Increased Leaf Nicotine Content by Targeting Transcription Factor Gene Expression in Commercial Flue-Cured Tobacco (Nicotiana tabacum L.).

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.

The conformational landscape of nicotinoids: solving the conformational disparity of anabasine.

The conformational landscape of the alkaloid anabasine (neonicotine) has been investigated by using rotational spectroscopy and ab initio calculations. The results allow a detailed comparison of the structural properties of the prototype piperidinic and pyrrolidinic nicotinoids (anabasine vs. nicotine). Anabasine adopts two most stable conformations in isolation conditions, for which we determined accurate rotational and nuclear quadrupole coupling parameters. The preferred conformations are characterized by an equatorial pyridine moiety and additional N-H equatorial stereochemistry at the piperidine ring (eq-eq; eq=equatorial). The two rings of anabasine are close to a bisecting arrangement, with the observed conformations differing by an approximately 180 degrees rotation of the pyridine subunit, denoted either syn or anti. The preference of anabasine for the eq-eq-syn conformation has been established by relative intensity measurements (syn/anti approximately 5(2)). The conformational preferences of free anabasine are directed by a weak N...H-C hydrogen bond interaction between the nitrogen lone pair at piperidine and the closest C-H bond in pyridine, with N...H distances ranging from 2.686 (syn) to 2.667 A (anti). Supporting ab initio calculations by using MP2 and the recent M05-2X density functional are provided, evaluating the predictive performance of both methods.

Nicotine and its metabolites in amniotic fluid at birth--assessment of prenatal tobacco smoke exposure.

Amniotic fluid was collected from 78 pregnant women at birth additionally with their urine prior to delivery as well as neonatal urine and meconium. The smoking markers, nicotine and its metabolites cotinine and trans-3'-hydroxycotinine (OH-cotinine), were determined using high-performance liquid chromatography (HPLC). The self-reported smoking status during pregnancy determined by means of a questionnaire was verified by measurement of maternal urine. In all smokers, nicotine metabolites were detected in amniotic fluid and in 80% of them nicotine as well. However, the sum of the nicotine metabolites (Sum(met)) was significantly lower (p < .001) in amniotic fluid (704 +/- 464 nmol/L) than in meconium (921 +/- 588 nmol/L), neonatal urine (1139 +/- 813 nmol/L) and maternal urine (4496 +/- 3535 nmol/L). Concentrations of nicotine metabolites in amniotic fluid correlated well (p < .001) with that in the other specimen types. After environmental tobacco smoke (ETS) exposure, no nicotine or nicotine metabolites were detectable in amniotic fluid but only in maternal and neonatal urine. Analysis of amniotic fluid at birth lends itself to verifying smoking habits during pregnancy and clearly discriminating from ETS exposure, but it is not a suitable approach to differentiating between ETS exposure and non-exposure.

Disparities in access to over-the-counter nicotine replacement products in New York City pharmacies.

OBJECTIVES: We surveyed the availability of tobacco products and nonprescription nicotine replacement therapy (NRT) in pharmacies in New York City, stratified by the race, ethnicity, and socioeconomic status (SES) of the surrounding neighborhoods to determine whether disparities in availability existed. METHODS: Surveyors visited a random sample of retail pharmacies to record the availability of tobacco products and nonprescription NRT. We used census data and geographic information systems analysis to determine the SES of each neighborhood. We used logistic modeling to explore relations between SES and the availability of NRT and tobacco products. RESULTS: Of 646 pharmacies sampled, 90.8% sold NRT and 46.9% sold cigarettes. NRT and cigarettes were slightly more available in pharmacies in neighborhoods with a higher SES. NRT was more expensive in poorer neighborhoods. CONCLUSIONS: Small disparities existed in access to nonprescription NRT and cigarettes. The model did not adequately account for cigarette access, because of availability from other retail outlets. These results may explain some of the excess prevalence of cigarette use in low-SES areas.

Cost-effectiveness of varenicline compared with bupropion, NRT, and nortriptyline for smoking cessation in the Netherlands.

OBJECTIVE: To examine the cost-effectiveness of varenicline, a new pharmacotherapy to support smoking cessation, compared with the currently available pharmacologic alternatives in the Netherlands. METHODS: The BENESCO-model was used to estimate the long-term health and economic benefits of smoking cessation for a cohort of smokers making a one-time quit attempt. The cohort represented the population of Dutch smokers with respect to gender, age, and prevalence of the smoking-related diseases included in the model: COPD, lung cancer, CHD, stroke, and asthma exacerbations. The model compared the cumulative incidence of smoking-related diseases, (quality-adjusted) life years, intervention costs, and direct medical costs between the cohort treated with varenicline and the same cohort either untreated (unaided cessation) or treated with bupropion, nortriptyline or NRT. The time horizon was lifetime. Future costs were discounted at 4%, health outcomes at 1.5%. RESULTS: The cost of varenicline per additional quitter ranged from 1030 Euro compared with NRT to 4270 Euro compared with nortriptyline. When including the savings due to the reduction in incidence of smoking-related diseases, varenicline generated net savings compared with bupropion and NRT. Compared with nortriptyline and unaided cessation, varenicline was estimated to cost 1650 Euro/QALY and 320 Euro/QALY gained, respectively. At a willingness-to-pay as low as 5000/QALY gained, the probability that varenicline was cost-effective was more than 80% compared to bupropion, NRT, and unaided cessation and about 60% compared to nortriptyline. CONCLUSION: Treatment with varenicline for smoking cessation is cost-effective compared with nortriptyline and unaided cessation and even cost-saving compared with bupropion and NRT.

The impact of pharmaceutical company funding on results of randomized trials of nicotine replacement therapy for smoking cessation: a meta-analysis.

AIMS: To assess whether source of funding affected the results of trials of nicotine replacement therapy (NRT) for smoking cessation. METHODS: We reviewed all randomized controlled trials included in the Cochrane review. There were insufficient non-industry trials of the newer products for these to be included. We included 90 trials of either the nicotine gum (52) or nicotine patch (38). They comprised 18 238 treatment and 16 235 control participants. Forty-nine showed evidence of industry support (18 gum, 31 patch). RESULTS: Industry (31 of 49, 63%) compared with non-industry (seven of 41, 17%, P < 0.001) supported a higher proportion of nicotine patch studies and had larger sample sizes (479 versus 268, P = 0.04). Twenty-five (51%) industry trials reported statistically significant (P < 0.05) results, compared with nine (22%) non-industry trials (OR = 3.70, 95% CI = 1.46-9.35). This difference was not explained by trial characteristics. Industry-supported trials had a pooled odds ratio of 1.90 (1.67-2.16), compared with 1.61 (1.43-1.80) for other studies (chi(2) = 3.6, P = 0.058). There was evidence of funnel-plot asymmetry among industry trials (t = 4.35, P < 0.001), but not among other trials, indicating that several small null-effect industry trials may not have reached publication. After imputation adjustment, the odds ratio for industry trials reduced to 1.64 (1.43-1.89) and the overall NRT odds ratio reduced from 1.73 (1.60-1.90) to 1.62 (1.49-1.77). CONCLUSIONS: Compared with independent trials, industry-supported trials were more likely to produce statistically significant results and larger odds ratios. These differences persisted after adjustment for basic trial characteristics. Although we had no data on the amount of funding for each trial, it is possible that more resources led to higher treatment compliance and therefore greater efficacy in industry-supported trials. Differences can also possibly be explained by publication bias with several small, null-effect industry studies not having reached publication. After adjustment for this possible bias, results for industry trials were lower and similar to non-industry results. Similarly, the overall estimate of the net effect for these products reduces to about 5% attributable 1-year successes. This remains of considerable public health benefit. Registration of clinical trials has become mandatory in many countries since most of the trials considered here were conducted, and this should reduce the potential for publication bias in future.

What to do with a patient who smokes.

Despite the reality that smoking remains the most important preventable cause of death and disability, most clinicians underperform in helping smokers quit. Of the 46 million current smokers in the United States, 70% say they would like to quit, but only a small fraction are able to do so on their own because nicotine is so highly addictive. One third to one half of all smokers die prematurely. Reasons clinicians avoid helping smokers quit include time constraints, lack of expertise, lack of financial incentives, respect for a smoker's privacy, fear that a negative message might lose customers, pessimism because most smokers are unable to quit, stigma, and clinicians being smokers. The gold standard for cessation treatment is the 5 As (ask, advise, assess, assist, and arrange). Yet, only a minority of physicians know about these, and fewer put them to use. Acceptable shortcuts are asking, advising, and referring to a telephone "quit line" or an internal referral system. Successful treatment combines counseling with pharmacotherapy (nicotine replacement therapy with or without psychotropic medication such as bupropion). Nicotine replacement therapy comes in long-acting (patch) or short-acting (gum, lozenge, nasal spray, or inhaler) forms. Ways to counter clinicians' pessimism about cessation include the knowledge that most smokers require multiple quit attempts before they succeed, that rigorous studies show long-term quit rates of 14% to 20%, with 1 report as high as 35%, that cessation rates for users of telephone quit lines and integrated health care systems are comparable with those of individual clinicians, and that no other clinical intervention can offer such a large potential benefit.

Substitutes for tobacco smoking: a behavioral economic analysis of nicotine gum, denicotinized cigarettes, and nicotine-containing cigarettes.

Both pharmacological and nonpharmacological stimuli may be responsible for the reinforcement and maintenance of tobacco smoking. The present study examined the self-administration of nicotine gum, denicotinized cigarettes, and nicotine-containing cigarettes utilizing a behavioral economic design in order to investigate the pharmacological and nonpharmacological aspects of cigarette smoking. Cigarette-deprived, dependent smokers worked for cigarette puffs and nicotine gum in daily operant sessions. In one phase, nicotine-containing cigarettes were available at increasing unit prices across sessions. Three phases replicated these sessions with nicotine gum, denicotinized cigarettes, or both, concurrently available at a constant unit price. As nicotine-containing cigarette unit price increased, consumption decreased. However, as nicotine-containing cigarette unit price increased, nicotine gum and denicotinized cigarette consumption increased. Consumption of nicotine gum, but not denicotinized cigarettes, diminished when all three reinforcers were concurrently available. Concurrently available denicotinized cigarettes, but not nicotine gum, caused a statistically significant reduction in nicotine-containing cigarette consumption. In another phase, denicotinized cigarettes were available at increasing unit prices across sessions while nicotine gum was concurrently available at a constant unit price. This phase demonstrated that nicotine content had no reliable effect on cigarette or nicotine gum consumption. These results suggest that denicotinized cigarettes are a more effective alternative reinforcer than nicotine gum, indicating that nonpharmacological stimuli of smoking merit attention in smoking cessation treatment. Furthermore, these findings indicate that alternative reinforcement would be most effective in smoking cessation treatment when combined with high prices for cigarettes.

Financial ties and conflicts of interest between pharmaceutical and tobacco companies.

Corporate diversification allows for well-hidden financial ties between pharmaceutical and tobacco companies, which can cause a conflict of interest in the development and marketing of pharmaceutical products. In our investigation of tobacco company documents released and posted on the Internet as a result of the Master Settlement Agreement, we have found that these financial ties have fostered both competition and collaboration between the tobacco and pharmaceutical industries. We present 3 case studies. One shows how tobacco companies pressured pharmaceutical companies to scale back their smoking cessation educational materials that accompanied Nicorette. The second shows how they restricted to whom the pharmaceutical company could market its transdermal nicotine patch. In the third case, we show how subsidiary tobacco and pharmaceutical companies of a parent company collaborated in the production of a nicotine-release gum. Thus, because tobacco cessation product marketing has been altered as a result of these financial conflicts, disclosure would serve the interest of public health.

Treatment of nicotine addiction.

The best available data show smoking to be by far the most important cause of disease and death in our society, contributing to an average of 1000 deaths every day. Although a large majority of current smokers express a desire to quit, the majority of "self-help" attempts to quit are not successful. Further, most smokers indicate never having received advice on cessation from healthcare providers. The combination of pharmacotherapy and behavioral interventions, even on a minimal level, have been shown to be effective in cessation. Such strategies are certainly highly cost-effective, given the enormous costs to society of smoking-related illnesses. Therefore, it is important that all healthcare providers provide at least some form of smoking cessation programs for their patients.

Smoking habits and cessation programme in an Australian teaching hospital.

BACKGROUND: Data on prevalence of cigarette smoking by hospital employees are limited in Australia, but anecdotal evidence suggests that many health sector employees continue to smoke despite abundant evidence regarding the harmful effects of this habit. Nicotine is an addictive drug and arguably this should be known better in the health industry than in any other industry. Despite having this knowledge at their disposal, health sector employers rarely provide assistance to employees, relying instead on restrictive policies to reduce smoking in the workplace. METHODS: To assist employees to quit smoking, we instituted a medium intensity Stop Smoking Programme, run by a clinical pharmacist offering nicotine patches and support on a weekly basis. A principal aim of the service was to redress the imbalance between the availability of cigarettes and the most effective nicotine replacement therapy, the trandermal nicotine patch. Following 18 months operation of this service, we surveyed hospital employees to ascertain smoking rates and views on smoking cessation in this South Australian teaching hospital. RESULTS: In the first 18 months of operation, 111 staff members availed themselves of the service. At the first follow up period (three months), 21 were not contactable, 29 were successful in not smoking and 61 were still smoking. Six of the 29 who were not smoking at three months resumed smoking by six months, and a further four resumed smoking by 12 months. At the time of this report, 12 of the remaining 19 non smokers had completed two years since quitting and a further three of these had resumed regular smoking by this time. The cost of providing the service was modest at approximately $180.00 per known successful quitter. Results from the survey showed that 12.4% of hospital employees were regular smokers. Smoking prevalence was not equally distributed with female employees being twice as likely to smoke as their male counterparts and employees in the catering department having the highest smoking prevalence (23.8%). CONCLUSIONS: Although the prevalence of cigarette smoking by employees of this teaching hospital is lower than for the general community, health sector employers can reduce smoking prevalence further by providing assistance to their employees to quit smoking. The Stop Smoking Programme we describe is effective and could be replicated by other hospitals and similar organisations.

The benefits and risks of over-the-counter availability of nicotine polacrilex ("nicotine gum").

Nicotine polacrilex ("nicotine gum") is effective in helping persons to quit smoking cigarettes. Because many persons try to quit without formal assistance, it may be an appropriate product for over-the-counter (OTC) purchase. Some smokers, however, might use such a product in lieu of more effective methods of cessation, and still others might use it to cope with enforced periods of nicotine abstinence (eg, at the work place) and thereby delay their decision to quit. The study's objective was to assess the public health benefits and risks of OTC availability of nicotine gum. A Markov model was developed and used to contrast two alternative policy scenarios: one in which nicotine gum was assumed to remain available only by prescription, and another in which it was assumed to be made available for OTC purchase. Various data sources were used to estimate the model, including the Health Promotion and Disease Prevention Supplement to the 1991 National Health Interview Survey and the 1986 Adult Use of Tobacco Survey. Primary outcome measures included the numbers of persons who would try to quit smoking, the numbers who would use various methods of smoking cessation, including OTC nicotine gum, and the numbers of current adult smokers who would be abstinent at the end of 10 years. Findings suggest that an average of 3 million persons each year would use OTC nicotine gum. As a consequence of OTC availability, an additional 450,000 smokers would be abstinent at the end of 10 years. These results are sensitive to assumptions regarding the effectiveness of OTC nicotine gum, as well as to the effect of OTC availability on the use of other methods of smoking cessation. The number of persons who would quit smoking, however, increases under a fairly wide range of assumptions. Over-the-counter availability of nicotine gum may confer significant public health benefits.

Cigarette smoking, nicotine addiction, and its pharmacologic treatment.

Cigarette smoking is the most prevalent modifiable risk factor for increased morbidity and mortality in the United States and perhaps the world. Not only does the smoker incur medical risks attributable to smoking, passive smokers and society also bear ill effects and increased economic costs attributable to the smoker's habit. These risks of morbidity and mortality have been shown to be related to the addictive component of tobacco smoke, so that pharmacologic therapies have been studied in an attempt to modify the addiction, aid in smoking cessation, and prevent relapse. Presently, nicotine polacrilex and transdermal nicotine show some efficacy. Clonidine's efficacy has been equivocal. In addition, the combination of nicotine substitution and clonidine may be useful but will need formal investigation. Nicotine agonists and antagonists have not proven helpful. Antidepressants are being studied at this time. Nonpharmacologic modalities are briefly mentioned but play a major role in helping the smoker quit.

Tobacco dependence and the nicotine patch. Clinical guidelines for effective use.

OBJECTIVE: A comprehensive review of transdermal nicotine treatment for tobacco dependence, with recommendations derived from the research literature. DATA SOURCES: English-language clinical trials. STUDY SELECTION: Clinical trials using placebo-controlled, double-blind methodology (11 studies) with at least 6 months of follow-up after treatment (eight studies) and biochemical verification of smoking status. DATA SYNTHESIS: Nicotine patches produce end-of-treatment smoking cessation rates that range from 18% to 77%; these rates are about twice those of placebo-treated subjects. Nicotine patches produced 6-month abstinence rates of 22% to 42%, while placebo patches produced quit rates of 5% to 28%. Nicotine patches appear to reduce some, but not all, nicotine withdrawal symptoms. For instance, while the patch reduces craving for cigarettes and negative moods, it does not appear to reduce hunger or weight gain. The clinical trials literature suggests that proper adjuvant smoking cessation counseling is crucial in determining successful long-term outcome with the nicotine patch, and suggests that 6 to 8 weeks of patch therapy may be an adequate duration for most patients. CONCLUSIONS: Nicotine patches are an effective aid to tobacco dependence treatment. However, success rates vary greatly across research studies and may be influenced highly by the nature and intensity of adjuvant smoking cessation counseling. More research is needed to identify optimal duration, dosage, and individualization of patch therapy. The impact of nicotine patches (more than 5 million users with $1 billion in sales for 1992) also raises important ethical and public health issues.

Patient characteristics and the effect of three physician-delivered smoking interventions.

BACKGROUND: This paper investigates individual patient characteristics predicting differential response to each of three physician-delivered smoking interventions after 6 months. METHODS: Participants were 1,286 currently smoking patients seen by 196 medical and family practice residents in five primary care clinics affiliated with the University of Massachusetts Medical School. Of the participants, 57% were female, 92% were white, their average age was 35 years, and they smoked an average of 23 cigarettes per day. Physicians were trained to provide the following interventions: advice only (AO), a brief (< 10 min) patient-centered counseling intervention (CI), and counseling plus prescription of the nicotine-containing gum Nicorette (CI+NCG). The CI+NCG condition included NCG only when appropriate and if acceptable to the patient. Patients were randomized to one of these three physician-delivered intervention conditions. RESULTS: Overall, stratified univariate analyses revealed that AO produced consistently lower cessation rates across most subgroups (generally 9-13%) but was somewhat more effective for certain groups of lighter smokers. Relative to AO, CI was somewhat more effective (about 20-24%) for less addicted smokers, for those with more previous quit attempts, and for those with fewer close associates who smoke, but generally failed to produce higher quit rates for harder core smokers or for women. CI+NCG had an overall pattern of greater effectiveness for both more addicted and less addicted smokers, with the highest absolute levels of cessation (about 27-30%) among less dependent smokers. Women in this group had cessation rates (20.6%) comparable to those of men (23.6%). Condition-stratified logistic regression analyses, controlling for a wide range of covariates, revealed associations similar to those observed in the univariate analyses: An overall logistic model in which intervention conditions were fitted as dummy variables produced the following significant main effects: sex, years smoked, contact with other smokers, symptoms, and CI+NCG condition. Significant interactions were observed for both CI and CI+NCG and smoking when feeling too ill to continue normal activities and CI+NCG and amount smoked. CONCLUSIONS: We observed significant main effects on cessation of variables related to addiction, sex, social factors, and physician counseling interventions. Specific interactions were observed between reported smoking when feeling ill and each of the counseling interventions as well as by amount smoked in the CI+NCG condition.