Effectiveness of ZYVERT® technology

Peer reviewed publication on ZYVERT® technology

— Lusso M, Gunduz I, Kondylis A, Jaccard G, Ruffieux L, Gadani F, Lion K, Adams A, Morris W, Danielson T, Warek U, Strickland J (2017) Novel approach for selective reduction of NNN in cigarette tobacco filler and mainstream smoke. Regulatory Toxicology and Pharmacology, 89, 101—111.

How TSNAs are formed

— Cooperative effort of University of Kentucky, University of Tennessee, Virginia Tech and NC State University. Burley and Dark Tobacco Production Guide [cited January 25th, 2018]. Available from: https://tobacco.ces.ncsu.edu/wp—content/uploads/2015/04/2015—2016—Burley—Production—Guide.pdf?fwd=no

— Lang, G., and Vuarnoz, A., 2015. Matrix—bound 4— (methylnitrosamino)—1—(3—pyridyl)—1—butanone in tobacco: quantification and evidence for an origin from ligninincorporated alkaloids. J. Nat. Prod. 78 (1), 85—92. http://dx.doi.org/10.1021/np500725a

— Moldoveanu, S. C., and M. Borgerding. 2008. “Formation of tobacco specific nitrosamines in mainstream cigarette smoke; Part 1, FTC smoking.” Beitrage zur Tabakforschung International/ Contributions to Tobacco Research no. 23 (1):19—31.

— Piadé J.J., Wajrock, S., Jaccard, G., Janeke, G., 2013. Formation of mainstream cigarette smoke constituents prioritized by the World Health Organization – yield patterns observed in market surveys, clustering and inverse correlations. Food Chem. Toxicol. 55, 329—347. http://dx.doi.org/10.1016/j.fct.2013.01.016

— Sophia, F., Spiegelhalder, B., Preussmann, R., 1989. Preformed tobacco—specific nitrosamines in tobacco— role of nitrate and influence of tobacco type. Carcinogenesis 10 (8), 1511—1517. http://dx.doi.org/10.1093/carcin/10.8.1511

Impact of weather, time and curing process on TSNA formation

— Fisher MT, Bennett CB, Hayes A, Kargalioglu Y, Knox BL, Xu D, Muhammad—Kah R, Gaworski CL (2012). Sources of and technical approaches for the abatement of tobacco specific nitrosamine formation in moist smokeless tobacco products. Food and Chemical Toxicology, 50, 942—948.

— LION K.; LUSSO M.; MORRIS W.; HART F. Tobacco specific nitrosamine (TSNA) levels of the U.S. domestic Burley crop and their relationship with relative humidity conditions during curing. CORESTA Meeting, Agronomy/Phytopathology, 2015, Izmir, Turkey, AP 17. https://www.coresta.org/abstracts/tobacco-specific-nitrosamine-tsna-levels-us-domestic-burley-crop-and-their-relationship

— LUSSO M.F.G.; HAYES A.; LION K.; HART F.; MORRIS W. Impact of curing management practices on leaf TSNA content in dark fire—cured tobacco. CORESTA Congress, Sapporo, 2012, Agronomy/Phytopathology Groups, AP 10. https://www.coresta.org/abstracts/impact-curing-management-practices-leaf-tsna-content-dark-fire-cured-tobacco-28792.html

— LUSSO M.F.; LION K.E.; ADAMS A.; MORRIS W.; WAREK U.; STRICKLAND J.A. NNN levels in stable reduced converter (SRC) and low converter (LC) lines cured under conditions that favor NNN formation. CORESTA Meeting, Agronomy/Phytopathology, 2017, Santa Cruz do Sul, AP 07. https://www.coresta.org/abstracts/nnn-levels-stable-reduced-converter-src-andlow-converter-lc-lines-cured-under-conditions

—MORTON M.J.; PHILLIPS D.J.; JORDAN J.L.; OLDHAM M.J.; LION III K.E.; LUSSO M.F.; FRANKE J.E.; STRICKLAND J.A. Comparison of intra—crop year variability in NNN in tobacco and NNN levels in smokeless tobacco products. CORESTA Meeting, Smoke Science/Product Technology, 2017, Kitzbühel, IG 01. https://www.coresta.org/abstracts/comparison-intra-crop-year-variability-nnn-tobacco-and-nnn-levels-smokeless-tobacco-1

— Saito H, Miyazaki M, Miki J. (2006) Role of nitrogen oxides in tobacco—specific nitrosamine formation in burley tobacco. CORESTA Congress, Paris, France, October, 15−20, 2006. https://www.coresta.org/abstracts/role-nitrogen-oxides-tobacco-specific-nitrosamine-formation-burley-tobacco-2254.html

— Shi H, Wang R, Bush LP, Zhou J, Yang H, Fannin N, Bai R (2013) Changes in TSNA contents during tobacco storage and the effect of temperature and nitrate level on TSNA formation. J. Agric. Food Chem. 61, 11588−11594.

Discovery of ZYVERT® technology

— Dewey, R.E., and R.S. Lewis., 2011. Compositions and methods for minimizing nornicotine synthesis in tobacco. Google Patents.

— Gavilano, L.B., Coleman, N.P., Burnley, L.E., Bowman, M.L., Kalengamaliro, N.E.,Hayes, A., Bush, L., Siminszky, B., 2006. Genetic engineering of Nicotiana tabacum for reduced nornicotine content. J. Agric. Food Chem. 54 (24), 9071a9078. http://dx.doi.org/10.1021/jf0610458

— Gavilano, L. B. & Siminszky, B. (2007). Isolation and characterization of the cytochrome P450 gene CYP82E5v2 that mediates nicotine to nornicotine conversion in the green leaves of tobacco. Plant Cell Physiol, 48, 1567—1574.

— Julio, E. (2008). Reducing the content of nornicotine in tobacco via targeted mutation breeding. Molecular Breeding 213, 369—381.

— Lewis, R.S.; Jack, A.M.; Morris, J.W.; Robert, V.J.M.; Gavilano, L.B.; Siminszky, B.; Bush, L.P.; Hayes, A.J.; Dewey, R.E., “RNA interference (RNAi)—induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves”. Plant Biotechnology Journal, 6(4): 346—354; 2008.

— Lewis, R.S., Bowen, S.W., Keog, M.R., Dewey, R.E., 2010. Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: functional characterization of the CYP82E10 gene. Phytochemistry 71, 1988—1998.

— Siminszky, B., Gavilano, L., Bowen, S.W., Dewey, R.E., 2005. Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proced. Natl. Acad. Sci. U. S. A. 102 (41), 14919e14924. http://dx.doi.org/10.1073/pnas.0506581102.

— Xu, D., Shen, Y., Chappell, J., Mingwu, C., Nielsen, M., 2007. Biochemical and molecular characterizations of nicotine demethylase in tobacco. Physiol. Plant. 129,307—319.

Scientific presentations on ZYVERT® technology

— XU Dongmei; SHEN Yanxin; LUSSO M.; BAILEY A.; NORMAN D.; DAVIS G.; HART F.; CUI Mingwu; NIELSEN M. Reducing nicotine conversion: stability of RNAi—mediated and mutant down—regulation of nicotine demethylase CORESTA Congress, Shanghai, 2008, AP 06. https://www.coresta.org/abstracts/reducing-nicotine-conversion-stability-rnai-mediated-and-mutant-down-regulation-nicotine

— NIELSEN M.; XU Dongmei; LUSSO M.; DAVIS G.; SHEN Yanxin; BAILEY A.; HART F.; CUI Mingwu; NORMAN D. Down—regulating the nicotine demethylase gene lowers NNN content levels with little or no effect on field performance of Burley and dark tobaccos. CORESTA Congress, Shanghai, 2008, AP 05. https://www.coresta.org/abstracts/down-regulating-nicotine-demethylase-gene-lowers-nnn-content-levels-little-or-no-effect

— HAYES A.; LUSSO M.; HAJALIGOL M.; LEWIS R. Reduction of nornicotine in experimental burley tobaccos and impact on N—nitrosonornicotine in lamina and smoke. CORESTA Meeting, Agronomy/Phytopathology, 2011, Santiago, AP 05. https://www.coresta.org/abstracts/reduction-nornicotine-experimental-b

— HAYES A.; LUSSO M.F.G.; LION K.; ADAMS A.; MORRIS W. Impact on N—nitrosonornicotine (NNN) of varying nitrogen rates for Burley TN90 isolines with variable genetic potential for nicotine to nornicotine conversion. CORESTA Congress, Sapporo, 2012, Agronomy/Phytopathology Groups, AP 08. https://www.coresta.org/abstracts/impact-n-nitrosonornicotine-nnn-varying-nitrogen-rates-burley-tn90-isolines-variable

— HAYES A.; LEWIS R.; LUSSO M.; LION K.; ADAMS A.; MORRIS W. From seed to smoke: N—nitrosonornicotine (NNN) level in smoke is significantly reduced in experimental Burley isolines bred to have stable low nornicotine content. CORESTA Meeting, Smoke Science/ Product Technology, 2013, Seville, ST 25. https://www.coresta.org/abstracts/seed-smoke-n-nitrosonornicotine-nnn-level-smoke-significantly-reduced-experimental-burley

— LUSSO M.; LION K.; ADAMS A.; LEWIS R.S.; MORRIS W.; DAVIS G. From seed to smoke: N-nitrosonornicotine levels in blended cigarettes containing Burley or flue-cured tobacco stable for low nornicotine content. CORESTA Congress, Quebec, 2014, Smoke Science/Product Technology Groups, ST 87. https://www.coresta.org/abstracts/seed-smoke-n-nitrosonornicotine-levels-blended-cigarettes-containing-burley-or-flue-cured

— LUSSO M.; LION K.; MORRIS W.; ADAMS A. N-nitrosonornicotine (NNN) level in lamina of experimental Burley isogenic lines bred to have stable low nornicotine content and cured under conditions favorable for TSNA formation. CORESTA Meeting, Agronomy/ Phytopathology, 2015, Izmir, Turkey, AP. 20.https://www.coresta.org/abstracts/n-nitrosonornicotine-nnn-level-lamina-experimental-burley-isogenic-lines-bred-have-stable

— LUSSO M.F.; ADAMS A.; LEWIS B.; POYNER T.; LION K.E.; DELOACH L.; DANIELSON T.; FRANKE J.E.; WAREK U.; STRICKLAND J.A. N-nitrosonornicotine reduction in dark tobacco varieties and smokeless product prototypes. CORESTA Meeting, Agronomy/ Phytopathology, 2017, Santa Cruz do Sul, AP 08. https://www.coresta.org/abstracts/n-nitrosonornicotine-reduction-dark-tobacco-varieties-and-smokeless-product-prototypes

— LUSSO M.F.; LION K.E.; ADAMS A.; MORRIS W.; WAREK U.; STRICKLAND J.A. NNN levels in stable reduced converter (SRC) and low converter (LC) lines cured under conditions that favor NNN formation. CORESTA Meeting, Agronomy/Phytopathology, 2017, Santa Cruz do Sul, AP 07.https://www.coresta.org/abstracts/nnn-levels-stable-reduced-converter-src-andlow-converter-lc-lines-cured-under-conditions

Patents & Trademark

— Nicotine Demethylase Patents www.zyvert.net