WO2012031328A1 - Treatments for substance abuse and addiction - Google Patents

Abstract

The present invention relates to methods for the prevention or inhibition of negative behaviours associated with substance abuse or addiction, such as substance use (self-administration) or substance seeking behaviour, the methods comprising administering to subjects in need thereof an antagonist of a relaxin-3 receptor. In particular embodiments the relaxin-3 receptor is the RXFP3 receptor, and in particular embodiments the antagonist is a modified relaxin polypeptide, optionally a selective antagonist of RXFP3.

Description

Treatments for substance abuse and addiction

Field of the Invention

The present invention relates generally to methods for the prevention of, and reduction in, negative behaviours associated with substance abuse and addiction, typically drug self- administration (consumption) and drug seeking or relapsing. Specifically the invention relates to the use of antagonists of relaxin-3 receptors in such methods.

Background of the Invention

The use and abuse of drugs cause enormous health, interpersonal, social and economic problems around the world. Dealing with these problems and combating the rise of substance use and addiction pose serious challenges for every society and every economy. Worldwide, there are estimated to be about 2 billion alcohol users, 1.3 billion smokers and 185 million illicit drug users, accounting for approximately 12% of deaths worldwide each year (WHO, 2002). The cost of substance abuse in the United States alone has been estimated at more than $500 billion per annum. Abuse of alcohol, one of the most readily available of all addictive substances, places enormous social and economic costs on society in terms of burdens on the health and criminal justice systems, damage to property, personal injury and lost productivity as well as being one of the leading 'lifestyle' causes of death in Western countries such as the United States.

Given the extent of this impact, much research is devoted to elucidating the mechanisms that drive the motivation to use alcohol and other drugs of abuse. Such information is clearly necessary for the improved targeting of therapeutics to assist in the overall management of patients presenting with substance abuse disorders.

Substance use and abuse leads to addiction, a neurobiological disease characterized by repeated episodes of compulsive drug seeking and use (see, for example, Jupp and Lawrence, 2010 for a review). Broadly speaking, characteristics of addiction include dependence on the addictive substance (drug use) and relapse (drug seeking) following withdrawal. Withdrawal occurs after cessation of drug taking and is typically associated with negative side effects including dysphoria, anxiety and irritability, which in turn tend to drive renewed drug seeking and relapse as a means of reducing these negative effects. Persistent drug craving and susceptibility to relapse of recovering or recovered addicts well after the end of a withdrawal syndrome is another feature of addiction (see Jupp and Lawrence, 2010). Factors including stress and certain environmental stimuli, or cues, contribute significantly to this.

Much effort has been devoted to the development of pharmacological agents for the treatment of substance abuse and addiction, and a number of therapeutic agents are in use or in clinical trials for the treatment of several drug addictions. However in most cases, these therapies are largely ineffective, suffering from problems including non-compliance and high relapse rates. For example in the treatment of alcohol addiction, relapse rates remain high under treatment with the currently used therapeutics of choice such as naltrexone, nalmefene, disulfiram and acamprosate, while new agents under development such as baclofen and topiramate appear to suffer from tolerance issues and side effects. Similar problems exist with therapeutics in use and under investigation for treating opiate addiction. For many other forms of addiction there remain no viable therapeutics.

There remains a clear and urgent need for the development of novel therapeutic approaches to the problem of substance abuse and addiction. In this regard, recent attention has moved to small neuropeptides and there is emerging a view that therapeutics useful for the treatment of alcohol addiction may also be useful for the treatment of opiate addiction, in particular agents that target GABAergic and stress related neuropeptide systems.

Summary of the Invention

In a first aspect the present disclosure provides a method for the prevention or inhibition of negative behaviour associated with substance abuse or addiction, the method comprising administering to a subject in need thereof an effective amount of an antagonist of a relaxin-3 receptor. Typically the negative behaviour comprises, but is not limited to, substance use (self- administration) and/or substance seeking behaviour. The substance may be any addictive substance. In particular embodiments the substance is selected from alcohol, nicotine, an opiate, a cannabinoid, a psychostimulant or an inhalant.

Typically the relaxin-3 receptor is selected from RXFP3 (GPCR135) and RXFP4 (GPCR142). More typically the relaxin-3 receptor is RXFP3. Even more typically the antagonist is a selective antagonist of RXFP3.

The relaxin-3 receptor antagonist may be a modified or chimeric relaxin-3 polypeptide. The chimeric polypeptide may comprise a B chain derived from relaxin-3 and an A chain derived from a different member of the relaxin superfamiiy. In one embodiment the A chain is derived from INSL5. In a further embodiment the relaxin-3 derived B chain includes a truncation of one or more amino acids at the C-terminus. For example the B chain may be derived from human relaxin-3 and may include a truncation of five amino acids from the C-terminus. The deleted C- terminal residues may be replaced by one or more amino acid residues. In a particular exemplary embodiment the relaxin-3 receptor antagonist is a selective polypeptide antagonist of RXFP3 comprising a B chain comprising an amino acid sequence as set forth in SEQ ID NO:2, or a variant or derivative thereof and an A chain derived from INSL5 comprising an amino acid sequence as set forth in SEQ ID NO:3, or a variant or derivative thereof. In a further particular exemplary embodiment the relaxin-3 receptor antagonist is a selective polypeptide antagonist of RXFP3 comprising only a relaxin-3 derived B chain wherein the B chain sequence is truncated by up to about 5 amino acids at the C-terminus compared to the native relaxin-3 B chain sequence and a basic amino acid residue is incorporated at the C-terminus of the relaxin-3 derived B chain. Typically the basic amino acid residue is arginine. The single chain polypeptide may be further modified such that one or more cysteine residues in the native relaxin-3 B chain sequence are replaced by neutral amino acids, typically serine or alanine, more typically serine. In a particular embodiment, where the B chain sequence is derived from human relaxin-3 the cysteine residues at positions 10 and 22 of the native human relaxin-3 sequence are replaced by serine residues. In a particular embodiment the single chain polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, or a variant or derivative thereof. ln a further particular exemplary embodiment the relaxin-3 receptor antagonist comprises: (i) a relaxin-3 derived A chain truncated by up to about 10 amino acids at the N-terminus compared to the native relaxin-3 A chain sequence; (ii) a relaxin-3 derived B chain truncated by about 5 amino acids at the C-terminus compared to the native relaxin-3 B chain sequence; and optionally (iii) a basic amino acid incorporated at the C-terminus of the relaxin-3 derived B chain. The basic amino acid may be arginine. In one embodiment, the C-terminal 5 amino acids from the native sequence of the relaxin-3 B chain are replaced by a terminal arginine residue. The B chain may comprise or consist of the amino acid sequence set forth in SEQ ID NO:14, or a variant or derivative thereof. In one embodiment the polypeptide comprises an A chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5, or a variant or derivative thereof and a B chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14, or a variant or derivative thereof, and wherein the polypeptide is an antagonist of the RXFP3 receptor. In embodiments in which the antagonist is a modified or chimeric relaxin-3 polypeptide, the antagonist may be administered to the subject in the form of a polypeptide, either in mature form or as a precursor polypeptide. Alternatively the subject may be administered a polynucleotide encoding the polypeptide antagonist. In a second aspect the present disclosure provides a method for the treatment of substance addiction in a subject, the method comprising administering to the subject an effective amount of an antagonist of a relaxin-3 receptor.

In a third aspect the present disclosure provides the use of an antagonist of a relaxin-3 receptor for the prevention or inhibition of negative behaviour associated with substance abuse or addiction or for the treatment of substance addiction.

In a fourth aspect the present disclosure provides the use of an antagonist of a relaxin-3 receptor for the manufacture of a medicament for the prevention or inhibition of negative behaviour associated with substance abuse or addiction or for the treatment of substance addiction.

Also provided are pharmaceutical compositions for the prevention or inhibition of negative behaviour associated with substance abuse or addiction, and for the treatment of substance addiction, wherein the compositions comprise an antagonist of a relaxin-3 receptor, optionally together with one or more pharmaceutically acceptable carriers.

Brief Description of the Drawings

The present invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings.

Figure 1. Operant responding for alcohol in alcohol-preferring (iP) rats and attenuation by the selective RXFP3 antagonist R3(BA23-27)/l5 (n = 18). All data are expressed as (± SEM). (A) Total ethanol lever presses under FR3 conditions after a 7 day recovery period and restabilisation of alcohol self-administration following intracerebroventricular cannulation. *p<0.05, **p<0.01 , ***p<0.001. (B) Total water lever presses showed no significant differences between treatment groups. All data were analysed by one-way repeated ANOVA and Tukey posf hoc test.

Figure 2. Extinction and cue-induced reinstatement of alcohol-seeking in iP rats (n = 18) following administration of the selective RXFP3 antagonist R3(BA23-27)/l5. (A) Active lever presses, with a statistical significance (p = 0.003) between vehicle (aCSF) and extinction responding. (B) Inactive lever presses showed no statistical difference between treatment groups. Statistical analysis of the data (mean ± SEM) was performed using one-way repeated ANOVA and Tukey post hoc tests.

Figure 3. Effect of R3B1-22R (3-30 μg icv) on responding for (A) alcohol (B, D) water, (C) sucrose. A dose-related reduction in alcohol self-administration occurs with no effect on water or sucrose. Baseline responding for alcohol and sucrose is similar. Water responding in B occurred during the alcohol test and that in D during the sucrose test. (Scale in D expanded to reveal low level of water responding that is stable across treatments). Number of rats marked inside columns. *P<0.05; **P<0.01 vs vehicle. Amino acid sequences of relaxin superfamily B chain and A chain sequences described herein are set forth in SEQ ID Nos: 1 to 14. The sequence provided in SEQ ID NO:1 represents the amino acid sequence of the B chain of human relaxin-3. The sequence provided in SEQ ID NO:2 represents the C-terminal modified amino acid sequence of the human relaxin-3 B chain as present in the selective RXFP3 antagonist R3(BA23-27)/l5 described herein. The sequence provided in SEQ ID NO:3 represents the amino acid sequence of the A chain of human INSL5. The sequence provided in SEQ ID NO:4 represents the A chain sequence of human relaxin-3 in which the cysteine residues at positions 10 and 15 responsible for intra-chain disulphide bond formation are replaced by alanine residues. The sequence provided in SEQ ID NO:5 represents the A chain sequence of SEQ ID NO:4 truncated by 10 amino acid residues at the N-terminus. The sequence provided in SEQ ID NO:6 is that of the single chain relaxin-3 B chain derived polypeptide B1-22R. The sequences provided in SEQ ID Nos:7 to 13 represent additional single chain relaxin-3 B chain derived polypeptides. The sequence provided in SEQ ID NO:14 is that of the relaxin-3 derived B chain present in the relaxin analogue designated herein as analogue 16.

Detailed Description of the Invention

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

The term "polypeptide" means a polymer made up of amino acids linked together by peptide bonds. The term "peptide" may also be used to refer to such a polymer although in some instances a peptide may be shorter (i.e. composed of fewer amino acid residues) than a polypeptide. Nevertheless, the terms "polypeptide" and "peptide" are used interchangeably herein. The term "modified" as used herein in the context of a relaxin polypeptide means a polypeptide that differs from a naturally occurring relaxin polypeptide at one or more amino acid positions in one or more peptide chains of such naturally occurring polypeptide. The term "polynucleotide" as used herein refers to a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof. The term includes reference to the specified sequence as well as to the sequence complimentary thereto, unless otherwise indicated, The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

The term "conservative amino acid substitution" as used herein refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (Glu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution. The nature of other conservative amino acid substitutions is well known to those skilled in the art.

As used herein the term "derived" in the context of relaxin A and B chains means that the A and B chain sequences correspond to, originate from, or otherwise share significant sequence homology with naturally occurring A and B chain sequences. Thus, for example, in accordance with embodiments described herein a relaxin B chain "derived" from a relaxin-3 polypeptide may be identical to the B chain sequence of a relaxin-3 polypeptide from any species, such as the human H3 relaxin, may be a modified version or variant thereof, or alternatively may share sequence homology with one or more relaxin-3 B chain sequences from any species. In the context of relaxin polypeptides the term "naturally occurring" refers to relaxin polypeptides as encoded by and produced from the genome of an organism. For example in humans three distinct forms of relaxin have been identified to date, H1 , H2 and H3. Each of these forms is considered herein as a different "naturally occurring" relaxin. Those skilled in the art will also understand that by being "derived" from a naturally occurring or native relaxin sequence, the sequence in the modified polypeptide need not be physically constructed or generated from the naturally occurring or native sequence, but may be chemically synthesised such that the sequence is "derived" from the naturally occurring or native sequence in that it shares sequence homology and function with the naturally occurring or native sequence.

As used herein the term "selective" when used in the context of the ability of a molecule to act as an antagonist of a relaxin-3 receptor means that the molecule antagonises the relaxin-3 receptor in question to a significantly greater extent than it antagonises other receptors. For example I the case of selective RXFP3 antagonists, the polypeptide antagonises RXFP3 to a greater extent than, for example, the RXFP1 receptor. Where the antagonist is a modified or chimeric relaxin polypeptide the polypeptide may bind to the receptor or otherwise interact with the receptor directly or indirectly at significantly higher frequency than it binds or interacts with other receptors. Embodiments described herein contemplate the selective antagonism of relaxin-3 receptors as compared with non relaxin-3 receptors and also contemplates the selective antagonism of a particular relaxin-3 receptor as compared with other relaxin-3 receptors. As used herein the terms "preventing" and "inhibiting" and the like refer to any and all uses which remedy, prevent the establishment or development of, or otherwise hinder, reduce, retard, or reverse one or more negative behaviours associated with substance abuse or addiction. Thus the terms "preventing" and "inhibiting" and the like are to be considered in their broadest context. For example, "prevention" does not necessarily mean that the subject will not eventually develop one or more of negative behaviours associated with substance abuse or addiction. Rather, "prevention" and "inhibition" encompass reducing the incidence or severity of negative behaviours or delaying the onset of such behaviours.

As used herein the terms "treating" and "treatment" refer to any and all uses which remedy an addiction, or otherwise prevent, hinder, retard, or reverse the progression of an addiction. Thus the terms "treating" and "treatment" are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery. Accordingly, treatment includes amelioration of one or more symptoms of, or characteristic behaviours associated with, an addiction.

As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount or dose of an agent or compound to provide the desired effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the nature and severity of the substance abuse or addiction, the identity of the substance used by the subject or to which the subject is addicted, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

A key problem with alcoholism, as with substance addiction in general, is the chronically relapsing nature of the disorder. This behaviour pattern can be effectively modelled in rodents, where numerous studies have demonstrated the ability of drug priming, psychological stress or the representation of cues previously associated with drug availability to reinstate drug-seeking behaviour following extinction, even in the absence of subsequent drug rewards. Moreover, despite differences between these means of reinstating previously extinguished behaviour, there is thought to be good general correspondence between animal studies of reinstatement and human experience of relapse (Shaham ef a/., 2003). The present inventors have developed models of cue-driven relapse to drug-seeking that collectively incorporate the varying human experience. The inventors have developed three models of relapse: (i) extinction-reinstatement; (ii) extinction followed by protracted withdrawal prior to reinstatement; and (iii) cue-conditioned drug-seeking following abstinence, with no extinction training. These models utilise the traditional operant self-administration paradigm, and in all cases relapse-like drug-seeking is precipitated by the re-exposure of rodents to cues previously associated with the availability of drug within the environment of the operant chamber. As exemplified herein, using such models the inventors have demonstrated the ability of a chimeric neuropeptide antagonist of the relaxin-3 receptor RXFP3 (the antagonist hereinafter referred as "R3(BA23-27)/l5") and of a single chain modified relaxin polypeptide antagonist (designated herein "B1-22R") to regulate self-administration of alcohol in alcohol-preferring rats and to inhibit the cue-induced reinstatement of alcohol-seeking in these rats. The findings of the inventors' present studies represent the first functional indications that the nucleus incertus/relaxin-3 pathway is implicated in alcohol and/or drug-seeking. Relaxin-3 is a heterodimeric peptide composed, in its mature form, of an A chain and a 6 chain linked via a disulphide bridge. Relaxin-3 is produced as a pro-peptide with a third amino acid (C) chain in the chain configuration B-C-A. Relaxin-3 is a member of a protein hormone superfamily which also includes insulin, insulin-like grown factors I and II (IGF-I and IGF-II), and the insulin- like hormones INSL3, 4, 5 and 6. The relaxin superfamily members have a wide range of biological activities which are well described in the art. Relaxin-3 has been conserved through vertebrate evolution and has been characterised in a large and diverse range of vertebrate species. Whilst in most species only two forms of relaxin have been identified (relaxin and relaxin-3), in humans three distinct forms of relaxin have been described and the genes and polypeptides characterised. These have been designated H1 , H2 and H3. Homologues of H1 and H2 relaxin have been identified in other higher primates including chimpanzees, gorillas and orangutans.

Of the three forms of relaxin in humans, the polypeptide encoded by the H2 gene is the major stored and circulating form. H2 relaxin is the only form known to be secreted in the blood. H1 relaxin expression is largely restricted to the decidua, placenta and prostate, whilst H3 relaxin expression is predominant in the brain. The differing expression patterns for H1 , H2 and H3 relaxin may suggest some differences in biological roles, however all three forms display similar biological activity as determined, for example, by their ability to alter cAMP levels in cells expressing relaxin family receptors, and accordingly share some biological functions in common.

The biological actions of relaxins are mediated through G protein coupled receptors (see Bathgate ef a/., 2006 for a review). To date, H1, H2 and H3 relaxins have been shown to primarily recognise and bind four receptors, LGR7 (RXFP1), LGR8 (RXFP2), GPCR135 (RXFP3) and GPCR142 (RXFP4). LGR7 binds each of H1 , H2 and H3 with high affinity. H1 and H2 relaxin also bind LGR8. H3 relaxin (relaxin-3) binds GPCR135 and GPCR142 and has moderate affinity for LGR7. Together with relaxin-3 GPCR135 has been shown to be highly expressed in brain tissue. Recent studies have demonstrated that a neural network seated in the midline pontine area of the human brain in a region known as the nucleus incertus expresses high levels of relaxin-3 (Bathgate ef a/., 2002) and is involved in the regulation of motivated behaviours, such as feeding, and is associated with reward/motivated behaviour pathways (see for example Ma ef a/., 2007). In one aspect, provided herein is a method for the prevention or inhibition of negative behaviour associated with substance abuse or addiction, the method comprising administering to a subject in need thereof an effective amount of an antagonist of a relaxin-3 receptor.

In accordance with embodiments disclosed herein the substance is typically an addictive substance wherein use or abuse of the substance typically results in or is associated with an addiction to the substance. Exemplary addictive substances to which embodiments of the disclosure relate include, but are not limited to alcohol, opiates, cannabinoids, nicotine, inhalants and psychostimulants such as cocaine, amphetamine and methamphetamine. In particular embodiments the substance is selected from alcohol and opiates. Addiction to substances such as alcohol, opiates, cannabinoids, nicotine and psychostimulants is typically associated with a number of adverse or negative behaviours exhibited by addicts, which behaviours may serve to exacerbate, prolong or induce relapse into use or abuse of the substance, reinforce or exacerbate the addiction, or induce relapse into addiction and addictive behaviour patterns. Embodiments of the present disclosure provide methods and compositions for the prevention and inhibition of such negative behaviours including, but not limited to the desire to consume the substance and substance-seeking behaviour. Other examples of negative behaviours associated with substance use or addiction include anxiety, dysphoria, stress reactivity and cue reactivity. Embodiments disclosed herein also provide for the treatment of the substance abuse or addiction.

Embodiments disclosed herein contemplate the antagonism of a relaxin-3 receptor in order to treat substance abuse or addiction, or prevent or inhibit negative behaviours associated with substance abuse or addiction. The relaxin-3 receptor is any receptor to which relaxin-3 binds and through which relaxin-3 initiates biological activity. Typically the receptor is selected from RXFP3 (GPCR135) and RXFP4 (GPCR142), more typically RXFP3. In particular embodiments the antagonist is a selective antagonist of RXFP3.

Those skilled in the art will appreciate that a relaxin-3 receptor antagonist for use in accordance with the embodiments disclosed herein may be any compound capable of blocking, inhibiting or otherwise preventing the relaxin-3 receptor from carrying out its normal biological function in the brain. Suitable antagonists may be proteinaceous or non-proteinaceous molecules. Suitable proteinaceous molecules include analogues or derivatives of relaxin-3 and antibodies, whilst suitable non-proteinaceous molecules include nucleic acid molecules encoding said relaxin-3 analogues and derivatives, and small molecule inhibitors. Analogues and derivatives of relaxin-3 may include manipulatbns of the amino acid sequence of the B chain and/or A chain of relaxin-3 such that the agonistic activity of wild-type or native relaxin-3 against a relaxin-3 receptor is replaced by antagonistic activity. Such manipulations may comprise, for example, altering the amino acid sequence at one or more locations (one or more amino acid residues) within the B chain and/or A chain, generating chimeric polypeptides in which one of the B chain or A chain is derived from relaxin-3 with the other chain being derived from a different relaxin superfamily member, and/or modifying the amino acid sequences of the B chain and/or A chain such as by incorporating unnatural amino acids and/or derivatives during peptide synthesis and using of crosslinkers or other approaches to impose conformational constraints on the polypeptide. The nature and suitability of particular modifications can be routinely determined by the person of skill in the art.

Amino acid changes may be effected by techniques well known to those persons skilled in the relevant art. For example, amino acid changes may be effected by nucleotide replacement techniques which include the addition, deletion or substitution of nucleotides (conservative and/or non-conservative), under the proviso that the proper reading frame is maintained. A conservative substitution denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norieucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids which can be substituted for one another include asparagine, glutamine, serine and threonine. The term "conservative substitution "also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Exemplary techniques for generating such amino acid insertion, deletion or substitution modifications include random mutagenesis, site-directed mutagenesis, oligonucleotide-mediated or polynucleotide-mediated mutagenesis, deletion of selected region(s) through the use of existing or engineered restriction enzyme sites, and the polymerase chain reaction. Such techniques will be well known to those skilled in the art.

Polypeptides can also be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention. The polypeptides can also be further modified to create polypeptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently-bound complexes can be prepared by cross-linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N- or C-terminus. Further, the polypeptides of the invention can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e. g., biotin or avidin). These are merely exemplary additional modifications that may be made to the modified polypeptides of the invention. Those skilled in the art will appreciate that further modifications may also be made so as to generate analogues of the polypeptides of the invention. By way of example only, illustrative analogues and processes for preparing the same are described in International patent application published as WO 2004/113381, the disclosure of which is incorporated herein by reference in its entirety. In particular embodiments the relaxin-3 receptor antagonist is a relaxin-3 derived polypeptide comprising at least a B chain sequence derived from native relaxin-3. The amino acid sequence of native human relaxin-3 (H3 relaxin) B chain is provided herein in SEQ ID NO:1. In exemplary embodiments modifications, including the addition, deletion and/or substitution of one or more amino acid residues, may be made to this sequence. Accordingly, the B chain amino acid sequences of relaxin polypeptides the subject of the present disclosure may be based on or derived from the amino acid sequence of the B chain of H3 relaxin, for example the sequence depicted in SEQ ID N0.1. However those skilled in the art will also appreciate that the amino acid sequences of B chains from which the modified polypeptides of the invention may be based, or from which the modified polypeptides may be derived, may include variants of this H3 relaxin B chain sequence.

In a particular embodiment the relaxin polypeptide is a chimeric polypeptide comprising a B chain derived from relaxin-3 and an A chain derived from a different member of the relaxin superfamily. The relaxin superfamily member from which the A chain is derived may be selected from relaxin- 1 , relaxin-2, insulin, IGF-I, IGF-II or insulin-like peptides (INSL) 3, 4, 5 or 6. The polypeptide sequences may be derived from any suitable species, but typically human where the antagonist is to be administered to a human subject. In a particular embodiment the A chain in a chimeric antagonist polypeptide is derived from INSL5. In exemplary embodiments the relaxin-3 derived B chain includes a truncation of one or more amino acids at the C-terminus. For example the B chain may be derived from human relaxin-3 and may include a truncation of five amino acids from the C-terminus. The deleted C-terminal residues may be replaced by one or more different amino acid residues. For example, the amino acid residue replacing the native C-terminal residues deleted may be an arginine residue. In a particular exemplary embodiment the relaxin-3 receptor antagonist is a selective polypeptide antagonist of RXFP3 comprising a B chain derived from relaxin-3 comprising an amino acid sequence as set forth in SEQ ID NO:2, or a variant or derivative thereof and an A chain derived from INSL5 comprising an amino acid sequence as set forth in SEQ ID NO:3, or a variant or derivative thereof. An antagonist consisting of the B chain sequence of SEQ ID NO:2 and the A chain sequence of SEQ ID NO:3 is termed herein R3(BA23- 27)/l5. This antagonist is described in US patent publication no. 20080051336, the disclosure of which is incorporated herein in its entirety by reference. In other exemplary embodiments of the present disclosure the antagonist comprises a relaxin A chain and a B chain derived from a relaxin superfamily member, typically relaxin-3, wherein the A chain comprises no intra-chain disulphide bonds and wherein the B chain comprises a truncation of one or more amino acids from the C-terminus compared to the corresponding native sequence. The B chain may be truncated by, for example, five amino acid residues at the C-terminus. The terminal five amino acid residues of the B chain of relaxin-3 may be deleted and replaced by a single arginine residue. In an embodiment the polypeptide comprises a B chain sequence as set forth in SEQ ID NO:2 and an A chain sequence derived from relaxin-3, said A chain sequence comprising one or more amino acid substitutions such that one or more cysteine residues are replaced by different amino acids thereby removing intra-chain disulphide bonds from the A chain but wherein the A chain retains the ability to form inter-chain disulphide bonds with a B chain. For example, the cysteine residues at positions 10 and 15 (or corresponding positions) of the human relaxin-3 A chain sequence may be replaced by alanine residues by site-directed mutagenesis, giving rise to the modified A chain sequence set forth in SEQ ID NO:4. In another embodiment the A chain sequence of SEQ ID NO:4 is truncated by up to 10 amino acids at the N terminus, giving rise for example to the modified A chain sequence set forth in SEQ ID NO:5. In a further particular embodiment the relaxin polypeptide is a selective polypeptide antagonist of RXFP3 comprising only a relaxin-3 derived B chain wherein the B chain sequence is truncated by up to about 5 amino acids at the C-terminus compared to the native relaxin-3 B chain sequence and a basic amino acid residue is incorporated at the C-terminus of the relaxin-3 derived B chain. Typically the basic amino acid residue is arginine. The single chain polypeptide may be further modified such that one or more cysteine residues in the native relaxin-3 B chain sequence are replaced by neutral amino acids, typically serine or alanine, more typically serine. The single chain polypeptide typically comprises a C-terminal amide or acid group, more typically a C- terminal amide group. The single chain polypeptide may further comprise a truncation of one or more amino acids from the N-terminus of the relaxin-3 B chain when compared to the native relaxin-3 sequence. In particular embodiments, the truncation is of up to about 4 amino acids from the N-terminus. In a particular embodiment, where the B chain sequence is derived from human relaxin-3 the cysteine residues at positions 10 and 22 of the native human relaxin-3 sequence are replaced by serine residues. In a particular embodiment the single chain polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, or a variant or derivative thereof. The antagonist having the sequence of SEQ ID NO:6 is described in co-pending patent application no. (and designated therein "B1-22R"), the disclosure of which is incorporated herein in its entirety by reference. Other single chain relaxin polypeptides having antagonist activity at RXFP3 are also described in this co-pending application (provided herein in SEQ ID Nos:7 to 13, and the present disclosure contemplates the administration of such antagonists in accordance with the methods described herein.

In a further particular embodiment the relaxin polypeptide comprises: (i) a relaxin-3 derived A chain truncated by up to about 10 amino acids at the N-terminus compared to the native relaxin-3 A chain sequence; (ii) a relaxin-3 derived B chain truncated by about 5 amino acids at the C- terminus compared to the native relaxin-3 B chain sequence; and optionally (iii) a basic amino acid incorporated at the C-terminus of the relaxin-3 derived B chain. The basic amino acid may be arginine. In one embodiment, the C-terminal 5 amino acids from the native sequence of the relaxin-3 B chain are replaced by a terminal arginine residue. The B chain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 14, or a variant or derivative thereof. In one embodiment the polypeptide comprises an A chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5, or a variant or derivative thereof and a B chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:14, or a variant or derivative thereof, and wherein the polypeptide is an antagonist of the RXFP3 receptor. The antagonist having an A chain of SEQ ID NO:5 and a B chain of SEQ ID ΝΟ.Ί4 is described in copending patent application no. (and designated therein "analogue 16"), the disclosure of which is incorporated herein in its entirety by reference.

Antagonistic relaxin polypeptides for use in accordance with the present disclosure may also comprise a relaxin C chain sequence. Relaxin C chain amino acid sequences are known to those skilled in the art. The present invention also contemplates the use of fragments and variants of the antagonistic polypeptides described above.

The term "fragment" refers to a polypeptide molecule that encodes a constituent or is a constituent of a polypeptide of the invention or variant thereof. Typically the fragment possesses qualitative biological activity in common with the polypeptide of which it is a constituent. The peptide fragment may be between about 5 to about 150 amino acids in length, between about 5 to about 100 amino acids in length, between about 5 to about 50 amino acids in length, or between about 5 to about 25 amino acids in length. Alternatively, the peptide fragment may be between about 5 to about 15 amino acids in length.

The term "variant" as used herein refers to substantially similar sequences. Generally, polypeptide sequence variants possess qualitative biological activity in common. Further, these polypeptide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. Also included within the meaning of the term "variant" are homologues of polypeptides of the invention. A homologue is typically a polypeptide from a different species but sharing substantially the same biological function or activity as the corresponding polypeptide disclosed herein. Further, the term "variant" also includes analogues of the polypeptides of the present disclosure, wherein the term "analogue" means a polypeptide which is a derivative of a polypeptide of the disclosure, which derivative comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide typically retains substantially the same function, for example in terms of receptor binding activity. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in a polypeptide although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences may include fusions with other peptides, polypeptides or proteins. Modifications may be made to relaxin polynucleotide sequences, for example via the insertion or deletion of one or more codons, such that modified derivatives of the relaxin polypeptide are generated. Such modifications are also included within the scope of the term "variant". For example, modifications may be made so as to enhance the biological activity or expression level of the relaxin or to otherwise increase the effectiveness of the polypeptide to achieve a desired outcome. In accordance with the present invention relaxin polypeptides may be produced using standard techniques of recombinant DNA and molecular biology that are well known to those skilled in the art. Guidance may be obtained, for example, from standard texts such as Sambrook et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989 and Ausubel ef a/., Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-lntersciences, 1992. Methods described in Morton ef a/., 2000 (Immunol Cell Biol 78:603-607), Ryan ef a/., 1995 (J Biol Chem 270:22037-22043) and Johnson et a/., 2005 (J Biol Chem 280:4037-4047) are examples of suitable purification methods for relaxin polypeptides, although the skilled addressee will appreciate that the present invention is not limited by the method of purification or production used and any other method may be used to produce relaxin for use in accordance with the methods and compositions of the present invention. Relaxin peptide fragments may be produced by digestion of a polypeptide with one or more proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested peptide fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. The purification of relaxin polypeptides may be scaled-up for large-scale production purposes. For this purpose a range of techniques well known to those skilled in the art are available. Relaxin polypeptides for use in accordance with the present disclosure, as well as fragments and variants thereof, may also be synthesised by standard methods of liquid or solid phase chemistry well known to those of ordinary skill in the art. For example such molecules may be synthesised following the solid phase chemistry procedures of Steward and Young (Steward, J. M. & Young, J. D, Solid Phase Peptide Synthesis. (2nd Edn.) Pierce Chemical Co., Illinois, USA (1984). In general, such a synthesis method comprises the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Typically, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected amino acid Is then either attached to an inert solid support or utilised in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected and under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next (protected) amino acid is added, and so forth. After all the desired amino acids have been linked, any remaining protecting groups, and if necessary any solid support, is removed sequentially or concurrently to produce the final polypeptide.

Embodiments of the present disclosure also contemplate the administration of nucleic acids encoding antagonistic polypeptides described herein. The polynucleotide sequence^) may be provided in a suitable vector system operably linked to a regulatory sequence(s) (typically promoters, enhancers and the like) which is capable of providing for the expression of the encoded polypeptide sequence(s) by a cell. Suitable vectors and regulatory sequences are well known to those skilled in the art. By way of example, the vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into eukaryotic cells and the expression of the introduced sequences. Typically the vector is a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences. Those skilled in the art will also appreciate that heterologous expression of polypeptides may be improved by optimising the codons for the particular species in which the relaxin polypeptide is to be expressed. Accordingly, polynucleotides encoding antagonistic polypeptides may be codon-optimised for expression in a particular species. Alternative approaches for the antagonism of relaxin-3 receptors are also contemplated herein, for example antibodies and small molecule inhibitors. Suitable antagonistic antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, humanized and single chain antibodies, Fab fragments, and Fab expression libraries. Typically suitable antagonistic antibodies are prepared from discrete regions or fragments of the relaxin-3 receptor polypeptide. Methods for the generation of suitable antibodies will be readily appreciated by those skilled in the art. For example, a monoclonal antibody may be prepared using the hybridoma technology described in Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N.Y. (1988). Screening for the desired antibody can also be accomplished by a variety of techniques known in the art. Assays for immunospecific binding of antibodies may include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, and the like (see, for example, Ausubel ef a/., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Similarly, small molecule antagonists can be designed, synthesized and screened by methods well known to those skilled in the art, including using combinatorial chemistry.

In general, suitable compositions for use in accordance with the methods of the present disclosure may be prepared according to methods and procedures that are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.

Compositions may be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intracerebroventricular, intraspinal, subcutaneous or intramuscular), oral or topical route. Administration may be systemic, regional or local. The particular route of administration to be used in any given circumstance will depend on a number of factors, including the nature of the condition to be treated, the severity and extent of the condition, the required dosage of the particular compound to be delivered and the potential side- effects of the compound.

In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and may include a pharmaceutically acceptable diluent, adjuvant and/or excipient. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

The compositions may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection. For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol. Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylceilulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Methods for preparing parenteral^ administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein. The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included. The compositions may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et sec/., the contents of which is incorporated herein by reference. The therapeutically effective dose level for any particular subject will depend upon a variety of factors including: the addiction being treated and the severity of the addiction; activity of the molecule or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the molecule or agent; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine. One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of agent or compound which would be required to treat applicable diseases and conditions.

Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about 1000mg per kg body weight per 24 hours; typically, about 0.001 mg to about 750mg per kg body weight per 24 hours; about 0.01 mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 500mg per kg body weight per 24 hours; about 0.1mg to about 250mg per kg body weight per 24 hours; about 1.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.Omg to about 200mg per kg body weight per 24 hours; about 1.0mg to about 100mg per kg body weight per 24 hours; about 1.0mg to about 50mg per kg body weight per 24 hours; about 1.0mg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours; about 5.0mg to about 15mg per kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500mg/m². Generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m², preferably about 25 to about 350mg/m², more preferably about 25 to about 300mg/m², still more preferably about 25 to about 250mg/m², even more preferably about 50 to about 250mg/m², and still even more preferably about 75 to about 150mg/m². Typically, in therapeutic applications, the treatment would be for the duration of the addiction or substance abuse by the subject. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the addiction or substance abuse, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques. It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Embodiments of the present disclosure also contemplate the administration of nucleic acid constructs encoding antagonistic polypeptides described herein. The nucleic acid construct to be administered may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers.

Those skilled in the art will appreciate that in accordance with the methods of the present disclosure antagonists as described herein may be administered alone or in conjunction with one or more additional agents. Thus, the present disclosure contemplates combination therapy using antagonists as described herein in conjunction with other therapeutic approaches to the treatment of substance abuse or addiction. For such combination therapies, each component of the combination therapy may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

The present invention will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention. Examples

Example 1 - Regulation of alcohol self-administration by the chimeric relaxin-3 peptide, R3(BA23-27)/l5.

To explore the involvement of endogenous relaxin-3 signalling in motivated drug-seeking behaviour the inventors tested the ability of the selective RXFP3 receptor antagonist, R3(BA23- 27)/l5 (Kuei et al. 2007), for effects on self-administration of alcohol in alcohol-preferring (iP) rats. R3(BA23-27)/l5 is a chimeric relaxin-3 peptide comprising the B chain of human relaxin-3 in which the C-terminal five amino acids (Gly23 - Trp27) are replaced by a single Arg residue and the A chain of INSL5. The amino acid sequence of the B chain of R3(BA23-27)/l5 is shown in SEQ ID NO:2. The amino acid sequence of the A chain of R3(BA23-27)/l5 is shown in SEQ ID NO:3.

Rats were trained to stably administer 10% ethanol during daily operant session (n = 18 rats) and then implanted with guide cannulae aimed at the lateral cerebral ventricle. Following a 7 day recovery period, the rats were re-stabilised on alcohol self-administration under fixed-ratio 3 (FR3) conditions and the effect of R3(BA23-27)/l5 was examined (Figure 1). Compared to vehicle (aCSF) and normal responding, intracerebroventricular (icv) injection of R3(BA23-27)/l5 (10 pg) produced a significant decrease in ethanol lever presses (Figure 1A), and importantly, responding for alcohol recovered when rats were tested 24 hours later (next day). In contrast, water lever responding remained unaffected (Figure 1 B), suggesting a degree of specificity within the effect. Previous studies indicate that R3(BA23-27)/l5 (10 \ig, icv) has no acute effect on locomotor activity.

Example 2 - Effect of R3(BA23-27)/\5 on relapse-like behaviour

Given that treatment with R3(BA23-27)/l5 reduced alcohol self-administration in iP rats (Example 1), the inventors then examined whether the relaxin-3 system is implicated in relapse-like behaviour. In this study, following established alcohol self-administration, iP rats received extinction training such that responding on the active lever was extinguished to levels similar to the inactive lever. Subsequently, rats were treated with either vehicle (aCSF) or R3(BA23-27)/l5 (10 pg) prior to being returned to the operant chambers for re-presentation of cues previously associated with alcohol availability. This caused a robust increase (reinstatement) in responding on the active lever (previously ethanol-paired) in vehicle treated rats (Figure 2).

Thus, reinstatement of responding on the active lever was associated with a significant difference between extinction and vehicle groups (p = 0.003; Figure 2A), indicating that rats had "relapsed" to alcohol-seeking. In contrast, there was no significant difference between extinction and R3(BA23-27)/l5 treatment (p = 0.701), suggesting that infusions of R3(BA23-27)/l5 into the lateral ventricle attenuate reinstatement precipitated by cues that were paired with drug availability. Responding on the inactive lever (Figure 2B) remained unaffected under all conditions (no significant difference), demonstrating the specificity of responding on the active lever.

Example 3 - Further analysis of the effect of R3(BA23-27)/15 on alcohol-seeking and self- administration

Additional studies can be conducted to further analyse the effect of selective antagonism of the RXFP3 receptor on drug-addictive behaviours. For example:

(i) Dose-response curve to R3(B 23-27)/l5

The effect of doses of 1 , 3 and 30 pg R3(BA23-27)/l5 in iP rats is determined (n=12 per treatment/dose). In all cases, responding on both alcohol and water levers is determined. Drug administration weeks are structured so that, for example, Monday, Tuesday and Friday are no- injection days, vehicle is injected Wednesday and drug administered on Thursday.

(//) Relaxin 3 and alcohol self-administratbn

The ability of an RXFP3 receptor agonist (for example R3/I5, at doses of 1, 3 and 10 g; Liu et al., 2005) to promote self-administration of alcohol is determined. In the event that the agonist is shown to promote alcohol self-administration, the effect of pre-treatment with R3(BA23-27)/l5 on agonist-stimulated responding is determined.

(///) Relaxin 3 and the motivational properties ofalcohoi

To determine whether relaxin-3 and its cognate receptor RXFP3 are implicated in integrating the motivational value of alcohol, the impact of RXFP3 antagonism on progressive ratio (PR) responding is evaluated. This paradigm varies from the fixed ratio responding in that each subsequent reward requires a progressively greater instrumental task. 'Breakpoint' represents the point at which an animal ceases to press the active lever for a drug infusion when the instrumental requirement is progressively increased. This reflects the motivation of an animal to self-administer a given drug. In this experiment, an optimal dose of R3(BA23-27)/l5 is chosen from the dose-response study (see (i) above). Similarly, if the agonist R3/I5 increases fixed ratio responding for alcohol, an appropriate dose of this peptide in a PR schedule is examined to determine if this increases the motivational value of alcohol.

(iv) Stress-induced reinstatement

Stress is another major cause of relapse and can be readily modelled in animals. These studies are of particular interest, given the direct influence of CRF signalling on relaxin 3 neurons in the nucleus incertus (Banerjee ef a/., 2010) and the intersection of relaxin-3 and CRF networks throughout the brain. In this experiment, rats are trained to self-administer alcohol under a fixed ratio schedule; and are subsequently extinguished. Following extinction of the instrumental response on the active lever, rats are placed back into the operant chambers and subjected to a stressor, such as foot-shock, or can be injected with yohimbine (1.25 mg/kg ip) prior to being returned to the operant chamber. In both cases, robust reinstatement of responding on the previously drug-paired lever is observed. In this experiment, an optimal dose of R3(BA23-27)/l5 will be chosen from the dose-response study (see (i) above). (v) Relapse alcohol-seeking following abstinence

Not all human addicts undergo extinction (rehabilitation) and the circuitry mediating relapse following abstinence has some differences to that involved following extinction. Therefore, it is also necessary to examine whether a dose of R3(BA23-27)/l5 that prevents reinstatement of alcohol-seeking following extinction has a similar effect on cue-conditioned alcohol-seeking following abstinence. This experiment employs a protocol developed by the CIA.

Example 4 - Regulation of alcohol self-administration by the single chain relaxin polypeptide antagonist H3 B1-22R

Co-pending patent application no. (the disclosure of which is incorporated herein in its entirety by reference) describes the generation of a single chain relaxin polypeptide comprising a human relaxin-3 (H3) derived B chain, designated H3 B1-22R. The B1-22R polypeptide is ":single chain" as it does not include a relaxin- or relaxin superfamily protein- derived A chain. In B1-22R the C-terminal 5 amino acid residues (GGSRW) from the native sequence of the human relaxin-3 B chain are replaced by a single terminal arginine (R) residue. This polypeptide further comprises two serine (S) residues in place of the cysteine (C) residues that occur at positions 10 and 22 of the native B chain sequence of H3 relaxin. Thus, the sequence of the single chain B1-22R polypeptide is the same as that of the B chain component of the R3(A23-27)/l5 antagonist described herein, with the exception that the two cysteine residues are replaced by serine residues in B1-22R.

As described in co-pending patent application no. , B1-22R specifically binds with high affinity to the RXFP3 receptor and displays antagonist activity at this receptor in in vitro assays. The inventors therefore sought to determine if the antagonistic activity of B1-22R is also observed in vivo and if this polypeptide has the same ability as R3(A23-27)/l5 to regulate alcohol self administration.

Experiments were carried out essentially as described above in Example 1. Rats were administered 3 μg₁ 10 μg or 30 μg of the B1-22R antagonist. As shown in Figure 3A, a significant decrease in ethanol lever presses was observed in rats injected with the B1-22R antagonist compared to normal and vehicle responding. The effect observed is dose-dependent. Importantly, this occurs at doses that have no impact upon water or sucrose self-administration, strongly suggesting specificity of action (Figure 3B-D). Moreover, these findings provide clear evidence of a putative role for RXFP3 in alcohol self-administration that apparently is not confounded by any generalised effect on appetitive or consummatory drive.

The inventors also carried out experiments to determine if the effects may be due to disruption in locomotor skills (to control for potential sedation) or hunger-stimulated feeding . The dose of 10 g R3B1-22R was chosen as this caused ~50% reduction in alcohol self-administration with no effect on water or sucrose (see Figure 3). No significant difference was observed between the locomotor skills of rats administered B1-22R (10 μg) compared to those administered vehicle, in terms of floor plane moves or floor plane distance over time periods between 0 and 45 minutes post administration (data not shown). There was also no significant difference between hunger- stimulated feeding of rats administered B1-22R (10 μg) compared to those administered vehicle (data not shown). These results indicate that, like R3(023-27)/l5, B1-22R does not effect locomotor activity, and support a specific role for RXFP3 in alcohol use. References

Banerjee et al. (2010) Neuropharmacology 58: 145-55

Ma et al. (2007) Neuroscience 144: 165-190

Bathgate et al. (2002) J Biol Chem 277: 1148-1157

Bathgate et al. (2006) Pharmacol Rev 58: 7-31

Jupp and Lawrence (2010) Pharmacology & Therapeutics 125: 138-168 Kuei ef al. (2007) J Biol Chem 282: 25425-25435

Shaham et al. (2003) Psychopharmacology 168: 3-20

Claims

Claims

1. A method for the prevention or inhibition of negative behaviour associated with substance abuse or addiction, the method comprising administering to a subject in need thereof an effective amount of an antagonist of a relaxin-3 receptor.

2. The method of claim 1 wherein the negative behaviour comprises substance use (self- administration) or substance seeking behaviour.

3. The method of claim 1 or 2 wherein the substance is an addictive substance.

4. The method of claim 3 wherein the substance is selected from alcohol, nicotine, an opiate, a cannabinoid, a psychostimulant or an inhalant.

5. The method of any one of claims 1 to 4 wherein the relaxin-3 receptor is RXFP3.

6. The method of any one of claims 1 to 5 wherein the antagonist is a selective antagonist of RXFP3.

7. The method of any one of claims 1 to 6 wherein the relaxin-3 receptor antagonist is a modified or chimeric relaxin-3 polypeptide.

8. The method of claim 7 wherein the chimeric polypeptide comprises a B chain derived from relaxin-3 and an A chain derived from a different member of the relaxin superfamily.

9. The method of claim 8 wherein the A chain is derived from INSL5.

10. The method of 8 or 9 wherein the relaxin-3 derived B chain includes a truncation of one or more amino acids at the C-terminus.

11. The method of claim 10 wherein the B chain is derived from human relaxin-3 and includes a truncation of five amino acids from the C-terminus.

12. The method of claim 10 or 11 wherein the deleted C-terminal residues are replaced by one or more amino acid residues.

13. The method of any one of claims 1 to 12 wherein the relaxin-3 receptor antagonist is a selective polypeptide antagonist of RXFP3 comprising a B chain comprising an amino acid sequence as set forth in SEQ ID NO:2, or a variant or derivative thereof and an A chain derived from INSL5 comprising an amino acid sequence as set forth in SEQ ID NO:3, or a variant or derivative thereof.

14. The method of any one of claims 1 to 7 wherein the relaxin-3 receptor antagonist is a selective polypeptide antagonist of RXFP3 comprising only a relaxin-3 derived B chain wherein the B chain sequence is truncated by up to about 5 amino acids at the C-terminus compared to the native relaxin-3 B chain sequence and a basic amino acid residue is incorporated at the C- terminus of the relaxin-3 derived B chain .

15. The method of claim 14 wherein the incorporated basic amino acid residue is arginine.

16. The method of claim 14 or 15 wherein the single chain polypeptide is further modified such that one or more cysteine residues in the native relaxin-3 B chain sequence are replaced by neutral amino acids

17. The method of claim 16 wherein the neutral amino acid is serine or alanine.

18. The method of any one of claims 14 to 17 wherein the B chain sequence is derived from human relaxin-3 the cysteine residues at positions 10 and 22 of the native human relaxin-3 sequence are replaced by serine residues.

19. The method of any one of claims 14 to 18 wherein the single chain polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, or a variant or derivative thereof.

20. The method of any one of claims 1 to 7 wherein the relaxin-3 receptor antagonist comprises: (i) a relaxin-3 derived A chain truncated by up to about 10 amino acids at the N- terminus compared to the native relaxin-3 A chain sequence; (ii) a relaxin-3 derived B chain truncated by about 5 amino acids at the C-terminus compared to the native relaxin-3 B chain sequence; and optionally (iii) a basic amino acid incorporated at the C-terminus of the relaxin-3 derived B chain.

21. The method of claim 20 wherein the basic amino acid is arginine.

22. The method of claim 20 or 21 wherein the C-terminal 5 amino acids from the native sequence of the relaxin-3 B chain are replaced by a terminal arginine residue.

23. The method of any one of claims 20 to 22 wherein the B chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:14, or a variant or derivative thereof.

24. The method of any one of claims 20 to 23 wherein the polypeptide comprises an A chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5, or a variant or derivative thereof and a B chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14, or a variant or derivative thereof, and wherein the polypeptide is an antagonist of the RXFP3 receptor.

25. A method for the treatment of substance addiction in a subject, the method comprising administering to the subject an effective amount of an antagonist of a relaxin-3 receptor.

26. Use of an antagonist of a relaxin-3 receptor for the prevention or inhibition of negative behaviour associated with substance abuse or addiction or for the treatment of substance addiction.

27. Use of an antagonist of a relaxin-3 receptor for the manufacture of a medicament for the prevention or inhibition of negative behaviour associated with substance abuse or addiction or for the treatment of substance addiction.