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Trtr•. CocncIa. FoH ToaACCO RESLARCH-[;.S.A., INC. March 10, 1975 Grant Application No. 869A PBARMACOIACY {i Kr IJSSOe,1' '!n: The ComIDittee comiprising Drs. Feldman, Meiet• and Sommers (Gardner) StIDJF7CT: Ronald P. Rubin, Ph.D., Medical College of Virginia, Richmond Continuation application No. 869A "The Action of Nicot.ine on the Adrenal Gland" (See Stone's memorandum of January 16, 1975) Dr. Gardner ccxmaented on this application that "the levels of nicotine used seem high --- 60-600 micro molar," and Dr. Lisanti as monitor, asked Rubin what considerations led his to adopt the levels used. b copy of his reply is attached. Comwents. Of course it is legitimate for apharmacologist to use large doses to find out what kinds of things can happen, but sooner or later he has to reduce the conditions to those that could occur in ordinary life, in order to ag1araise their actual significance. Because cf the way Rubin reports his experiments, it is difficult to determine the nicotine concentrations actually present in the reaction media. A 6 micro molar solution of nicotine contains ~.y734 g'. of nicotine per liter or a0009734 g. per ml. A 60 micro molar solution contains 9.734 g./liter or 0•009734 g•%• (9•734 mg./ml) and a 600 micro molar solution contains 97.34g./liter or o.09734 g./ml. We are told that the test media contained 250,0D0 cells per milliliter but are not given the volumes of solution at outset nor the volumes of nicotine solutions added. Rubin states that his nicotine concentrations were "in thQ order of 1-10 micrograms/ml -- approximating those found in blood end tissue after chronic exposure to tobacco smoke" citing Larson and Silvettea, Tobacco , Suppl. II. (I do not find the figure he quotes in the reference cited. Wolf et al. working back in 1948 with the la,borious steam distillation - cyanogen bromide technique, found nicotine levels of 0.1-0.2B mcg./ml in the blood of human smokers. Desoille et al•., using the :azne method in 1963 reported 1 to 9.8 meg/ml. His values havie been considered doubtful because they are so far out of line with many others. None of the earlier workers, however, appears to have been fully aware of the rapidity with which nicotine is metabolized and excreted. Their methods not have very adequately aoroimnodated the time factor in my case. Armitage et al., in 1974, reported that four human subjects who inhaled habitually, achieved arterial bbod nicotine levels of 30-40 ng./m1 (0.03-0.04 mcg./ml) when smoking, as normally as possible, one radin].abelled cigarette, at one puff per minute for 9-1.1 total pui'fs. (They were estimat:~d to have absorbed from 1.83 to 2.62 mg. of totpl n•s.--tiae from the cipnrette, or 19% of that originally presert.)

-2- March 10, 1975 NKqlJh5031 Isaac and Rand, (1969) in a smoke inhalation study employing dogs and a GLC method of analysis, found peak bbod levels of 0.15 to 0.25 mcg/ml io:mediately after, each pu#Y', and noted the rapid drop in concentration, between puffs. This was ore of the early attempts at a kinetic study, though the methods were laborious. 9chieve3bein and Grundke (1968), using a GLC method, found 0.005 to 0.064 mcg/ml in human venous blood ten minutes after the smoking of one cigarette. it appears that Rubin is probably citing a report based upon Desoille's f'igures. These appear to be very high as comp'ared to the others given above. The need for a radioiasnuno asss4y that is rapid, sensitive, specific, reliable and applicable to smal7l bbod and tissue samples is manifest, to provide better infor- matioti on the absorption rates, peak blood levels atb;ined, tissue distribution, disappearance rates and rapidity of excretion of nicotine during smoking (and chewing) as practised in ordinary life conditions. Such a method, apparently now nearly within grasp, should considerably assist in the design and interpretation of such pharmacological studies as this. It.C.H. 1tCH,/1p cc: David Stone V. F. Lisanti J. H. Kreisher Sumnsry Blood levels of nicotine after smoking Schievelbein 0.005 - 0.064 mcg./ml (196$ ~ human (venous) Armitage 0.03 - 0.04 mcg./ml (197~+ human (arterial) Wolf 0.10 - 0.28 mcg./ml (1948) human Issac 0.15 - 0.25 mcg./ml 1969 dogs Desoi]1e 1.0 - 9.8 mcg./ml ~1963~ human