USE OF INHIBITORS OF THE Y2 RECEPTOR OF NEUROPEPTIDE Y IN THE TREATMENT OF ALCOHOLISM The present invention relates to the use of a substance that inhibits the binding of neuropeptide Y (NPY) or related molecules to the Y2 receptor receptor for NPY, that is a pharmacological NPY-Y2 receptor antagonist, for the preparation of a pharmaceutical composition for treatment of alcoholism. The invention also relates to a composition comprising the substance.

Technical background.

Neuropeptide Y (NPY) is a major neuromodulator in the mammalian central nerv- ous system (CNS). It is widely distributed in the brain, expressed at high levels in regions involved in regulation of motivation and emotionality, and produces a num- ber of striking behavioural effects: it is one of the most potent orexigenic agents known (21), produces marked anti-stress effects (7; 24), while its expression is regulated in response to stress (23; 25). Central NPY administration of high NPY doses induces suppression of locomotor activity (8).

NPY may also play a role in alcohol dependence. Ethanol consumption is increased in mice with a null mutation of the NPY gene, but decreased in transgenic NPY- overexpressing subjects (22). In addition, rat strains selected for high and low alco- hol preference have been reported to differ in NPY peptide concentrations in spe- cific brain regions (2; 4; 11) and in their electrophysiological response to NPY (3).

Despite these observations, NPY has not been shown to modulate ethanol intake, and the possible involvement of specific NPY receptors in the control of ethanol intake has until now not been elucidated.

Effects of NPY are mediated by heterogeneous receptor populations. Several re- ceptor classes for NPY have been cloned and pharmacologically characterised e. g.

Y1, Y2 and Y5. NPY receptors cloned to date all belong to the superfamily of G- protein coupled receptors, but differ in their ligand affinity profiles. The NPY-Y1 receptor (5; 9; 13) requires the intact NPY sequence for recognition and activation, and appears to be the subtype mediating anti-anxiety actions of NPY (1 ; 6; 7; 19;

26). The Y2 receptor subtype is also activated by C-terminal fragments of NPY, such as NPY, 3 36 (20). The highest number of NPY-bindning sites, predominantly of the Y2-subtype, is found within the hippocampus. Activation of Y2-receptors within this structure has been shown to suppress hippocampal glutamatergic transmission through presynaptic mechanisms (14; 16). No information is presently available whether signalling through any of the known NPY-receptors is involved in modula- tion of alcohol intake, and/or in alcoholism.

Summary of the invention.

It has now been found that NPY-Y2 receptor antagonists reduce ethanol self- administration (see the example). Thus, NPY-Y2 receptor antagonists produced a robust suppression of operant responding for ethanol.

Detailed description of the invention.

The present invention relates to the use of a substance that inhibits the Y2 receptor for neuropeptide Y for the preparation of a pharmaceutical composition for treat- ment of alcoholism. Any substance that inhibits the binding of NPY or related molecules to the Y2 receptor, i. e. acts as a pharmacological NPY-Y2 antagonist may be used according to the invention.

By NPY we understand NPY from any species, especially mammal, preferably hu- man. By Y2 receptor we understand aY2 receptor from any species, especially mammal, preferably human. Any substance or fragment of a substance that inhibits the Y2 receptor or the binding of NPY to the Y2 receptor and that lowers or stops alcohol consumption in humans may be used according to the invention. The fol- lowing substances are examples of compounds that may be used.

Suitable substances may be chosen from aminoacid derivatives with the general formula I R1 O O <BR> <BR> <BR> <BR> N-C-A-C-B-G<BR> Razz

or pharmaceutically acceptable salts thereof, wherein R'and R'may be elected from the same group or different groups, which may be chosen from hydrogen, (Cl-C6)- alkyl, mono-or disubstituted (Cl-C6)-alkyl, (wherein the substituent (s) is (are) phenyl or mono-or disubstituted phenyl), (wherein the substituent (s) may be chosen from halogen, nitro, Cl-C3-alkoxy or CF3), piperidinyl, pyrrolidinyl, morpholino, perhydroazepinyl, amino, (C1-C6)-alkylamino or di (C1-C6)alkylamino, or R'and R2 represent phenyl or mono-or disubstituted phenyl, (wherein the substituent (s) may be halogen, nitro, Cl-C3-alkoxy or CF3) or the group R' (R2) N- represent the ring wherein n is 2 or 3 and R3 represents hydrogen, (C1-C6)-alkyl, mono-or disubstituted (Cl-C6)-alkyl, (wherein the substituent (s) may be chosen from di (C1-C6)-alkylamino, (C3-C6)- cycloalkyl, phenyl or mono-or disubsituted phenyl, (wherein the substituents may be halogen, nitro, Cl-C3-alkoxy or CF3) or methylendioxyphenyl), or R3 represents (C3-C7)-cycloalkyl, phenyl or mono-or disubstituted phenyl, wherein the substitu- ent (s) may be halogen, nitro, C1-C3-alkoxy or CF3, or R3 represents amidino or N1,N2-dicyclohexylamidino, or R3 represents a group with the general formula

wherein Q and U are CH2, C=O or NH, and wherein Q and U only can be the same when they represent CH2 ; A represents (a) a saturated or an unsaturated ring comprising from 3 to 6 atoms, which ring may contain a bridge, wherein all ring atoms are carbon atoms or one of the ring at- oms is O, S or N and the other ring atoms are carbon atoms, whereby 2 adjacent carbon atoms in the ring are bound to the carbonyl groups that are bound to group A; (b) a group or a group (c) a group-CH2-W-CH2, wherein W represents a bond O, S, NR6, wherein R6 is (Cl-C6)-alkyl or phenyl- (C,-C3)-alkyl), a group R4 and R'independently represent hydrogen, (Cl-C6)-alkyl or phenyl, or W represents a group wherein m represents 2, 3, 4, 5 or 6; B represents

Arg, homo-Arg, Lys, His or Orn in L-or D-configuration, possibly with protected side chains, G represents one of the following groups: (a)-O-(Cl-C4) alkyl<BR> <BR> (b)-NH2 (c) NH (C,-C4) alkyl, wherein alkyl may be substituted by phenyl or p-aminophenyl (e)-NH- (CH2) 2 or 3-OH or-NH-(CH2) 2 or 3-0- (Cl-C4) alkyl wherein E represents C2-C6-alkylen, C2-C6-alkylen substituted with phenyl or I represents a group wherein the methylen group may be in the ortho, meta or para position, X and Y represent independently CH2, CH-C6H5, N-C6H5 or-X-Y-represent to- gether 1,2-phenylen, and pharmaceutically acceptable salts thereof.

The amino acids comprises natural occurring and synthetically produced amino ac- ids and both their D-and L-form. Halogen may be fluor, chlor, brom and jod.

Preferably (S)-N2-//1-/2-/4-/(R, S) -5, 11-Dihydro-6 (6H) -oxodibenz/b, e/azepin-11-yl/- 1-piperazinyl/-2-oxoethyl/-cycloplentyl/acetyl/-N-/2-/1, 2-dihydro-3,5 (4H) -dioxo- 1, 2-diphenyl-3H-1, 2, 4-triazol-4-yl/-ethyl/-argininamide (BIIE0246) is used. These substances may be produced as described in DE 19816929.

N-acetyl-/Leu28, Leu31/-NPY24-36 may also be used. This substance may be ob- tained as described in J. Auton. Nerv. Syst. (1998), 73 (2,3), 80-85.

Compounds of formula (II) below can also be used according to the invention. wherein R'is aryl1-CH-aryl2, aryl1-CH(aryl2)Ch2, aryl1-CH(aryl2)CH2Ch2, aryl1-C(aryl2)- aryl3 or aryl'-CH=C-aryl2, wherein aryl', aryl2 and aryl3 are the same or different and are optionally substituted by one or more OH, NH2, NHC1-6 alkyl, N (Cl 6 alkyl) 2, NO2, CN, halo, or C1-6 alkoxy or any combination thereof; X'and X2 are the same or different and are C=O, C=S, S02 or CH2 ; Q'and Q2 are the same or different and are NH, NC1-6 alkyl or CH2 ; R2 and R2a are the same or different and are H or Cl 6 alkyl ; R3 is H, CH=NH or C (NH2) =NH ; m is an integer of 0 to 3;

n is an integer of 1 to 4; when R'is aryl'-CH-aryl2, aryl'-CH (aryl2) CH2, aryl'-CH (aryl2) CH2CH2, or aryl'- C (aryl')-aryP, then R4 is CONR2bR2C, COOR2b, or Cl 6 alkyl optionally substituted by one or more OR2b, COR2b, CONR2bR2c, COR2b, NR2bR2c, or NHOH, wherein R2b and R2c are the same or different and are H or C1-6 alkyl ; when R'is aryl'-CH=C-aryl2, then R4 is H, CONR2bR2C, COOR2b, or Cl 6 alkyl op- tionally substituted by one or more OR2b, COR2b, COR, COR2b, NR2bR2c, or NHOH, wherein R2b and R2 are the same or different and are H or C1-6 alkyl ; and Rus ils aryl or heteroaryl each optionally substituted by one or more OH, NR2dR2e (wherein R2d and R2e are the same or different and are H or Cl 6 alkyl), NO2, halo, C1-6 alkyl, C1-6 alkoxy, benzyloxy or substituted benzyloxy or any combination thereof; or a salt, solvate or physiologically functional derivative thereof.

A compound of formula (1) or a salt, solvate or physiologically functional deriva- tive thereof can be used according to the present invention.

A pharmaceutically acceptable salt of these compounds is also included. Suitable pharmaceutically acceptable salts include acid addition salts when the peptide is suf- ficiently basic, i. e. contains one or more basic residues.

A suitable pharmaceutically acceptable acid addition salt of a compound of the pre- sent invention may be formed with an inorganic acid, for example, hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, sulphonic and sulphuric acids or with an organic acid, for example acetic, benzenesulphonic, benzoic, citric, ethane- sulphonic, fumaric, gluconic, glycollic, isothionic, lactic, lactobionic, maleic, malic, methanesulphonic, succinic, p-toluenesulphonic, tartaric and trifluoroacetic acids.

The chloride salt is particularly preferred for medical purposes.

Compounds of formula (I) may form solvates, in particular hydrates or partial hy- drates, and such solvates are also included within the scope of the invention.

The term"physiologically functional derivative"as used herein refers to any physiologically acceptable derivative of a compound of the present invention, for example, an ester, which upon administration to a mammal, such as a human, is ca- pable of providing (directly or indirectly) such a compound or an active metabolite thereof.

These substances may be produced as described in WO 96/22305.

Y2 antagonists may also be used according to the invention. As used herein, the term"antagonist"means any substance capable of inhibiting Y2 receptor normal functional activity. An antagonist may be identified by exposing a mammalian cell comprising and expressing an isolated DNA molecule which encodes a human Y2 receptor with the substance and a known Y2 receptor agonist such as NPY or PYY, under conditions permitting the activation of a functional response, detecting an in- hibition of ligand bidning and/or a decrease in Y2 receptor activity, and thereby determining which substances act as Y2 receptor antagonists. The DNA in the mammalian cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1 of USP 5,545, 549 or in (18).

Preferably, the mammalian cell is nonneuronal in origin. An example of a nonneu- ronal mammalian cell is a COS-7 cell. Other examples of a non-neuronal mammal- ian cells that can be used for functional assays with human receptors are the 293 human embryonic kidney cells and L-M (TK-) cells. The preferred method for de- termining whether a substance is capable of binding to the Y2 receptor comprises contacting a transfected nonneuronal mammalian cell (i. e. a cell that does not natu- rally express any type of NPY receptor, thus will only express such a receptor if it is transfected into the cell) expressing a Y2 receptor on its surface, or contacting a membrane preparation derived from such a transfected cell, with the ligand under

conditions which are known to prevail, and thus to be associated with, in vivo binding of the substance to a Y2 receptor, detecting the presence of any of the sub- stance being tested bound to the Y2 receptor on the surface of the cell, and thereby determining whether the substance binds to, activates or inhibits the activation of the Y2 receptor. Thus, this method may especially be used to identify substances that bind to the Y2 receptor and inhibit the binding of NPY thereto.

This response system is obtained by transfection of isolated DNA into a suitable host cell containing the desired second messenger system such as phospholipase C, adenylate cyclase, guanylate cyclase or ion channels. Such a host system is isolated from pre-existing cell lines, or can be generated by inserting appropriate compo- nents of second messenger systems into existing cell lines. Such a transfection sys- tem provides a complete response system for investigation or assay of the activity of human Y2 receptors with substance as described above. Transfection systems are useful as living cell cultures for competitive binding assays between known or can- didate drugs and ligands, which bind to the receptor and which, are labelled by ra- dioactive, spectroscopic or other reagents. Membrane preparations containing the receptor isolated from transfected cells are also useful for these competitive binding assays. Functional assays of signal transduction pathways in transfection systems determine a ligand's efficacy of activating the receptor. A transfection system con- stitutes a"drug discovery system"useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the Y2 receptor. The transfection system is also useful for determining the affinity and efficacy of known drugs at the Y2 receptor sites.

That a substance lowers or prevents the consumption of alcohol can be tested as is done in the example below.

The substances according to the invention may be used in pharmaceutical composi- tions against alcohol consumption. The composition comprises an effective concen- tration of at least one substance with Y2 receptor inhibiting activity in mixture or

otherwise together with at least one pharmaceutically acceptable carrier or excipi- ent.

By the expression"comprising"we understand including but not limited to. Thus, other non-mentioned substances, additives or carriers may be present.

The pharmaceutical compositions are prepared in a manner known to a person skilled in the pharmaceutical art. The carrier or the excipient could be any standard pharmaceutical carriers such as a solid, semi-solid or liquid material that could serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are known in the art. The pharmaceutical composition could be adapted to oral, paren- teral, intravaginal, or topical use and could be administered to the patient as tablets, capsules, suppositories, solutions, suspensions or the like.

The pharmaceutical compositions could be administered orally, e. g. with an inert diluent or with an edible carrier. They could be enclosed in gelatine capsules or be compressed to tablets. For oral therapeutic administration the compounds according to the invention could be incorporated with excipients and used as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. The amount of the active ingredient that is contained in compositions is so high that a unit dosage form suitable for administration is obtained.

The tablets, pills, capsules, lozenges and the like could also contain at least one of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatine, excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch, and the like, lubricants such as magnesium stearate or Sterotex, glidants such as colloidal silica dioxide, and sweetening agents such as saccharose or saccharin could be added or flavourings such as peppermint, methyl salicylate or orange flavouring. When the unit dosage form is a capsule it could contain in addition to the type above a liquid carrier such as polyethylene glycol or a fatty oil. Other unit dosage forms could contain other different materials that modify the physical form of the unit dosage form, e. g. as coatings. Accordingly, tablets or

pills could be coated with sugar, shellac or other enteric coating agents. A syrup could in addition to the active ingredient contain saccharose as a sweetening agent and some preservatives, dyes and flavouring agents. Materials that are used for preparation of these different compositions should be pharmaceutically pure and non-toxic in the amounts used.

For parenteral administration the compounds according to the invention could be incorporated in a solution or suspension. Parenteral administration refers to the ad- ministration not through the alimentary canal but rather by injection through some other route, as subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intravenous, intranasal, intrapulmonary, through the urinary tract, through eye drops, rectal or intravaginal (e. g. as a suppository, a vagitorium, a cream or an ointment). The amount of the active ingredient that is contained in such compositions is so high that a suitable dosage is obtained.

As used herein, the term"pharmaceutically acceptable carrier"encompasses any of the standard pharmaceutical carriers, The solutions or suspensions could also comprise at least one of the following adju- vants: sterile diluents such as water for injection, saline a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and vari- ous types of wetting agents, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylene diamine tetraacetic acid, buffers such as acetates, citrates or phosphates, and agents for adjustment of the tonicity such as sodium chloride or dextrose. The parenteral preparation could be enclosed in ampoules, disposable sy- ringes or multiple dosage vessels made of glass or plastic.

For topical administration the compounds according to the invention could be in- corporated in a solution, suspension, ointment, or gel. These preparations could contain at least 0. 1% by weight of an active compound according to the invention

but could be varied to be approximately 0.1-50% thereof by weight. The amount of the active ingredient that is contained in such compositions is so high that a suitable dosage is obtained. The administration could be facilitated by applying touch, pres- sure, massage, heat, warms, or infrared light on the skin, which leads to enhanced skin permeability. (10) describes how to enhance the transport of a drug via the skin using the driving force of an applied electric field. Preferably, iontophoresis is ef- fected at a slightly basic pH.

Other administration forms are inhalation through the lungs, sublingually, buccal administration via the mouth and enteral administration via the small intestine that could be effected by means known by a person skilled in the art.

The substance may be administered in an amount of 0. 01-100 ng/kg body weight of the patient, preferably in an amount of 0.1-10 ng/kg body weight of the patient.

All publications mentioned herein are hereby incorporated by reference.

The invention will now be described with reference to the following figures of which Fig. 1A shows significantly reduced responding for ethanol following 1.0 nmol in- tracerebroventricular injection of the selective NPY-Y2 receptor antagonist BIIE0246 (n=13, mean SEM ; p=0.013).

Fig. 1B shows a detailed analysis of ethanol-reinforced responses over time and in- dicates a highly significant treatment x time interaction (p « 0. 001), such that the suppressive effect of BIIE0246 is present during min 10-30 (***: p<0.001 vs. saline controls) but not 0-10 of the 30 min session.

The invention will be illustrated by the following Example, which is only intended to describe and not restrict the invention in any way.

Example Subjects and surgery Male Wistar rats (Charles River, Hamburg, Germany), appr. 225g at start of ex- periment were used for the experiments from which data are presented. For a repli- cation experiment (see Results), Wistar males of the same age but delivered by Mollegard, Ry, Denmark, were used. Subjects were housed on a reversed light-cycle (lights off 11 am, on 11 pm). Following training for operant responding (see below), surgery was carried out for unilateral cannula implantation. Rats were anaesthetized with ketamine/xylazine (80 mg/kg and 15 mg/kg) and placed in a stereotaxic appa- ratus (Kopf). Stainless steel guide cannulas (23 gauge, PlasticOne, Roanoke, Va) aimed at the lateral ventricle were implanted and secured to the skull with stainless screws and dental cement. Final injection coordinates were: A=-0. 8, L=1. 4, V=-4.3 from Bregma (15). Animals were allowed to recover for one week, during which time they were only repeatedly handled, in order to reduce the stress of subsequent intracerebroventricular injections. They were then reintroduced to the operant task (see below), and allowed to re-establish their rates of operant responding for two weeks. Finally, the actual Latin-square design was initiated, and carried out over the course of three weeks.

Injections BIIE0246 (0.3 or 1.0 nmol; kind gift of Boehringer Ingelheim) or saline was in- jected i. c. v. via an injection cannula (28-gauge) connected to a 10 ul Hamilton sy- ringe with a low-volume plastic tubing. These doses were selected on the basis of preliminary experiments, in which a dose of 3 nmol produced a marked sedative ef- fects. The drug was dissolved in 0.9% NaCl and injected 45 min prior to onset of sessions, in a volume of 10 ul over a period of 2 min interval. The injection cannula was left in place for an additional minute to prevent back flow.

Operant training and self-administration Training and self-administration was as in (12), except saccharin was not faded out after training, since it helps maintain response rates, and it has recently been shown that the presence of this sweetener does not affect the reinforcing properties of etha- nol (17).

Following completion of training, rats were subjected to surgery, allowed to recover for one week and to stabilize their baseline responding for two weeks, after which a Latin Square design was initiated and carried out over three weeks. During each of these weeks, rats were run Monday-Friday, and drug administration was on Fri- day. Due to failed injections, some subjects did not receive all three treatments (0, 0.3 and 1.0 nmol BIIE0246). In order to retain a balanced design, the analysis pre- sented here includes only those subjects, which completed all treatments. An analy- sis based on all subjects, with missing values for failed injections, produced a virtu- ally identical result.

Results.

Total responding for ethanol was significantly reduced by BIIE0246 (One way ANOVA, n=13 ; F [2, 24] =5. 1; p=0. 013 ; fig. la). Post hoc analysis revealed that this was due to a reduction by appr. 40% at the 1.0 nmol dose, which was significantly lower than both the saline control and the 0.3 nmol dose (p<0.05 for both). Re- sponding for saccharin was unaffected by treatment (F [2,24] =1. 5; p=0.25 ; data not shown).

To allow a more detailed analysis, cumulative response rates for ethanol were ex- amined in 5 min intervals over the 30 min duration of the self-administration ses- sions, using two way ANOVA for time and treatment effects. This analysis repro- duced the main effect indicated by the analysis of total response rates (overall treatment effect F [2,40] =3.83 ; p=0. 03), but additionally suggested a highly signifi- cant treatment x time interaction (F [10,200] =5.05, p<<0. 001). Subsequent post hoc

analysis using Tukey HSD indicated that the 1.0 nmol dose group responded for ethanol significantly less during the last 20 but not during the initial 10 min of test- ing (fig. lb ; p<<0. 001) Since we have found that basal rates of operant ethanol self-administration differ between substrains of Wistar rats originating from different colonies, these results were independently reproduced in a separate experiment utilizing Wistar subjects delivered from Mollegard, Ry, Denmark. A virtually identical, 40% suppression of responding for ethanol with no effect on the saccharin solution was found in that experiment (data not shown).

Locomotor activity in previously non-habituated subjects showed the expected de- cline over time indicative of habituation (time effect: horisontal activity- F [2, 30] =21. 1; p<<0. 001; vertical activity/rearings-F [2,30] =29.3 ; p<<0. 001).

Neither horisontal (n=6 ; F [2,15] =0.3 ; p=0. 74) nor vertical activity (F [2, 15] =2. 0; p=0. 17) was affected by BIIE0246 treatment. Summary data for the 30 min test pe- riod are shown in table 1.

Table 1.

Unaffected exploratory locomotor activity following intracerebroventricular admini- stration of BIIE0246 (n=6 ; mean. SEM).

BIIE0246 (nmol) Horizontal Vertical 0.0 2503.0 380. 0 877.2 202. 6 0.3 2873.8 572.0 756.8 122.9 1.0 2630.2 480. 0 503.3 113. 4

Reference List 1. Broqua P, Wettstein JG, Rocher MN, Gauthier-Martin B, Junien JL. Behav- ioral effects of neuropeptide receptor agonists in the elevated plus-maze and fear-potentiated startle procedure. Behavioural Pharmacology 1995; 6: 215-22.

2. Ehlers CL, Li TK, Lumeng L, Hwang BH, Somes C, Jimenez P, Mathe AA.

Neuropeptide Y levels in ethanol-naive, alcohol-preferring, and non- preferring rats and in Wistar rats after ethanol exposure. Alcoholism Clinical & Experimental Research 1998; 22 (8): 1778-82.

3. Ehlers CL, Somes C, Cloutier D. Are some of the effects of ethanol mediated through NPY ? Psychopharmacology 1998; 139 (1-2): 136-44.

4. Ehlers CL, Somes C, Li TK, Lumeng L, Hwang BH, Jimenez P, Mathe AA.

Calcitonin gene-related peptide (CGRP) levels and alcohol. Internation Journal of Neuropsychopharmacology 1999; 2: 173-9.

5. Eva C, Keinanen K, Monyer H, Seeburg P, Sprengel R. Molecular cloning of a novel G protein-coupled receptor that may belong to the neuropeptide receptor family. Febs Letters 1990; 271: 81-4.

6. Heilig M. Antisense inhibition of neuropeptide Y (NPY)-Y1 receptor expres- sion blocks the anxiolytic like action of NPY in amygdala and para- doxically increases feeding. Regulatory Peptides 1995; 59 (2): 201-5.

7. Heilig M, McLeod S, Brot M, Heinrichs SC, Menzaghi F, Koob GF, Britton KT. Anxiolytic-like action of neuropeptide Y: mediation by Y1 recep- tors in amygdala, and dissociation from food intake effects. Neuropsy- chopharmacology 1993; 8: 357-63.

8. Heilig M, Murison R. Intracerebroventricular neuropeptide Y suppresses open field and home cage activity in the rat. Regulatory Peptides 1987; 19: 221-31.

9. Herzog H, Hort YJ, Ball HJ, Hayes G, Shine J, Selbie LA. Cloned human neu- ropeptide Y receptor couples to two different second messenger sys- tems. Proceedings of the National Academy of Sciences of the United States of America 1992; 89: 5794-8.

10. Hirvonen J, Kalia YN, Guy RH. Transdermal delivery of peptides by iontopho- resis. Nature Biotechnology 1996; 14 (13): 1710-3.

11. Hwang BH, Zhang JK, Ehlers CL, Lumeng L, Li TK. Innate differences of neuropeptide Y (NPY) in hypothalamic nuclei and central nucleus of the amygdala between selectively bred rats with high and low alcohol preference. Alcoholism Clinical & Experimental Research 1999; 23 (6): 1023-30.

12. Hyytia P, Koob GF. GABA (A) receptor antagonism in the extended amygdala decreases ethanol self-administration in rats. European Journal of Pharmacology 1995; 283 (1-3): 151-9.

13. Larhammar D, Blomqvist AG, Yee F, Jazin E, Yoo H, Wahlestedt C. Cloning and functional expression of a human neuropeptide Y/peptide YY re- ceptor of the Y1 type. Journal of Biological Chemistry 1992; 267: 10935-8.

14. McQuiston AR, Colmers WF. Neuropeptide Y2 receptors inhibit the frequency of spontaneous but not miniature EPSCs in CA3 pyramidal cells of rat hippocampus. Journal of Neurophysiology 1996; 76 (5): 3159-68.

15. Paxinos G; Watson C. The rat brain in stereotaxic coordinates. 2 ed. San Di- ego: Academic Press; 1986.

16. Qian J, Colmers WF, Saggau P. Inhibition of synaptic transmission by neuro- peptide Y in rat hippocampal area CA1 : modulation of presynaptic Ca2+ entry. Journal of Neuroscience 1997; 17 (21): 8169-77.

17. Roberts AJ, Heyser CJ, Koob GF. Operant self-administration of sweetened versus unsweetened ethanol: effects on blood alcohol levels. Alcohol- ism Clinical & Experimental Research 1999; 23 (7): 1151-7.

18. Rose PM, Fernandes P, Lynch JS, Frazier ST, Fisher SM, Kodukula K, Kien- zle B, Seethala R. Cloning and functional expression of a cDNA en- coding a human type 2 neuropeptide Y receptor. Journal of Biological Chemistry 1995; 270 (39): 22661-4.

19. Sajdyk TJ, VandergriffMG, Gehlert DR. Amygdalar neuropeptide Y Y-1 re- ceptors mediate the anxiolytic-like actions of neuropeptide Y in the so- cial interaction test. European Journal of Pharmacology 1999; 368 (2- 3): 143-7.

20. Sheikh SP, Håkansson R, Schwartz TW. Y, and Y2 receptors for neuropeptide Y. Febs Letters 1989; 245: 209-14.

21. Stanley BG. Wahlestedt C, Colmers WF, editors. The Biology of Neuropeptide Y. Totowa: Humana Press; 1993; Neuropeptide Y in multiple hypotha- lamic sites controls eating behavior, endocrine and autonomic systems for body energy balance. p. 457-509.

22. Thiele TE, Marsh DJ, Ste M, Bernstein IL, Palmiter RD. Ethanol consumption and resistance are inversely related to neuropeptide Y levels. Nature 1998; 396 (6709): 366-9.

23. Thorsell A, Carlsson K, Ekman R, Heilig M. Behavioral and endocrine adap- tation, and up-regulation of NPY expression in rat amygdala following repeated restraint stress. Neuroreport 1999; 10 (14): 3003-7.

24. Thorsell A, Michalkiewicz M, Dumont Y, Quirion R, Caberlotto L, Rimondini R, Mathe AA, Heilig M. Behavioral insensitivity to restraint stress, ab- sent fear suppression of behavior and impaired spatial learning in trans- genic rats with hippocampal neuropeptide Y overexpression. Proceed- ings of the National Academy of Sciences of the United States of America 2000; 97 (23): 12852-7.

25. Thorsell A, Svensson P, Wiklund L, Sommer W, Ekman R, Heilig M. Sup- pressed neuropeptide Y (NPY) mRNA in rat amygdala following re- straint stress. Regulatory Peptides 1998; 75-6 : 247-54.

26. Wahlestedt C, Pich EM, Koob GF, Yee F, Heilig M. Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides.

Science 1993; 259: 528-31.