Introduction Tobacco dependence is understood to be a complex process that is primarily caused by the pharmacological effects of nicotine which activate nicotinic acetylcholine receptors in the brain leading to release of the neurotransmitter dopamine into the mesolimbic area, corpus striatum and frontal cortex.1–6 Dopamine release induces rewards, including pleasure, arousal, mental acuity and modulation of mood.1 Since the 1980s, nicotine is believed to play a central role in biological reinforcement, tolerance and physical dependence, and withdrawal symptoms on discontinuation of intake.7 However, substantial evidence exists to suggest that nicotine's reinforcing effects alone are not sufficient to account for the intense addictive properties of tobacco smoking and the high relapse rates among smokers after quitting even when provided nicotine in forms other than tobacco.8–16 Further evidence that tobacco dependence entails more than addiction to nicotine includes the drug's limited ability to induce self-administration in animals;17 ,18 lack of positive mood effects of pure nicotine in abstinent smokers;19–21 lack of findings that nicotine in any other form than tobacco was preferred to placebo in normal smokers;22 ,23 de-nicotinised cigarettes were as effective as regular cigarettes, and more than nicotine in any other delivery mode, in relieving withdrawal and craving;24–27 and essential role of non-nicotine factors in cigarette addiction.24 ,28 The release of tobacco industry documents in the 1990s and investigation by the US Food and Drug Administration (FDA) brought to light tobacco manufacturers’ research and development of the use of additives and ingredients besides nicotine which led to the increased appeal, attractiveness and addictiveness of products.3–5 ,29 Independent scientific evidence has demonstrated that conditioned cues produced by tobacco non-nicotine ingredients and smoke constituents are instrumental in maintaining tobacco use.6 ,7 ,16 ,22 ,28 ,30–35 Therefore, current models of tobacco product abuse potential recognise nicotine as the primary drug of addiction, and that non-nicotine tobacco constituents and sensory stimuli from packaging and environmental cues also contribute to tobacco dependence.36 Two major determinants of abuse potential are (A) dynamic pharmacokinetics of nicotine delivery and (B) learned behaviour effects triggered by sensory cues associated with use.36 ,37 A smoker may feel the need to puff in order to attain threshold doses of nicotine and elicit the hedonic effects attributable to dopaminergic system reward pathways.38 Nicotine delivery and its perception may be related to ease of the drug's administration and the ‘impact’ of tobacco smoke on posterior pharynx nociceptors, which is proposed to occur primarily by free nicotine.39 ,40 Puff volume, speed of delivery, lung deposition, frequency of dosing, arterial absorption and other parameters affect the efficiency of nicotine delivery.41 Tobacco manufacturers modified the design of products by directly adding constituents to cigarettes that stimulate gustatory, tactile and olfactory nerve receptors and create chemosensory effects that could enhance elasticity in nicotine dosing as well as strengthen sensory cueing to optimise the ‘pleasure’ in smoking.38 ,42–51 Pyrazines, a class of chemosensory agents, comprise 15 of the 599 compounds on the list of cigarette ingredients provided by manufacturers to the US Department of Health and Human Services in 1994,52 8 of the compounds on the list of additive ingredients provided by manufacturers to the FDA in 201153 and 10 of the compounds presently listed on cigarette manufacturers’ website as cigarette ingredients”54–56 (box 1). The present study explores tobacco industry research that first identified pyrazines in tobacco smoke and was followed by the introduction of pyrazines in ‘light’ cigarettes and subsequent incorporation into Marlboro Lights and eventually in many other cigarette brands. It further examines their possible role in abuse potential. Box 1 Pyrazine compounds in manufacturers’ reports of cigarette ingredients 2·acetyl·3·ethylpyrazine,52 Acetylpyrazine,52 ,53 ,55 ,56 2,3·diethylpyrazine,52–55 2,3-dimethylpyrazine,52 2,5-dimethylpyrazine,52–55 2,6·dimethylpyrazine,52 2·ethyl(or methyl)·(3,s and 6)-methoxypyrazine,52 2·ethyl·3,(s or 6)-dimethylpyrazine,52 ,53 ,55 ,56 2·ethyl·3·methylpyrazine,52 ,53 ,55 2-isobutyl·3·methoxypyrazine,52 Methoxypyrazine,52 2-methylpyrazine,52 ,53 ,55 ,56 (Methylthio)methylpyrazine,52 2,3,5,6·tetramethylpyrazine,52–56 2,3,5-trimethylpyrazine,52–56 Methoxymethylpyrazine,52 ,54 ,56 52Manufacturers’ 1994 cigarette ingredients report, 53Manufacturers’ 2011 report to the Food and Drug Administration (FDA), 54Philip Morris, Inc. web site, 55Lorillard, Inc. web site, 56R.J. Reynolds Tobacco Company web site.

Methods More than 7 000 000 tobacco industry documents have been disclosed by the major tobacco companies during litigation processes and made public as a result of the Minnesota Tobacco Trial and the Master Settlement Agreement of 1998.57 ,58 We searched online internal tobacco industry document databases housed at Tobacco Documents Online (http://www.tobaccodocuments.org), the British American Tobacco Document Archive (http://bat.library.ucsf.edu) and the Legacy Tobacco Documents Library (http://legacy.library.ucsf.edu). Standard methods used for document analysis have been described in detail elsewhere.59 ,60 Document identification was performed using an index-based word search of titles, authors, recipients and other document characteristics (such as date, document type, original file location), as well as keywords and abstracts. Whenever available, full-text optical character recognition was also used. We used a snowball sampling method to first search the databases using an initial set of key words (eg, pyrazines, flavorant, flavoring, flavor, chemosensory, sensory, low-tar, stimulation, attributes, perception, effects, taste, smoothness and product development) and relevant combinations of these terms, and to generate further search terms from the documents identified. Relevant documents were abstracted and indexed. The resulting document set was surveyed for recurring authors, keywords, codes or project names that would suggest further avenues for retrieval. A number of unique difficulties associated with the use of internal industry documents as a source of scientific information must be considered. Industry research was not generally subjected to careful peer review, and details regarding the experimental methods used and the resulting quality of the data are often unavailable, making it difficult to assess the reliability of the science. In addition, the available documents do not always represent the totality of the internal research that was conducted on a particular topic—as indicated by the existence of many partial reports and memos. Finally, within each given company, the documents are authored by numerous different researchers from a range of departments over tens of years, and so findings are sometimes inconsistent and occasionally even contradictory. Comparisons of the documents reveal real company-to-company differences in approach to the engineering of tobacco product design, a finding that must be taken into account. For these reasons and to inform the findings in internal industry documents, we conducted this research in conjunction with a systematic review of evidence from the current scientific literature indexed in databases including PubMed (http://www.pubmed.gov) and Web of Science (http://thomsonreuters.com/web-of-science) using the same search strategies.

Results Introduction of pyrazines in cigarettes The first US Surgeon General Report in 1964, which greatly increased concerns about the dangers of smoking, and the decline in cigarette sales beginning for the first time since World War II gave a major impetus to the tobacco industry’s efforts to increase product appeal.61 Tobacco manufacturers introduced new cigarette brands in response to these concerns, using filter ventilation, which lowered tar and nicotine yields or altered ratios measured under a standardised machine-based testing protocol.38 The ‘low-tar’ cigarettes were found to have diminished taste, aroma and flavour and a weaker impact on receptors in the throat.62 ,63 Facing a decrease in smoking and continuing 2% annual decline in cigarette sales, Philip Morris (PM) endeavoured to develop cigarettes with even lower tar yields, yet with taste and flavour that would satisfy smokers’ ‘palates and needs’.62 PM achieved a major breakthrough in this area by developing a ‘full-flavour, low-tar product’, marketed under the MERIT brand, which was the first ‘light’ cigarette.62 The company accomplished this by first selecting out components of the volatile fraction of the particulate phase that contributed the greatest odour intensity from among the multitudes of aromatic chemicals and substances in tobacco smoke.62 The gas chromatographic fractions of approximately 100 distinctive tobacco smoke flavourants were selected on the basis of high odour intensity as perceived by human participants using vapour dilution olfactometry.62 The molecular structures of these compounds were then tentatively identified by high resolution mass spectrometry and by comparing the ‘cracking’, a term used by PM for fragmentation patterns, with known reference spectra. PM then incorporated the flavourants of highest intensity into the variety of compounds to be added to the reconstituted tobacco sheet. The reformulated cigarette flavour systems provided the taste, flavour and aroma qualities of the low-tar delivery cigarettes.62 Finally, the company used panels of trained flavour experts to evaluate the smoke flavour of prototypes. PM's research and development resulted in a cigarette yielding less than 9 mg tar with a smoke flavour of much higher tar yielding products.62 An extensive consumer testing programme of the new MERIT product was conducted, including blind interviews with nearly 3000 smoker panellists.62 ,64 The majority of consumer participants reported that the new MERIT was equal or superior in taste to brands that delivered 60% more tar. Advertisements touted the product’s ‘enriched flavor’ and described, “After twelve years of intensive research, Philip Morris scientists isolated certain key ingredients in smoke that deliver taste way out of proportion to tar.” 65 (figure 1). This brand went on to capture a significant share of the low-tar cigarette market following its national launch in 1976.62 Figure 1 Advertisement for new ‘Enriched Flavor’ MERIT cigarettes (1976). PM called the new flavour formulation ‘Super Juice’, which contained 2,6-dimethyl pyrazine, tetramethyl pyrazine and trimethyl pyrazine as well as acetic acid, cyclotene, maltol, isobutyric acid and 1-methyl indole.66 Reverse engineering and research by British American Tobacco of PM products, MERIT, MERIT Menthol, Marlboro and Marlboro Lights identified at least six pyrazines: 2-methylpyrazine, methylethylpyrazine, dimethylethylpyrazine, 2,3-dimethylpyrazine, 2,6-dimethylpyrazine, trimethylpyrazine and tetramethylpyrazine, and found that pyrazines contribute to the burley flavour, which is a common characteristic of many PM brands (isomers noted only where referenced in document).67 In the USA in the late 1970s, ‘Super Juice’-like compounds were added to Marlboro Lights, which is now called ‘Marlboro Gold’. Ingredients also included essential oils, inorganic acids and other constituents, added to a reconstituted tobacco sheet with diammonium phosphate, which appears to have allowed better control of constituent release.68 Marlboro Lights have since become the leading selling cigarette brand. Pyrazine flavour profiles Pyrazines are heterocyclic aromatic organic compounds with the underlying chemical formula C 4 H 4 N 2 . They are formed under pyrolytic conditions (temperatures ≥100°C) via the Maillard Browning reaction between amines and carbonyl compounds (generally sugars)69 ,70 (figure 2), which occurs during the curing of tobacco leaf and during the smoking process.71 ,72 Numerous pyrazine compounds have been detected in foods, which arise from the common practice of heating and Maillard Browning reaction of sugars with protein and ammonia, providing a distinctive flavour. Other pyrazine compounds have been synthesised and promoted as flavouring agents because of their unique organoleptic properties and flavour and aroma profiles.73–77 Pyrazines are 1 of 18 chemical classes of flavouring materials used in combustible tobacco products as described by Leffingwell et al.78 They have been said to be among the most important compounds characterising the aroma and flavour of tobacco and tobacco smoke, contributing the ‘brown notes’ in general, and at least in some cases the cocoa, nutty or popcorn-type flavour notes.79 Figure 2 Graphic representation of the Maillard Browning reaction in the formation of pyrazines. Chemosensory effects Pyrazines are known to act on chemoreceptors, sensory receptors that transduce chemical signals into action potentials.80 In addition to conveying the classical senses of taste and smell in humans, the mouth, nose and airways also contain chemosensory nerve endings of the trigeminal nerve.81 These can be activated by physical stimuli as well as by a large array of chemical agents, leading to sensations such as burning, cooling and tingling, and contributing to flavour even in the absence of an olfactory percept.82 Chemosensory effects of some other additives to cigarettes have been described, including essential oils (eg, menthol)83 and organic acids (eg, levulinic acid).84 A report by the Tobacco Product Scientific Advisory Committee to the FDA described menthol's actions on transient receptor potential (TRP) channels, in particular TRPM8, which produce cooling and analgesia at low doses, irritation and pain at high doses, and desensitisation of the receptors with prolonged stimulation.85 The report described how the addition of menthol in cigarettes creates perceptions of smoothness at low levels and analgesia at high levels and reduces the discomfort of smoking in long-term users. Results of population studies cited in the report showed youth being more likely to initiate smoking with a low menthol brand (eg, Newport), and older adults being less likely with a high menthol brand (eg, Kool).85 An earlier review of internal tobacco industry documents reported the addition of levulinic acid to cigarettes to increase nicotine yields while enhancing perceptions of smoothness and mildness.84 ,86 Important chemosensory effects of pyrazines identified by the industry include smoothing, which may enhance the ease of inhalation and nicotine deposition by reducing the harshness and irritating effects of nicotine and other tobacco smoke constituents in the airways.87 PM's internal documents of 1990 pertaining to the company's chemical senses research programme describe how a “chain of events from stimulation in the mouth, the throat and at the olfactory level leads to trans-membrane electrical signals which are integrated in the brain.”88 According to these documents, diffusion and binding of constituents to receptors at sites of action, generation of action potentials, transmembrane signalling and integration of the diverse stimulus signals result in percepts (perceptions), which the company attempted to balance in order to promote high consumer acceptance and continued use as opposed to rejection of the product.89 Pyrazines and learned behaviour Pyrazine stimulation of olfactory receptors may enhance learned behaviour, either by acting alone or in combination with other sensory modality stimuli.90 ,91 Human responses to chemosensory and olfactory effects that are associated with emotionally significant experiences can become constitutional through neuroplastic changes in the olfactory pathways to the limbic system as well as other areas of the brain associated with hedonic perception.92 ,93 Such events can reinforce smoking through associative learning and become cues for increased hedonic valence of stimuli94 and motivate increased desire or wanting, or even unrestrained consumption.