6 *20. Currently, the very rare CYP2B6 *13, CYP2B6 *19, and CYP2B6 *20 have not yet been found in Caucasians; lower activity is suggested by expression studies (Klein et al. 2005; Lang et al. 2004). The CYP2B6 *7 allele is infrequent in Caucasians (1.1–3.0%) (Hesse et al. 2004; Klein et al. 2005) and the in vivo functional impact is unknown; the expected impact of the CYP2B6 *7 allele in our population is minimal compared with that of the decreased function CYP2B6 *6 allele, which occurs with much higher frequency. A haplotyping assay was developed to detect the CYP2B6 polymorphisms G516T and A785G using a two-step allele-specific polymerase chain reaction (PCR). CYP2B6 primers were ordered from ACGT Corporation (Toronto, Ontario, Canada). Reagents used in the assays were purchased as a kit from Fermentas (Burlington, Ontario, Canada) with the Taq polymerase enzyme. The deoxyribonucleotide triphosphate (dNTP) set and 1-kb Gene Ruler DNA ladder were also purchased from Fermentas.
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Caucasians, CYP2A6 *2, CYP2A6 *4, CYP2A6 *9, and CYP2A6 *12, was performed as previously described (Malaiyandi et al. 2005; Schoedel et al. 2004).
Table 1. Primer Sequences for CYP2B6*6 (G516T and A785G) Haplotyping Assay First Amplification Primers 2B6ln3F-1 (intron 3 forward primer) 2B6ln5R-1 (intron 5 reverse primer) Second Amplification Primers 2B6G516-F (G516T forward primer wild-type) 2B6T516-F (G516T forward primer variant) 2B6A785-R (A785G reverse primer wild-type) 2B6G785-R (A785G reverse primer variant)
5’-GGTCTGCCCATCTATAAAC-3’ 5’-CTCTCCCTGTCTCAGTTCC-3’
Results
5’-CCCACCTTCCTCTTCCAG-3’ 5’-CCCACCTTCCTCTTCCAT-3’ 5’-AGGTGTCGATGAGGTCCT-3’ 5’-AGGTGTCGATGAGGTCCC-3’
Polymerase chain reaction amplifications were carried out on PTC-200 Peltier Thermal Cycler (BioRad, Toronto, Ontario, Canada). Agarose was purchased from ONBIO Inc. (Richmond Hill, Ontario, Canada). For the first gene-specific amplification, each sample reaction contained 2.5 L of 10 X Taq PCR buffer containing 100 mmol/L Tris-HCl (pH 8.8 at 25°C), 500 mmol/L potassium chloride (KCl), and .8% Nonidet P40, .2 L of a dNTP mixture containing 25 mmol/L of each nucleotide, 1.7 L of 25 mmol/L magnesium dichloride (MgCl2,) .125 L each of the forward and reverse primers at 12.5 m, .25 L of Taq, 1.0 L of the sample DNA (5 to 50 ng/L stock concentrations), and 19.1 L of water. The conditions were an initial denaturation at 95°C for 1 min, 31 cycles at 95°C for 15 sec, 52°C for 30 sec, 72°C for 2 min, followed by a final extension at 72°C for 7 min. For the second haplotype-specific amplification, each reaction contained 2.5 L of 10 X Taq PCR buffer, .1 L of the dNTP mixture, 1.2 L of 25 mmol/L MgCl2, .25 L each of the forward and reverse primers at 12.5 m, .15 L of Taq, .8 L of the sample DNA, and 19.75 L of water. The conditions were an initial denaturation at 95°C for 1 min, 16 cycles at 95°C for 15 sec, 60°C for 30 sec, 72°C for 1 min and 30 seconds, followed by a final extension at 72°C for 7 min. A list of primers for the haplotyping assay is given in Table 1. Genotyping for the common reduced function alleles in
In our sample, 423 participants were successfully genotyped, including 342 Caucasians. The CYP2B6*9 allele was not found. The Caucasian genotype frequencies were CYP2B6*1/*1 (52.3%), CYP2B6*1/*6 (36.3%), CYP2B6*6/*6 (6.7%), CYP2B6*1/*4 (4.1%), and CYP2B6*4/*6 (.6%). The Caucasian CYP2B6*6 and CYP2B6*4 allele frequencies were 25.2% and 2.3%, respectively (n ⫽ 342 total); both alleles were in Hardy-Weinberg equilibrium (p ⫽ .96 and p ⫽ .53, respectively). Smokers with a CYP2B6*1/*4 genotype, indicating increased rates of bupropion metabolism (Kirchheiner et al. 2003; Loboz et al. 2006), were omitted from subsequent analyses due to low power to assess the impact. Smokers with the CYP2B6*4/*6 genotype were omitted, as the effect of this genotype on bupropion metabolism is unclear (Loboz et al. 2006). The final sample of Caucasians (n ⫽ 326) was grouped into a CYP2B6*1 (n ⫽ 179) and a CYP2B6*6 group (n ⫽ 147). There were no significant differences across genotype groups in any sociodemographic variables, treatment group assignment, or Fagerström nicotine dependence scores (Table 2). At the end of treatment (8 weeks following the target quit date), the overall rate of biochemically verified smoking cessation was 27.9% (91 of 326 participants). In the bupropion arm, the abstinence rate was 31.6% (56 of 177) compared with 23.5% (35 of 149) in the placebo arm [2(1) ⫽ 2.67, p ⫽ .10]. At 6 months, the abstinence rate was 22.1% [26.0% in the bupropion arm compared with 17.4% in the placebo arm; 2(1) ⫽ 3.43, p ⫽ .064]. At both EOT and at 6 months, abstinence was not significantly associated with CYP2B6 genotype [at EOT, 2(1) ⫽ 2.24, p ⫽ .13; at 6 months, 2(1) ⫽ .02, p ⫽ .89], sex, educational level, marital status, BMI, age, age at smoking initiation, number of cigarettes smoked per day at baseline, or baseline nicotine dependence (all p ⱖ .10). In the logistic regression model of abstinence at EOT, there was a significant CYP2B6 genotype ⫻ treatment arm interaction (odds ratio [OR] ⫽ 2.97, confidence interval [CI] ⫽ 1.05– 8.40, p ⫽
Table 2. Demographic Characteristics by CYP2B6 Genotype Mean (SD) Among Variable Baseline Nicotine Dependencea Cigarettes Per Day at Baseline Age at Smoking Initiation (Years) Body Mass Index Age (Years)
CYP2B6*1
CYP2B6*6
t Value
p Value
5.31 (2.21) 22.3 (10.5) 16.7 (3.0) 27.3 (4.8) 44.9 (11.0)
5.25 (2.04) 21.1 (7.3) 17.2 (3.8) 26.5 (4.7) 43.7 (12.3)
.24 1.18 ⫺1.29 1.48 .91
.81 .24 .20 .14 .36
N (%) Among Variable
CYP2B6*1
CYP2B6*6
2
p Value
Female Subjects Marital Status Never Married Married Divorced/Separated/Widowed Education No College Some College College Graduate
94 (52%)
84 (57%)
.70 .52
.40 .77
40 (23%) 87 (49%) 50 (28%)
33 (23%) 76 (52%) 36 (25%) .25
.88
33 (19%) 66 (37%) 78 (44%)
29 (20%) 56 (39%) 60 (41%)
a
Fagerström Test for Nicotine Dependence score.
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638 BIOL PSYCHIATRY 2007;62:635– 641
A.M. Lee et al.
Table 3. Logistic Regression Models of Abstinence at EOT and 6-Month Follow-Up Variable End of Treatment CYP2B6 Genotype (CYP2B6*1 ⫽ 0, CYP2B6*6 ⫽ 1) Treatment (Placebo ⫽ 0, Bupropion ⫽ 1) CYP2B6 Genotype by Treatment 6-Month Follow-Up CYP2B6 Genotype (CYP2B61 ⫽ 0, CYP2B66 ⫽ 1) Treatment (Placebo ⫽ 0, Bupropion ⫽ 1) CYP2B6 Genotype by Treatment
Odds Ratio
Confidence Interval
p Value
.36 .97 2.97
.16, .82 .51, 1.83 1.05, 8.40
.02 .93 .04
.54 1.03 2.98
.22, 1.30 .50, 2.10 .98, 9.06
.17 .94 .054
Each of these models includes a significant interaction between genotype and treatment; therefore, the p-values for the component main effect variables will not correspond to the results of bivariate analyses (2 tests) testing associations of those variables with abstinence. EOT, end of treatment.
.04); the interaction was similar in magnitude and close to significance at 6 months (OR ⫽ 2.98, CI ⫽ .98 –9.06, p ⫽ .054) (Table 3). Within the CYP2B6 *6 group, bupropion significantly increased abstinence rates compared with placebo at EOT [32.5% vs. 14.3%, 2(1) ⫽ 6.68, p ⫽ .01] and at the 6-month follow-up [31.2% vs. 12.9%, 2(1) ⫽ 7.06, p ⫽ .008] (Figure 1A and 1B). Thus, bupropion had a large benefit over placebo for 45% of the sample, the CYP2B6 *6 group. Within the CYP2B6 *1 group, bupropion did not increase abstinence rates compared with placebo at EOT [31.0% vs. 31.6%, 2(1) ⫽ .01, p ⫽ .93] or at the 6-month follow-up [22.0% vs. 21.5%, 2(1) ⫽ .01, p ⫽ .94]. Thus, for 55% of the sample, the CYP2B6 *1 group, bupropion did not provide a benefit over placebo. The CYP2B6 *6 group did not have a higher abstinence rate on bupropion compared with the CYP2B6 *1 group at EOT [32.5% vs. 31.0%, 2(1) ⫽ .04, p ⫽ .84] but had a nonsignificantly higher abstinence rate than the CYP2B6 *1 group at the 6-month follow-up [31.2% vs. 22.0%; 2(1) ⫽ 1.90, p ⫽ .17]. The abstinence rate in the CYP2B6 *6 group treated with bupropion did not decrease between EOT and the 6-month follow-up (32.5% vs. 31.2%; p ⫽ .99 by McNemar test); however, the abstinence rate decreased significantly from EOT to the 6-month follow-up in the CYP2B6 *1 group (31.0% vs. 22.0%; p ⫽ .04). The CYP2B6 *6 group had a smaller change in the abstinence rate between the EOT and the 6-month follow-up compared with the CYP2B6 *1 group (1.3% vs. 9%; p ⫽ .13 by longitudinal logistic regression) but this did not reach significance. These data suggest there may be an impact of the CYP2B6 *6 allele at the 6-month follow-up with respect to the maintenance of abstinence. The CYP2B6 *1 group achieved a significantly higher abstinence rate on placebo compared with the CYP2B6 *6 group at
EOT [31.6% vs. 14.3%, 2(1) ⫽ 6.22, p ⫽ .01] (Figure 1A). The results were similar at 6-month follow-up [21.5% vs. 12.9%, 2(1) ⫽ 1.93, p ⫽ .16], although not statistically significant (Figure 1B). There was a gene-dose effect on placebo abstinence rates at EOT. The placebo abstinence rates were 31.6% for homozygous CYP2B6 *1, 16.4% for heterozygous CYP2B6 *1/ *6, and 0% for homozygous CYP2B6 *6 (p ⫽ .03). The 6-month abstinence rates were 21.5%, 14.8%, and 0%, respectively (p ⫽ .21). Gender differences in abstinence rates have been observed in bupropion clinical trials (Lerman et al. 2002; Swan et al. 2003), including an earlier analysis indicating that in female subjects only, there was decreased abstinence on placebo with the rarer CYP2B6 C1459T variant (Lerman et al. 2002). In the current study, there was no main effect of gender on abstinence at EOT or at the 6-month follow-up. The CYP2B6 genotype distribution and the gene-specific effect of treatment arm were similar between male subjects and female subjects at the end of treatment and at the 6-month follow-up (the sex ⫻ CYP2B6, sex ⫻ treatment arm, and sex ⫻ CYP2B6 ⫻ treatment arm interactions were not significant at either time point (all p ⬎ .10). To investigate potential mediators of the difference in abstinence rates between genotype groups, we examined associations of CYP2B6 genotype with pretreatment and midtreatment smoking-related variables. There were no differences between CYP2B6 genotype groups in reported age of smoking initiation, cigarettes per day at baseline, and baseline scores on nicotine dependence in the placebo group or among bupropion-treated participants (all p ⬎ .15, results not shown). In addition, we examined the main and interacting effects of CYP2B6 genotype and treatment arm on midtreatment withdrawal symptoms, urge to smoke, and total adverse effects using multivariate analysis of Figure 1. Significant CYP2B6 genotype by drug treatment effects on abstinence rates. The CYP2B6*6 group (n ⫽ 147) had a significantly higher abstinence rate on bupropion compared with placebo at the end of treatment and at the 6-month follow-up. The CYP2B6*1 group (n ⫽ 179) did not benefit from bupropion compared with placebo at the end of treatment (A) or at the 6-month follow-up (B). (C) The sample was restricted to normal nicotine metabolizers with a CYP2A6*1/*1 genotype (n ⫽ 274). The pattern of abstinence between the CYP2B6*1 and the CYP2B6*6 groups is similar to that found in the whole group (B). Numbers within the bars indicate the percent of the subsample that was abstinent.
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A.M. Lee et al. variance. No significant effects of genotype or genotype by treatment arm interactions were found and adverse events did not appear to mediate the abstinence rates in either the CYP2B6 *6 or CYP2B6 *1 group. CYP2A6 metabolizes nicotine (Messina et al. 1997) and can contribute to altered hepatic nicotine metabolism (Xu et al. 2002) and smoking behaviors (Schoedel et al. 2004); therefore, we examined the impact of common CYP2A6 decreased activity alleles (*2, *4, *9, and *12) on abstinence. There was no significant main effect of CYP2A6 genotype on abstinence at EOT or 6-month follow-up (p ⱖ .15), nor was there a CYP2A6CYP2B6 gene-gene interaction at either time point (p ⱖ .14). However, there was a significant CYP2A6 gene ⫻ treatment arm interaction at EOT (OR ⫽ .20, 95% CI ⫽ .04 –.95; p ⫽ .04). Among cases with normal CYP2A6 metabolism (n ⫽ 274), similar EOT abstinence rates were seen compared with the general population on placebo (21.5%) and bupropion (34.6%) [2(1) ⫽ 5.70, p ⫽ .02]. In contrast, among the small proportion of cases with intermediate or slow CYP2A6 metabolism (16%, n ⫽ 51), EOT abstinence rates were high on placebo (32.1%) and low on bupropion (13.0%) [2(1) ⫽ 2.56, p ⫽ .11]. The CYP2A6 gene ⫻ treatment arm interaction was not significant at 6-month follow-up (OR ⫽ .38, 95% CI ⫽ .06 –2.46; p ⫽ .31). We repeated the analysis of abstinence by CYP2B6 among participants with CYP2A6 *1/ *1 genotype only (those with normal CYP2A6 metabolism, n ⫽ 274) and found that the abstinence rates within the CYP2B6 *1 and CYP2B6 *6 groups (Figure 1C) were similar to those seen in the total sample, which included CYP2A6 intermediate and slow metabolizers (Figure 1B).
Discussion This study illustrates the importance of investigating genetic variation in drug-metabolizing enzymes to predict smoking cessation and therapeutic response to bupropion. Without investigating CYP2B6 genetics, the overall effect on abstinence of bupropion is modest. However, when CYP2B6 genotype is incorporated, bupropion is significantly more efficacious than placebo for ⬃45% of the population (those in the CYP2B6 *6 group) but not in the remaining ⬃55% (those in the CYP2B6 *1 group). At EOT, the CYP2B6 *6 group had low abstinence rates on placebo but responded well with bupropion, whereas the CYP2B6 *1 group had high abstinence rates on placebo and these were not significantly enhanced by bupropion (Figure 1). The CYP2B6 *1 and CYP2B6 *6 groups achieved maximal abstinence rates of approximately 33% on bupropion at EOT, similar to other studies (Hurt et al. 1997; Tonnesen et al. 2003). Thus, the CYP2B6 *6 group does not have an advantage over the CYP2B6 *1 group when examining only the response to bupropion treatment, and the observed interaction is due to differences in placebo response. The change in abstinence rates from EOT to 6-month follow-up also appears to be modified by CYP2B6 genotype. Abstinence rates after the termination of treatment tend to drop as participants relapse (Hurt et al. 1997). Among participants treated with bupropion, the abstinence rate was maintained at 6 months by the CYP2B6 *6 group (Figure 1), suggesting that bupropion may be more efficacious for preventing relapse in the CYP2B6 *6 group. Among placebo-treated participants in the CYP2B6 *6 group, there was also no change in abstinence, but these rates were low at both time points. The CYP2B6 *1 group did not maintain abstinence at the 6-month follow-up compared with EOT on either bupropion or placebo, suggesting that the
BIOL PSYCHIATRY 2007;62:635– 641 639 maintenance of abstinence is specific to the CYP2B6 *6 group. The maintenance of abstinence at 6 months by the CYP2B6 *6 group may be due to an intermediate outcome measure that was not assessed with our current self-report questionnaires. Although bupropion has been shown to decrease withdrawal and craving (Brody et al. 2004; Shiffman et al. 2000; Teneggi et al. 2005), there were no significant differences by genotype in midtreatment withdrawal symptoms or adverse effects in either the placebo or bupropion groups. This suggests that the decreased abstinence on placebo in the CYP2B6 *6 group, and the enhanced response to bupropion in this group, is not mediated by these variables. Further, the data suggest that these effects may not be mediated by differences in bupropion metabolism, because we did not see genotype associations in adverse effect scores in the bupropion-treated group. The high placebo abstinence rates (Hurt et al. 1997; Tonnesen et al. 2003) observed in the CYP2B6 *1 group may be due to other factors. Firstly, the increased placebo abstinence rate may reflect a function of CYP2B6 that is currently unknown. CYP2B6 is expressed in extrahepatic locations, such as lung (Gervot et al. 1999) and brain (Miksys et al. 2003), and metabolizes endogenous substrates such as testosterone (Imaoka et al. 1996). The structurally altered CYP2B6 *6 has also been associated with increased nicotine metabolism (Ring et al, unpublished data) compared with CYP2B6 *1. CYP2B6 *1 may be involved in a mechanism of smoking that provides good short-term abstinence (occurring during the behavioral counseling treatment) compared with the CYP2B6 *6 in the placebo-treated groups, resulting in the higher abstinence rate. The advantage in quitting on placebo conferred by CYP2B6 *1, however, appears to be unaltered by bupropion treatment. Conversely, CYP2B6 *6 may be involved in a mechanism of smoking that contributes to decreased abstinence in the placebo-treated arm but which is responsive to bupropion. Secondly, CYP2B6 *1 may be in linkage disequilibrium with another gene conferring high abstinence rates. We investigated the CYP2A6 gene, as the CYP2A6 enzyme metabolizes nicotine (Messina et al. 1997), and can contribute to differences in hepatic nicotine metabolism (Xu et al. 2002) and smoking behavior (Schoedel et al. 2004). It is located in close proximity to CYP2B6 on chromosome 19 (Hoffman et al. 2001). For example, slow CYP2A6 metabolism has been previously associated with shorter smoking durations (Schoedel et al. 2004) and increased quit rates (Gu et al. 2000). Although there was a significant CYP2A6 gene ⫻ treatment arm interaction at EOT, there was no significant main effect of CYP2A6 genotype on abstinence and no detectable CYP2A6-CYP2B6 gene-gene interaction. In addition, when restricted to CYP2A6 normal metabolizers, the abstinence rates within the CYP2B6 *1 and CYP2B6 *6 groups were similar to those in the total sample (Figure 1C). CYP2B6 *1 may be in linkage disequilibrium with another unknown gene that confers an advantage in quitting on placebo compared with the CYP2B6 *6 group; however, on inspection of the chromosomal region, no other obvious candidates were identified. One limitation of the study was that the population was restricted to persons of European ancestry. Further studies will be required to confirm these effects in other ethnic groups. Another limitation was the lack of plasma bupropion and hydroxybupropion levels to confirm the effect of the CYP2B6 genotype. Subsequent prospective clinical trials to test the findings observed here should also include the measurement of bupropion and hydroxybupropion plasma levels. If the present CYP2B6 genotype results are replicated, genetic testing prior to www.sobp.org/journal
640 BIOL PSYCHIATRY 2007;62:635– 641 treatment would provide an opportunity to match smokers with a treatment that would maximize abstinence rates. We have shown that the CYP2B6 *1 group achieves high quit rates on placebo and does not benefit from bupropion therapy for smoking cessation. This subgroup of smokers may be better treated with an alternative therapy, such as behavioral counseling. In contrast, the CYP2B6 *6 group did poorly on placebo and exhibited a significant benefit of bupropion treatment at EOT, an effect that was maintained at 6-month follow-up. In the future, if these findings are validated and as genetic testing becomes more economical, it could be advantageous to genotype smokers for CYP2B6 and offer bupropion treatment for those with the CYP2B6 *6 genotype. This work was supported by a Canadian Tobacco Control Research Initiative student grant 16803 (AML), Canadian Institute of Health Research (CIHR) Tobacco Use in Special Populations Fellowship (AML), grants from the National Cancer Institute and National Institutes on Drug Abuse, P5084718, RO1 CA63562 (CL) and DA020830 (RFT, CL), CIHR grant MOP53248 (RFT), and a Canada Research Chair in Pharmacogenetics (RFT). Study medication was provided at no cost by GlaxoSmithKline. CL has served as a consultant to GlaxoSmithKline. RFT holds shares in Nicogen Inc., a company focused on creating novel smoking cessation treatments. No funding for this study was received from Nicogen and no benefit to the company was obtained. Brody AL, Mandelkern MA, Lee G, Smith E, Sadeghi M, Saxena S, et al. (2004): Attenuation of cue-induced cigarette craving and anterior cingulate cortex activation in bupropion-treated smokers: A preliminary study. Psychiatry Res 130:269 –281. Cho JY, Lim HS, Chung JY, Yu KS, Kim JR, Shin SG, et al. (2004): Haplotype structure and allele frequencies of CYP2B6 in a Korean population. Drug Metab Dispos 32:1341–1344. Damaj MI, Carroll FI, Eaton JB, Navarro HA, Blough BE, Mirza S, et al. (2004): Enantioselective effects of hydroxy metabolites of bupropion on behavior and on function of monoamine transporters and nicotinic receptors. Mol Pharmacol 66:675– 682. Faucette SR, Hawke RL, Lecluyse EL, Shord SS, Yan B, Laethem RM, et al. (2000): Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 28:1222–1230. Gervot L, Rochat B, Gautier JC, Bohnenstengel F, Kroemer H, de Berardinis V, et al. (1999): Human CYP2B6: Expression, inducibility and catalytic activities. Pharmacogenetics 9:295–306. Gonzales D, Rennard SI, Nides M, Oncken C, Azoulay S, Billing CB, et al.(2006): Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs. sustained-release bupropion and placebo for smoking cessation: A randomized controlled trial. JAMA 296:47–55. Gu DF, Hinks LJ, Morton NE, Day IN (2000): The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit. Ann Hum Genet 64:383–390. Haas DW, Ribaudo HJ, Kim RB, Tierney C, Wilkinson GR, Gulick RM, et al. (2004): Pharmacogenetics of efavirenz and central nervous system side effects: An Adult AIDS Clinical Trials Group study. AIDS 18:2391–2400. Hesse LM, He P, Krishnaswamy S, Hao Q, Hogan K, Moltke LL, et al. (2004): Pharmacogenetic determinants of interindividual variability in bupropion hydroxylation by cytochrome P450 2B6 in human liver microsomes. Pharmacogenetics 14:225–238. Hoffman SM, Nelson DR, Keeney DS (2001): Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 11:687– 698. Hsyu PH, Singh A, Giargiari TD, Dunn JA, Ascher JA, Johnston JA (1997): Pharmacokinetics of bupropion and its metabolites in cigarette smokers versus nonsmokers. J Clin Pharmacol 37:737–743. Hughes J, Stead L, Lancaster T (2004): Antidepressants for smoking cessation. Cochrane Database Syst Rev CD000031.
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