Andy Z. X. Zhu

Cannabinoid receptor stimulation increases motivation for nicotine and nicotine seeking.

The cannabinoid system appears to play a critical facilitative role in mediating the reinforcing effects of nicotine and relapse to nicotine‐seeking behaviour in abstinent subjects based on the actions of cannabinoid (CB) receptor antagonists. However, the effects of CB receptor stimulation on nicotine self‐administration and reinstatement have not been systematically studied. Here, we studied the effects of WIN 55,212‐2, a CB1/2 agonist, on intravenous nicotine self‐administration under fixed‐ratio (FR) and progressive‐ratio (PR) schedules of reinforcement in rats. The effects of WIN 55,212‐2 on responding for food under similar schedules were also studied. In addition, the effects of WIN 55,212‐2 on nicotine‐ and cue‐induced reinstatement of nicotine seeking were also studied, as well as the effects of WIN 55,212‐2 on nicotine discrimination. WIN 55,212‐2 decreased nicotine self‐administration under the FR schedule. However, co‐administration of WIN 55,212‐2 with nicotine decreased responding for food, which suggests that this effect was non‐selective. In contrast, WIN 55,212‐2 increased both nicotine self‐administration and responding for food under the PR schedule, produced dose‐dependent reinstatement of nicotine seeking, and enhanced the reinstatement effects of nicotine‐associated cues. Some of these effects were reversed by the CB1 antagonist rimonabant, but not by the CB2 antagonist AM630. In the drug discrimination tests between saline and 0.4 mg/kg nicotine, WIN 55,212‐2 produced no nicotine‐like discriminative effects but significantly potentiated discriminative stimulus effects of nicotine at the low dose through a CB1‐receptor‐dependent mechanism. These findings indicate that cannabinoid CB1‐receptor stimulation increases the reinforcing effects of nicotine and precipitates relapse to nicotine‐seeking behaviour in abstinent subjects. Thus, modulating CB1‐receptor signalling might have therapeutic value for treating nicotine dependence.

CYP2B6 and Bupropion's Smoking‐Cessation Pharmacology: The Role of Hydroxybupropion

Bupropion is indicated to promote smoking cessation. Animal studies suggest that the pharmacologic activity of bupropion can be mediated by its major metabolite, hydroxybupropion. We measured plasma bupropion and its metabolite levels in a double‐blind, placebo controlled, randomized smoking‐cessation trial. Among the treatment‐adherent individuals, higher hydroxybupropion concentrations (per μg/ml) resulted in better smoking‐cessation outcomes (week 3, 7, and 26 odds ratio (OR) = 2.82, 2.96, and 2.37, respectively, P = 0.005–0.040); this was not observed with bupropion levels (OR = 1.00–1.03, P = 0.59–0.90). Genetic variation in CYP2B6, the enzyme that metabolizes bupropion to hydroxybupropion, was identified as a significant source of variability in hydroxybupropion formation. Our data indicate that hydroxybupropion contributes to the pharmacologic effects of bupropion for smoking cessation, and that variability in response to bupropion treatment is related to variability in CYP2B6‐mediated hydroxybupropion formation. These findings suggest that dosing of bupropion to achieve a hydroxybupropion level of 0.7 μg/ml or increasing bupropion dose for CYP2B6 slow metabolizers could improve bupropions cessation outcomes.

The ability of plasma cotinine to predict nicotine and carcinogen exposure is altered by differences in CYP2A6: the influence of genetics, race, and sex.

Background: Cotinine, a nicotine metabolite, is a biomarker of tobacco, nicotine, and carcinogen exposure. However, a given cotinine level may not represent the same tobacco exposure; for example, African-Americans have higher cotinine levels than Caucasians after controlling for exposure. Methods: Cotinine levels are determined by the amount of cotinine formation and the rate of cotinine removal, which are both mediated by the enzyme CYP2A6. Because CYP2A6 activity differs by sex (estrogen induces CYP2A6) and genotype, their effect on cotinine formation and removal was measured in nonsmoking Caucasians (Study 1, n = 181) infused with labeled nicotine and cotinine. The findings were then extended to ad libitum smokers (Study 2, n = 163). Results: Study 1: Reduced CYP2A6 activity altered cotinine formation less than cotinine removal resulting in ratios of formation to removal of 1.31 and 1.12 in CYP2A6 reduced and normal metabolizers (P = 0.01), or 1.39 and 1.12 in males and females (P = 0.001), suggesting an overestimation of tobacco exposure in slower metabolizers. Study 2: Cotinine again overestimated tobacco and carcinogen exposure by 25% or more in CYP2A6 reduced metabolizers (≈2-fold between some genotypes) and in males. Conclusions: In people with slower relative to faster CYP2A6 activity, cotinine accumulates resulting in substantial differences in cotinine levels for a given tobacco exposure. Impact: Cotinine levels may be misleading when comparing those with differing CYP2A6 genotypes within a race, between races with differing frequencies of CYP2A6 gene variants (i.e., African-Americans have higher frequencies of reduced function variants contributing to their higher cotinine levels), or between the sexes. Cancer Epidemiol Biomarkers Prev; 22(4); 708–18. ©2013 AACR.

Pharmacogenetics of Drug Dependence: Role of Gene Variations in Susceptibility and Treatment

Drug dependency is a highly prevalent mental health disorder that imposes a significant burden on those directly affected, health care systems, and society in general. There is substantial heritability in the susceptibility to drug addiction, which indicates that there are genetic risk factors. Variation in the human genome is abundant and can directly affect drug dependency phenotypes, for example, by altering the function of a gene product or by altering gene expression. Pharmacogenetic studies can assess the effects of genetic variation on the risk for a particular phenotype (e.g., being an alcoholic). In addition, pharmacogenetic variability in treatment efficacy and adverse reactions can be investigated to identify particular genetic variants associated with altered responses. This review highlights examples of genetic variations that are important in the development and maintenance of specific drug dependencies as well as those that affect the response to treatment.

Influence of CYP2B6 genetic variants on plasma and urine concentrations of bupropion and metabolites at steady state

Background Bupropion, an antidepressant and smoking cessation medication, is metabolized to hydroxybupropion (HB), an active metabolite, primarily by CYP2B6. Objectives To compare plasma concentrations of bupropion and metabolites at steady state in healthy volunteers with and without CYP2B6 genetic variants. Methods In a genotype-guided study of 42 healthy individuals, we measured the plasma and urine concentrations of bupropion and its metabolites, HB, threohydrobupropion, and erythrohydrobupropion after 7 days of sustained-release bupropion dosing. Results CYP2B6*6 and *18 gene variants were associated with ∼33% reduced concentrations of HB, with no effects on concentrations of bupropion or other metabolites. We could account for 50% of the variation in HB concentrations in a model including genotype and sex. Conclusion As HB is active and its steady-state concentrations are more than 10 times higher than bupropion, CYP2B6 variants are likely to affect pharmacological activity. Because of the large individual variation within the genotype group, the use of therapeutic drug monitoring for dose optimization may be necessary.

Association of CHRNA5-A3-B4 SNP rs2036527 with smoking cessation therapy response in African-American smokers.

Associations between CHRNA5‐A3‐B4 variants and smoking behaviors exist; however, the association with smoking abstinence is less understood, particularly that among African Americans. In 1,295 African Americans enrolled in two clinical trials, we investigated the association between CHRNA5‐A3‐B4 and smoking abstinence. The rs2056527(A) allele was associated with lower abstinence with active pharmacotherapy (during treatment: odds ratio (OR) = 0.42, P < 0.001; end of treatment (EOT): OR = 0.55, P = 0.004), or with nicotine gum alone (during treatment: OR = 0.31, P < 0.001; EOT: OR = 0.51, P = 0.02), but not significantly with bupropion, although similar directions and magnitudes were observed (during treatment: OR = 0.54, P = 0.05; EOT: OR = 0.59, P = 0.08). In addition, the rs588765(T) allele was associated with abstinence with gum during treatment (OR = 2.31, P < 0.01). The SNP rs16969968 occurred at a low frequency and was not consistently associated with abstinence. CHRNA5‐A3‐B4 variants were not associated with tobacco consumption, and adjustments for smoking behaviors did not alter the associations with smoking abstinence. Together, our data suggest that among African Americans, CHRNA5‐A3‐B4 variants are not associated with baseline smoking but can influence smoking abstinence during active pharmacotherapy.

CYP2A6 and CYP2B6 genetic variation and its association with nicotine metabolism in South Western Alaska Native people

ObjectivesAlaska Native (AN) people have a high prevalence of tobacco use and associated morbidity and mortality when compared with the general USA population. Variations in the CYP2A6 and CYP2B6 genes, encoding enzymes responsible for nicotine metabolic inactivation and procarcinogen activation, have not been characterized in AN and may contribute toward the increased risk. MethodsAN people (n=400) residing in the Bristol Bay region of South Western Alaska were recruited for a cross-sectional study on tobacco use. They were genotyped for CYP2A6*1X2A, *1X2B, *1B, *2, *4, *7, *8, *9, *10, *12, *17, *35 and CYP2B6*4, *6, *9 and provided plasma and urine samples for the measurement of nicotine and metabolites. ResultsCYP2A6 and CYP2B6 variant frequencies among the AN Yupik people (n=361) were significantly different from those in other ethnicities. Nicotine metabolism [as measured by the plasma and urinary ratio of metabolites trans-3′-hydroxycotinine to cotinine (3HC/COT)] was significantly associated with CYP2A6 (P<0.001), but not CYP2B6 genotype (P=0.95) when controlling for known covariates. It was noteworthy that the plasma 3HC/COT ratios were high in the entire Yupik people, and among the Yupik CYP2A6 wild-type participants, they were substantially higher than those in previously characterized racial/ethnic groups (P<0.001 vs. Caucasians and African Americans). ConclusionYupik AN people have a unique CYP2A6 genetic profile that associated strongly with in-vivo nicotine metabolism. More rapid CYP2A6-mediated nicotine and nitrosamine metabolism in the Yupik people may modulate the risk of tobacco-related diseases.

Lack of associations of CHRNA5-A3-B4 genetic variants with smoking cessation treatment outcomes in Caucasian smokers despite associations with baseline smoking

CHRNA5-A3-B4 variants, rs16969968, rs588765 and rs578776, are consistently associated with tobacco consumption among smokers, but the association with smoking cessation is less consistent. Among the studies that reported significant associations with cessation, the effects were observed in smokers treated with placebo treatment in some studies and conversely in those receiving active pharmacological therapy (bupropion and nicotine replacement therapies) in others. Thus, it remains unclear whether CHRNA5-A3-B4 is a useful marker for optimizing smoking cessation. Using data from 654 Caucasian smokers treated with placebo, nicotine patch or varenicline, we investigated whether CHRNA5-A3-B4 variants were associated with smoking cessation outcomes, and whether there were significant genotype-by-treatment or haplotype-by-treatment interactions. We observed no significant associations between CHRNA5-A3-B4 variants and smoking cessation, despite replicating previous associations with baseline tobacco consumption. At end of treatment the effect size on smoking cessation in the placebo, patch and varenicline groups for rs16969968 [GG vs. GA+AA] was OR = 0.66 (P = 0.23), OR = 1.01 (P = 0.99), and OR = 1.30 (P = 0.36) respectively, of rs588765 [CC vs. CT+TT] was OR = 0.96 (P = 0.90), OR = 0.84 (P = 0.58), and OR = 0.74 (P = 0.29) respectively, and for rs578776 [GG vs. GA+AA] on smoking cessation was OR = 1.02 (P = 0.95), OR = 0.75 (P = 0.35), and OR = 1.20 (P = 0.51) respectively. Furthermore, we observed no associations with cessation using the CHRNA5-A3-B4 haplotype (constructed using rs16969968 and rs588765), nor did we observe any significant genotype-by-treatment interactions, with or without adjusting for the rate of nicotine metabolism (all P>0.05). We also observed no significant genetic associations with 6 month or 12 month smoking abstinence. In conclusion, we found no association between CHRNA5-A3-B4 variants and smoking cessation rates in this clinical trial; however, as expected, significant associations with baseline tobacco consumption were replicated. Our data suggest that CHRNA5-A3-B4 gene variants do not exhibit a robust association with smoking cessation and are unlikely to be useful for clinically optimizing smoking cessation pharmacotherapy for Caucasian smokers.

Variation in Trans-3′-Hydroxycotinine Glucuronidation Does Not Alter the Nicotine Metabolite Ratio or Nicotine Intake

Background CYP2A6 metabolizes nicotine to its primary metabolite cotinine and also mediates the metabolism of cotinine to trans-3′-hydroxycotinine (3HC). The ratio of 3HC to cotinine (the “nicotine metabolite ratio”, NMR) is an in vivo marker for the rate of CYP2A6 mediated nicotine metabolism, and total nicotine clearance, and has been associated with differences in numerous smoking behaviors. The clearance of 3HC, which affects the NMR, occurs via renal excretion and metabolism by UGT2B17, and possibly UGT2B10, to 3HC-glucuronide. We investigated whether slower 3HC glucuronidation alters NMR, altering its ability to predict CYP2A6 activity and reducing its clinical utility. Methods Plasma NMR, three urinary NMRs, three urinary 3HC glucuronidation phenotypes and total nicotine equivalents were examined in 540 African American smokers. The UGT2B17 gene deletion and UGT2B10*2 were genotyped. Results The UGT2B17 gene deletion, but not UGT2B10*2 genotype, was associated with slower 3HC glucuronidation (indicated by three 3HC-glucuronidation phenotypes), indicating its role in this glucuronidation pathway. However, neither lower rates of 3HC glucuronidation, nor the presence of a UGT2B17 and UGT2B10 reduced function allele, altered plasma or urinary NMRs or levels of smoking. Conclusions Variation in 3HC glucuronidation activity, including these caused by UGT2B17 gene deletions, did not significantly alter NMR and is therefore unlikely to affect the clinical utility of NMR in smoking behavior and cessation studies. This study demonstrates that NMR is not altered by differences in the rate of 3HC glucuronidation, providing further support that NMR is a reliable indicator of CYP2A6 mediated nicotine metabolism.

Genome-Wide Meta-Analysis of Cotinine Levels in Cigarette Smokers Identifies Locus at 4q13.2.

Genome-wide association studies (GWAS) of complex behavioural phenotypes such as cigarette smoking typically employ self-report phenotypes. However, precise biomarker phenotypes may afford greater statistical power and identify novel variants. Here we report the results of a GWAS meta-analysis of levels of cotinine, the primary metabolite of nicotine, in 4,548 daily smokers of European ancestry. We identified a locus close to UGT2B10 at 4q13.2 (minimum p = 5.89 × 10−10 for rs114612145), which was consequently replicated. This variant is in high linkage disequilibrium with a known functional variant in the UGT2B10 gene which is associated with reduced nicotine and cotinine glucuronidation activity, but intriguingly is not associated with nicotine intake. Additionally, we observed association between multiple variants within the 15q25.1 region and cotinine levels, all located within the CHRNA5-A3-B4 gene cluster or adjacent genes, consistent with previous much larger GWAS using self-report measures of smoking quantity. These results clearly illustrate the increase in power afforded by using precise biomarker measures in GWAS. Perhaps more importantly however, they also highlight that biomarkers do not always mark the phenotype of interest. The use of metabolite data as a proxy for environmental exposures should be carefully considered in the context of individual differences in metabolic pathways.