22nd Century holds exclusive worldwide rights to nucleic acid sequences that encode enzymes involved in alkaloid biosynthesis pathways, including their use for genetically engineering levels of nicotine and other alkaloids in plants. This provides 22nd Century with the ability to regulate the production and content of nicotine and other alkaloids in plants, and in particular, to genetically engineer tobacco plant lines to contain a wide range of specific desired alkaloid contents.

The company's technology platform also includes (a) novel plant enzymes for the production of alkaloids including nicotinic alkaloid analogs for use in the production of pharmacological therapeutics, (b) methods for starch/sugar enhancement in plants, (c) reduced-exposure tobacco products and (d) uses of Verfolia™ (nicotine-free tobacco biomass) for production of proteins, biofuels and animal feed.


NOVEL PLANTS AND PRODUCTS PRODUCED FROM 22ND CENTURY'S TECHNOLOGY

Smoking Cessation Aid

In 1997, 22nd Century's president conceived the product concept of a series of cigarettes having progressively reduced nicotine content for use in smoking cessation. Vector 21-41, a very low nicotine tobacco variety, was developed for and is exclusively licensed to 22nd Century by one of the company's university research collaborators. This variety contains only about five (5) percent of the amount of nicotine in conventional commercial tobacco varieties (a 95% reduction) (Xie et al. 2004). It is protected by various international patents and in the U.S. and Canada by Plant Variety Certificate Nos. 200100039 and 2357. This variety was the first GE tobacco variety deregulated in the U.S. by the Animal Plant & Health Inspection Service (APHIS) of the United States Department of Agriculture. Since the development of Vector 21-41, 22nd Century has obtained exclusive worldwide rights to a number of additional nicotine biosynthesis genes. Expression of each of these has been suppressed in separate tobacco lines and in certain combinations, resulting in numerous, novel very low nicotine tobacco lines.

Research studies demonstrate that very low nicotine ("VLN") cigarettes, also referred to as "nicotine-free" and "denicotinized," are an effective tool in smoking cessation because they (a) relieve cravings for and withdrawal symptoms from conventional cigarettes and (b) extinguish the reinforcing value of smoking by breaking the association of sensory and behavioral cues of cigarette use with the rapid delivery of nicotine (Gross et al. 1997; Rose 2006; Donny et al. 2007).

"Reduced-nicotine cigarettes" (containing a blend of very low nicotine tobacco and conventional tobacco) and VLN cigarettes, all containing Vector 21-41, were among the study materials of a successful FDA-reviewed phase II clinical trial for smoking cessation. Use of these cigarettes in combination with nicotine replacement therapy (NRT) was more effective than use of NRT alone in achieving 4 weeks of continuous abstinence, and use of these cigarettes without NRT yielded an abstinence rate similar to that of NRT (Becker et al. 2008).

The company's R&D and results of published research studies to date demonstrate that there is an important distinction among (a) conventional "light" or "ultra light" (low-yield ) cigarettes that have reduced tar and nicotine yields as measured by smoking machines mainly due to smoke dilution by ventilation (b) reduced-nicotine cigarettes, which contain blends of low nicotine tobacco and conventional tobacco, and (c) VLN cigarettes, which have an extraordinarily low nicotine content and independent of ventilation and machine yield (e.g. 0.06 mg nicotine/9 mg "tar" or 0.02 mg nicotine/3 mg "tar"), have a very low ratio of delivery of nicotine compared to "tar."

VLN cigarettes provide a smoking experience that differs in significant ways from that provided by low-yield or reduced-nicotine cigarettes due to the lack of nicotine impact. Smoking low-yield or reduced-nicotine cigarettes sometimes causes what is known as "compensation" or "compensatory smoking," meaning over smoking by using more cigarettes, taking larger puffs or more puffs per cigarette, and/or inhaling deeper (Gori et al. 1985). When smokers switch exclusively to VLN cigarettes, smoking is less satisfying and smokers do not engage in compensatory smoking; rather they smoke fewer cigarettes per day and have higher quit rates (Donny et al. 2007; Benowitz et al. 2007; Becker et al. 2008). 22nd Century is continuing the clinical development of VLN cigarettes for smoking cessation.

Nicotine-Free Tobacco for Biomass

Conventional tobacco is widely cultivated throughout the world. Tobacco agronomy is as understood as that of any popular crop. Biomass tobacco, without limitations of factors affecting quality for traditional tobacco products such as cigarettes, may have an even broader growing range than conventional tobacco. The widespread use of tobacco as a model system for plant biology and genetic engineering means that extensive information about the tobacco genome and the functions of a number of tobacco genes already exists. This body of knowledge is quite useful for development of innovative crops through incorporation of additional traits into 22nd Century's genetically engineered reduced-alkaloid tobacco lines for more efficient, sustainable production of a variety of specific products.

North Carolina State University has demonstrated over three consecutive years of field trials that very low nicotine tobacco grown and handled as a forage crop, with multiple harvests from a single planting, produces about 50 tons per acre of biomass per growing season in North Carolina. Higher volumes than those obtained in North Carolina may be achieved in geographical regions with longer growing seasons.

For every acre of tobacco biomass grown from tobacco with conventional nicotine levels, hundreds of pounds of alkaloids including nicotine would have to be dealt with. 22nd Century's GE very low nicotine tobacco lines alleviate this problem. One of 22nd Century's short-term goals is to reduce nicotine levels in a tobacco biomass variety to equal the trace amounts found in related plants, such as green peppers, tomatoes and potatoes (Andersson et al. 2003; Sheen 1988).

Since tobacco biomass is a green leaf crop, protein production and quality are impressive. Total protein production is about 1 ton per acre (DW), half of which is soluble, extractable protein with an excellent balance of essential amino acids. A crystalline protein fraction, consisting of a protein involved photosynthesis (RibUlose BIsphosphate Carboxylase-Oxygenase or "Rubisco"), can be easily prepared from tobacco. Purified Rubisco is a pharmaceutical grade protein that is tasteless, odorless, and colorless when mixed with water. With the exception of slightly lower levels of the amino acid methionine, Rubisco's content of essential amino acids equals or exceeds that of the FAO [Food and Agriculture Organization (of the United Nations)] Provisional Pattern (Ershoff et al. 1978). Sugars, starches, cellulose and other components remaining after extraction of protein and/or other products can be used directly or for production of ethanol, butanol or industrial feedstocks by fermentation.

Tobacco with Higher Nicotine Content to Reduce Smoke Exposure

The dependence of many smokers on tobacco is largely due to the addictive properties of nicotine, but the adverse effects of smoking on health are mainly due to other substances present in tobacco smoke (for a review, see Slade et al., 2000).

Studies demonstrate when nicotine is added to conventional cigarette tobacco, smokers inhale less smoke (Woodman et al. 1987; Fagerström 1982; Armitage et al. 1998; Russell 2000).

22nd Century has developed tobacco plants with increased nicotine content in the leaf. This tobacco is for use in potential reduced risk-cigarettes that deliver no more nicotine than conventional cigarettes and potentially much lower amounts of most other smoke components. The objective of the company's research and development on increasing nicotine levels in cigarette tobacco is to reduce smoke exposure in smokers who fail to quit or refuse to quit. 22nd Century expects that evaluation of these cigarettes in human exposure studies will confirm that exposure to "tar" and carbon monoxide is significantly reduced when smoking low tar-to-nicotine ratio cigarettes compared to currently marketed cigarettes.





References

Andersson C, Wennström P, Gry OJ. 2003. Nicotine alkaloids in Solanaceous food plants. TemaNord 531: 1-37.
http://www.norden.org/pub/ebook/2003-531.pdf

Armitage AK, Alexander R, Hopkins R, Ward C. 1988. Evaluation of a low to middle tar/medium nicotine cigarette designed to maintain nicotine delivery to the smoker. Psychopharmacology 96: 447-453.
http://www.ncbi.nlm.nih.gov/pubmed/3149764

Becker KM, Rose JE, Albino AP. 2008. A randomized trial of nicotine replacement therapy in combination with reduced-nicotine cigarettes for smoking cessation. Nicotine Tob Res 10(7):1139-48.
http://www.ncbi.nlm.nih.gov/pubmed/18629723

Benowitz NL, Hall SM, Stewart S, Wilson M, Dempsey D, Jacob P 3rd. 2007. Nicotine and carcinogen exposure with smoking of progressively reduced nicotine content cigarette. Cancer Epidemiol Biomarkers Prev. 16(11):2479-85.
http://www.ncbi.nlm.nih.gov/pubmed/18006940

Donny EC, Houtsmuller E, Stitzer ML. 2007. Smoking in the absence of nicotine: behavioral, subjective and physiological effects over 11 days. Addiction 102:324-34.
http://www.ncbi.nlm.nih.gov/pubmed/17222288

Ershoff BH, Wildman SG, Kwanyuen P. 1978. Biological Evaluation of Crystalline Fraction I Protein from Tobacco. Society for Experimental Biology and Medicine. 157:626-630.

Fagerström KO. 1982. Effect of a nicotine-enriched cigarette on nicotine titration, daily cigarette consumption, and levels of carbon monoxide, cotinine, and nicotine. Psychopharmacology 77:164-167.
http://www.ncbi.nlm.nih.gov/pubmed/6812134

Gori GB, Lynch CJ. 1985. Analytical Cigarette Yields as Predictors of Smoke Bioavailability. Regulatory Toxicology and Pharmacology 5:314-326.
http://www.ncbi.nlm.nih.gov/pubmed/4059592

Gross J, Lee J, Stitzer ML. 1997. Nicotine-containing versus denicotinized cigarettes: effects on craving and withdrawal. Pharmacol Biochem Behav 57:159-165.
http://www.ncbi.nlm.nih.gov/pubmed/9164567

Rose JE. 2006. Nicotine and nonnicotine factors in cigarette addiction. Psychopharmacology 184:274-285.
http://www.ncbi.nlm.nih.gov/pubmed/16362402

Russell MAH. 2000. Public Health and Levels of Nicotine: Should nicotine be minimized or maximized? In: Nicotine and Public Health Ferrence R, Slade J, Room, R, Pope M, eds., Washington D.C.: American Public Health Association, pp. 265-284.
http://www.xxiicentury.com/downloads/chapterxv1030005.1.pdf

Sheen SJ. 1988. Detection of Nicotine in Foods and Plant Materials. J. Food Sci. 53(5):1572-1573.

Slade J, Bickel WK, Crowley TJ, Ferrence R, Henningfield JE, Houston TP, Mitchell B, Room R, Sweanor DT, Warner KE. 2000 The Role of Managing Nicotine in Controlling the Tobacco Epidemic. In: Nicotine and Public Health. Ferrence R, Slade J, Room, R, Pope M, eds., Washington D.C.: American Public Health Association, pp. 483-501.

Woodman G, Newman SP, Pavia D, Clarke SW. 1987. The separate effects of tar and nicotine on the cigarette smoking manoeuvre. Eur J Respir Dis. 70:316-321.
http://www.ncbi.nlm.nih.gov/pubmed/3609190

Xie J, Song W, Maksymowicz W, Jin W, Cheah K, Chen W, Carnes C, Ke J, Conkling MA. 2004. Biotechnology: A Tool for Reduced Risk Tobacco Products - The Nicotine Experience From Test Tube to Cigarette Pack. Recent Advances in Tobacco Science 30:17-37.