WO2010083445A1 - Use of u18666a compounds for smoking cessation and inhibition of nicotine receptor function - Google Patents

Abstract

The present invention relates to the use of a nAChR antagonist. In one embodiment, the present invention provides a method of treating a nicotine addiction in an individual by administering a therapeutically effective amount of U18666A compound, or a derivative, pharmaceutical equivalent, analog and/or salt thereof. In another embodiment, the present invention provides a method of characterizing and/or profiling one or more nicotinic acetylcholine receptors in a cell by administering a therapeutically effective amount of U18666A compound, or a derivative, pharmaceutical equivalent, analog and/or salt thereof, where the U18666A compound inhibits nicotinic acetylcholine receptors with the following rank order of potency for nAChR subtype: α4β2 > α3β2 > α4β4 > α7

Description

USE OF U18666A COMPOUNDS FOR SMOKING CESSATION AND INHIBITION OF

NICOTINE RECEPTOR FUNCTION

GOVERNMENT RIGHTS This invention was made with U.S. Government support on behalf of the National

Institutes of Health by NIH Grants NS40417 and DAO 15389. The U.S. Government may have certain rights in this invention.

BACKGROUND All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Nicotinic acetylcholine receptors (iiAChRs) are ligand-gated ion channels. As neurotransmitter receptors, nAChRs have served as models for the establishment of concepts regarding the mechanisms of drug action and synaptic transmission, and the structure and function of transmembrane signaling molecules (Lukas et al., 1999). Mammalian nAChRs are composed of varying combinations of different subunits encoded by at least 16 genes (αl-7, α9- 10, β 1-4, γ, δ, and ε). Different combinations of subunits give rise to nAChR subtypes, with varying pharmacology, distribution, and physiological roles. In addition to their role at the neuromuscular junction (αlβl(γ/ε)δ subtype), a diverse family of neuronal nAChRs play important roles in nervous system function. Some neuronal nAChRs are involved in nicotine dependence and the pathophysiology of neuropsychiatric conditions. α4 and β2 subunits assemble to form the most abundant nAChR subtype in the brain (α4β2-nAChR; Gopalakrishnan et al., 1996), which has a high affinity for nicotine and can respond to levels of nicotine found in smokers' plasma (Fenster et al., 1997). The activation of α4-containing nAChRs by nicotine is sufficient for nicotine-induced sensitization, reward and tolerance (Tapper et al., 2004). Furthermore, in β2-nAChR knockout mice, nicotinic agonists fail to increase midbrain dopaminergic neuronal discharge frequency or striatal dopamine release,

1

DWT 13836226v2 0048135-0ϋ3WO0 and nicotine self-administration ceases quickly (Picciotto et al,, 1998; Marubioet al., 2003). Therefore, α4β2-nAChRs in the brain appear to play major roles in the mediation of nicotinic reinforcement and dependence. Studies have also suggested that <x4β2-nAChRs are involved in several prevalent disorders such as Parkinson's disease, Alzheimer's disease, and epilepsy (Cordero-Erausquin et al., 2000; Nakamura et al., 2001 ; O'Neill et al., 2002; Quik, 2004).

Since nAChRs exist as a family of different subtypes their characterization is complex. Differences in ligand sensitivity of nAChR subtypes can be used to study the roles of these receptors in general as well as the subtypes themselves. Also, the pharmacological profiles of nAChRs can be utilized to distinguish between subtypes and more clearly define their roles in both health and disease.

Cholesterol plays an important role in the construction of brain cells, and it also supplies these cells with substrates for proper metabolism. Also, it is a precursor for the synthesis of steroid hormones and lipids. The synthesis of cholesterol in liver cells is complex, and some compounds can exert negative effects on its direct synthesis or transportation into cells. One of these compounds, (3β)-3-[2-(Diethylamino) ethoxy]androst-5-en-17-one dihydrochloride

(Ul 8666A), inhibits both cholesterol synthesis by suppressing desmosterol Δ24-reductase and cell cholesterol transport.

Evidence suggests that the function of nAChRs is influenced by their lipid microenvironment (see reviews by Barrantes, 2001, 2003), and both endogenous and exogenous lipids, acting as hydrophobic non-competitive inhibitors, may have some effects on AChRs (Jones and McNamee, 1988; Rankin et al., 1997; Addona et al., 1998; Baenziger et at, 2000). Cholesterol's interaction with lipids in general has already been well described. For example, cholesterol can modulate organization of the γM4 transmembrane domain of the muscle-type nAChR (Rodrigo et al., 2004). By using novel fluorescence techniques, the nongenomic effects of cholesterol-derived steroids on nAChR membrane has been assayed (Francisco et al., 2000), and the interactions of spin- labeled androstanol and cholestane with nAChRs have already been demonstrated (Francisco et al., 2000). Cell-surface trafficking of nAChRs is dependent on metabolic cholesterol (Pediconi et al., 2004). Numerous groups have explored the putative cholesterol-binding sites of the AChR (Gonzalez-Ros et al., 1982; Barrantes et al., 1989; Marsh and Barrante, 1978; Simmonds et al., 1982; Jones et al., 1988), and it has been shown that the

DWT ϊ3836226v20048135-003WO0 cholesterol-binding sites of AChRs are not accessible to phospholipids (Narayanaswami et al., 1993).

Because drugs which are used to manipulate cholesterol levels may, themselves, have direct effects on nAChRs, the effects of U 18666A on selected human nAChRs heterologously expressed in the SH-EPl cell-line (or on native α3-containing nAChRs from the undifferentiated human neuroblastoma cell line SH-SY5Y) was evaluated. There is a need in the art for greater understanding of nAChRs, both in their functional characterization and pharmacological profiling, as well as the development of novel smoking cessation treatments.

SUMMARY OF THE INVENTION

Various embodiments include a method of treating an individual for a disease and/or condition, comprising administering a therapeutically effective amount of a composition comprising a compound of the formula:

(Formula 1) or a pharmaceutical equivalent, derivative, analog and/or salt thereof, to the individual. In another embodiment, the disease and/or condition is mediated by nicotinic acetylcholine receptor (nAChR) signaling. In another embodiment, the disease and/or condition comprises nicotine addiction. In another embodiment, the disease and/or condition comprises atherosclerosis. In another embodiment, the disease and/or condition comprises smoking addiction. In another embodiment, the composition is administered to the individual in a water solubilized solution. In another embodiment, the composition is administered to the individual intravenously, by direct injection, and/or orally.

Other embodiments include a method of inhibiting and/or reducing nicotinic signaling in a quantity of cells, comprising administering a therapeutically effective amount of a composition comprising a compound of the formula:

3

DWT 13836226v20048i35-003WOO

(Formula 1) or a pharmaceutical equivalent, derivative, analog and/or salt thereof, to a quantity of cells. In another embodiment, the quantity of cells comprise SH-EPl human epithelial cells. In another embodiment, the quantity of cells express nicotinic acetylcholine receptors (nAChRs). In another embodiment, the quantity of cells express α4β2 nAChRs. In another embodiment, the therapeutically effective amount of composition comprises 0.001 to 500 μM of the compound of Formula 1, or the pharmaceutical equivalent, derivative, analog and/or salt thereof. In another embodiment, the therapeutically effective amount of composition comprises 5 to 30 nM of the compound of Formula I₅ or the pharmaceutical equivalent, derivative, analog and/or salt thereof. Other embodiments include a method of selectively inhibiting and/or decreasing nicotinic acetylcholine receptor (nAChR) function, comprising administering a therapeutically effective amount of composition comprising a compound the formula:

(Formula 1) or a pharmaceutical equivalent, derivative, analog and/or salt thereof, to a sample comprising one or more cells expressing a plurality of nAChR subtypes. In another embodiment, the plurality of nAChR subtypes comprises α4β2 nAChR, α3β2 nAChR, α4β4 nAChR and/or nAChR α7. In another embodiment, the therapeutically effective amount of composition comprises about 10

DWT 1383ό22όv2 G048135-003 WOO nM of the compound of Formula 1, or the pharmaceutical equivalent, derivative, analog and/or salt thereof.

Various embodiments include a method of inhibiting and/or reducing nicotinic signaling in a quantity of cells, comprising administering a therapeutically effective amount of a composition comprising a compound of the formula:

• 2HC!

(Formula 2) or a pharmaceutical equivalent, derivative, analog and/or salt thereof, to the quantity of cells, In another embodiment, the quantity of cells comprise SH-EP 1 human epithelial cells. In another embodiment, the quantity of cells express nicotinic acetylcholine receptors (nAChRs).

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. Figures IA and IB depict chemical structures of U18666A and AY9944, respectively.

Figure 2 depicts subtype selectivity of U 18666 A inhibition of human nAChRs. A: Representative effects of U18666A (1 μM. 2-min pretreatment) on α4β2- (Aa, 3 μM nicotine), α3β2 (Ab, 40 μM nicotine), α4β4- (Ac, 1 μM nicotine), and α7-nAChR (Ad, 3 mM choline)- mediated currents. B: Bar graph indicates U 18666A -mediated inhibition for the indicated nAChR subtype (abscissa) during exposure to 1 μM U18666A (solid bars) or after 4 min washout of U18666A (open bars). C; U18666A concentration -dependent inhibition of human α4β2-O), α3β2-(β), α4β4-( A) or α7-( Y) nAChR function, (ordinate, peak whole-cell current responses normalized to those in the absence of U18666A, n - 6-9 cells). **p < 0.01.

Figure 3 depicts U18666A suppressed whole-cell currents mediated by human α4β2- nAChR. A: Under the conditions of whole-cell voltage-clamp recording, rapid application of 3

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DWT 13836226v2 0048135-003 WOO μM nicotine to the recorded cell induced an inward current consisting of peak (I_p) and steady- state (I_s) components (left trace), Exposure to 10 μM U 18666A alone did not induce detectable current (middle trace). B: Co-application of 10 μM U 18666A and 3 μM nicotine reduced both peak and steady-state current components and accelerated the decay time constant from the peak to the steady-state current (middle trace). For these and all subsequent traces, current amplitude bar is indicated, the V_H was -60 mV unless indicated otherwise, and the duration of ligand exposure is indicated by the bars above each trace. C: Statistical analysis shows that 10 μM U18666A significantly inhibits α4β2-nAChR function represented (ordinate, normalized values to 3 μM nicotine-induced current (horizontal dashed line) as peak whole-cell current (l_p, open bar) and steady-state current (I_s, solid bar). Each column (or symbol) is the average from 7 cells tested. In this and all following figures, unless specifically mentioned, the vertical bars indicate ± SEM. A single asterisk (^*) represents/? < 0.05, and double asterisks (^**) represent/? < 0.01 compared with responses to 3 μM nicotine.

Figure 4 depicts effects of different U 18666A application modes on nicotinic responses. A: Typical traces recorded from the same SH-EPl cell are represented. In these experiments, the U18666A was applied to cell in three different ways: co-application with nicotine (Aa), pretreatment for 2 min then exposure to nicotine aione (Ab), and pretreatment for 2 min then co- application with nicotine (Ac). The effects of these three application modes on peak (B) and steady-state (C) of nicotine-induced current are summarized (n = 7 for each column). Figure 5 depicts concentration-dependent inhibition of α4β2-nAChR function by

U18666A . A: Superimposed 3 μM nicotine-induced current with (black traces), and without (gray traces), U 18666A at the indicated concentrations with co-application (Aa) or with 2-min pretreatment then co -application (Ab). B: A comparison of the effects of U18666A on the peak current of nicotine-induced inward current with (o) or without (B) 2-min pretreatment and co- application. The ordinate presents a normalized peak amplitude (nicotine-induced peak current at ICT⁶ μM U18666A as 100%, n = 7-9 cells). C: Concentration-dependent inhibition of nicotinic response (peak ■ and steady-state •) by U 18666A (with 2-min pretreatment) at the indicated concentrations (abscissa, log micromolar scale). The ordinate presents normalized amplitudes (nicotine-induced peak or steady-state current at 10^"6 μM U18666A as 100%, n = 9 cells). Figure 6 depicts the mechanism of U 18666A-induced inhibition of α4β2-nAChR function. A: Representative whole-cell current traces induced by different concentrations of

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DWT 13836226v2 0048135-003 WOO nicotine with (black traces) and without (gray traces) U18666A. For these experiments, 10 nM U18666A was applied with 2-min pretreatment and then co-application with nicotine. Each ceil was only exposed to U18666A and Ul 8666A plus one concentration of nicotine. Thus, the four recoding trace pairs presented in (A) were obtained from different cells. B: Concentration- response curves for nicotine-induced currents with (o) and without (■) U18666A. All symbols were normalized to the peak amplitude of 100 μM nicotine-induced current (a maximally- effective concentration, indicated by asterisk), and each symbol was averaged from 6 cells tested. When the symbols were normalized to the peak amplitude of 100 μM nicotine-induced current (B) and to the peak amplitude of 100 μM nicotine plus U 18666A-induced current (•), respectively (indicated by asterisk), the concentration-response curves shown in C were generated, in which there is again no significant change of nicotine EC₅₀ values in the absence or presence of U 18666 A.

Figure 7 depicts the inhibition of Ul 8666A on nicotinic responses is voltage-dependent. A: The superimposed traces show whole-ceil current responses of SH-EP l-hα4β2 cells to 3 μM nicotine alone (gray traces) or with 2-min pretreatment and then co-application with 10 nM

U 18666A (black traces) at the indicated F_HS. B: Statistical comparison of effects of U18666A on nicotine-induced currents at different Vn^~ s. The abscissa values were normalized to 3 μM nicotine-induced currents at different V^s, respectively (n = 6 for each column).

Figure 8 depicts U 18666A inhibits nicotinic responses in a use-dependent manner, A: Recorded cell was repetitively exposed to 3 μM nicotine (4-s exposure at 2-min intervals) during the continual presence of 10 nM U 18666A for 10 min (a) or nicotine was applied at the beginning and at the end of 10-min exposure to 10 nM U 18666A, respectively (b). The traces in Aa and Ab were recorded from the same cell. B: Bar graph shows the comparison of the effects of U18666A during repetitive exposure to nicotine (a, solid bars, n = 8) or at the beginning and end of nicotine exposures (b, open bars, n = 8). The ordinate values were normalized to the responses to nicotine without the presence of U18666A.

Figure 9 depicts effects of intracellular application of U18666A on α4β2-nAChR function. A: Repetitive applications of 3 μM nicotine (2-s exposure at 20-s intervals) induced inward current responses without functional rundown. B: Co-application of 10 μM U18666A plus nicotine clearly diminished nicotinic responses, C: Addition of 100 μM Ul 8666A into the recording pipette followed by maintenance of whole-cell configuration for 20 min (allow a full

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DWT 13836226v20048135-003 WOO exchange between the intracellular environment and pipette solution), then repetitive applications of 3 μM nicotine using the same protocol as (A) induced current responses without rundown. However, extracellular co-application of 10 μM U18666A plus nicotine induced profound rundown of current responses (D). E: Summary of effects of U 18666A supplied under conditions specified in A (■), B (Δ), C (A), or D (•) on peak current responses (ordinate, normalized to the response to 3 μM nicotine alone; mean± S. E.; n - 7 cells).

Figure 10 depicts effects of intracellular GDP-βS on U18666A-mediated inhibition of α4β2-nAChR function. A: Typical whole-cell inward current induced by 3 μM nicotine at 1 (Aa) and 25 min (Ab) after converting into whole-cell configuration in the presence of 600 μM GDP- βS in the recording pipette. (B) After preloading of 600 μM GDP-βS for 25 min, repetitive applications of 3 μM nicotine plus 10 μM U18666A (2-s exposure at 20-s intervals) induced dramatic rundown of nicotinic responses. C: Summary of the effects of 10 μM U18666A on responses to 3 μM nicotine with (U) and without (>. :) intracellular loading of 600 μM GDP-βS (n - 8). Figure 1 1 depicts concentration-dependent inhibition of α4β2-nAChR function by AY-

9944. A: Superimposed 3 μM nicotine-induced current with and without, U18666A at the indicated concentrations with co-application (A) or with 2-min pretreatment then co-application (B). C: Concentration-dependent inhibition of nicotinic response (peak: ■ and steady-state: read •) by AY-9944 (with 2-min pretreatment) at the indicated concentrations (abscissa, log micromolar scale). The ordinate presents normalized amplitudes (nicotine-induced peak or steady-state current at 10^"5 μM AY-9944 as 100%, n = 5-6 cells).

DESCRIPTION OF THE INVENTION

AU references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton el ai, Dictionary of Microbiology and Molecular Biology 3^rd ed. , J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^ih ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed. , Cold Spring Harbor Laboratory Press (Cold Spring

DWT !383622όv2 ϋG48135-003 WGG Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As used herein, the term "U18666A" means Formula 1 and also includes (3β)-3-[2- (Diethylamino) ethoxy]androst-5-en-17-one dihydrochloride.

(Formula 1) As used herein, the term "Ul 8666A compound" refers to a U 18666A molecule, as we!l as its derivatives and/or additional molecular components.

As used herein, the term "ΑY9944" means Formula 2 and also includes 366-93-8, 366- 93-8 (DIHYDROCHLORIDE), AIDS060324, A1DS-060324, AY9944, AY 9944, AY 9944 (^Dihydrochloride*), AY-9944 (*Dihydrochloride*), CPD-4521, NCGC00025242-01, NSCl 23019, NSC123019 (DIHYDROCHLORIDE), Tocris-1639.

• 2HCt

(Formula 2) As disclosed herein, the effects of U 1 8666A, a cholesterol synthesis/ transporter inhibitor, on selected human neuronal nAChRs heterologously expressed in the SH-EPI cell-line was evaluated using whole- cell patch-clamp recordings. The results indicate that with 2-min pretreatment, U18666A inhibited different nAChR subtypes with a rank-order of potency (IC₅₀ of whole-cell peak current): α4β2 (8.0 ± 3.0 nM) >α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (>100 μM),

9

DWT S3S36226v2 0048135-003 WOO showing this compound to be more selective to α4β2-nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on α4β2-nAChRs were further investigated in detail. U18666A suppresses both peak and steady-state components of whole-cell currents mediated by human α4β2-nAChRs in response to nicotine. In nicotine- induced concentration- response curves, U 18666A reduces nicotine-induced current at maximally effective agonist concentrations without influencing nicotine's EC50 value, support for a non-competitive inhibition. U18666A-induced inhibition of nAChR function is concentration-, voltage- and use- dependent, support for an open channel block. Considering about 10,000-fold enhancement of the potency of U 18666A after 2-min pretreatment, this compound also likely inhibits α4β2- nAChRs through a close channel block, In addition, the U18666A-induced inhibition in α4β2- nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data demonstrate that U18666A is a potent antagonist of α4β2-nAChRs and will be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as for smoking cessation. In one embodiment, the present invention provides a method of treating a disease mediated by a nicotinic acetylcholine receptor signaling in an individual by administering a therapeutically effective amount of a cholesterol reducer compound to the individual, where the inhibition of nicotinic acetylcholine receptor signaling by the compound results in treatment of the disease. In another embodiment, the cholesterol reducer compound inhibits cholesterol synthesis. In another embodiment, the cholesterol reducer compound inhibits cell cholesterol transport. In another embodiment, the compound that reduces cholesterol is a Ul 8666A compound. In another embodiment, the nicotinic acetylcholine receptor includes subtypes α4β2, α3β2, α4β4 and/or α7. In another embodiment, the individual is human.

In one embodiment, the present invention provides a method of treating a nicotine addiction in an individual by administering a therapeutically effective amount of U 18666A compound, where the U 18666A compound acts as an α4β2-nAChR antagonist. In another embodiment, the nicotine addiction includes tobacco smoking and/or chewing.

In another embodiment, the present invention provides a method of characterizing and/or profiling one or more nicotinic acetylcholine receptors in a cell by administering a therapeutically effective amount of U18666A compound, where the U18666A compound inhibits nicotinic acetylcholine receptors with the following rank order of potency for nAChR

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DWT 13836226v20048135-003WOO subtype: α4β2 > α3β2 > α4β4 > α7. In another embodiment, the U18666A compound inhibits nicotinic acetylcholine receptors by altering cell cholesterol homeostasis. In another embodiment, nicotine is co-administered with the U 18666A compound. In another embodiment, the administration of the U ! 8666A compound is preceeded by a pretreatment of Ul 8666A. fn another embodiment, the pretreatment of U 18666A is approximately 2 minutes at a concentration of approximately 10 μM. In another embodiment, the cell is SH-EPl ceils.

The present invention is also directed to a kit to prepare a U18666A compound, SH-EPl cells solution, as well as the delivery of the U 18666A compound to an individual, and may include a pipette, pipette solution, standard external solution for SH-EPl cells, and combinations thereof. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including a therapeutically effective dosage of U18666A compound, as described above.

The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of delivering a therapeutically effective dosage of U18666A compound to a eel! or cell culture, or mammalian subjects, such as, but not limited to, human subjects, farm animals, domestic animals, and laboratory animals. Other embodiments, for example, are configured fox preparing & therapeutically effective dosage of Ul 8666A compound to a cell or cell culture, or mammalian subjects, such as, but not limited to, human subjects, farm animals, domestic animals, and laboratory animals.

Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to prepare a solution for SH-EPl cells or a U 18666A compound and/or deliver a therapeutically effective dosage of U 18666A compound to treat a nicotine addiction. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophϊUzed form; they can be

1 1

DWT 1383622όv2 0048135-003WO0 provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term

"package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing a solution of U18666A compound or components thereof. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.

In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of U 18666A compound. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to pipette, intravenous injection, aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

The U18666A compound according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another

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DWT !383ό22όv2 0048135-003WO0 tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

The Ul 8666A compound according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

The preparations of the U 18666A compound are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

The Ul 8666A compound according to the invention may be delivered in a therapeutically effective amount The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for

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DWT 13836226v20048135-0Q3WG0 instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Typical dosages of nicotinic compositions can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as the responses observed in the appropriate animal model. In one embodiment, a therapeutically effective dosage of U18666A compound administered to a cell culture ranges from 0.001 μM to 500 μM, with a preferable dosage of i0 μM, or the effective equivalent when administered to the respective mammal or human.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which couid be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1 Generally The effects of U18666A, a cholesterol synthesis/ transporter inhibitor, on selected human neuronal nAChRs heterologously expressed in the SH-EPl cell-line was evaluated using whole-

14

DWT 13836226v2 0048135-003WO0 cell patch-clamp recordings. The results indicate that with 2-min pretreatment, U18666A inhibited different nAChR subtypes with a rank-order of potency (IC₅₀ of whole-cell peak current): α4β2 (8.0 ± 3.0 nM) >α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (>100 μM), showing this compound to be more selective to α4β2-nAChRs. Thus, the pharmacological profiles and mechanisms of U 18666A acting on α4β2-nAChRs were further invest) gated in detail. U18666A suppresses both peak and steady-state components of whole-cell currents mediated by human α4β2-nAChRs in response to nicotine. In nicotine-induced concentration- response curves, U 18666A reduces nicotine-induced current at maximally effective agonist concentrations without influencing nicotine's ECso value, support for a non-competitive inhibition. U18666A-induced inhibition of nAChR function is concentration-, voltage- and use- dependent, support for an open channel block. Considering about 10,000-foId enhancement of the potency of U18666A after 2-min pretreatment, this compound also likely inhibits α4β2- nAChRs through a close channel block. In addition, the Ul 8666A-induced inhibition in α4β2- nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data demonstrate that Ul 8666A is a potent antagonist of α4β2-nAChRs and will be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as for smoking cessation.

Example 2 Expression of human neuronal nAChRs in SH-EPl human epithelial cells.

Heterologous expression of human α4β2- α4β4- or α7-nAChRs has been previously described in detail (Eaton et al., 2003; Zhao et al., 2003; Wu et al., 2004b; Wu et a!., 2006). Briefly, human nAChR α4 and β2 subunits, subcloned into pcDNA3.1-zeocin or -hygromycin vectors, respectively, were introduced (Puchacz et al., 1994; Peng et al., 1999) into native nAChR-null SH-EPl cells (Lukas et al.. 1993) to create the SH-EP l-hα4β2 cell-line. Cells were maintained as low passage number (1-26 from our frozen stocks) cultures in medium augmented with 0.5 mg/mL zeocin and 0.4 mg/mL hygromycin and were passaged once weekly by splitting just-confluent cultures 1/10 to maintain cells in proliferative growth. To evaluate the effects of U18666A on α3-containing nAChRs, the native α3-containing nAChRs from the undifferentiated human neuroblastoma cell line SH-SY5Y were used.

15

DWT 13836₂26v₂ 0048135-003 WOO Example 3

Patch-clamp whole-cell current recordings and data acquisition. Conventional whole-cell current recordings were performed combined with a fast drug application system allowing for fast application and removal of drugs as previously described (Zhao et al., 2003; Wuet ah, 2004a, Wu et al., 2006). Briefly, three kinds of transfected SH-EPl cells were prepared in 35-mm culture dishes without poly(lysine) coating and then plated on the bottom of the dishes and later placed on the stage of an inverted microscope (Axiovert 200; Zeiss, Germany). Cells were perfused with standard external solution (2 mL/min). Glass microelectrodes with 3-5 MΩ resistance between the pipette and extracellular solution were used to form tight seals (>2GΩ) on the surface of the cells, and standard whole-cell current recording was initiated by suitable suction and then waiting for 5 to 10 min to allow for exchange of the pipette solution and the cytosol. Thereafter, recorded cells were lifted up gently from the bottom of the culture dishes, which allows for improved solution exchange and more accurate evaluation of differences in the kinetics of agonist-induced whole-cell currents (Wu et al., 2006). Before capacitance and resistance compensation, access resistance (Ra) was measured and accepted for experiments if less than 20 MΩ. Whole-cell capacitance was minimized and series resistance was compensated routinely to 80%. Recorded cells were voltage-clamped at a holding potential of -60 mV, and inward currents induced by nicotine were measured (Axopatch 200B amplifier; Molecular Devices, Sunnyvale, CA). Current signals were typically filtered at 2 kHz, acquired at 10 kHz, and displayed and digitized on-line (Digidata 1440A series AID board; Molecular Devices, Sunnyvale, CA). Data acquisition and analyses were performed using pClamplO.O (Molecular Devices, Sunnyvale, CA), and results were plotted using Origin 5.0 (OriginLab Corp., North Hampton, MA). All experiments were performed at room temperature (22 ± 1°C).

Example 4

Solutions and drug application.

The standard external solution for SH-EPl eel Is contained (in mM): 120 NaCl, 5 KCl, 2 MgCl₂, 2 CaCl₂, 25 D-glucose, and 10 HEPES; pH 7.4 (Tris-base). For conventional whole-cell recording, the pipette solution contained (in mM): 1 10 Tris-phosphate dibasic, 28 Tris-base, 1 1 EGTA, 2 MgCl₂, 0.5 CaCl₂, and 4 Na-ATP; pH 7,3. In most experiments, nicotine was used as the test agonist and induced whole-cell current responses. Nicotine was quickly perfused to

16

DWT 13S36226v2 0048135-003WOO recorded cells using a computer-controlled perfusion system (SF-77B Perfusion Fast Step, Warner Instruments Incorporated, Harvard Bioscience Company, Hamden, CT), and cells were completely surrounded by applied drugs within 20 ms. The interval between drug applications (2 min) was optimized specifically to ensure stability of nAChR responsiveness (i.e., no functional rundown). Drugs used in these experiments were (-)-nicotine, choline, guanosine 5'-O-(2-thio) diphosphate (GDP-βS) trilithium salt (all purchased from Sigma Chemical, St. Louis, MO), and U18666A and AY9944 (Tocris Cookson Inc., Ellisville, MO). The chemical structures of U18666A and AY-9944 are shown in Fig. 1. Since U18666A-induced inhibition in nAChRs was maintained for several tens min (data not shown), the data collection was more laborious than usual because many recordings were performed from cells exposed only once to the ligand, especially when high concentrations of U 18666 A were applied.

Example J Data analysis and statistics. To assess nAChR whole-cell current responses, both peak and steady-state components of inward currents were measured. Data are presented as means ± standard errors. Statistical analysis was performed using paired /-tests when evaluating data obtained from a single cell or Student's t test (unpaired values) or one-way ANOVA with Duncan's multiple comparison when comparing data obtained from different cells. Values ofp less than 0.05 were considered significant. Curve fitting for agonist and antagonist concentration-response data were performed (Origin 5.0 software; OriginLab Corp.) using the logistic equation to provide fits for maximal and minimal responses, EC₅₀ or ICso values, and Hill coefficients.

Example 6 Different effects of U18666A across nAChR subtypes.

Initial experiments were designed to test the effects of U18666A on multiple nAChR subtypes. The inventors compared the effects of U 18666A on heterologous Iy expressed α4β2-, α4β4-, and α7-nAChRs in human SH-EPl cells, and also on native α3-containing nAChRs from the human neuroblastoma cell line SH-SY5Y. In undifferentiated SH-SY5Y cells, the main subunit combination is α3β2 nAChR (Wang et al., 1996). Figure 2A illustrates the effects of 1 μM Ul 8666A with 2 min pretreatment on different type of nAChRs. The individual subtype of

17

DWT i 3 B3622όv2 0048 i 35-003 WOG nAChRs were activated by nicotine (or choline) at a concentration closed to its ECso (i.e., α4β2 3 μM, α3β2 40 μM, α4β4 1 μM nicotine and α7 3 mM choline). The results showed that 1 μM U18666A suppressed α4β2-(Fig. 2Aa) or α3β2-nAChR-mediated current (Fig. 2Aa) but not α4β4-(Fig. 2Ac) and α7-nAChRs (Fig. 2Ad). The recovery from inhibition by 1 μM U18666A after 4 min washout was not complete in both α4β2- and α3β2-nAChRs (Fig. 2B). Figure 2C summarizes the concentration-dependent inhibition of different nAChR subunit combinations by U18666A and shows that the order of potency (IC ₅₀) for inhibitory effects of 1 μM U18666A on whole-cell peak current was α4β2 (8.0 ± 3.0 nM) > α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (>100 μM), suggesting that U18666A is more selective to α4β2-nAChRs. Therefore, the inventors evaluated pharmacological profiles and possible mechanisms of U 18666A on α4β2 nAChRs in more detail.

Example 7 U 18666 A suppresses a4β2-nΛChR-medϊated whole-cell currents. The inventors examined the effects of U 18666A on α4β2 -nAChR function in detail.

Under conventional whole-cell recording configuration in voltage-clamp mode, the recorded cell was lifted (Wu et a!., 2006, Liu et a!., 2008) and nicotine was applied for 4 seconds via a rapid drug application system. As shown in Fig. 3A, exposure to 3 μM (-EC₅₀ concentration for α4β2-nAChR, Wu et a!., 2006 ) nicotine induced an inward current at a holding potential of -60 mV, which consisted of peak (I_p) and steady-state (I_s) components. The application of 10 μM Ul 8666A alone failed to induce detectable current (Fig. 3A). Co-application of U18666A (10 μM) and nicotine (3 μM) reduced both peak (to 82.7 ± 7.4% of control, n = 8, p < 0.05) and steady-state (to 7.8 ± 1.8% of control, n = 8, p < 0.001, Fig. 3B₅C) components. To compare the effect of different application modes of U18666A on nAChR function, the inventors examined the effects of co-application of Ul 8666A plus nicotine (Fig. 4Aa), pretreatment with U 18666A for 2 min then nicotine alone (Fig. 4Ab) and pretreatment with U 18666 A for 2 min, then co- application of U18666A plus nicotine (Fig. 4Ac). In these experiments, the inventors used a much lower concentration (10 nM) of U 18666A because our results showed that with an appropriate pretreatment, the potency of Ul 8666 A was dramatically increased (see Fig. 5). In the acute co-application group (Fig. 4Aa, n = 10), the peak and steady-state components of inward currents induced by 3 μM nicotine were reduced to 89.7 ± 4.9% (p < 0.05) and 84.2 ± 8.2% (Fig.

18

DWT 1383622όv2004S135-00SWO0 4B,C,p < 0.05). In the 2-min pretreatment, then nicotine-alone stimulation group (Fig. 4Ab, n = 10), peak and steady state currents were 74.6 ± 6Λ% (p < 0.01) and 62.3 ± 10.2% (Fig. 4B,C,ρ < 0.01) of control, respectively. Finally, in the 2-min pretreatment then nicotine and U18666A co- application group (Fig. 4Ac, n = 10), peak and steady-state values were reduced to 51.2 ± 7.3% (p < 0.01) and 49,7 ± 8.9% (Fig. 3B₅C, p < 0.01 ) of control, respectively. These results indicate that Ul 8666A suppressed human α4β2-nAChR-mediated currents even at a low concentration (10 tiM), that the inhibitory effect was more profound with 2-min pretreatment, and that pretreatment and co -application resulted in the most potent functional effect.

Example 8

U 18666 A suppresses a4β2-nAChR-mediated currents in a concentration- dependent manner.

The inventors also determined the concentration- inhibition relationship for UI 8666A effects on α4β2-nAChR-mediated currents. In these experiments, the effects of different concentrations of U18666A on an EC₅₀ concentration (3 μM) nicotine-induced current were examined. Co-application of U18666A at different concentrations (0.001-100 μM) with nicotine (3 μM) showed concentration-dependent suppression of α4β2-nAChR function (Fig. 5Aa). In 7 cells tested, the IC50 values and Hill coefficient for U18666A-induced suppression without pretreatment were 95.3 ± 15.8 μM and 1.1 ± 0.2 for peak component, and were 1.6 ± 0,6 μM and 1.0 ± 0.3 for steady-state component. With 2-min pre-treatment followed by co-application with nicotine, U18666A showed more profound effect on 3 μM nicotine-induced currents (Fig. 5Ab). In 9 cells tested, the IC50 values and Hill coefficient for U18666A-induced suppression with 2 min pre-treatment were 8.0 ± 3.0 nM and 0,6 ± 0.1 for peak component, and were 1.0 ± 1.0 nM and 0.4 ± 0.1 for steady-state component. Figure 5B and C summarized the concentration- dependent inhibition of α4β2-nAChR- mediated currents by U18666A. These results demonstrate that U 18666 A inhibits α4β2-nAChR function in a concentration-dependent manner, and interestingly, with 2-min pretreatment, the apparent affinity of U18666A-induced inhibition is increased about i 0000-fold.

Example 9 Mechanism of Ul 8666A-induced suppression of a4β2-nAChR function

DWT 13836226\2 0048135-003WOO In order to identify whether the U 18666A-induced suppression of α4β2-nAChR function is mediated through a competitive or non-competitive mechanism, the inventors tested nicotine concentration-response relationship with and without 10 nM U18666A (close to fCso with 2 min pretreatment). Figure 6 A showed the superimposed typical traces of nicotine (gray traces) and nicotine plus i 0 nM U 18666A (black traces) following 2 min U18666A pretreatment. Nicotine potency was indistinguishable in the absence or presence of U18666A (EC₅₀ = 3.5 ± 0.4 μM, n - 6 vs. EC₅₀ of 2.8 ± 0.3 μM, n = 6, respectively; EC₅₀ values obtained by assessing whole-cell peak current amplitudes;/? > 0.05; Fig 6B). However, the inhibitory effect of 10 nM U18666A increased as the nicotine concentration increased (Fig. 6B). When normalized to the maximal response in nicotine or nicotine plus U i 8666A, the concentration-response curves yielded ECso values of 3.5 ± 0.4 or 3.6 ± 0.4 μM, respectively (Fig. 6C, n = 6,p > 0.05). Therefore, U18666A reduced current responses induced by high concentrations of nicotine without altering EC₅₀ values, suggesting a non-competitive mechanism of antagonism.

Example 10

U 18666 A -induced inhibition in a4β2-nAChRs is dependent on holding potentials. When charged ligands block ion channels, their residence in transmembrane regions is affected by the transmembrane potential. Here, whole-cell inward currents induced by nicotine plus 10 nM U i 8666A were recorded at the holding potentials (F_HS) of -100, -60 or 0 mV, respectively (Fig. 7A). Results demonstrated that U18666A exhibited more profound inhibition on nAChR function at the V_n of -100 mV than that at 0 mV, suggesting a voltage-dependent mechanism (Fig. 7B). Comparison to the whole-cell peak current response to nicotine plus U18666A at the V₁₁ of -100 mV (42.5 ± 9.6%, n = 6), that at the -60 and 0 mV were 47.8 ± 8.0% (n = 6, p > 0.05) and 65.7 ± 1 1.6% (n - 6,p < 0.05), respectively. These results indicate that the inhibition of α4β2-nAChR-mediated current by U 18666A is voltage-dependent.

Example 11

U 18666A-induced inhibition in a4β2-nAChRs is use-dependent. To test whether LJ 18666A inhibits α4β2-nAChRs in a use-dependent manner, the inventors repetitively applied the nicotine and U 18666A to recorded cell. Repetitive applications of nicotine (3 μM) alone for 4s at 2-min intervals showed that α4β2-nAChR-mediated currents

20

DWT I3836226v2 0048135-003WOO did not exhibit functional rundown (data not shown). Under the same conditions, repetitive applications of nicotine (3 μM) in the continuous presence of 1O nM U 18666A led to gradual decreases in responses induced by nicotine (Fig. 8Aa). In contrast, after continual application of U 18666A for 10 min without exposure to nicotine, there was less inhibition of 3 μM nicotine- induced responses (Fig. 8Ab). After exposure to U 18666A for 10 min, the peak component of nicotine-induced current by repetitive challenges of nicotine was reduced to 21.4 ± 6.8% (Fig, 8Aa, n - 8) and that by non-repetitive challenges of nicotine was reduced to 40,4 ± 8,6% (Fig. 8Ab, n = 8, p < 0.01). These results indicate that U18666A-induced inhibition of α4β2-nAChR function is use-dependent. Considering the recovery of inhibitory effect on nAChR-mediated currents by U18666A is relatively slow (-80% recovery after 4 min washout from 40 sec

U18666A exposure) and to avoid any 'hangover effect' of U18666A when the protocol switched from Aa to Ab, the inventors continuously washed after experiment Aa for at least for 20 min. After washout of U 18666A, the inventors typically monitor nicotinic response every 2 min until they record two similar current responses, ensuring that recovery is complete, then start experiments shown in Ab,

Example 12

U18666A-induced inhibition in a4β2-nAChRs is not through intracellular sites. To test whether U 18666A inhibits α4β2-nAChRs mediated via intracellular sites, a high concentration (100 μM) of U 18666A was added to the pipette solution and applied to ceils intraceliularly throughout whole-cell recordings. In the control experiment (no Ul 8666A in the pipette solution), repetitive applications of 3 μM nicotine induced stable responses (2-s exposure at 20-s intervals; Fig. 9A). However, these responses continually decreased when nicotine and 10 μM U 18666 A were co-applied extraceliulariy to the same recorded ceil (Fig. 9B). In contrast, when 100 μM Ul 8666A was added to the pipette solution, after conversion to whole- cell recording configuration for more than 20 min, which allowed for full loading of U18666A into the cell, no decline in nicotine response was seen (Fig. 9C). In the same recorded cell, bath- application of nicotine plus 10 μM U 18666 A resulted in persistent reductions in responses (Fig. 9D). Thus, the results demonstrate that U18666A-induced inhibition in α4β2-nAChRs is not mediated through intracellular sites (Fig, 9E).

21

DWT 13836226v2 0048!35~ϋU3WO0 Example 13

U18666A-induced inhibition ofa4β2-nAChRs is not due to receptor internalization. Since endocytosis or exocytosis of several transmembrane receptors occurs on a time scale of seconds to minutes through the processes modulated by agonists and antagonists, we tested whether U18666A inhibits α4β2-nAChR-mediated current via an internalization mechanism. To reach this goal, 600 μM GDP-βS (added to the pipette solution) was preloaded into recorded cell by conversion to whole-cell recording configuration for 20 min. GDP-βS has been reported to prevent internalization of AMPA and GABA_A receptors (Luscher etal., 1999; Blair et al., 2004). The results showed that pre-loading cells with GDP-βS for 20 min neither affected nicotine-induced currents (Fig, 10A) nor prevented U18666A-induced inhibition in α4β2-nAChR function (Fig. 10B,C). These results indicate that U18666A-induced inhibition in α4β2-nAChRs is not through a receptor internalization mechanism.

Example 14 U 18666 A inhibits n AChR function with subunil selectivity

A major finding by the inventors is that a cholesterol reducer, U18666A, inhibits nAChR function with subunit selectivity, exhibiting more profound inhibition on α4β2-nAChRs than that on α4β4-, α7- or α3-containing nAChR. Acting on human α4β2-nAChRs, U18666A reduces the maximal current response of α4β2 nAChRs to nicotine without changing the nicotine EC₅₀ value. This indicates a non-competitive inhibitory mechanism. U18666A inhibits α4β2-nAChRs in a concentration-, voltage- and use-dependent manner, suggesting an open channel block mechanism. U 18666 A also inhibits α4β2-nAChRs without channel activation, and with 2-min pretreatment, the inhibitory efficacy is increased more than 10000-fold, suggesting a closed- channel block mechanism. In addition, U 18666A-induced inhibition of α4β2-nAChRs is mediated neither through intracellular sites, nor by receptor internalization. Collectively, with its high potent and subunit selectivity, the U18666A exhibits high potential to be developed as a novel α4β2-nAChR antagonist for study of nAChR pharmacology and also for treatment of some smoking-associated disorders.

Example 15

U 18666 A inhibits human nAChRs

22

DWT ϊ383ό22όv2 0048B5-003WO0 U18666A blocks desmoslerol Δ24-reductase, and consequently inhibits intracellular cholesterol trafficking. By chronic exposure to U 18666 A at early postnatal age in animal studies, brain extracellular cholesterol levels were significantly reduced (Cortez et al., 2002). For this reason, this drug has commonly been used to establish an atypical absence epilepsy model in Long-Evens rats (Bierkamper and Cenedella, 1978; Snead et al., 1999; Wu et al., 2004c). Sub-chronic exposure (i.e., 24 h) of cultured cortical neurons to U18666A induced neuronal apoptosis (Koh et al., 2006). However, direct, acute effects of U18666A on neuronal receptor/ion channel functions are largely unknown. It was reported that acute exposure to U18666A increase insulin release (Ikeda et al., 2005). in the present study, we demonstrate for the first time that U 18666A is a potent human nAChR antagonist. At low nano-molar concentrations, U 18666A significantly suppressed α4β2-nAChR-mediated current with 2-min pretreatment. The Ul 8666A-induced inhibition of α4β2 -nAChR function appears not to be mediated through altering cell cholesterol homeostasis since under the same experimental conditions (i.e., 10 nM with 2-min pretreatment), another cholesterol synthesis inhibitor, AY9944 showed much lower affinity (~ 1000-fold lower than U 18666A) for inhibition α4β2- nAChR function. However to induce absence epilepsy, both drugs exhibited comparable potency (Smith and Fisher, 1996; Wu et al., 2004c). Intracellular application of U18666A (added U18666A in the recording pipette) at very high concentration (100 μM) failed to affect α4β2-nAChR-mediated current, suggesting that U 18666 A acts on extracellular site(s) to exert its antagonism on α4β2-nAChRs. Furthermore, the preloading cell with GDP-βS for 20 min via recording pipette also failed to prevent U18666A-induced inhibition of α4β2-nAChRs via extracellular application, suggesting that U 18666A inhibits α4β2-nAChRs through a mechanism that may not involve receptor internalization. Further, the rapid onset of U18666A effects on nAChR function argue against indirect effects via cholesterol synthesis inhibition and/or nAChR internalization.

Example 16

Pharmacological mechanisms of U 18666 A-induced inhibition on a4β2-nAChRs Since U 18όόόA's effects occurred in a use-dependent manner, this suggests its suppression of nicotine-induced currents involves an agonist-induced transition of nAChRs to conformations having higher affinity for the antagonist and/or allowing free access of it to its

23

DWT !383ό22όv2 0048 I 35-003WO0 binding sile(s) (Zhao et at., 2004), and il is reasonable to conclude that this transition is to an open channel state. When a relatively low concentration of U18666A was co-applied with nicotine, the peak current of whole-cell current responses was much less altered compared to the steady-state component (Fig. 2B), again suggesting it engaged in open channel block with relatively slow associate rate. However, pre-treatment with U 18666A induced a more profound inhibition of nicotinic responses, suggesting its binding sites at nAChRs are at least partially accessible in the closed state. It is possible there may be more than one type of binding site for U18666A at nAChRs in the closed state, with each site having a different affinity for the antagonist (Hill coefficients for suppression by U18666A when co-applied with agonist were ~1). This would make fractional occupancy at a given concentration less than complete, but once receptors are exposed to U18666A, it would induce conversion of receptors to a state where binding site affinities for the antagonist are uniformly higher (Hill coefficients for suppression by Ul 8666A following pre-treatment were ~0.5), as is fractional occupancy.

Interestingly, although the effects of U 18666A on peak current amplitudes were reduced in the presence of higher concentrations of nicotine, steady-state components of current responses in the presence of U 18666 A were dramatically decreased. One reasonable explanation for this is the channel activation rate is slow and channels activate asynchronous Iy when exposed to low concentrations of nicotine, which would give U18666A enough time to block the channel. This is consistent with the observed larger fractional suppression of peak current amplitude by Ul 8666A and its minimal effects on steady-state components in the presence of low concentrations of nicotine, whereas at very high concentrations of nicotine, the agonist association rate (binding rate) and channel activation rate should be much faster than the association with U 18666A (at a fixed concentration), so more channels will be activated synchronously before block, which is evident by the observed lower fractional inhibition of peak current and greater suppression of steady-state current responses and acceleration of α4β2- nAChR desensitization by Ul 8666 A at high concentrations of nicotine. In addition, an alternative explanation is that these phenomena could be due to enhanced desensitization., Since low agonist concentrations do not favor extensive desensitization, the drug may be less efficacious on steady-state phase. Collectively, these findings suggest U 18666A has access to the α4β2-nAChR in the resting and activated states.

24

DWT 13836226v2 G04813S~0Q3WO0 Example 17 Pharmacological and clinical significance of U18666A-induced inhibition of nAChR function

U18666A was initially designed and synthesized as a water-soluble inhibitor of cholesterol transport. To date, a complete description of its pharmacological effects has not been undertaken, and no studies have evaluated its effects clinically. In the present study, the inventors provide evidence that U 18666A suppressed the function of human α4β2-nAChRs, which provides additional insights into more fully understanding the pharmacological bases of U18666A's action in studies employing various in vivo and in vitro preparations.

Tobacco smoking is a strong risk factor for cardiovascular morbidity, including accelerated atherosclerosis and increased risk of heart attacks, and a leading cause of atherosclerosis is hypercholesteremia. U 18666A serves as an inhibitor of cholesterol transport, so it potentially could be used clinically to significantly decrease the concentration of cholesterol in the blood of patients. Furthermore, our observation that UI 8666A suppressed the function of nAChRs in a relatively subtype-specific manner suggests its potential use in the development of anti-smoking therapies. In addition, it is of interest to determine if U18666A also affects α3- containing nAChRs, since it also could be exploited to serve as a cardiovascular medicine similar to mecamylamine. which is a nicotinic antagonist that was initially developed as an effective antihypertensive drug (Young et al.₅ 2001), and could have double utility in control of blood pressure variability and atherogenetic lipid profiles (Shytle et al., 2002), At the least, U 18666 A may be used for the characterization of different nAChR subtypes.

While the description above refers to particular embodiments of the present invention, it should be readily apparent to people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the

25

DWT 13836226v2004SI 35-003WO0 ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description, The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "Open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as '"having at least," the term "includes'¹ should be interpreted as "includes but is not limited to," etc.)- It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "'at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" {e.g., "a" and/or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In

26

DWT 13S36226v2004S 135-00 WOO addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations). Accordingly, the invention is not limited except as by the appended claims.

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Baenziger JE, Morris ML, Darsaut TE and Ryan SE (2000) Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor. J. Biol Chem. 275, 777-784.

Barrantes FJ (1989) The lipid environment of the nicotinic acetylcholine receptor in native and reconstituted membranes. Crit. Rev. Biochem. MoI. Biol. 24, 437-478.

Barrantes FJ (2001 ) Fluorescence studies of the acetylcholine receptor: structure and dynamics in the membrane environment. J. Fluorescence. 1 1, 273-285.

Barrantes FJ (2003) Transmembrane modulation of nicotinic acetylcholine receptor function. Curr, Opin. Drug Disc. Develop. 6, 620-632. Bierkamper GG and Cenedella RJ (1978) Induction of chronic epileptiform activity in the rat by an inhibitor of cholesterol synthesis. U18666A. Brain Res 150, 343-51.

Blair RE, Sombati S, Lawrence DC, McCay BD and DeLorenzo RJ (2004) Epileptogenesis causes acute and chronic increases in GABA_A receptor endocytosis that contributes to the induction and maintenance of seizures in the hippocampal culture model of acquired epilepsy. J Pharmacol Exp Ther 310, 871 -880.

Cordero-Erausquin M, Marubio LM, Kl ink R, and Changeux J-P (2000) Nicotinic receptor function: perspectives from knockout mice. Trends Pharmacol Sci 21, 21 1-217.

Cortez MA, Cunnane SC and Snead OC 3rd. (2002) Brain sterols in the AY-9944 rat model of atypical absence seizures. Epilepsia 43, 3-8. Eaton JB, Peng J-H, Schroeder KM, George AA, Fryer JD, Krishnan C, Buhlman L, Kuo

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Claims

1, A method of treating an individual for a disease and/or condition, comprising providing a composition comprising a compound of the formula:

(Formula 1) or a pharmaceutical equivalent, derivative, analog and/or salt thereof; and administering a therapeutically effective amount of the composition to the individual.

2. The method of claim ] , wherein the disease and/or condition is mediated by nicotinic acetylcholine receptor (nAChR) signaling.

3. The method of claim 1. wherein the disease and/or condition comprises nicotine addiction.

4. The method of claim 1 , wherein the disease and/or condition comprises atherosclerosis.

5. The method of claim 1, wherein the disease and/or condition comprises smoking addiction.

6. The method of claim 1 , wherein the composition is administered to the individual in a water solubilized solution.

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DWT 13836226*.2 0048 ! 35-003 V> CXJ

7. The method of claim 1 , wherein the composition is administered to the individual intravenously, by direct injection, and/or orally.

8. A method of inhibiting and/or reducing nicotinic signaling in a quantity of cells, comprising: providing a composition comprising a compound of the formula:

(Formula 1) or a pharmaceutical equivalent, derivative, analog and/or salt thereof; and administering a therapeutically effective amount of the composition to a quantity of cells.

9. The method of claim 8, wherein the quantity of cells comprises SH-EPl human epithelial cells.

10. The method of claim 8, wherein the cells express nicotinic acetylcholine receptors (nAChRs).

1 1. The method of claim 8, wherein the cells express α4β2 nAChRs.

12. The method of claim 8, wherein the therapeutically effective amount of the composition comprises 0.001 μM to 500 μϊvl of the compound of Formula 1, or the pharmaceutical equivalent, derivative, analog and/or salt thereof.

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13. The method of claim 8, wherein the therapeutically effective amount of the composition comprises 5 to 30 nM of the compound of Formula I , or the pharmaceutical equivalent, derivative, analog and/or salt thereof.

14. A method of selectively inhibiting and/or decreasing nicotinic acetylcholine receptor (nAChR) function, comprising: providing a composition comprising a compound of the formula:

(Formula 1) or a pharmaceutical equivalent, derivative, analog and/or salt thereof; and administering a therapeutically effective amount of the composition to a sample comprising one or more cells expressing a plurality of nAChR subtypes.

15. The method of claim 14, wherein the plurality of nAChR subtypes comprises α4β2 nAChR, α3β2 nAChR, α4β4 nAChR and/or nAChR α7.

16. The method of claim 14, wherein the therapeutically effective amount of composition comprises about 10 nM of the compound of Formula 1 , or the pharmaceutical equivalent, derivative, analog and/or salt thereof.

17. A method of inhibiting and/or reducing nicotinic signaling in a quantity of cells, comprising; providing a composition comprising a compound of the formula:

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DWT 13836226v2 0u48135-003 W OO

^{* 2HCi} (Formula 2) or a pharmaceutical equivalent, derivative, analog and/or salt thereof; and administering a therapeutically effective amount of the composition to the quantity of cells.

18. The method of claim 17, wherein the quantity of cells comprises SH-EPl human epithelial cells.

19. The method of claim 17, wherein the cells express nicotinic acetylcholine receptors (nAChRs).

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DWT 13836226v2 0048535-003 WOO