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To: Dr. D. H. Piehl From: W.H. Henley • A summary of the major points developed during the October 25, 1976 discussion on nicotine is presented below. The primary goals of this discussion were: (a) to review our current k~owledge concerning nicotine, (b) to dlscus~. . partlcu]ar research needs in terms of .problems to be solved and questions td be ans~red and (c) to establish, among staff personn~]~ a firmer base for stlmulatin~ dialogue and cross-ferCilization of ideas in this area. In evalpat|ng progress toward the ~bove goals, the author be|laves that the participants made excellent progress toward.the first goa~. while progress: toward the second goal, due to a lacR. df time, w~"~6~y ]Imlted. As assessment of p~ogresg, toward the third goal is much more speculative but is regarded as having been excellent. If this evaluation is reasonably accurate, then further discussions dlrected toward the second goad might be worthwhile. P~"inclpa]p~rticipants and their tcplcs are nete~ ~elow: : R. F. Moates -, C. L~ Neumann C. E. Rix . J. P. Dickerson 1,-+ Phys|ologtcal Actton of I~lcottne 2. -Smok~hg a~d Health Aspects 3. Taste• of Nlcutlne 4. Factors Influencing Presence in Leaf and S~oke ITRIAL EXHIBIT 12,673 5. Nicotine and Tobacco Substitutes C. L,. Reumann 6. Nicotine ~alogs and Mimics C..L. Neumann " .--. : Rodgman, R~:L. Rowla~'d, D. L.Roberts, b. H. Piehl, Other participants were: A. R. Green, R. A. Lloyd, M. E~' Sto~e, Do Ly~m, R.~E. S?ackelford, W. M. flenl~y. Ph~siolo91cal Action ~f Nicotine (R. F. Moates) • -:~- l. Site of Action "; ' Nicotine interacts with cholinoceptlve receptdrs at neural ~unction.~nd ~hus initiates normal.neural impulses. Those functions of the body which ape normally under neu~al'control by a steady-state normal rate of nerve impulses will thus be increased When nicotine reacts with these cho)inoceptive nerve junctions. A nerve ImpUlse is normally transmitted across a neural Junction by the chemical acetylcholine~-,,,,- ~icotine is able to very effectively imitate the action of acetylcholine. ~icotine thus augments the sti~u]ation of those tissues which have chollnoceptive receptors.

The body functions which are control.led by cholinergic nerves are mostly parts of the autonomic nerve system. Included in these functions are the gastrointestinal tract (stomach, intestinal muscle contractions), heart rate control, peripheral blood vessel constriction and skeletal (postural) muscle control. The influence of nicotine on these functions leads to the following symptoms: I. Elevated heart rate. 2. E%evated coronary flow 3. Elevated blood sugar level 4. Lowered cutaneous temperature at extremities 5. Increased blood flow in skeletal muscles 6. Reactive release of adrenalin 7. Alteration of brain electrical potential pattern 8. Inhibition of patellar reflex • These last th.ree symptoms are general~y believed to be the results of nicotine's action on the central nervous system.'-M6~tstudies of nicotine's effect on the CNS are not conclusive, due to the difficulty of isolating a single neural function known to originate in the brain. Furthermore, the brain is a mixture of cholinoceptive and adrenoceptive receptors, of t#hich nicotine affects only the cholinoceptive receptors. Subjectively, ho~'ever, nicotine is said to c~use bct;~ mental arousal and relaxation. 2. Absorp~tion, Metabolism and Excretion P~o~bly the most effective method of administerln~ nicotine to the boCy . is by inhalation of cigarette smoke. This is due to the fact that unionized nicotine is readily soluble in both hydrophilic and lipophilic solvents, f.bout 60-90% of the nicotine in a puff of smoke is absorbed upon inhalation. The buffering action of oral and lung tissue, along with its large surface area, aid in the absorption of any nicotine hound in a salt fo~. ~hus a high concentration of nicotine is suddenly produced in the pulmona)~ veins, which is then distributed to the brain and many parts of the body within a fet~ seconds. Efforts to reproduce~ this concentration of blood nicotine by intravenous injection usually require about twice the amount of nicotine injected vs inhaled to produce a given physiological response. The responses most easily measured in both man and animals are heart .~. rate and blood pressure. The half-life of nicotine in the body is about 20-.30 minutes. Nicotine in blood is readily excreted in urine and metabolized in the liver. Carbon-l~ studies have shown that nicotine becomes concentrated in the brain about 5 minutes after ~: ~injection. This study also illustrates nicotine's ready solubi]ity in body tissues and ability to cross the blood-brain barrier. Nicotine is readily excreted in urine, especially as an acid salt. Urinary excretion of nicotine can be hastened if the urinary pH is lowered by ingestion of NH4CI. Conversely, excretion can be slowed if urinary pH is raised by ingestion of bicarbonate. Nicotine is readi detoxified in the liver by oxidation to cotinine. ~ost other nicotine metabclites are derived from cotinine b~ further oxidation and ring-opening of the pyrroli~one ring. The blood concentration of cotinine peaks about 2 hours after nicotine absoprtion, indicating the much slower metabolism and excretion of thisconpound.

The physiological effects of cotinine are not kn~vn at this time, but are believed to be minor. Habituated smokers, both male and female, metabolize nicot%ne more rapidly than non-s,~okers, indicating the bodily metabolic acclimation tonicotine. However, male non-smokers excrete more cotinine and less nicotine than fen~le non-smokers, indicating a sex-dependent metabolic difference in humans. In contrast to the lung absorption of nico.tine mentioned above, absorption • through the mouth is very~uch slov/er and inefficient. The nicotine in cigar smoke (pH 8.5) is almost all unionized and readily absorbed by the oral membraDes, if contact is made. But due to the slower absorption and transport, the concen- tration of blood nicotine achieved is never equivalent to that of lung absorption. The absorption of nicotine from an aqueous solution in the mouth is also pH dependent. The amount of free nicotine is a buffered solution held in the mouth thus determines the physiological response. Similar results are reported for ., nicotine absorbed from chewing gum. Also nicotine injected into other body tissues requires a 20-I00 times larger quanti~v, to ~roduce similar physiological responses, again due to ~uch slower transport wi~hin-t~su~ If. Snw)king_a._n_d__Healt_____h As~ (C. L. Neumann) T~o basic schools of thought exist currently: A. Reduce both "tar" and nicotine to achieve safety B.- Reduce "tar" and increase (or hold level) nicotine Classic~ skin painting experiments have tacitly assu~ed nicotine to be har~..less, as animals (~i~e) are usually conditioned to nicotine toxicity before ti~e zero. ~ Wynder and Floffmann report nicotine to be a very low level ciliastat: "Nicotine~ in the concentration present in cigarette smoke was not found to be ciliatoxic by ....... Boche and Quill~igan" (195g). .............. Beck has reported (1976 AACR meeting) lo~ to moderate concentrations of nicotine ,. act as a cocarcinogen stimulus when applied to mouse skin. During our discussion, ~ ........... the accuracy of the results of Beck (and of all the NCl skin painting studies) was ~ questioned. Possibly an explanation of nicotines promoter activity may be due to .: ...... its ability to rapidly transport across the skin barrier, an effect not relevant in inhalation studies. .~. III. Taste of Nicotine {C. E. Rix) ~ ~ Observations concerning the ta{te of aqueous nicotine are described below: Concentratio~ 10-5 M (1.67 ~,g/ml) 10-4 M lO-I M lO"l 14 Taste Perception None Some Taste (foul, rotten rubber) No Irritation Definite Burning Irritation l ~,l Puff: Strong Tongue Sting and Throat Grab

III 4 Sensitivity to nicotine occurs only on the front tip of the tongue. Nicotine is definitely an irritant in smoke and its taste must be blended out or modified by other constituents in the TPM to make the smoke acceptable. Propylene glycol and sucrose can reduce nicotine irritancy, but large amounts are necessary. "Neutralization" with acids also reduces irritancy somewhat and appears most promising as a taste modifier, IV, Factors Influencing Presence in Leaf and Smoke_(J, P, Dickerson} Nicotine is produced in the root system of the tobacco plant. The amount of nicotine produced depends on genetics, climate and cultural practices. Burley tobacco generally contains more nicotine than flue-cured. However, geneticists have produced low-alkaloid burley'varieties with nlcotine, contents as low as 0.2 percent. Similar flue-cured varieties are apparently not available. Experi- mental flue,cured varieities with hig~_nicotine levels, ca. 6 percent, have been produced. Annual variations in nicotine contentof burley and flue-cured tobaccos are primarily due to weather conditions. In dry weather the tobacco plant produces an extensive root system and as a result nicotine production increases. Excessive rainfall produces a smaller root system and also leaches nitrogen, which is necessary for nicotine production, from the soil. Fertilization and topping height are cultural factors which influence the amount of nicotine in tobacco. ~!icotine production in the plant can be increased by increasing the amount of available nitrogen in the soil. Nicotine content is inversely related to topping height. As the numbe~ ~f leaves on the plant is reduced by lowering the topping height, the ni~cotine content of the plant increases. Therefore, a low topping heiDht such as that employed in the loH-profile harvest system results in a nicotine level which is greater than that of conventionally gro~vn tobacco. The nicotinecontent of RJR domestic purchases varies from year to year. The 1969 and Ig70 hurley crops ivere exceptionally high in nicotine. In Ig71 the nicotine content of the burley tobacco dropped to one of the lowest levels that have been observed. During the 1971-1973 period both burley and flue-cured crops had low to average nicotine contents. The 1974.-1976 flue-cured cr~ps have been high in nicotine. Nicotine content of the flue-cured crops has increased each year since 1973. This increase has been due to lack of adequate rainfall. The hurley crops have produced reasonable levels of nicotine since 1972. The nicotine content of the 1976 burley crop is not known at this time, Nicotine content of both flue-cured and burley tobaccos varies with stalk position. Nicotine in the leaf and smoke increases from the bottom to the top of • the stalk. FTC "tar" values follow the same pattern. The "tar"/nicotine ra~lOS decrease from the bottom to the top of the stalk. The lowest "tar"/nicotlne ratios are observed in the tobaccos with the hiohest "tar" deliveries. company flue-cured and burley grades show variations in leaf and smoke nicotine which are related to stalk position, The flue-cured "straight" grades, which come from the lower half of the tobacco stalk, give lo~er nicotine analyses than the "X" grades from the upper stalk positions. Variations between grades in the "straight" g~ades series are relatively small with nicotine in leaf and smoke

increasing slightly es quality increases. Larger differences between grades are observed in the "X" grades and nicotine decreases with increasing quality. The burley grades are divided into three groups according to stalk position.' Within each group quality increases as nicotine increases. The transfer of leaf nicotine to smoke appears to follow a pattern similar to that of smoke pH in both burley.and flue-cured tobaccos. In the hurley tobacco both nicotine transfer and smoke pH increase with increasing height of the leaf on the tobacco plant. In flue-cured tobacco, both of these characteristic~ decrease from the bottom of the plant to mid-stalk and then begin to increase. Nicotine transfer in flue-cured is roughly inversely proportional to sugar content._,~ lhe major portion of nicotine in the smoke of ~IINSTON comes from flue-cured and burley tobaccos. Preliminary data from a study of WINSTON. blend components indicate that Turkish tobacco and G-7.-are responsible for less than 20 percent of the nicotine delivery of WINSTON blen~i~ Flue-cured tobacco contributes around 6D percent of the smoke nicotine and~eremaining.nicotine comes from hurley tobacco. The flue-cured appears to contribute more nicotine per gram of tobacco than the burley. This is due to the denicotinization of burley and the use of casing. In addition, nicotine content of the flue-cured which is used in current blends is relatively high. Additional studies of the relationship between nicotine delivery of WINSTON blend and nicotine delivery of individual blend components are in progress. !be Koo]:,.b]~end contains the most nicotine of the four major competit!ve brands (WI~N, SALEM, Marlboro). Marlboro contains the least. Nicotine deliveries of KooI,~~ALE~ and WINSTON have fluctuated between 1.2 and 1.4 mg for the past t~ years. Theli.~i~Iboro delivery has been consistently lower d~ring this period. Philip ~orrls~as maintained a relatively constant nicotine delivery of around 1.1 mg in t~arlboro for the past two years. ~he relatively low nicotine delivery ofi~ii~Iboro is partially due to the filter and rod characteristics of this cigarettd~ The pressure.drop of the Marlboro rod is lower than thet of ~-IINSTOI~ and the filter pressure drop ~s higher. The Marlboro filter ~s more efficient than~ that of WINSTON. An 85 mm filter cigarette from ~IIt~STOM blend with the Marlboro filter and tobacco rod characteristics delivers less "tar" and nicotine than ~!If&STO~ The transfer of leaf nicotine to smoke is lower in Marlboro than in WINSTON. The difference bet~veen the two blends is partially due to blend characteristics of the Marlboro which have not yet been defined. titerature reports indicate that as smoke pH increases the efficiency of nicotine filtration of cellulose acetate filters increases. In-house experiments indicate that this is not the case. Sugar in burley casing r~duces the nicotine delivery. A response surface (RSt!) study which is designed to define the relationship between casing and nicotine delivery is in progress. Additional information concerning nicotine in Company tobaccos and competitive brands is available in formal reports. RDR, 1976, No. 14 reports ~ited therein describe the relationship bebveen Company flue-cured grades and nicotine delivery for the 1972-1975 period. RD~, 1976, No. 16 contains competitive brand data for

6 1975. A review of in-house nicotine data is reported in RDR, 1975, No. 9. V. Nicotine and ~obacco Substitutes (C. L. Neumann) I. Sutton ] Material and Nicotine Deliver~iC Original RJR notebook data .showed two thin.o,s for nicotine transfer in Sutton I material, first that mixtures of tobacco and Sutton ] material did not transfer nicotine in proportion to the tobacco content, but rather, the expected amount was decreased as Sutton material increased. Secondly, nicotine or its salts added to Sutton material transferred to mainstream smoke at a rate about half that found for tobacco (9% vs 17-1g%). 2. Current Substitg.tes and_Nic.o.tine Deliver~ Extrapolating the data bore to otl~rlsubstitutes such as Cytrel, NS!4, J-lO, we may reas6~:'~hat they will transfe~:~cotine..to .smoke in a tobacco-like manner, as the available data shows that mixtures of these substitutes with tobacco have smoke nicotine levels proportional to tobacco content. 3. Inert. Extenders,. G-7L and N.i.coti.n.e Delivery_ Tobacco ~n~rporating inert extenders (CaCO3) transfer nicotine much like tobacco, at ~:~ transfer rate of ca, 17%; G-TL n~terial transfers nicotine at a so~)~'c lo)~<percent {I~..%). Both rates are corrected for cigarette burn rate.

I II I 7 V__$._._ ll__ico.~t_i~_e_.A_nalo~ and llimics (C. t. l(eumann) Standard relative activity on froo rectus r.}uscle preparations; ~ "~CH3 ~3 1.0 F =' 0.2 F = 0.00 0.5 F = 0.07 F = 0.015 ~re~bl e~ ~icotine ..... in ~ode 6) ~tion and aiso st~n"~d relative activity or C-,uinea piD ileum (nicotine = 1.0): "I~ = 0.00 N- cetyl nornicotlne is reported to have ca. nornicotine, 1/12 the activity of )(-ethyl

Conformational studies and x-ray diffraction show nicotine to have the following conforn~ation. This correlates vlith p~ost data. ~latural nicotine mimic~ are relatively abundant in nature. Those belovz shovz nicotine-%ike activity. They have widely varying activiKv levels and toxicity levels, however. Nicotine: found in Nicotiana species, (1~bI1i~.ii) h..oD.~too_~.l.lt, L_j(_co_o.gIL~_dium species, E.~_q~isetum arvense, ;~ucuna I)ruriens Anabasine: ~in Nicotiana, Anabasis a_p~lla, .C_~h_e_n_oDodiaceae Nornicotine*: in Ni___c~o.ti_a_na, Duborsia h_9.pwoodii-~d"and I forms) also dialcohDl and diketone active Cyt~~e ~ Conine Pelletierine.~, ~ Spartei ne ~ from hemlock 120 mg fatal t? man ’ .5 mg/kg = active dose from rootbark of pomegranite tree from yello~ and black lupin beans .......... *~-noro~cot,ne is ~eported to be 2.5X toxic as nicotine to rats inote that this means a 240Ib man has active and fatal dose equal Considering the wide abundance of nicotinic drugs, tobacco has assumed n very unique role in society. I do notthink the ranlflcations of nicotine being classed a~ a drug by FDA and other (foreign) agencies should be overlooked.

g ~/|I. What We Need to Know About Nicotine Due to a time limitation, this topic received virtually no attention. Several important questions were raised, however. C. R. Green and D. Lynm raised the questions concerning the minimum level of nicotine required for smoker satisfaction. R. t. Rowland asked if every possible variable had been investigated for its effect upon nicotine delivery to the smoker. It may be generally accepted • that the delivery of nicotine is changed by changing the type of tobacco leaf which is used in the cigarette. But, holding constant the tobacco which makes up the cigarette, are we cognizant of all other factors in cigarette manufacture which would change the nicotine delivery, particularly any factors which would allow a decrease in tar delivery without the accompanying ~roportional decrease in nicotine delivery. .... C.R. Greefi asked if nicotine in ~E~k~~as "free" or "bound" or some mixture of these two fo~s. Also, the question was raised if some physical response could be measured todetermine nicotine effect or satisfaction. :ki Xc: Dr. Alan R~dgman • Dr. D. L. Roberts ...... . Dr. M. E.r'.Sto~ " Dr, R..L. ~owland ~~r-"~ Dr. C. R. ~reen ~;~:' Dr. R. A. Lloyd ~ ~ Dr. Dwo tynm " Mr. R. E. Shackelford Dr. R. F. Moates Dr. C. L. Neumann ~- Dr. C. E. Rix :~" Dr, J. P. Dickerson