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Pharmaceutical Sciences Faculty Patents Pharmaceutical Sciences

7-11-2000 Use of Compounds in the Treatment of Central Nervous System Diseases and Pathologies Peter A. Crooks University of Kentucky, [email protected]

Linda P. Dwoskin University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits oy u.

Follow this and additional works at: https://uknowledge.uky.edu/ps_patents Part of the Pharmacy and Pharmaceutical Sciences Commons

Recommended Citation Crooks, Peter A. and Dwoskin, Linda P., "Use of Lobeline Compounds in the Treatment of Central Nervous System Diseases and Pathologies" (2000). Pharmaceutical Sciences Faculty Patents. 94. https://uknowledge.uky.edu/ps_patents/94

This Patent is brought to you for free and open access by the Pharmaceutical Sciences at UKnowledge. It has been accepted for inclusion in Pharmaceutical Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. US006087376A United States Patent [19] [11] Patent Number: 6,087,376 Crooks et al. [45] Date of Patent: *Jul. 11,2000

[54] USE OF LOBELINE COMPOUNDS IN THE Scherman, D., “ binding and monoam TREATMENT OF CENTRAL NERVOUS ine uptake in mouse brain regions,” J. Neurochem. 47, SYSTEM DISEASES AND PATHOLOGIES 331—339 (1986). Scherman, D. et al., “The regionaliZation of [3H]dihydrotet [75] Inventors: Peter A. Crooks; Linda P. DWoskin, rabenaZine binding sites in the mouse brain and its relation both of Lexington, Ky. ship to the distribution of monoamines and their metabo lites,” Brain Res. 370, 176—181 (1986). [73] Assignee: University of Kentucky Research Scherman, D. et al., “[3H]Dihydrotetrabenazine, a neW in Foundation, Lexington, Ky. vitro monoaminergic probe for human brain,”J. Neurochem. 50, 1131—1136 (1988). Standaert, D.G. et al., “Treatment of central nervous system [*1 Notice: This patent is subject to a terminal dis degenerative disorders,” The Pharmacological Basis of claimer. Therapeutics, 9th ed. (Hardman J .G., Limberd L.E., Moli noff P.B., Ruddon R.W., and Gilman A.G., eds.), pp. 503—519, McGraW—Hill, NeW York (1996). [21] Appl. No.: 09/089,420 Olin, B.R. et al., “Smoking Deterrents,” In Drug Facts and Comparisons. 1995 edition, ed. by B.R. Olin et al., pp. 3087—3095, St. Loius, MO: J.B. Lippincott Co., 1995. [22] Filed: Jun. 3, 1998 Sloan, J .W. et al., “The comparative binding characteristics of nicotinic ligands and their pharmacology,” Pharmacol. Related US. Application Data Biochem. Behav., 30:255—267 (1988). [63] Continuation-in-part of application No. 08/795,852, Feb. 5, Hamann, S.R. et al., “Hyperalgesic and analgesic actions of 1997, Pat. No. 5,830,904. , U50—488, , and (—)lobeline in the rat brainstem,” Pharmacol. Biochem. Behav., 47:197—201 [51] Int. Cl.7 ...... A61K 31/445 (1994). [52] US. Cl...... 514/317; 514/331 Brioni, J .D. et al., “Nicotinic receptor exhibit [58] Field of Search ...... 514/317, 331 anxiolytic—like effects on the elevated plus—maZe test,” Eur. J. Pharmacol., 238:1—8 (1993). [56] References Cited Decker, M.W. et al., “Effects of lobeline, a nicotinic receptor , on learning and memory,” Pharmacol. Biochem. U.S. PATENT DOCUMENTS Behav., 45:571—576 (1993). 3,901,248 8/1975 Lichtneckert et al...... 131/2 4,971,079 11/1990 Talapin et al...... 131/359 (List continued on next page.) 5,272,144 12/1993 Melloni et al...... 514/227.5 5,403,595 4/1995 Kitchell et al...... 424/501 Primary Examiner—Phyllis G. Spivack 5,414,005 5/1995 Schneider et al. 514/343 Attorney, Agent, or Firm—McDermott, Will & Emery 5,468,755 11/1995 Cincotta et al. .. 514/288 5,486,362 1/1996 Kitchell et al. 424/426 [57] ABSTRACT 5,536,503 7/1996 Kitchell et al. 424/449 5,552,429 9/1996 Wong et al...... 514/415 Lobeline and evoke [3H]over?oW from rat striatal 5,576,321 11/1996 Krushinski, Jr. et al...... 514/255 slices preloaded With [3H] ([3H]DA). The lobeline-evoked over?ow is calcium-independent and not FOREIGN PATENT DOCUMENTS antagonized by , suggesting a mechanism of action other than the stimulation of nicotinic receptors. WO92/19241 11/1992 WIPO . Whereas nicotine stimulates nicotinic receptors, lobeline OTHER PUBLICATIONS inhibits [3H]DA uptake into synaptic vesicles and striatal synaptosomes. The results suggest that different mecha Levin, E.D., Drug Dev. Res., 38(3—4), 188—95 (abstract), nisms are responsible for the increase in striatal DA release 1996. evoked by lobeline and nicotine. [3H]-DihydrotetrabenaZine BarloW, RB. et al., “Relationship betWeen structure and [3H]DTBZ), used routinely to probe a high-af?nity binding nicotine—like activity: X—ray crystal structure analysis of site-on the vesicular (VMAT 2) binds (—) and (—)lobeline hydrochloride and a comparison to vesicle membranes from rat striatum. Lobeline inhibits With (—)nicotine and other nicotine—like compounds,” Br J. [3H]DTBZ binding With an IC5O of 0.90 nM, consistent With Pharmacol., 98:799—808 (1989). its IC5O of 0.88 nM for inhibition of [3H]DA uptake into BroWnstein, M.J. et al., “Neurotransmitter transporters,” vesicles. These results suggest that the action of lobeline is Rec. Prog. Hormone Res., 49:27—42 (1994). similar to that of and that it speci?cally interacts With DTBZ sites on VMAT2 to inhibit DA uptake Debler, EA. et al., “Ascorbic acid and striatal transport of into synaptic vesicles. d-amphetamine inhibits [3H]DTBZ [3H]1—methyl—4—phenylpyridine (MPP+) and [3H]dopam binding to vesicle membranes With an IC5O of 39.4 nM, a ine,” Life Sci., 42:2553—2559 (1988). concentration 20 times greater than reported for inhibition of Floor, E. et al., “Dynamic storage of dopamine in rat brain VMAT2 function, suggesting that d-amphetamine interacts synaptic vesicles in vitro,” J. Neurochem. 64, 689—699 With a different site than lobeline on VMAT2 to inhibit (1995). monoamine uptake. These results suggest the use of lobeline Liu Y. et al., “A molecular analysis of vesicular amine and analogs thereof in treating individuals for diseases and transporter,” Behav. Brain Res. 73, 51—58 (1996). pathologies of the central nervous system. 6,087,376 Page 2

13 Claims, 12 Drawing Sheets OTHER PUBLICATIONS Nunn—Thompson et al., “Pharmacotherapy for smoking ces sation,” Clin. Pharmacy, 8:710—720 (1989). Prignot, J., “Pharmacological approach to smoking cessa tion,” Eur. Respir J., 2:550—560 (1989). KalyuZhnyy, V.V., “The treatment of nicotinism With the aid of lobeline and its in?uence on vegetative and vascular reactions,” J. Neural. Psychiat., 68:1864—1870 (1968). Stolerman, I.P. et al., “Dissociation betWeen the locomotor and depressant effects of nicotinic agonists in rats,” PsychopharmacoL, 117:430—437 (1995). Fudala, R]. et al., “Further studies on nicotine—induced conditioned place preference in the rat,” Pharmacol. Bio chem. Behav., 25:1041—1049 (1986). Geller, I. et al., “Effects of nicotine monomethiodide, lobeline, , meprobamate and on a discrimination task in laboratory rats,” Psychopharmacol. (Berl.), 20:355—365 (1971). Schechter, MD. et al., “Nicotine as a discriminative cue in rats: inability of related drugs to produce a nicotine—like cuing effect,” Psychopharmacol. (Berl.), 27:379—387 (1972). Reavill, C. et al., “Behavioral and pharmacokinetics studies on nicotine, and lobeline,” NeuropharmacoL, 29(7):619—624 (1990). Romano, C. et al., “Sterespeci?c nicotine receptors on rat brain membranes,” Science, 210:647—650 (1980). Decker, M.W. et al., “Diversity of neuronal nicotinic ace tylcholine receptors: lessons from behavior and implications for CNS therapeutics” Life Sci., 56:545—570 (1995). Yamada, S. et al., “Brain nicotinic receptors biochemical characterization by neosurugatoXin,” Mol. PharmacoL, 28:120—127 (1985). Lippiello, PM. et al., “The binding of L—[3H]nicotine to a single class of high affinity sites in rat brain membrane,” Mol. Pharmacol., 29:448—454 (1986). S. Martin et al., “ and nicotinic medellary hyperal gesic in?uences in the decerebrated rat,” Pharmacol Bio chem and Behav., 29 725—721 1988. Broussolle, E.P. et al., “In vivo binding of 3H—nicotine in the mouse brain,” Life Sci., 44:1123—1132 (1989). Bhat, R.V. et al., “Regulation of brain nicotinic receptors by chronic agonist infusion,” J. Neurochem, 56(6):1932—1939 (1991). Sakurai, Y. et al., “Enhancement of [3H]dopamine release and its [3H]metabolites in rat striatum by nicotinic drugs,” Brain Res., 242:99—106 (1982). Takano, Y. et al., “Presynaptic modulation of the release of dopamine from striatal synaptosomes: difference in the effects of High K+ stimulation, and nico tinic drugs,” Brain Res., 279:330—334 (1983). Grady, S. et al., “Characterization of nicotine receptor—me diated 3H—dopamine release from synaptosomes prepared from mouse striatum,” J. Neurochem, 59:848—856 (1992). U.S. Patent Jul. 11,2000 Sheet 1 0f 12 6,087,376

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FIG. I 3 6,087,376 1 2 USE OF LOBELINE COMPOUNDS IN THE and IWamoto, 1986). Although initially lobeline Was shoWn TREATMENT OF CENTRAL NERVOUS to generaliZe to nicotine in discrimination studies (Geller et SYSTEM DISEASES AND PATHOLOGIES al., 1971), most subsequent studies have failed to reproduce this original ?nding (Schechter and Rosecrans, 1972; REFERENCE TO RELATED APPLICATION Reavill et al., 1990; Romano and Goldstein, 1980). This application is a continuation-in-part of US. patent Nicotine has been reported to be avidly self-administered application Ser. No. 08/795,852, ?led Feb. 5, 1997 US. Pat. by rats (Corrigal et al. 1992, 1994; Donny et al., 1996); No. 5,830,904. hoWever, the ability of lobeline to support self administration has not been investigated. Based on the FIELD OF THE INVENTION 10 differential effects of lobeline and nicotine in behavioral studies, it appears that these drugs may not be acting via a The present invention relates to the use of lobeline and common CNS mechanism, even though lobeline is often analogs thereof in the treatment of diseases and pathologies considered to be a (Decker et al., 1995). of the central nervous system (CNS). The invention also relates to the treatment of drug abuse and WithdraWal The positive reinforcing effect of nicotine is believed to be 15 due to the activation of central dopaminergic systems therefrom, as Well as eating disorders, such as obesity. (BoWell and Balfour, 1992; Corrigal et al., 1992, 1994). BACKGROUND OF THE INVENTION Presynaptic nicotinic receptors have been found on dopam ine (DA)-containing nerve terminals (Giorguieff-Chesselet Lobeline (ot-lobeline) is a lipophilic, non-pyridino, alka et al., 1979; Clarke and Pert, 1985). Nicotine binds to loidal constituent of Indian ( in?ata). As 20 nicotinic receptors With high affinity (Kd=1—7 nM) shoWn by the folloWing formulas, no obvious structural (Lippiello and Fernandes, 1986; Reavill et al., 1988; Romm resemblance to S(—)nicotine is apparent: et al., 1990; Bhat et al., 1991; Loiacono et al., 1993; Anderson and Arneric, 1994). Also, lobeline has been reported to displace [3H]nicotine binding from central nico 25 tinic receptors With high affinity (Ki=5—30 nM) (Yamada et al., 1985; Lippiello and Fernades, 1986; Banerjee and Abood, 1989; Broussolle et al., 1989). Chronic treatment With nicotine results in an increase in

30 the number of nicotinic receptors in many regions of rat and mouse brain (Collins et al., 1990; Bhat et al., 1991, 1994; Marks et al., 1992; Sanderson et al., 1993). An increase in the number of nicotinic receptors in postmortem human brain tissue obtained from smokers also has been reported 35 (BenWell et al., 1988). In contrast, chronic lobeline admin istration did not increase the number of nicotinic receptors in mouse brain regions in Which increases Were observed folloWing chronic nicotine administration (Bhat et al., 1991). Structure-function relationships betWeen nicotine and 40 Nicotine evokes DA release in in vitro superfusion studies lobeline do not suggest a common pharmacophore (BarloW using striatal slices (Westfall, 1974; Giorguieff-Chesselet et and Johnson, 1989). Nonetheless, lobeline has been reported al., 1979; Westfall et al., 1987; Harsing et al., 1992) and to have many nicotine-like effects including tachycardia and striatal synaptosomes (Chesselet, 1984; RoWell et al., 1987; hypertension (Olin et al., 1995), bradycardia and hypoten Rapier et al., 1988, 1990; Grady et al., 1992, 1994; RoWell sion in urethane and anesthetiZed rats (Sloan et 45 and Hillebrand, 1992, 1994; RoWell, 1995), and in in vivo al., 1988), hyperalgesia (Hamann and Martin, 1994), mid studies using microdialysis in striatum (Imperato et al., olytic activity (Brioni et al., 1993), and improvement of 1986; Damsma et al., 1989; BraZell et al., 1990; Toth et al, learning and memory (Decker et al., 1993). Moreover, 1992). Nicotine-evoked DA release is calcium-dependent, lobeline has been used as a substitution therapy for tobacco mecamylamine-sensitive and mediated by nicotinic recep (Nunn-Thompson and Simon, 1989; tors (Giorguieff-Chesselet et al., 1979; Westfall et al, 1987; Prignot, 1989; Olin et al., 1995); hoWever, its effectiveness Rapier et al., 1988; Grady et al., 1992). Mecamylamine is a is controversial as re?ected by both positive (Dorsey, 1936; noncompetitive nicotinic , Which more KalyuZhnyy, 1968) and negative reports (Wright and effectively blocks the ion channel of the receptor (Varanda Littauer, 1937; Nunn-Thompson and Simon, 1989). et al., 1985; Loiacono et al., 1993; Peng et al., 1994). Similar Furthermore, only short-term usage of lobeline as a smoking 55 to nicotine, lobeline has been reported to increase DA deterrent has been recommended due to its acute toxicity release from superfused rat and mouse striatal synaptosomes (nausea, severe heartburn and diZZiness) and the lack of (Sakurai et al., 1982; Takano et al, 1983; Grady et al., 1992). information concerning its long-term usage (Wright and Based on these neurochemical studies, lobeline Was sug Littauer, 1937; Olin et al., 1995). gested to be an agonist at nicotinic receptors (Decker et al., In behavioral studies, nicotine has been shoWn to increase 60 1995). It is difficult to reconcile that nicotine and lobeline locomotor activity (Clarke and Kumar, 1983a, 1983b; similarly release DA and displace [3H]nicotine binding; Clarke, 1990; Fung and Lau, 1988), and to produce condi hoWever, the observed upregulation of nicotinic receptors tioned place preference (Shoaib et al., 1984); Fudala et al., folloWing chronic nicotine administration is not observed 1985) in rats. HoWever, the results of the latter studies are folloWing chronic lobeline administration. controversial (Clarke and Fibiger, 1987). In contrast, 65 Earlier studies of the pharmacokinetic properties of lobeline does not increase locomotor activity (Stolerman et lobeline have centered on its proposed use in the treatment al., 1995) or produce conditioned place preference (Fudala of nicotinism. For example, US. Pat. Nos. 5,536,503; 5,486, 6,087,376 3 4 362; 5,403,595; and PCT Publication WO 92/19241 are all in treating dependencies on drugs of abuse is implicated by related to a drug delivery system and method for treating the present studies. In particular, the treatment of dependen . US. Pat. Nos. 5,414,005; 4,971,079; cies on such drugs as , , caffeine, and 3,901,248 also discuss the use of lobeline in the context , , , , of treating nicotine abuse and/or addiction. A scienti?c cannabinoids, hallucinogens, and is implicated. article has studied the actions of morphine, lobeline, and Also, the treatment of eating disorders, such as obesity, is other drugs in inducing “analgesia” in rats (S. Hamann et al. implicated. In a preferred aspect of the invention, the 1994). HoWever, these Workers did not equate their ?nding method of treatment reduces an individual’s desire for the of an “analgesic” response for lobeline to a reduction of the drug of abuse or for food by at least one day. pain response in man, nor did they propose the use of 10 A lobeline compound of the present invention is contem lobeline in treating drug abuse, WithdraWal from addiction, plated primarily for use in the treatment of diseases and and the like. pathologies associated With the CNS. Thus, cognitive Similarly, to the present inventors’ knowledge, the use of disorders, head or brain trauma, memory loss, psychosis, lobeline in the treatment of eating disorders has not been sleep disorders, obsessive-compulsive disorders, panic proposed. This is in spite of the Widely accepted ability of 15 disorders, myasthenia gravis, Parkinson’s disease, AlZhe nicotine to suppress appetite (see, e.g., Remington’s Pharm. imer’s disease, schiZophrenia, Tourette’s syndrome, Hun Sci., 18th ed., p.891) and the previously proposed associa tington’s disease, and attention de?cit disorder, and related tion of obesity With reduced bioavailability of dopamine conditions are considered to be susceptible to treatment With (US. Pat. Nos. 5,552,429; 5,576,321; 5,272,144; and 5,468, a lobeline compound of the present invention. 755). 20 As shoWn by the results of the studies described herein, The present study further elucidates the mechanism of and contrary to conventional belief, lobeline is found to act at higher concentrations primarily not as a nicotinic agonist, DTBZ),action of a structurallobeline usinganalog [3H]dihydrotetrabenaZine of (TBZ), Which but by a different mechanism than is observed for nicotine. binds to a single class of high-af?nity sites on the vesicular The present studies also suggest that lobeline may be monoamine transporter-2 protein (VMAT2) to inhibit effective in inhibiting uptake of extracellular dopamine by vesicular DA uptake (Pletscher et al., 1962; Scherman et al., cells of the CNS, perhaps by blocking dopamine receptors 1986; Kilbourn et al., 1995; Liu et al., 1996). Of note, TBZ on the cells. Either or both mechanisms can thereby Work to does not alter spontaneous efflux of [3H]DA from rat brain increase the extracellular concentration of dopamine. Many vesicles (Floor et al., 1995). Taken together, TBZ appears to respects in Which the actions of lobeline are similar to those block [3H]DA uptake into vesicles but does not promote of amphetamine have been identi?ed. [3H]DA release from vesicles. In the present study, the effect of lobeline is compared BRIEF DESCRIPTION OF THE FIGURES With that of d-amphetamine, a psychostimulant and lipo FIGS. 1A and 1B depict the time course of nicotine philic Weak base reported to inhibit DA uptake into striatal 35 evoked fractional release (A) and concentration-dependence synaptic vesicles (Philippu and Beyer, 1973; Ary and of nicotine-evoked total [3H]over?oW (B) from rat striatal Komiskey, 1980) and to inhibit monoamine uptake into slices preloaded With [3H]DA (3,4-dihydroxyphenylethyl-2 human VMAT2 expressed in CV-1 cells (Erickson et al., [N-3H]-amine). Nicotine Was added to the superfusion 1996). d-Amphetamine has also been reported to release DA buffer after the second sample (as indicated by the arroW) from synaptic vesicles of the Planorbis corneas giant DA 40 and remained in the buffer until the end of the experiment. cell, increasing DA concentrations in the cytosol and pro The data in FIG. 1A are presented as means:S.E. fractional moting reverse transport of DA via DAT (SulZer and release, Which represents the tritium in the sample as a Rayport, 1990; SulZer et al., 1995). Furthermore, percentage of the total tritium in the slice at the time of d-amphetamine has been reported to inhibit [3H]DTBZ sample collection. The data in FIG. 1B are presented as binding to rat striatal homogenates (Rostene et al., 1992) and 45 mean:S.E. total [3H]over?oW, Which represents the area human VMAT2 expresed in COS cells (GonZaleZ et al., under the curve of the corresponding nicotine concentration 1994), but With loW potency. The ability of lobeline to evoke response as a function of time. §P<0.05, different from basal [3H]DA release from rat striatal synaptic vesicles preloaded (5—10 min), When fractional release Was collapsed across With [3H]DA is also assessed in the present study. nicotine concentration; *P<0.05, signi?cantly different from SUMMARY OF THE INVENTION 0—0.01 pM and 1—100 pM; **P<0.05, different from 0—0.1 pM and 100 pM; ***P<0.05, different from 0—10 pM; The present invention is for a method of treating an Duncan’s NeW Multiple Range Test. n=4—9 rats. individual Who suffers from a disease or pathology of the FIGS. 2A and 2B depict the time course of lobeline central nervous system (CNS). The method comprises evoked fractional release from rat striatal slices preloaded administering to the individual an amount of a lobeline 55 With [3H]DA. Lobeline Was added to the superfusion buffer compound, i.e., lobeline, analogs, and derivatives thereof, after the collection of the second sample (as indicated by the including pharmaceutically acceptable salts. The amount of arroW) and remained in the buffer until the end of the lobeline compound administered is effective to alleviate at experiment. Data are presented as mean:S.E. fractional least one of the symptoms of the individual’s condition. release, Which represents the tritium in the superfusate The lobeline compound can be administered alone, com 60 sample as a percentage of the total tritium in the slice at the bined With an excipient, or coadministered With a second time of sample collection. FIG. 2A illustrates the time course drug having a similar or synergistic effect. The compound or of the fractional release evoked by loW concentrations composition is preferably administered subcutaneously, (0.01—3 pM) of lobeline, and FIG. 2B illustrates that evoked intramuscularly, intravenously, transdermally, orally, by high concentrations (3—100 pM). *P<0.05, different from intranasally, or rectally. 65 basal out?oW; +P<0.05, different from the peak responses at The utility of lobeline, analogs, and derivatives thereof, 25 min for 0.01—3 pM and 30—300 pM; §P<0.05, different e.g., those that form lobeline upon metabolism by the body, from the peak responses of 0.01—10 pM and 100 pM; 6,087,376 5 6 #P<0.05, different from the peak responses of 0.01—30 pM; FIG. 8 depicts lobeline inhibition of [3H]DTBZ binding to Fisher’s LSD post hoc test. n=6 rats. rat striatal vesicles. Data represent the rneanzSEM prnol FIG. 3 depicts the concentration-dependence of lobeline [3H]DTBZ bound/rng protein. Control represents the evoked total [3H]over?oW from rat striatal slices preloaded amount of [3H]DTBZ bound in the absence of lobeline. With [3H]DA. Data are presented as rnean:S.E. total [3H] *p<0.05, signi?cantly different from control; Fisher least over?ow, Which represents the area under the curve of the signi?cant difference post hoc test. n=5 experirnents. corresponding lobeline concentration-response as a function of time. The inset illustrates the total [3H]over?oW evoked FIG. 9 shoWs that d-arnphetarnine inhibits [3H]DTBZ by the loWer concentrations (0.01—1 nM) of lobeline. Con binding to rat striatal vesicles. Data represent the trol slices Which Were superfused With buffer in the absence 10 rneanzSEM prnol [3H]DTBZ bound/rng protein. Control of lobeline did not evoke [3H]over?oW (i.e. fractional represents the amount of [3H]DTBZ bound in the absence of release Was not different from basal during the course of d-arnphetarnine. *p<0.05, signi?cantly different from con superfusion). *P<0.05, different from control and each of the trol; Fisher least signi?cant difference post hoc test. n=5 other lobeline concentrations; Duncan’s NeW Multiple experirnents. Range Test. n=6 rats. 15 FIG. 10 shoWs the time course of spontaneous [3H]DA FIG. 4 depicts the time course of the effect of rnecarny ef?ux frorn [3H]DA-preloaded striatal vesicles in the larnine to inhibit nicotine(10 nM)-evoked fractional release absence of drug. Data are expressed as rneanzSEM [3H]DA of [3H]DA frorn preloaded rat stratal slices. For clarity of ef?ux as a percent of total [3H]DA content of the vesicles at graphical presentation, only signi?cant effects of the loWest time 0. n4 experirnents. and highest concentration, 0.01 and 100 pM, respectively, of rnecarnylarnine are illustrated. Data are presented as FIG. 11 shoWs that d-arnphetarnine evokes [3H]DA rnean:S.E. fractional release as percentage of basal out?oW. release from synaptic vesicles preloaded With [3H]DA. Experiments were performed as described in Table 2 here d-Arnphetarnine-evoked vesicular [3H]DA release Was inbeloW. The time course begins at the time of nicotine (10 expressed as percent of control content. The total amount of 25 pM) addition to the superfusion buffer containing rnecarny [3H]DA present in control samples Was 4.37:1.06 prnol/rng larnine. The control represents fractional release in the protein. *p<0.05, signi?cantly different from control; Fisher absence of either rnecarnylarnine or nicotine in the super least signi?cant difference post hoc test. n3 experirnents. fusion buffer. Duncan’s NeW Multiple Range Test revealed a signi?cant inhibitory effect of 0.01 pM rnecarnylarnine, FIG. 12 depicts lobeline inhibition of nicotine-evoked When the data Were collapsed across time of superfusion. [3H]doparnine release from rat striatal slices. Striatal slices n=8 rats. Were obtained from rat brain, preincubated With [3H] FIG. 5 depicts the effects of nicotine (0.01—1000 pM) and doparnine (0.1 nM) for 30 min and subsequently superfused lobeline (0.01—1000 pM) on rat striatal synaptosornal and With Kreb’s buffer for 60 rnin. FolloWing the initial period synaptic vesicular [3H]DA uptake. III nicotine, synaptoso of superfusion, slices Were superfused With buffer contain rnal [3H]DA uptake; I nicotine, vesicular [3H]DA uptake; O ing various concentrations of lobeline for 30 min and lobeline, synaptosornal [3H]DA uptake; O lobeline, vesicu subsequently various concentrations of nicotine Were lar [3H]DA uptake. Data are presented as rnean:S.E. per included in the buffer. Superfusate samples were collected to centage of total [3H]DA uptake. Total [3H]DA uptake for determine the ability of lobeline to inhibit the nicotine synaptosornes and vesicles Was 10919.80 prnol/rnin/rng and induced response. 40 13401717 prnol/rnin/rng, respectively. Non-speci?c [3H] FIG. 13 illustrates the current understanding of the pri DA uptake in synaptosornal and vesicular experiments was rnary mechanism of the action of lobeline in the central 2% and 20%, respectively, of total [3H]DA uptake as deter nervous system. mined by incubation With 10 pM GBR and incubation at 0° C., respectively. Experirnents examining the effect of nico 45 DETAILED DESCRIPTION OF THE tine on synaptosornal uptake included a loW concentration INVENTION range (0.001—1 nM), hoWever, no effect Was observed and for clarity of graphical presentation these results are not As used herein, the term “lobeline” refers to a compound illustrated. *P<0.05, different from total [3H]DA uptake; having the general chemical formula 2-[G-([3 Dunnett’s post hoc test. n=3—6 rats. hydroxyphenethyl)-1-rnethyl-2-piperidyl]-acetophenone. FIG. 6 depicts the endogenous DA and DOPAC The term “lobeline” as used herein refers to the above (dihydroxyphenylacetic acid) content in rat striatal slices compound in its free form, or as a salt thereof, Which has the superfused With high concentrations (30—100 nM) of physiological activity addressed. Inasmuch as a compound lobeline. Endogenous DA and DOPAC content Were deter having this formula has three chiral centers, eight optical mined after 60 rnin superfusion With various concentrations 55 isomers of the compound can exist. HoWever, particular of lobeline. Data are presented as rnean:S.E. ng/rng protein. optical isorner(s) are not intended herein unless speci?cally *P<0.05, different from control, P<0.05, **P<0.001, differ mentioned. ent from control; Dunnett’s post hoc test. n=8 rats. The term “lobeline analogs” and equivalents thereof, as FIG. 7 depicts equilibrium binding of [3H]DTBZ to rat used herein, refers to chemical derivatives of lobeline, such striatal vesicles. Striatal vesicles Were incubated for 10 min 60 as those obtained by oxidation or reduction of lobeline, at 25° C. in the absence and presence of [3H]DTBZ (0.5—10 nM) Nonspeci?c binding (0) was determined using 20 pM others obtained by esteri?cation of lobeline and its redox TBZ. Speci?c binding (O) Was de?ned as the difference derivatives, as Well as various substitutions at the N-position betWeen total binding (not shoWn) and the nonspeci?c of the piperidinyl group in the lobeline rnolecule. binding. Data are rneanzSEM prnol/rng protein. Inset: Scat 65 Preferred lobeline analogs, Which may act as of chard transformation of the mean speci?c [3H]DTBZ bind lobeline itself When metabolized by the body, include those ing data from the saturation analyses. n=4 experiments. contemplated by formula (I) (Without regard to chirality): 6,087,376 8 Apharmaceutical composition containing a lobeline com (I) pound of the invention is also contemplated, Which may include a conventional additive such as a stabiliZer, buffer, salt, preservative, ?ller, ?avor enhancer, and the like, as knoWn to those skilled in the art. Representative buffers include phosphates, carbonates, citrates, and the like. Exem plary preservatives include EDTA, EGTA, BHA, BHT, and the like. A composition of the invention may be administered by inhalation, i.e., intranasally as an aerosol or nasal formula 10 tion; topically, i.e., in the form of an ointment, cream or Where R1 and R2 each independently represents hydrogen, lotion; orally, i.e., in solid or liquid form (tablet, gelcap, time loWer alkyl, loWer alkenyl, loWer alkylcarbonyl, release capsule, poWder, solution, or suspension in aqueous arylcarbonyl, e.g., phenylcarbonyl, aralkylcarbonyl, e.g., or non-aqueous liquid); intravenously as an infusion or alkylphenylcarbonyl, loWer alkoxycarbonyl, loWer injection, i.e., as a solution, suspension, or emulsion in a alkylaminocarbonyl, higher alkylcarbonyl, and poly 15 pharmaceutically acceptable carrier; transdermally, e.g., via (alkyleneoxide)carbonyl; R3 and R4 each independently a transdermal patch; rectally, as a suppository, and the like. represents hydrogen or combines With R1 and , Generally, it is expected that a pharmacologically effec respectively, to form a double bond; and X represents H or tive dose of a present compound Will require its adminis loWer alkyl. Whenever a carbonyl-containing substituent is tration in an amount less than 1x10‘3 mg/kg of body Weight provided as R1 or R2, it is understood that the carbonyl group per day. The amount to be administered depends to some is covalently bonded to the respective O atom appearing in extent on the lipophilicity of the speci?c compound selected, formula Thus, in the instances Where the substituent is an since it is expected that this property of the compound Will alkoxycarbonyl or alkylaminocarbonyl, a carbonate or car cause it to partition into fatty deposits of the subject. The bamate linkage is present in the molecule. precise amount to be administered can be determined by the Preferred substituents for R1 and R2 include methylcar bonyl (acetyl), phenylcarbonyl (benZoyl), natural fatty acid skilled practitioner in vieW of desired dosages, side effects, groups, e.g., palmitoyl, oleyl, linoleyl, stearyl, and lauryl, 25 the medical history of the patient, and the like. It is antici and polyethyleneglycol (PEG) covalently bonded to the pated that the compound Will be administered in an amount molecule via a carbonate linkage. Long chain moieties such ranging from about 1x10“5 to about 1x10“3 mg/kg/day. as a PEG group in a lobeline enhance transdermal The present study Was performed to determine the delivery of the molecule, Which may be metaboliZed to involvement of nicotinic receptors in lobeline-evoked [3H] lobeline and derivatives thereof. over?oW from rat striatal slices preloaded With [3H]DA. The As used herein, the terms “loWer alkyl”, “loWer alkenyl”, calcium-dependency of the effect of lobeline and the ability “loWer alkoxy”, and the like, refer to normal, branched and of mecamylamine to inhibit the lobeline response Were cyclic hydrocarbyl groups containing 1 to 6 carbon atoms. determined. To assess the contribution of potential effects on The term “higher alkyl” includes alkyl groups containing 7 DA uptake, the effect of nicotine and lobeline to inhibit to about 20 carbon atoms. The term “aryl” refers to a 35 [3H]DA uptake into striatal synaptosomes and synaptic hydrocarbon group containing one or more aromatic rings, vesicle preparations Was also determined. Based on the optionally substituted With one or more heteroatoms. The present results of the in vitro superfusion studies, striatal term “aralkyl” refers to an aryl group covalently bonded to dopamine (DA) and dihydroxy phenylacetic acid (DOPAC) a loWer alkyl group. content Were also determined after lobeline superfusion in It is, of course, contemplated that certain lobeline analogs vitro, and after lobeline administration in vivo. having the above formula may be converted into a different Effect of Nicotine on Superfused Rat Striatal Slices Pre molecule upon metabolism by the body. For example, When loaded With [3H]DA. ever an acetyl group is present at R1 and/or R2 in the In a concentration-dependent manner, nicotine evoked an compound, the acetyl group may be removed by metabolic increase in the fractional release of tritium over the time processes, e.g., such as occur in the gastrointestinal tract or course of the superfusion experiment (FIG. 1A). Repeated the liver. The choice of substituents is subject to consider 45 measures, tWo-Way AN OVA (analysis of variants) revealed ations of toxicity, side effects, dosage, and the like. a signi?cant main effect of nicotine concentration (F(8>429)= Particularly preferred lobeline analogs are those in Which 29.45, P<0.0001) and a signi?cant main effect of time (i) both R1 and R2 are H (i.e., a lobelanidine compound), (ii) (F10)429)=9.76, P<0.0001), but the concentration>