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Date. . Nameoflnvestigotbr(s)e(inciudeTitleandDegrees) Herbert McKennis, Jr., Ph.D., Professor of Pharmacology z Uutttutton & • Address: i`ledical College of Virginia, Richmond, Virginia 23219 Factors Controlling the Biological Disposition of Pyridine .;Coupounds of Tobacco Smoke {~. . . _ :• I. ProposedStartingDate: July 1, 1967' =` S. AnGcipoted Duration ofithis Specific Study: 1967-197D (3 years) 'it has been possible to•describe the broad outlines of many of the routes involved in the 6. Brief Descripton of Objectives or Specific Aims: From a series of experiments with support of the Couneid for Tobacco Researeh-U.S.A., .:~r -__c:;, i, ..c;.•oammalian metabolism of nicotine, nornicotine, and 3-acetylpyridine -- three of the many ; pyridine compounds that occur in tobacco and tobacco smoke. [The general nature of the 4igiRf. FI previous studies are given in the publications which are enumerated in an attached 1ist.) Through. isolation or total synthesis it has been possible to supply many of the mammalian .::pharmacologica3 properties of many nicotine metabolites. •' metabolites to biological investigators and. to assist them in a variety of work on the +=" , It is now desirable for us and interesting to us to expand our knowledge of the factors ~.='~'''which determine the disposition of pyrid3ne compounds arising from tobacco smoke and eaptore oiocnemicai1 inieraccions OeLWCeID Yml1AC WNpvucuw. rLC16 a~s aireauy in tne puo- lished literature some data that suggests that desensitization or tachyphylaxis to nicotine arises in part through a stimulation of the enzymes that are concerned with, the : aetabolism-of nicotine. It has been suggested that this stimulation of metabolism is ; produced by nicotine and:that other substances have a similar capacity to enhance the metabolism-of nicotine. Direct studies on nicotine metabolism-are however very limited ., in their scope. "determine the nature of stimulation or inhibition of metabolism at almost all- of the key C points that are involved in mammalian systems. Similarly, it should be possible to determine individual differences in the capacity to metabollize nicotine and related pyridine compounds. (continued on sheet la attached). 7. Give a Brief Statement of your,Working Hypothesis: Current data on the effect of various smoke components on the metabolism and transfer of nicotine and other alkaloids are limited or apparently contradictory. A study of these factors should be helpful to an- understanding of the.biological effects of tobacco smoking. have already described,, and through synthetic routes which make possible the labelLing of nicotine and related in almost any desired position, we believe that it is now possible to Through the current availability of synthetic routes to nicotine metabolites, which we [o-`mm a- CiAW 49MM ~ ~

Brief Description of Objectives or Specific Aims: (continued) ~.,_ . ~ . .. . . -~~. ..',. -.. ,. , . . .-. In the case of some of the simpler metabolites of nicotine, such as methylamine, 'it is "i relatively easy to obtain a broad picture of the nature of compounft which interfere with metabolic events. Work with 1`'C-methylamine in our own laboratory (The Pharmacologist, Fall 1967, in press) and others illustrates that interference with oxidation of rz ;}` T methylamine maY be produced by some types of monoamine-oxidase inhibitors. The data " suggest, however, that the inhibition may be referrable to-a so-called methylamine : oxidase which has not been fully characterized. Liver preparations obtained from rats after administration of monoamine-oxidase inhibitors have thus far been shown to be as fully capable as control livers in tests which, involve a spectrophotometric measurement of the disappearance of•nicotine. From another series of experiments, the data now suggests that the oxidation of nicotine can be impeded by the presence of large quantities of cotinine. It would, be desirable to study possible effects of other pyridine compounds 'T of smoke on the metabolism of nicotine and related substances. The.need for a biologically. , oriented study appears to be re-emphasized by apparently conflicting chemical data on the _ oxidation of nicotine. For instance, the pyrolidine-N-oxide of nicotine has been considered; (by J. C. Craig) to be the logical key intermediate in the biological.formation of cotinine, while Linnell has reported that the same oxide in vitro inhibits the oxidation ;..;: ~f nicotine to cotinine. 0

1 8. Details offiExperimentat Design and Procedures: (Attach Separate Pages) With the aid of isotopically labelled.nicotine and its metabolites it is feasible to study by a number of different methods factors influencing the disposition of nicotine. It is anticipated that during the current studies both in vivo and!in vitro methods will be employed. _ Initial experiments to determine the suitability of the rat as an experimental animal for initial studies have already beem conducted. Liver microsomal preparations from both :young and old rats of the >:,'?Ystar strain have been examined for their capacity to metabolize . ;;;,y nicotine in vitro. The general picture currently presented is that equivalent fractions " by weight of liver that are obtained from older animals (250-550g body weight) do not ~: metabolize nicotine as effectively as do the respective fractions from younger animals (150-175 g body weight). It is not currently:known whether or not the observed deficiency •in the older animals can be attributed to a lesser amount of metabolizing cellular •'•.fractions per unit of liver weight, or an overa•11 lessening of metabolic ability that exists throughout the animal. It would-be desirable to settle this point and to determine ; whether or not the elimination of unchanged nicotine by the older animals suffers any ` impairment. The additional matter of elimination can.be settled without modification of the experimental design of the metabolic experiments. Questions of this type can be .•"answered by comparing the ability of groups of young and old'animals to'metabolize (continued--on sheet 2a attached) • . 9. • Physical Facilties Available (Where Other than Administering Organization Indicate Geographical Location) Laboratories and animal facilities are.located on the fourth and fifth floors of McGuire Hall at the Medical College of Virginia. Gas chromatography and isotope counting facilities are available in areas convenient to the working laboratories but separate from these. The equipment list includes a preparative ultracentrifuge, polarimeter, Wa bu a lratus, hydrogenation apparatus, and!the general equipment of chemical, and 1~ A~citiortequirements: biological laboratories. , Additional requirements, if any, would depend!upon the outcome of the research. J • 11. Biographical sketches of all:principal and professional personnel (append) 12. List of publications: (Five most recent as pertinent) (append) -~~ _..,...,.....- .._.KS,.r. ~ . . • ... .. ... ._ . ..- . _.._ .. .._ .~.._....._._,.. .__ .. ...._.,_..,...- .~.~.,.~,.._,..~ ......_

• 2a. 8. Details of Experimental Design,and Procedures: (continued) 1'+C-labelled nicotine and to eliminate unchanged alkaloid. For all of this isotopic material is now available.as a result of our synthetic work and previous nutriculture: nicotine-1`'C (random), nicotine-14C-methyl, and nicotine-2-14C. -"`'In additiom, six of the mammalian metabolites of (-)-nicotine are available in both• _aF . "' isotopic and'non-isotopic form so that quantitative data can be obtained. 0 .

R: REDACTED MATERIAL •- E. Consvmabie Supplies (iist by catepories) --Chemi.cal Reagents .Isotopes Glass ware : Animals and feed Year 2 Year 3 C. Other: Expenses Qtemize) Reprints and page charges •Travel (Presentation of reports and conferences) E.. Overhead (] 5% of A+ B+ C) EstimatedFuture Requirements: Salaries Consumable Suppl. It is understood thotthe applica in applying fona grant have re the Council's "Statement oi. Pot and?erms Under Which Project Sub Totai I OtherExpenses PermanentEquiR. 250 500 Ovencead Total S,S00 _ 1,000 1,000 6,675 52,175 6,000_ 1,000 1,000 7,050 55,050 // ~ Siqnature 1'..~- ~ t. / / /y~.( r t and institutionai officen 644-9851 xt. 8153 ~ ad and found accept661t - cy Containing Conditions Signature Telephone Grants Are Made-`° wu ~aoflk..ohhtlmGru6°n ~•1NJE. ~L [;~: ;S VICE Te4ephone n i ML VJ ~a );h W 'L•-•- "~ +~ at meetings Ar=

i , List financial support fea research from oPl sources, tndudinp own instituNon, for this and/or related research projects. . Quantitative Methods for the'Detesmination*of the Distribution of Nicotine and Its Congeners in Biological Systems Institution supplies salary of principal-investigator and all secretarial help currently obtained through other funds None other than this application for the Council for Tobacco Research - U.S.A. VD99PSC00t

R: REDACTED MATERIAL B'iographical'Inyormation on HERBERT I•icKEtJi+tll S , JR. '9-.. ~- _ _ ~ Attended elementary schools in Scarsdale, N.Y. and Loomis School, Windsor, Conn. , Harvard S~.II . , ~ and Cornel•-l. Fh..D-. Chemist at Nuodex Products Co., 1938-1939, and Ciba Pharmaceutical Products Co,, 1940-1942, Appointed Assistant in Biochemistry, Cornell University Medical College, 1942; Assistant Professor of Chemistry, Medical College of Virginia, 1945; Instructor in Physio- logical.Chemistry, the Johns H'opkins-University, 19:46; Associate Professor of-Biochemistry, Medical College of Virginia, 1948; fie•ad, Basic Sciences Res'earch Department, Naval C. E. Laboratory, 1949; Associate-Professor-, 1953, Professor of Pharmacology, Medical College-of Virginia, 1955. Biochemist; specialist in intermed!iary metabolism, anti- biotics, alkaloids, polymeric compoundis. U..S. and-foreign patents: soil stabilization and antispasmodic. Has done a wide variety of scientific-advisory and technical consulting work for industrial firms, government agencies,' scientific organizations, and universities. Visiting Professor•, University of Chile, 1960; Hono•ra•ry member-, Faculty of Medicine, University of Chile, Sociedad de Biologia, de Santiago. Sigma Xi. Phi Lambda Upsilon. American-Chemical Society, American Society of Biological Chemists, The Society for Experimental Biology and Medicine, International Oceanographic R'oundation, The New York Academy of Sciences, American Society for Pharmacology and Experi- mental Therapeutics, American Associ!_&ti:orn for the Advancement of Science, Society of American, Mil-itary Engineers, American Institute of Chemists, Virginia Academy of Science, Society of Toxicology.

s- Edward R. Bowman Born: West Virginia 1927 Medical C.>llege of Virginia Citizen U.S.A. Richmond, Virginia Education: -Concord College B.S. 1952 (Biology - Chemistry). West Virginia University 1953 (Physi:ology). Duke University 1955-56• (Graduate Student-in Physiology). %=. Medical College of Virginia PheD. 1963 (Pharmacology). Experience: 1961 - present • 19'56 - 1958 1955 - 1956 1954 - 1955 ~ 1952 - 1953 1952 195 0 - 1951 •.19'47 - 1950 •1944 - 1946 Research Associate Department of Pharmacology Medical College of Virginia Richmond, Virginia Graduate Student, Major - Pharmacology Minor - Physiology & Biochemistry Medical College of Virginia, Richmond., Virginia Research Assistant Department of Pharmacology Medical College of Virginia Richmond, Virginia Graduate Student, Major - Physiology Minor - Anatomy Duke University Durham, North Carolina Bacteriologist State Department of Health Richmond, Virginia Graduate Student, Physiology West Virginia University Morgantown, West Virginia Student, Biology & Chemistry Concord College N Athens West Vir inia O , g O U.S. Army ~JI EA Student, Biology & Chemistry Concord College ~ Athens , West Vir inia g Q? U.S. Arm.y

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PUBLICATIONS OF PROJECT WORK SUPPORTED BY THE COUNCIL FOR TOBACCO RESEARCH - U.S.A. 1. Synthesis and properties of pyridylalanines. Herbert McKennis, Jr., and Edward R. Bowman. Virginia Journal of Science, 8, 314 (1957). 2. Metabolism of y-(3-pyridyl)-Y-oxobutyric acid. Lennox B. Turnbull, Edward R. Bowman, and Herbert McKennis, Jr. Federation Proceedings, 17, 325 (1958). 3. Y-(3-Pyrid)rl)-Y-methylaminobutyric acid as a urinary metabolite "of nicotine. Herbert McKennis, Jr., Leiinox B. Turnbull, and Edward R. Bowman. Journal of the American Chemical Society, 79, 6342 (1957). 4. Metabolites of nicotine and a synthesis of nornicotine. Herbert McKennis, Jr., Lennox B. Turnbull, Harvey N. Wingfield, Jr., and Lovell J. Dewey. Journal of the American Chemical Society, 80, 1634 (1958). 5. The role of cotinine in nicotine metabolism Herbert McKennis, Jr., Lennox B•. Turnbull, and Edward R. Bowman. Abstracts of Communications, IV. International Congress of Biochemistry, Vienna, Sept. 1-6, 1958. 6. 7. 8. A constant rate infusion apparatus. Quentin S. McKennis, Edward R. Bowman, and Herbert McKennis, J Toxicology and Applied Pharmacology, 1, 61 (1959). Metabolism of nicotine to (+)-Y-(3-pyridyl)-y-methylaminobutyric acid. Herbert McKennis, Jr., Lennox B. Turnbull,*and Edward R. Bowman. Journal of the American Chemical Society, 80, 6597 (1958). Metabolism of nicotine in the human and excretion of pyridine compounds by smokers. Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. The Journal of Pharmacology and Experimental Therapeutics, 127, 92 (1959). Demethylation of cotinine in vivo.. N O ~ Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and Einosuke P+ada. Journal of the American Chemical Society, 81, 3951 (1959). GJ CA ~,. Oxidation of cotinine in vivo. ~ Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. ~ ~ Federation Proceedings, 18, 371 (1959).