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/d'V7 TIII; COL:NC'ILFOItTOIiACCO RI:SF.ARCIi-U.S.A., I:1C. 110 EAST 59TH STREET NEW YORK. X. Y. 10022 Appl cation for Research Grant (Use exrro pages as needed) 1. Principal Investigator (give title and degrees). ,r% rq ersw es.. iv q A 47M In ~J U L 7 -1975 Date 6/30/75 ,. ---. Mario D. Aceto, Ph.D., Associate Professor of Pharmacology 2. Institution & address: Medical College of Virginia Virginia Commonwealth University Health Sciences Division MCV, Box 726 Richmond, VA 23298 3. Department(s) where research will be done or collaboration provided: Department of Pharmacology b. Short title of study; Antinicotinic Effects and Antianxiety Agents 5. Proposed starting date: J a n. 2 , 19 7 6 6. Estimated time to complete. 2 years 7. Brief descript on of specific research aims: 1) Determine the relative localization of nicotine-14C over a wide dose range in selected rat brain areas. 2) Determine the subce11u1ar distribution of nicotine-14C in the rat brain. 3) Study the effects of antianxiety agents such as iibriuinn and mepYoba_ mate upon nicotine-14C localization. 4) Attempt to ascertain a functional role for the central nicotinic ner- vous system as it relates to the mechanism of action of antianxiety agents. . . •:; • . I l

8. Brief statement of working hypothesis: ` Aithouqh it has been shown that cholinergic receptors in the central nervous `;ystem (CNS) may be either nicotinic or muscarinic (Eccles, 1964), little work has been done on the localization of nicotine in the brain and on its subcellu- lar distribution (Larson and Silvette, 1964, 1968, 1971). Even less is known about the role of the nicotinic nervous system and its interactions with CNS drugs. Studies by Hansson and 5chmiterlow (1962) have shoaln that soon after the administration of nicotine-methyl-14C in mice, very high concentrations of nicotine appeared in the CNS. Studies by the investigator (Aceto, 1967) showed that for the intraventricular (intracerebral) route of administration that a direct relationship between ganalion blocking potency and blockinq of nicotine extensor convulsions existed and that the site of nicotine extensor convulsions is central in origin and is associated with brain areas near the ventricles. Later in 1971, Benesova and Nahunek reported a correlation between the degree of antinicotine convulsant activity and the clinical efficiency of antidepres- sants in agitated depression. These studies encouraged the author to examine the possible relationship of the antinicotine effects of a wide variety of CNS agents to their clinical properties. A good relationship was found between blockage of nicotine extensor convulsions and sedative antianxiety properties (Aceto, 1975, accepted for publication in Pharmacology). This relationship was especially good for drugs designated as antidepressants, antipsychotics and anti anxiety agents. Because it was shown that for the druas classified as antianxi- ety agents, there was a direct relationship between the recommended therapeutic dose in man and antinicotine potency in the mouse, this study will focus on this relationship. - C . Details of experimental desian and procedures: The brain areas which we propose to investigate are the cortex, and cerebellum. The subcellular fractionation procedures described below are cur- rently being used in this laboratory, and are primarily based on those reported by DeRobertis et al., 1962; Mule et al., 1961; and Hokin and Hokin, 1958. I. SUBCELLULAR FRACTIONATION OF BRAIN TISSUE (DeRobertis et al., 1962; Mule et al., 1967) Brain tissue is homogenized with a teflon pestle for two minutes at a speed of 400 rpm. The homogenate is diluted with 0.32 M sucrose (Ca++) to give a final concentration of 1 9 of brain per 10 ml. An aliquot representing 10% of the total homogenate Is removed and labelled homogenate. The remainina homogen- ate is centrifuged at 900 times g for 10 minutes at 0•C in a Sorvall RCZ-B cen- trifugc. This centrifugation yields a crude nuclear pellet. The supernatant is decanted and the crude nuclear pellet is washed twice. The resulting sus- pension is then centrifuged at 900 times g for 10 minutes at 0•C. The superna- tant is decanted and cnmbined with the other supernatant. The final crude nu- clear peilet consists of nuclei, myelin, membrane fractions and tissue debris. The combined crude nuclear supernatants are centrifuged in the Sorvall RC2-B centrifuge at 11,500 times g for 20 minutes at 0•C to yield a crude mitochon- drial pellet which contains mitochondrial nerve endings, membrane fragments and myelin. The supernatant is decanted, and the pellet is washed once with 0.32 N C ucrose (Ca++) and centrifuged at 11,500 times g. The pellet wash is added to .he supernatant. The final crude mitochondrial pellet is resuspended in a vol- ~ ~ d aais ~ ~ .. . _ W

ume of 0~32„Msucrosetiequivalent to l/3 of`the origtnal~homogenate volume:+_,The `z d su erntant' combin if t d _ . p e m cros ma , ~ellet,-ts"'resuspended in 0.32 M sucrose (Ca++) for marker assays or in 0.1 N~- , „ . r o as l'. ~_he final'°soluble~supernatant fraction ellet "''whtch is th The i o ntrifugeat 124,OOO timesg. , The supernatant is decanted and eferred L p e s are cen uge r for 30 minutes In the Beckmn L350lt- a- ura Nc I t o r raatoactlve counttng• density gradient is prepared by layering 0.8 M, 1.0 M, 1.2 M and 1.4 M sucrose in,17-m1 cellulose nitrate tubes. An aliquot of the crude mitochondrial frac- tion Is layered on top. The tubes are placed in the Beckman L3-50 ultracentri- fuge•°and spun at 81,000 times g for 120 minutes. The following layers are ob- tained:: myelin, membrane fractions, cholinergic nerve endings, non-cholinergic nerve endings, and free mitochondria. IIOSMOTIC SHOCK OF NERVE ENDINGS AND ISOLATION OF SYNAPTIC VESICLES `,: 3~ya-~''#~~~'r'^ cles (Mp). The supernatant Is considered to be the final soluble supernatant _ 20 minutes at 0•C. The pellet (Ml) consists of swollen mitochondria, myelin = fragments, and the subsynaptic web. The supernatant is centrifuged at 124,000 times g for 30 minutes at 0'C. The pellet consists primarily of synaptic vesi- ; se solution. The dtluted CM Is homogenized for 2 minutes at 400 rpm. The . su r ~Ehomogena-te is centrifuged In the Sorvall RC2-8 centrifuge at 11,500 times g for o c ~'G••iorder to s.In isolate pure nuclei, a discontinuous sucrose density gradient `-y'- the crude mitochondrial (CM) fraction with 10 uM CaC12 to make a final 0.32 M~; B AC IO TI IV~` ~''with 0.8 M and 1.2 M sucrose is prepared in a 17 ml cellulose nitrate tube by a?'`adding 6.0 ml of 1.2 M sucrose and carefully layering 6.0 ml of 0.8 M sucrose ,~• won'top. 5.0 ml or less of the crude nuclear pellet which has been resuspended 4 c~ es J~- The pellet N3 contains whole ceTls, tissue debris and blood cells. `:p- . y , p y , P'of nuclei, but contains some mitochondria, myelin fragments and synaptic vesi- ° • i SU NA FR T ON OF THE CRUDE NUCLEAR PELLET . irr 0.32 M sucrose Is then layered on top and spun in the Beckman ultracentri-•,:', fuge at 81,000 tines g at 0•C for 120 minutes. The top layer, designated N', _ consists of large myelin fragments The middle la er N2 consists rimaril ~ 1~ing'subcellular fractions are mixed with an equal volume of 10% TCA in a 45 ml ,~ polyprcpylene centrifuge tube. The samples are centrifuged in the Sorvall t 'RC2-8 at 12,000 times g for 5 minutes at 0'C. The liouid is discarded by de-,, ~. rcantation and the pellets washed four times with cold 5% TCA. Each washing 1. onsists of adding cold 5% TCA and resuspending the pellet by gentle stirring II_C._ . y p - n an m o eac of t e rema n - BRAIN UB~FRACT ON~ATION~OF~THE CRUDE MITOCHONDRIA Li«..'><^w.i;q`:~#: r.'_ •J'.~i`/N-=`:-t":G?-<-".r: i s..T' - In order to isolate nerve endings and mitochondria, adiscontlnuous sucrose ..'T~+x Synaptic vesicles are isolated from nerve endings by diluting an aliquot of .Lf r' 7`ISOLATION OF DNA, RNAAND PHOSPHOLIPID FROM THE SUBCELLULARFRACTION OF R Brain tissue for marker assays is homogenized and subfracfionated as des- • :r~ crtbed above Twent ml of the su ernata d 5 t 10 l h f i h ' with a plastic rod. The suspension is centrifuged at 12,000 times q for 5 nutes and the liquid discarded. To the final pellets are added cold 5% TCA,`';;: d the pellets are resuspended by gentle stirring with a rod lastic stirrin k_.. p ,- g One-half of this suspension is removed and placed in a 15 ml polyethylene cen-'<: •trifuge tube for DNA assay. The remaining suspension is centrifuged at 12,000 '_ " ' tim f 5it Thtt i dtdd didd Thllt es gor mnues.e supernaansecane anscaree pee ~,.,, } ; 4,'' , n:~••• . .M7 ~ o W L't V

is then suspended in ethanol and CHC13 is added. The samples are stirred by CVortex until all material dissolves. The tubes are tightly stoppered and tored overnight at 4°C. One then adds 0.1 N HC1 (cold) and shakes to form an emulsion. The emulsion is centrifuged in the Sorvall RC2-B at 30,000 times g for 5 minutes at 0°C. The aqueous layer contains the RNA, and the chloro- fo.rm layer contains the phospholipids. VI. CHEMICAL AND ENZYMATIC MARKER ASSAYS In order to verify the morphology of each fraction, the following chemical and enzymatic assays are performed: 1. Protein is determined by the method of Lowry (1951). 2. DNA is estimated by the diphenylamine reaction described by Burton (1956). 3. RNA is estimated by Schneider's RNA assay (1957). 4. Phospholipid phosphorus is determined by the method described by Bartlett (1959). 5. Succinic deh drogenase activity is determined by the method of Bonner (1955~. 6. NADPH-cytochrome-C-reductase activity is determined as described by Mule (1967). DNA, RNA and phospholipid are isolated for assay as described previously. VII. DETECTION OF RADIOACTIVITY AND IDENTIFICATION OF NICOTINE An aliquot of the crude mitochondrial fraction is subjected to osmotic shock, rehomogenization and centrifugation in order to isolate synaptic vesi- cles. An aliquot of each fraction that contains radioactivity is oxidized in a Packard Tri-Carb Sample Oxidizer. The samples are counted in a Beckman scintillation counter and corrected for quenching by external standardization. To determine how much of the radioactivity is due to unchanged drug or metabo- lites, thin-layer and gas chromatography of organic extracts is done. ,_ - VIII. ANIMAL STUDIES The research plan is to first determine the disposition of nicotine-14C in male rats. Nicotine-14C will be given intravenously to six naive animals per dose and at lea t three doses of nicotine will be studied. At 5 and 20 minutes after nicotine-~4C, the animals will be sacrificed and the brain levels and subcellular disposition of nicotine will be determined. In the drug studies, animals will also be injected subcutaneously with selected antianxiety agents such as diazepam, chlordiazipoxide and meprobamate (each drug will be given at three dose levels and at two different time periods; namely, 2 and 2 hours) before receiving radioactive nicotine. Saline controls will be run with each drug group and the mean concentration of nicotine-14C in the various brain areas and subcellular fractions will be determined and expressed as pmol/Q + S.D. or as % of control and an analysis of variance of the data will be done with each drug. These results will be used to interpret the possible involve- ment and function of the nicotine nervous system as it relates to antianxiety agents. For the localization studies, 50 to 100 mg portions of selected brain areas will be ~Kidized'in the Packard Oxidizer and the radioactivity counted in the Beckmann scintillation nstrument. In the subcellular experiments, the selected brain areas will be pooled to yield sufficient tissue for the studies (2 g.). 1003540751

REFEltElCES Aceto, 3.U., Bentley, H.C. and Dembins4;i, J.R. (1969). Effects of ganglion blocking agents on nicotine extensor convulsions and lethality in mice. Br. J. Pharmac., 37, 104-111. Aceto.(1974). Effects of CUS agents on nicotine extensor convulsion and Ietiiality ttt mice and their sedative-antianxiety effects in man. Accepted for publication by Pnarmacoiogy, 1975. Bartlett, G.R., (1959). Phorphorus assay in column chromatograph. J. Biol. Chen., 234, 4GG-4GB. Benesova, 0. and flahunek, K. (1971). Correlation between the experimntal data from animal studies and therapeutic effects of antidepresant ; drugs. Psychopharnacologia, 20, 337-347. C Bonner, Fl.U. (1955). Succinie dehydrogenase. In t1ETHODS Itt EtfZYi10L0GY, eds. by ..P. Colowich and tl.0. Kaplin, Vol. 1, 722-729. Academic Press, tl.Y., N.Y. Biochm. J. 62, 315-323. Burton, K. (195G). A study of the condition and mechanism of the dyhenylanine reaction for the colorinetric estir.lation of deo;cyribonucleic acid. Curtis, H.R. (1963). The pharmacology of central and peripheral inhibitions. • Pharmac. Rev., 15, 333-364. Dale, H.H. (1914). The action of certain esters and ethers of choline, and their relation to muscarine. J. Pharmc. Exp. Ther., 6, 147-190. Uettoberti s, C., Pel l egri no De Iral di ,!1. , Rodri quez de Lores A., and Solganicoff, L.. Cholinergic and non cholinergic nerve endings in rat brain-Isolation and subcellular distribution of acetyl chain and acetyl cholinesterase. (1962). J. of Ueurochem., 9, 23-35. . EcciEs, J.C. THE PHYSIOLOGY OF SYPI/1PSES. Springer-Verlag, Berlin; Academic Press, Inc., Netit York, 1964. Hansson, E. and Schmiterlow, C.G. (1:362). J. Phat•macol, 137, 91. Hokin, L.E., and Hokir., 11.R. (1958). Phosphoinositides and Protein Secretions in Pancreatic Slices. J. Biol. Chen., 233, 805-810. Langley, J.H., (1905). On the reaction of cells and of nerve-endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and curare. J. Physiol., Lond., 33, 374-413. Larson, P.S., and Silvette, Tobacco Experimental and Clinical Studies. A Comprehensive Account of the World Literature. William and Wilkin, Baltir,wre, 1961, and Supp. I, 1968 and Supp. II, 1971. 193, 265-275. Protein measurement with the Folin phenol reagent. J. Biol. Chem., Lowry, O.H., Rosenbrough, tl.F., Farr, A.L., and'Randall, R.J. (1951).

Mule, S.J., Kednan C.11., and Tlesher, J.11., (1967). Intracellular dis- position of H~-r•iorphine in the brain and liver of nontolerant and ~ tolerant guinea pigs. J. Pharmacol. Exp. Therap., 157, 459-472. Schneider, W.C.,•(1957). Determination of nucleic acids on Tissues by Pentose llnalysis. METHODS OF EiIZYN0LOGY, ed. by S.P. Cotowich and ; N.O. Kaplan, Academic Press, N.Y., N.Y., Vol. 3, 680. Silvette, fi., Hoff, E.C., Larson, P.S. and ffaag, H.B., (19G2). The actions •- •: of nicotine on central nervous system function. Pharmac. Rev., 14, •_ " 137-173.

3. 10. Space and facilities available (when elsewhere than item 2 indicates, state location): r, . • . , The Department of Pharmacology is currently occupying 20,000 square feet of •space. The department has a library which makes the important journals availa- ble for the members. In addition, a new Health Sciences Library has been built in close proximity to the Department of Pharmacology. The Department of Pharmacology has its own animal facilities and they have recently been refurnished. Attendents for the care of the animals are supported by the department. _ The CNS Division of the Department of Pharmacology occupies a space of 8,000 square feet with office and research space available. These laboratories are well supplied with pharmacological equipment. The proposed study will be con- ducted in one of these laboratories. ~ 11. Additional facilities required: None •- - , .. . .> _ 12. Biographical sketches of invest;gotor(s) and other professional personnel (append): 13. Publications: (five most recent and pertinent of investigator(s); append list, and provide reprints if avoilable).

R: REDACTED MATERIAL Biographical Sketch C - Name: Aceto, 4lario D. Role in-Project: Principal Investigator Date of Birth: REDACM tiarital Status: RWACTE® ` Education: University of Rhode Island, Kingston, R.I. - I3.S. Pharmacy - _ University of Maryland, College Park, MD. -;q.S. Pharmacology - 1955 University of Connecticut, Storrs, Conn. - Ph.D. Pharmacology - 1959 Professional Experience: 1973 - Associate Professor of Pharmacology, !ledical College of VA. 1973 - 1973 Project tlead, Sterling-l9inthrop Research Institute 1967 - 1973 CiIS Secti on llead and Pro ject Leader, Sterl i ng-tli nthrop 196G - 1973 Research Institute Senior Research Biologist (Pharmacology) Sterling-llinthrop 1964 - 1966 Research Institute Research Biologist (Pharnacology) Sterling-Winthrop Research 1963 - 196G Institute Group Leader (Pharmacolagy) Sterling-Winthrop Research Institute 1964 - 1972 Lecturer in Pharmacology, Albany 1•tedical College 1962 - 1963 Associate Research E~iologist (Pharmacology) Sterling-Winthrop 1959 - 19G2 Research Institute Assistant Professor, University of Pittsburgh 1958 - 1959 Instructor, Pharmacology, University of Pittsburgh 1956 - 1953 Graduate Assistant, University of Connecticut 1953 - 1956 Graduate Assistant, University of Maryland C Honors: Honor Achievement Award granted by the American College of Angiology for the top animal study published during a 5 year period in Angiology in June, 1966. REDACTED Publications: Aceto, 11.U., Harris, L.S., Dewey, W.L. and Balster, R.L.: Dependence studies of new compounds in rhesus monkeys. Committee on Problems of Drug Dependence, 1975. (In Press). t Aceto, M.D. and Harris, L.S.: Comparative study of the effects of two narcotic antagonists naloxone arid nalorphine on developing dependence in rhesus monkeys. Accepted for publication in J. Pharmac. Exptl. Ther. (1975). Aceto, t1.D.: Effects of CUS agents on nicotine extensor conculsions and lethality in mice and their sedative-antianxiety effects in man. Accepted for publication in Pharmacology (1975). Aceto, ti.D., Harris, L.S., Balster, R.L., and Dewey, tI.L.: Evaluation dependence liability of narcotic agonist and antagonist. Committee on of Drug Dependence 77-78, 1974. C of ~.S Problems Q O ..W . .~ ~ - IA . C1t 3 ` • • 4 U- ,l n 5

C ` ) Publications (continued) Clarke, R.L., Daum, S.H., Gambino, A.J., Aceto, M.D., Pearl, J., Leavitt, M., Cuminsky, W.R., and Bogado, E.F.: Compounds affecting the central nervous system 4. 3B-Phenyltropane-2-carboxylic esters and analogs. J. Med. Chem., 16: 1260-1967, 1973. Daum, S.J., Aceto, M.D., and Clarke, R.L.: Compounds affecting the central nervous system 3. 1 3S-Phenyltropan-2-ols. J. Med. Chem., 16: 667-670, 1973. Daum, S.J., Gambino, A.J., Aceto, M.D.G., and Clarke, R.L.: Compounds affecting the central nervous system. J. Med. Chem. 15: 509-514, 1972. _ P.T.: Pharmacologic properties and mechanism of action of amfonelic acid. Eur. J. Pharmac., 10: 344-354, 1970. Aceto, M.D.G., Botton, I., Levitt, M., Martin, R., Bentley, H.C., and Speight, ists on the straub tail reaction in mice. Brit. J. Pharmac., 36: 225-239, 1969. Aceto, M.D.G., McKean, D.B., Pearl, J.: Effects of opiates and opiate antagon- Aceto, M.D.G., Bentley, H.D., and Dembinski, J.R.: Effects of ganglion blocking agents on nicotine extensor convulsions. Brit. J. Pharmac., 37: 104-111, 1969. Pearl, J., Aceto, M.D.G., and Fitzgerald, J.: Differences in antiwrithing activity of morphine and nalorphine over time and in slopes of the dose-response lines. Psychopharmacologia, 13: 341-345, 1968. Pearl, J., Aceto, M.D.G., and Fitzgerald, J.: Stimulant drugs and temporary increase in avoidance responding. J. Comp. Physiol. Psych. 65: 50-54, 1968. Pearl, J., Harris, L.S., and Aceto, M.D.G.: Prevention of writhing and other effects of narcotic and narcotic antagonists in mice. J. Pharmacol. and Ex.p Therap., 160: 217-230, 1967. Aceto, M.D_, Harris, L.S., Lesher, G.Y., Pearl, J., and Brown, T.G.: Pharmacologic studies with 7-Benzy1-l-ethyl-1, 4-dihydro-4-oxo-1, 8-naphthy ridine-3-carboxylic acid. J. Pharmacol. Exp. Therap., 158: 286-293, 1965. Pearl, J., Aceto, M.D.G., and Fitzgerald, J.J.: Drugs and avoidance performance Psychon. Sci., 6: 41-42, 1966. Harris, L.S., Pearl, J., and Aceto, M.D.G.: Similarities in effects of barbiturates and mild tranquilizers on activity in mice. Psychon. Sci., 4: 267- 268, 1966.

R: REDACTED MATERIAL C Biographical Sketch Name: Dewey, William L. - Role in Project: Date of Bi rth: REDACTED Marti a1 Status : Education: St. Bernardine of Siena College,= - Loudonville, N.Y. - B.S. College of Saint Rose; Albany, N.Y. - M.S. Univ. of Connecticut, Storrs, Conn. -Ph.D. Professional Experience: Co-Investigator REDACTEfl Biology - 1957 Biology - 1964 Pharmacology - 1967 1973 - Associate Professor Dept. of Pharmacology, Medical College of Va., Richmond, Va. 1969 - 1973 Assistant Professor of Pharmacology, University of North Carolina, Chapel Hill, N.C. 27514 1971 - 1973 Consultant, Sharps Associates, 7678 Concord Ave., Cambridge, Mass. 1968 - 1969 Instructor of Pharmacology, University of North Carolina, Chapel Hill, N.C. 27514 - 1967 - 1970 Consultant, Arthur D. Little Inc., Cambridge, Mass. 1967 - 1968 Postdoctoral Research Trainee. Neurobiology Program (MH-1107- 01) University of North Carolina, Chapel Hill, N.C. 1966 - 1967 Postdoctoral Research Fellow, Dept. of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, N.C. 27514 1964 - 1966 Graduate Assistant, School of Pharmacy, University of Connecticut 1959 - 1964 Assistant Research Biologist, Sterling-Winthrop Research Institute Rensselaer, N.Y. . - Honors : REDAC;ED Publications: F~- 0 0 G7 ' C!t ~ O ~ C!t Aceto, M.D., Harris, L.S., Dewey, W.L., and Balster, R.L.: Dependence studies ~ of new compounds in the rhesus monkey (Submitted for publication.) Dewey, William L., Patrick, Graham A. and Harris, Louis S.: Annual Report: Narcotic Antagonists in the rat infusion technique (submitted for publication.) Dewey, William L. (Book Review) Narcotics and the hypothalamus, Kroc Foundation Symprsia No 2. Zimmerman and Gerge editors. Amer. J. Pharm. Ed. (in press). ~ Spaulding, T.C' and Dewey, W.L.: Some effects of the behaviorally active drug, NnenI i.rvne d purpurLeu nasnrsn anu cXn anLayurn5L, un oraln noraareneryrc c+nu serotonergic systems. Res. Comm. Chem. Path. and Pharm. (in press).