WO2006095167A1 - Appetite-influencing medicaments - Google Patents

Abstract

The present invention relates to a method for identifying compounds which will be useful as agents for the control of appetite and/or food intake in a mammal, comprising contacting a candidate molecule with GPCR 135 and monitoring the interaction of the candidate molecule and the GPCR135. The invention finds application, amongst others in the field of obesity and cachexia.

Description

APPETITE-INFLUENCING MEDICAMENTS

Introduction

The present invention relates to agents for use in control of appetite and/or food intake, and to devices and methods for identification of such agents. The invention is applicable in particular, but not solely, in the field of obesity.

Background of the Invention

One of the diseases with the highest incidence but which lacks effective treatment is obesity. It is a debilitating condition which reduces quality of life and substantially increases the risk of other diseases.

In the USA 25% of the adult population is now considered to be clinically obese. It has been estimated that $45 billion of US healthcare costs, or 8% per annum of total healthcare spend, is a direct result of obesity. In Europe the problem is increasing. It was predicted that without new approaches over 20% of the UK population would be clinically obese by 2005. The fact that obesity is a metabolic disease is being increasingly recognised by the medical profession and the health authorities. There is, however, a shortage of effective and safe drugs which can be used in conjunction with diet and exercise for the long-term management of obesity.

Other conditions associated with appetite include those in which the patient is underweight as a result of loss of appetite. The condition anorexia nervosa has become increasingly widespread in the industrial world.

Thus, there is a widespread need for agents both for suppression of and stimulation of appetite.

It is an object of the present invention to provide means to identify and develop further drugs for use in the control of appetite and/or food uptake, and to provide medicaments comprising the drugs. The relaxin peptides belong to the insulin superfamily, a group of structurally related hormones whose precursors have a domain arrangement similar to that of pro-insulin (Hudson P, Haley J, John M, Cronk M, Crawford R, Haralambidis J, Tregear G, Shine J, Niall H 1983 Structure of a genomic clone encoding biologically active human relaxin. Nature 301:628-631). The insulin superfamily comprises functionally diverse peptides with a common structure: A and B chains with interchain disulphide bridges. Relaxin- 1 in mice and rats and the human homologue reIaxin-2 were among the first hormones described. Until recently, a single relaxin gene had been described in mice and rats, Ml and Rl respectively, (Soloff MS, Gal S, Hoare S, Peters CA, Hunzicker-Dunn M, Anderson GD, Wood TG 2003 Cloning, characterization, and expression of the rat relaxin gene. Gene 323:149-155; Fowler KJ, Clouston WM, Fournier RE, Evans BA 1991 The relaxin gene is located on chromosome 19 in the mouse. FEBS Lett 292:183- 186) and two relaxin genes, Hl and H2, had been described in humans ( Hudson P, John M, Crawford R, Haralambidis J, Scanlon D, Gorman J, Tregear G, Shine J, Niall H 1984 Relaxin gene expression in human ovaries and the predicted structure of a human preprorelaxin by analysis of cDNA clones. EMBO J 3:2333-2339). The H2 gene is the homologue of the Ml and Rl genes. The H2 gene product is secreted by the corpus luteum in early pregnancy and primarily associated with female reproductive physiology. It is only recently that an additional relaxin peptide, relaxin-3, and its receptors have been identified. A relaxin gene, relaxin-3, has now been identified in humans (H3) ( Bathgate RA, Samuel CS, Burazin TC, Layfield S, Claasz AA, Reytomas IG, Dawson NF, Zhao C, Bond C, Summers RJ, Parry LJ, Wade JD, Tregear GW 2002 Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family. J Biol Chem 277:1148-1157; hereafter Bathgate et al 2002), mice (M3) and most recently in rats (R3) ( Burazin TC, Bathgate RA, Macris M, Layfield S, Gundlach AL, Tregear GW 2002 Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain. J Neurochem 82:1553-1557; hereafter Burazin et al). H3, M3 and R3 retain their insulin-like peptide structure and are highly homologous. Whilst Rl and Ml are expressed in many tissues, R3 and M3 mRNA expression is localised to the nucleus incertus of the brainstem (Burazin et al) which has extensive projections to the hypothalamus ( Goto M, Swanson LW, Canteras NS 2001 Connections of the nucleus incertus. J Comp Neurol 438:86-122). Relaxin-like immunoreactivity has been described in the hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei ( Bathgate RA, Samuel CS, Burazin TC, Gundlach AL, Tregear GW 2003 Relaxin: new peptides, receptors and novel actions. Trends Endocrinol Metab 14:207-213; hereafter Bathgate et al 2003).

Unlike insulin, relaxin peptides signal via G-protein coupled receptors to modulate intracellular cAMP. Rl and Ml act via two leucine-rich repeat-containing receptors, LGR7 and LGR8 ( Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJ 2002 Activation of orphan receptors by the hormone relaxin. Science 295:671-674). LGR7, expressed predominantly in reproductive tissues but also in the CNS, binds relaxin-3 with high affinity. However, relaxin-3 is the cognate ligand for two previously orphan G-protein-coupled receptors, GPCRl 35 and GPCR142 ( Liu C, Eriste E, Sutton S, Chen J, Roland B, Kuei C, Farmer N, Jornvall H, Sillard R, Lovenberg TW 2003 Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein- coupled receptor GPCR135. J Biol Chem 278:50754-50764; Liu C, Chen J, Sutton S, Roland B, Kuei C, Farmer N, Sillard R, Lovenberg TW 2003 Identification of relaxin- 3/INSL7 as a ligand for GPCRl 42. J Biol Chem 278:50765-50770, hereafter Liu et al). Whilst GPCR142 expression is absent in the rat (Chen J, Kuei C, Sutton SW, Bonaventure P, Nepomuceno D, Eriste E, Sillard R, Lovenberg TW, Liu C 2004 Pharmacological Characterization of Relaxin-3/INSL7 Receptors GPCRl 35 and

GPCR142 from Different Mammalian Species. J Pharmacol Exp Ther; hereafter Chen et al), GPCR135 niRNA is highly expressed in the rat brain, particularly the PVN (Liu et al; Chen et al).

Summary of the Invention

The invention is based on the unexpected observation that relaxin-3 can significantly increase food intake in satiated animals. It has been further unexpectedly observed that relaxin-3 has no significant effect on energy expenditure.

The invention provides a method for identifying compounds which will be useful as agents for the control of appetite and/or food intake in a mammal, comprising contacting a candidate molecule with GPCR135 and monitoring the interaction of the candidate molecule and the GPCR135.

The term "GPCR135" as used herein includes human GPCR135 and peptides having a substantial degree of homology, for example at least 75%, advantageously at least 90%, especially at least 95%, more especially at least 97%, preferably at least 98% and most preferably at least 99% homology, with human GPCRl 35, and in particular may include GPCR135 of other mammals. Other mammalian GPCR135 having high levels of homology with human GPCR135 includes rat GPCR135 and mouse GPCR135. The term "GPCR135" is further to be understood as including peptides which have a relatively low homology with human GPCRl 35, for example at least 25% homology, whilst nonetheless substantially retaining the functionality of human GPCR135 in mediating the effects of relaxin-3 on food intake. Furthermore it is to be understood that the term GPCRl 35 as used herein includes fragments, derivatives and modifications of GPCR135, provided that functionality is retained. The term GPCRl 35 as used herein is not limited to naturally occurring products but includes synthetically produced GPCRl 35 (including functionally equivalent fragments, derivatives and modifications) and other synthetically generated peptides that are functionally equivalent to naturally occurring human GPCR135. It will be appreciated that in the foregoing definition "functionally equivalent" refers to substantial equivalence in the function of mediating the effects of relaxin-3 on food intake.

The invention also provides a method for identifying compounds which will be useful in the treatment of obesity, comprising contacting a candidate molecule with GPCRl 35 and monitoring the interaction of the candidate molecule and the receptor.

Moreover, the invention provides a method for identifying compounds which will be useful as agents for the control of appetite and/or food intake, comprising ascertaining the binding affinity of a candidate molecule to GPCRl 35 receptor. The invention further provides the use of a compound which is an antagonist of GPCR135, or a physiologically acceptable salt, solvate or derivative thereof, in the manufacture of a medicament for suppression of appetite and/or food uptake.

The invention also provides the use of a compound which is an agonist of GPCR135, or a physiologically acceptable salt, solvate or derivative thereof, in the manufacture of a medicament for enhancement of appetite and/or food uptake.

Additionally, the invention provides a method for the treatment of an eating disorder in a mammal, including a human, comprising administration of a compound which agonises GPCR135.

Moreover, the invention provides a method for the treatment of obesity in a mammal, including a human, comprising administration of a compound which antagonises GPCR135.

The present invention provides a method for cosmetic weight loss in a mammal, the method comprising administering a composition comprising a GPCRl 35 antagonist to a mammal. In this circumstance, the weight loss is purely for the purposes of cosmetic appearance.

Suitable tests for determining binding affinity to GPCR135, and for determining agonist and antagonist behaviour are described further below.

Brief Description of the Drawings

Fig. IA is a graphical illustration of the effect of ICV administration of relaxin-3 (18-180 pmol) in satiated male Wistar rats on 1 h food intake * = p < 0.05 vs vehicle;

Fig. IB is a graphical illustration of the effect of relaxin-3 (18-180 pmol) in satiated male Wistar rats on cumulative food intake over 4 h. & = p < 0.05 at 18 pmol vs vehicle, * = p < 0.05 at 54 pmol vs vehicle, # = p < 0.05 at 180 pmol vs vehicle; Fig. 2A is a graphical illustration of the effect of iPVN administration of relaxin-3 (1.8- 18 pmol) in male Wistar rats on 1 h food intake in early light phase;

Fig. 2B is a graphical illustration of the effect of relaxin-3 (18 pmol) in male Wistar rats on 1 h food intake in early dark phase; * = p < 0.05 vs vehicle; Fig. 3 is a graphical illustration of the effect of IP administration of relaxin-3 (0.03- 0.3nmol/g) on Ih food intake in satiated C57BL/6 mice;

Fig. 4 is a graphical illustration of the effect of iPVN administration of equimolar doses of relaxin-3 (H3) and relaxin-2 (H2) on 1 h food intake in satiated male Wistar rats. * = p < 0.05 vs vehicle. Fig. 5 is a graphical illustration of the effect of acute iPVN administration of H3 (180-1620 pmol) on 1 hour food intake in satiated male Wistar rats in the early light phase. * = p < 0.05 vs vehicle

Fig. 6 is a graphical illustration of the effect of acute iPVN administration of H3 (540 pmol) on plasma TSH at 15 and 30 minutes post-injection. * = p< 0.05 vs vehicle Fig. 7 A is a graphical illustration of the effect of repeated iPVN administration of vehicle or H3 (180 pmol/injection) in ad libitum fed rats on 24 hour food intake on day 1 and day 7, * = p < 0.05 vs vehicle

Fig. 7B is a graphical illustration of the effect of repeated iPVN administration of vehicle or H3 (180 pmol/injection) in ad libitum fed rats on 1 hour food intake in the early light phase on day 1 and day 7, * p < 0.05 vs vehicle

Fig. 8 is a graphical illustration of the effect of cumulative food intake after repeated iPVN administration of vehicle, H3 in ad libitum fed rats (180 pmol/injection) or H3 in pair-fed (PF) rats for 7 days. White circles = vehicle; plus sign = H3 ad libitum fed group; black triangle = H3 pair fed group, * = p < 0.05 vs vehicle Fig. 9 is a graphical illustration of the effect of repeated iPVN administration of vehicle, H3 in ad libitum fed rats (180 pmol/injection) or H3 in pair-fed ( PF) for 7 days on plasma TSH, *p< 0.05 vs vehicle

Fig. 10 is a graphical illustration of the protein sequences of the human, mouse and rat GPCR135 receptors. Detailed Description of the Invention

Sequences of GPCR135 are known and documented in the art. The GPCR135 human sequence SEQ ID NO: 1 is as follows: MQMADAATIATMNKAAGGDKLAELFSLVPDLLEAANTSGNASLQLPDLWWELGLELPDGA PPGHPPGSGGAESADTEARVRILISVVYWVVCALGLAGNLLVLYLMKSMQGWRKSSINLF VTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCKIVSMVTSMNMYASVFFLTAMSVTR YHSVASALKSHRTRGHGRGDCCGRSLGDSCCFSAKALCVWIWALAALASLPSAIFSTTVK VMGEELCLVRFPDKLLGRDRQFWLGLYHSQKVLLGFVLPLGIIILCYLLLVRFIADRRAA GTKGGAAVAGGRPTGASARRLSKVTKSVTIVVLSFFLCWLPNQALTTWSILIKFNAVPFS QEYFLCQVYAFPVSVCLAHSNSCLNPVLYCLVRREFRKALKSLLWRIASPSITSMRPFTA TTKPEHEDQGLQAPAPPHAAAEPDLLYYPPGVVVYSGGRYDLLPSSSAY

The GPCRl 35 rat sequence SEQ ID NO: 2 is as follows: MPKAHLSMQVASATTAAPMSKAAAGDELSGFFGLIPDLLEVANRSSNASLQLQDLWWELG LELPDGAAPGHPPGSGGAESADTEARVRILISAVYWVVCALGLAGNLLVLYLMKSKQGWR KSSINLFVTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCKIVSMVTSMNMYASVFFL TAMSVARYHSVASALKSHRTRGHGRGDCCGQSLGESCCFSAKVLCGLIWASAAIASLPNV IFSTTINVLGEELCLMHFPDKLLGWDRQFWLGLYHLQKVLLGFLLPLSIISLCYLLLVRF ISDRRVVGTTDGATAPGGSLSTAGARRRSKVTKSVTIVVLSFFLCWLPNQALTTWSILIK FNVVPFSQEYFQCQVYAFPVSVCLAHSNSCLNPILYCLVRREFRKALKNLLWRIASPSLT SMRPFTATTKPEPEDHGLQALAPLNATAEPDLIYYPPGVVVYSGGRYDLLPSSSAY

The GPCR135 mouse sequence SEQ ID NO: 3 is as follows: MQVASATPAATVRKAAAGDELSEFFALTPDLLEVANASGNASLQLQDLWWELGLELPDGA APGHPPGGGGAESTDTEARVRILISAVYWVVCALGLAGNLLVLYLMKSKQGWRKSSINLF VTNLALTDFQFVLTLPFWAVENALDFKWPFGKAMCKIVSMVTSMNMYASVFFLTAMSVAR YHSVASALKSHRTRGRGRGDCCGQSLRESCCFSAKVLCGLIWASAALASLPNAIFSTTIR VLGEELCLMHFPDKLLGWDRQFWLGLYHLQKVLLGFLLPLSIISLCYLLLVRFISDRRVV GTTDAVGAAAAPGGGLSTASARRRSKVTKSVTIVVLSFFLCWLPNQALTTWSILIKFNAV PFSQEYFQCQVYAFPVSVCLAHSNSCLNPILYCLVRREFRKALKNLLWRIASPSLTNMRP FTATTKPEPEDHGLQALAPLNAAAEPDLIYYPPGVVVYSGGRYDLLPSSSAY

The inventors have shown for the first time that ICV relaxin-3 significantly increases food intake in satiated animals (see Example 1 below). Similarly, relaxin-3 injection into the PVN, an area with a high level of expression of GPCR135, also stimulated food intake and was able to potentiate nocturnal feeding (see Examples 2 and 3 below). Behavioral studies show a significant increase in feeding behavior and no change in other behaviors following iPVN relaxin-3 administration. These studies were performed using human relaxin-3. There is, however, a high level of homology among the relaxin-3 peptides of different species: the mouse and rat peptides are identical and share 92% sequence identity to human relaxin-3 (Chen et al). At the present time, only human relaxin-3 is commercially available but this binds with high affinity to rat GPCRl 35 (Chen et al).

The inventors have also demonstrated that acute administration of iPVN relaxin-3 has no significant effect on metabolic parameters (see Example 10) and that 7 day repeated iPVN administration of relaxin-3 has no significant effect on the body weight of vehicle- treated animals compared to H3 -treated animals pair-fed to the food intake of the vehicle- treated animals (see Example 11). The inventors have therefore shown for the first time that relaxin-3 can significantly increase food intake in animals while having no significant effect on energy expenditure.

In view of their showing of the association between relaxin-3 and feeding behaviour, the inventors propose that the GPCRl 35 receptor will provide a suitable basis for identifying compounds which have appetite suppressant or appetite stimulating activity. In a preferred embodiment of the invention, candidate molecules for use as agents for control of appetite and/or food intake in a mammal comprises contacting a candidate molecule with GPCRl 35 and monitoring the effect of the candidate molecule on the activity of GPCR135. In accordance with certain embodiments of the invention, a method for identifying compounds which will be useful as agents for the control of appetite and/or food intake comprises ascertaining the binding affinity of a candidate molecule to GPCR135, and/or determining whether a candidate molecule is an agonist or an antagonist for GPCRl 35. Suitable assays for determining the binding affinity, and for determining agonist or antagonist behaviour, in relation to GPCRl 35 are as follows:

Binding affinity to GPCR135 may be determined by any suitable method, one suitable method being an ELISA assay. An example of an ELISA assay is as described in the following steps:

1. Coat microwells of immuno-detection plate with purified GPCR135 receptor.

2. Incubate at 4°C overnight.

3. Wash unbound receptor off the plate using an appropriate wash solution. 4. Block non-specific binding by adding an appropriate volume of 1% BSA/PBS to each microwell.

5. Incubate at RT for 30-60 minutes.

6. Repeat washing step.

7. Prepare appropriate dilution series of relaxin-3 conjugated to an enzyme such as alkaline phosphatase or horseradish peroxidase.

8. Add appropriate volume of conjugated relaxin-3 to each microwell and incubate for 1 hour.

9. Repeat washing step.

10. Prepare appropriate substrate solution for conjugated enzyme. 11. Add appropriate volume of substrate to each microwell.

12. Incubate at RT for 1 hour.

13. Add stopping solution if appropriate.

14. Read plates on an ELISA plate reader.

Another suitable method for determining binding affinity to GPCR135 is a radioligand saturation binding assay. An example of such an assay is described in the following steps: 1. Transiently transfect appropriate cells (e.g. 293T cells) with a plasmid expressing GPCR135.

2. Plate the transiently transfected cells in a 24-well plate for whole cell binding assay.

3. Remove media and wash with PBS.

4. Pre-incubate cells in binding buffer (e.g. 300μl of 20 mM HEPES, 50 mM NaCl, 1.5 mM CaCl₂, 1% BSA, 0.1 mg/ml lysine, 0.01% NaN₄, pH 7.5).

5. Prepare a dilution series of radiolabeled ligand. 6. Add an appropriate amount (e.g. 100 μl) of each dilution of ligand to separate wells of the 24-well plate.

7. Add an excess of radiolabeled ligand to a further well to determine non-specific binding.

8. After incubation, wash cells with PBS, recover cells from the plates using an appropriate amount (e.g. 500 μl) of an appropriate concentration (e.g 1 M) of

NaOH and transfer to scintillation vials.

9. Add liquid scintillation mixture to the vials (e.g. Ultima Gold, Packard).

10. Count vials in a liquid scintillation analyser (e.g. Packard 1900 TR).

A GPCR135 agonist is a peptide, small molecule, or chemical compound that preferentially binds to the GPCRl 35 receptor and stimulates the same biological activity as does relaxin-3. In one embodiment, an agonist for the human GPCR135 receptor binds to the receptor with an equal or greater affinity than human relaxin-3. GPCRl 35 agonists include relaxin-3 related peptides and peptides that result from natural or synthetic enzymatic or chemical processing of relaxin-3 peptide or a related peptide.

Agonist activity in respect of GPCRl 35 may be determined by any suitable method, one suitable method being a cell-based assay. For example, such an assay may be a GTPγS- binding assay as described in the following steps: 1. Transfect suitable cells (e.g. CHO-Kl) with GPCR135 expression vector.

2. Harvest cells after 2 days and prepare cell membranes by homogenisation in a suitable buffer (e.g. 50 nM Tris-HCl, 5 mM EDTA),

3. Pellet the cell membranes via centrifugation and remove supernatant. 4. Add a suitable GTPγS binding buffer (e.g. 50 mM Tris-HCl, pH 7.4, 10 mM

MgCl₂, 10 μM GDP, 1 mM EDTA, pH 8.0 and 100 mM NaCl) and rehomogenise the pellet in the buffer.

5. Add a suitable protease inhibitor to the sample.

6. Prepare a dilution series of the potential agonist and an appropriate negative control.

7. Add each dilution of the potential agonist and negative control to a separate sample of cell membrane expressing GPCRl 35 in a single well on a 96-well plate.

8. Incubate at RT for 20 min.

9. Add the appropriate amount of ³⁵S-labelled GTPγS to each well to reach a concentration of 200 pM.

10. Allow reaction to proceed for 1 hour at RT.

11. Filter the plate through a 96-well filter plate.

12. Wash with a suitable cold washing buffer (e.g. 50 nM Tris-HCl, pH 7.4, 10 mM MgCl₂). 13. Add an appropriate amount of liquid scintillation cocktail (e.g. Microscint-40 from Perkin-Elmer) to each well. 14. Count the plate on an appropriate apparatus.

A GPCRl 35 antagonist is a peptide, small molecule, or chemical compound that binds to the GPCR135 receptor and inhibits the mediation thereby of the effects of relaxin-3 on food intake. In one embodiment, an antagonist for the human GPCRl 35 receptor binds to the receptor with an equal or greater affinity than human relaxin-3. Antagonist activity in respect of GPCRl 35 may be determined by any suitable method, one suitable method being a cell-based assay. For example, such an assay may be a cAMP -stimulation assay as described in the following steps:

1. Transfect suitable cells (e.g. CHO-Kl) with GPCRl 35 expression vector.

2. Culture cells using an appropriate selection.

3. Seed the receptor expressing cells into 96 well plates at an appropriate density (e.g. 30,000 cells/well).

4. After 24 h, replace the cell culture medium with an appropriate test medium (e.g. Dulbecco's modified Eagle's medium/F-12 (Invitrogen) plus 2 mM isobutylmethylxanthine (Sigma).

5. Add an appropriate amount of test compound to each well.

6. Incubate at RT for 25 min.

7. Add an appropriate amount of 0.5 N HCl to halt the reaction and extract accumulated cAMP .

8. Assay the cAMP concentrations in the extracted media using an appropriate method (e.g. cAMP Flash Plates (Perkin Elmer Life Sciences)).

An alternative example is to assay antagonist activity by performing a competition assay using a modified version of the GTPγS-binding assay described above. In the modified version, both relaxin-3 and a potential antagonist molecule are both added to the wells in step 7. The antagonist activity of the candidate molecule is assessed by measuring its ability to compete with relaxin-3 and thus reduce the GTP -binding activity that is promoted by relaxin-3.

Agonist or antagonist behaviour can be demonstrated in vivo by administering an intracerebroventricular injection of candidate compound in a suitable vehicle, for example 10% acrylonitrile in 0.9% saline, to satiated rats and comparing subsequent food intake at 2 hours and 4 hours following administration with a control group of rats injected with vehicle alone. The doses of relaxin-3 required to elicit a significant feeding response are in the picomolar range and similar to effective doses of other orexigenic peptides such as ghrelin. For example, a significant orexigenic response with iPVN ghrelin has been seen at 30 pmoles ( Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR 2001 Ghrelin causes hyperphagia and obesity in rats. Diabetes 50:2540-2547; hereafter Wren et al) compared to 18 pmoles of H3 relaxin. Similarly, the lowest dose of the potent orexigenic peptide NPY to significantly stimulate feeding in the PVN is 24 pmol ( Stanley BG, Kyrkouli SE, Lampert S, Leibowitz SF 1986 Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peptides 7:1189-1192). As with NPY and ghrelin, the effect of relaxin-3 occurs in the first hour following administration but cumulative food intake remains elevated for several hours.

The present inventors also investigated the maximum orexigenic response elicited by H3. Acute iPVN administration of high dose H3 significantly increased food intake in the first and second hour in satiated rats (180-1620 pmol, see Figure 5), and cumulatively up to 8 hours following injection. The maximum orexigenic response was achieved by a dose of 540 pmol H3 with no further increase at the higher dose of 1620 pmol H3. Although the increase in food intake in the first hour following injection of 540 pmol H3 was almost seven-fold that following vehicle administration, it was only half the orexigenic response seen following administration of an equimolar dose of NPY. However, high dose H3 continued to stimulate food intake for longer than NPY since by the end of the second hour the orexigenic response to H3 almost matched that of NPY.

It is known that several peptides, such as NPY and AgRP, both stimulate food intake and also alter energy expenditure (C.J.Small, Y.L.Liu, S.A.Stanley, I.P.Connoley, A.Kennedy, MJ.Stock, S.R.Bloom, Chronic CNS administration of Agouti-related protein (Agrp) reduces energy expenditure IntJ.Obes.Relat Metab Disord. 2003;27:530- 533; C.J.Billington, J.E.Briggs, M.Grace, A.S.Levine, Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism AmJ.Physiol 1991;260:R321-R327; M.Egawa, H.Yoshimatsu, GA.Bray, Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats Am. J.Physiol 1991;260:R328-R334.). The inventors also examined the acute effects of H3 on both plasma TSH and other parameters of energy expenditure (VO2, VC02, RER and ambulatory activity, see Examples 9 & 10). Acute iPVN administration of H3 significantly suppressed plasma TSH at 15 and 30 minutes post injection. However, there were no significant changes in VO2, VCO2, RER or activity when the effects of acute iPVN administration of H3 were examined in the CLAMS system. Energy expenditure is comprised of several components: thyroid hormone activity, BAT activity, activation of the sympathetic nervous system and both locomotor and non-locomotor muscle activity. Thus, although TSH was suppressed, there may be compensation by other components with the net result being no overall change in energy expenditure in response to H3. The inventors have therefore demonstrated that, surprisingly, the effects of acute administration of H3 differ from those of NPY and AgRP in that H3 stimulates food intake without altering energy expenditure.

The inventors also investigated the effects of repeated iPVN administration of H3 on food intake and body weight over a 7 day period (see Example 11). Three groups were studied: 1) vehicle-treated, ad libitum fed control animals, 2) H3-treated ad libitum fed animals and 3) H3 -treated animals pair-fed to the median food intake of the vehicle-treated animals.

Repeated 7-day iPVN administration of H3 significantly increased cumulative food intake in the ad libitum fed, H3-treated animals compared with the vehicle-treated ad libitum fed controls and cumulative body weight change was increased in the H3-treated group. Further, one hour food intake following injection was significantly increased in the first hour following administration in the early light phase on both day 1 and day 7 with no evidence of tachyphylaxis. These results thus demonstrate that H3 is effective at increasing food intake and increasing body weight over a 7 day period. Plasma leptin was significantly elevated in ad libitum fed H3 treated animals and though epididymal fat pad weight was higher in the ad libitum fed H3 treated animals this did not achieve statistical significance.

A comparison of the results from the vehicle-treated control animals and the pair-fed H3- treated animals showed that although there was an increase in cumulative body weight change in the vehicle-treated animals compared to the pair-fed H3 -treated animals, this was not significant at the end of the study. This would suggest that there was no significant effect of H3 on energy expenditure and is in keeping with the results of acute H3 administration on metabolic parameters. In keeping with this, there was no difference in BAT UCP-I mRNA expression between the treatment groups.

There was, however, a significant difference in cumulative body weight change between ad libitum fed H3 treated and H3 pair- fed animals by the end of day 7. Again, plasma TSH was significantly suppressed by repeated iPVN injection of H3. In keeping with this, a suppression of TRH release and an increase in SRIF release was seen from hypothalamic slices following administration with 10 nM H3.

The inventors have thus demonstrated that the effects of repeated H3 administration differ from those of other orexigenic peptides such as NPY, AgRP and ghrelin. Repeated administration of NPY, AgRP and ghrelin significantly increases food intake and decreases energy expenditure, with the combined effect resulting in body weight gain. In contrast, while administration of H3 significantly increases food intake, it does not significantly decrease energy expenditure

After repeated iPVN H3 administration, no significant change was observed in testicular or adrenal weight, plasma LH, prolactin or corticosterone. This is in contrast to the effects of chronic NPY administration (A.Sainsbury, I.Cusin, F.Rohner-Jeanrenaud, B.Jeanrenaud, Adrenalectomy prevents the obesity syndrome produced by chronic central neuropeptide Y infusion in normal rats Diabetes 1997;46:209-214; J.Wang, A.Akabayashi, J.Dourmashkin, H. J. Yu, J.T.Alexander, H.J.Chae, S.F.Leibowitz, Neuropeptide Y in relation to carbohydrate intake, corticosterone and dietary obesity Brain Res. 1998;802:75-88) and leptin deficiency (ob/ob and db/db mice) (J.A.Edwardson, C.A.Hough, The pituitary-adrenal system of the genetically obese (ob/ob) mouse LEndocrinol. 1975;65:99-107; D.L.Coleman, D.L.Burkart, Plasma corticosterone concentrations in diabetic (db) mice Diabetologia 1977; 13:25-26) which results in profound hypercorticosteremia in rodents and alteration in the gonadal axis.

In conclusion, the inventors have demonstrated that acute iPVN administration of high dose H3 increases food intake with similar efficacy to NPY, suppresses plasma levels of TSH but does not alter energy expenditure. The inventors have also shown for the first time that repeated iPVN administration of H3 is effective for 7 days and increases cumulative food intake, body weight and plasma leptin. Repeated iPVN H3 treatment results in a suppression of plasma TSH without alteration in plasma T3. This effect is independent of food intake, since it also occurs in pair-fed animals.

Active agents of the invention having GPCRl 35 antagonist activity can be used as a prophylaxis to prevent excess weight gain or can be used as a therapeutic to lose excess weight. The excess weight is typically obesity, although the mammal need not be certified as clinically obese in order to be suffering from excess weight. The agent may be in liquid, solid or semi-solid form.

In today's society, the prevention or treatment of excess weight in a mammal is a real need. Preferably the mammal is a human, although it may also include other mammalian animals, such as horses, canine animals (in particular domestic canine animals), feline animals (in particular domestic feline animals) as well as mammals which are produced for meat, such as porcine, bovine and ovine animals. The present invention can be used to prevent excess weight in such animals in order to maximise lean meat production. Throughout this text, the term "prevention" in relation to excess weight means any effect which mitigates any excess weight, to any extent. Throughout this text, the term "treatment" in relation to excess weight means amelioration of excess weight, to any extent.

Active agents of the invention having GPCRl 35 agonist activity, including relaxin-3, can be used as a prophylaxis to prevent weight loss or can be used as a therapeutic in the treatment of appetite disorders to promote weight gain. For example, the agents can be used in the treatment of anorexia nervosa.

In another aspect of the invention, an active agent having GPCRl 35 agonist activity, including relaxin-3, can be used as a therapeutic in the treatment of weight loss due to underlying disease (cachexia). Weight loss due to underlying disease, often termed "cachexia", occurs in patients with a wide variety of diseases including acquired immune deficiency syndrome (AIDS), liver cirrhosis, chronic obstructive pulmonary disease, chronic renal failure, chronic infections including pneumonia, cancer (cancer cachexia), diabetes and heart disease including hypertension and chronic heart failure (CHF)

(cardiac cachexia). Cachexia may also occur idiopathically. In all cases, cachexia may be an indicator of a poor prognosis and its reversal, stopping or at least slowing down, is desirable. Indeed, a strong relationship between weight loss and mortality has been found for many conditions.

This aspect of the invention provides a method of treating weight loss due to underlying disease in a patient the method comprising administering to the patient an effective amount of a GPCRl 35 agonist, a preferred example of which is relaxin-3, which increases appetite, food intake, weight gain, rate of weight gain, or stabilises weight loss, or rate of weight loss.

In treating weight loss due to underlying disease in a patient it is useful if the weight loss is reversed or stopped or at least slowed down. Agonists of GPCR135, a preferred example of which is relaxin-3, are useful for the treatment or prevention of weight loss due to underlying disease (cachexia). These underlying diseases include, for example, but are not restricted to, AIDS, liver cirrhosis, chronic obstructive pulmonary disease with or without emphysema, chronic renal failure, chronic infections (like pneumonia), cancer (i.e. cancer cachexia), and heart disease including hypertension and chronic heart failure (i.e. cardiac cachexia), and idiopathic cachexia (i.e. cachexia due to unknown disease).

Therapeutic agents according to the invention, that is in particular relaxin-3 or GPCRl 35 agonists or antagonists, can be provided in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or diluent. Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration). The agent can be provided as a crystalline solid, a powder, an aqueous solution, a suspension or in oil.

The compositions according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral, rectal, parenteral eg intravenous, intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual, nasal, subcutaneous or transdermal administration, including administration by inhalation, and the compositions adapted accordingly.

For oral administration, the compositions can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.

Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule.

Typical parenteral compositions, including compositions for subcutaneous administration, comprise a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or non-aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.

Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or nonaqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump- atomiser.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

Compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas.

Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections.

Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule.

Relaxin-3 and other therapeutic agents according to the invention may be administered peripherally at a dose of, for example, 0.01 nmoles or more per kg body weight of the subject, for example, 0.02 nmoles or more, for example, 0.05 nmoles or more, for example, 1 nmole or more, for example, 2 nmoles or more, up to 12 nmoles per kg body weight. The amount used may be up to 5 nmoles, for example, up to 4 nmoles, for example, up to 3 nmoles, for example, up to 2 nmoles, for example, up to 1 nmoles, for example, up to 0.5 nmoles, for example, up to 0.4 nmoles, for example, up to 0.2 nmoles per kg body weight. The dose is generally in the range of from 0.01 to 12 nmoles per kg body weight, for example, within any combination of upper and lower ranges given above. A dose may be calculated on an individual basis or on the basis of a typical subject, often a 70 or 75 kg subject. Dosages may alternatively be calculated on the basis of body surface area. The dose may be administered before each meal. For subcutaneous administration, a dose of therapeutic agent within the range of from 10 nmol to 500 nmol, which dose is calculated on the basis of a 75 kg subject, may be administered, generally before meals.

A pharmaceutical preparation in unit dosage form for peripheral administration preferably comprises an amount of therapeutic agent calculated on the basis of the per kg doses given above. Typically, the dose may be calculated on the basis of a 70 or 75 kg subject. A composition for subcutaneous administration, for example, may comprise a unit dose of therapeutic agent within the range of from 10 nmol to 500 nmol, calculated on the basis of a 75 kg subj ect.

It is preferable to administer the agent via a peripheral route of administration, that is to say, via a route other than directly to the brain. Examples of such routes include oral, rectal, parenteral e.g. intravenous, intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual, nasal, subcutaneous or transdermal administration, including administration by inhalation. Unlike relaxin-1, relaxin-3 is expressed in few peripheral tissues and only at low levels. Whilst peripheral administration of relaxin-1 is able to elicit central effects, such as drinking, initial indications are that peripheral administration of relaxin-3 did not alter food intake (see Example 4 below). In the case of relaxin-3, and of other agents that are peptides, injection into the paraventricular nucleus or intracerebroventricular injection has been shown to be advantageous. Based on that observation, preferred methods of administration will be those which are effective in delivery of the active agent into the brain. In the case of some active agents, in particular those which are small molecules, that will be achievable by means of peripheral administration of the molecule per se or a prodrug. For certain other active agents, for example polypeptides, peripheral administration will be less preferred.

The present invention provides a pharmaceutical composition comprising a therapeutic agent according to the invention and a pharmaceutically suitable carrier, in a form suitable for oral, rectal, parenteral eg intravenous, intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual, nasal, subcutaneous or transdermal administration, including administration by inhalation. If in unit dosage form, the dose per unit may be calculated on the basis of the per kg doses given above.

If desired, one or more other agents, such as, but not limited to, an additional appetite suppressant, may also be administered. Specific, non-limiting examples of an additional appetite suppressant include amfepramone (diethylpropion), phentermine, mazindol and phenylpropanolamine, fenfluramine, dexfenfluramine, and fluoxetine.

When used in combination with another agent, active compounds of the invention may be administered simultaneously or substantially simultaneously as the other agent, or sequentially, in either order. Active compounds of this invention and the other agent may be administered in a single pharmaceutical composition or in separate compositions, and they may be administered by the same route or by different routes. It is generally more convenient to administer all the active agents in a single composition. However, in some cases it may be necessary or appropriate to administer the active agents by different routes. For example, peptides are generally not stable on oral administration unless modified or formulated in a special way, so must generally be administered via a non-oral route. Some agonists are chemical compounds that are stable when administered orally. It may be appropriate to administer active agents of the invention non-orally and the other component by a non-oral route or vice versa.

All preferred features given above also apply to the application of the invention in cosmetic weight loss.

In contrast to relaxin-3, equimolar doses of human relaxin-2 did not elicit any increase in feeding (see the Example 5 below). Since both relaxin-2 and relaxin-3 bind LGR7 receptors with high affinity but only relaxin-3 binds GPCRl 35 with similar affinity, this would suggest the GPCRl 35 receptor may mediate the effects of relaxin-3 on food intake. In keeping with this, neither relaxin-1 null mice nor LGR7 null mice have any reported abnormality of gross phenotype, such as body weight ( Kamat AA, Feng S, Bogatcheva NV₅ Truong A, Bishop CE, Agoulnik AI 2004 Genetic targeting of relaxin and insulin-like factor 3 receptors in mice. Endocrinology 145:4712-4720).

The action of some orexigenic peptides, for example ghrelin, is mediated via NPY, AgRP and the melanocortin system ( Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S 2004 Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti -related protein. Endocrinology 145:2607-2612). Central administration of ghrelin upregulates the expression of NPY and AgRP mRNA in the hypothalamus after 4 hours ( Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi T, Inoue G, Hosoda K, Kojima M, Kangawa K, Nakao K 2001 Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Yl receptor pathway. Diabetes 50:227-232; Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi 12000 Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression. Endocrinology 141:4797-4800). In contrast, relaxin-3 did not alter hypothalamic NPY, POMC or AgRP mRNA expression. This suggests that the effects of relaxin-3 on food intake may be mediated by an unknown mechanism or act downstream of NPY, AgRP and POMC.

In summary, these results suggest that ICV and PVN administration of relaxin-3 stimulate feeding in male rats and that this effect is mediated via the GPCRl 35 receptor. The mechanism for the orexigenic action of relaxin-3 remains to be established but does not appear to be via regulation of hypothalamic NPY, POMC or AgRP.

The present invention is now described by way of example only in the following non- limiting Examples. Materials and Methods

Materials

Human relaxin-3 (H3) used in the Examples was purchased from Phoenix Pharmaceuticals (Belmont, CA) and human relaxin-2 (H2) from Dr A. Parlow, National Hormone and Peptide Program (Torrance, CA). Reagents for Ribonuclease Protection Assay studies were purchased from Ambion (Austin, TX). Reagents for hypothalamic explant studies were supplied by BDH (Poole, Dorset, UK).

Animal studies Male Wistar rats (250-300 g) were maintained in individual cages for all studies.

Intraperitoneal (IP) feeding studies were performed in C57BL/6 mice (25-30 g) maintained in individual cages.

All animals were kept under controlled temperature (21-23°C) and light (12 h light, 12 h dark, lights on at 0700 h) with ad libitum access to food (RMl diet, SDS, UK) and water. AU procedures undertaken were approved by the British Home Office Animals Scientific

Procedures Act 1986 (project license 70/5516).

Food intake studies

Male Wistar rats underwent third ventricle (ICV) or intra-paraventricular nucleus (iPVN) cannulation 7-10 days before feeding studies and were habituated to regular handling and injection, as previously described (Wren et al). Central injections (5μl (ICV) or lμl (iPVN)) were administered via stainless steel injectors (27-gauge (ICV) or 31 -gauge (iPVN)), placed in and projecting 1 mm below the end of the cannula, over 1 minute. IP injections were given in a volume of 100 μl. All compounds were dissolved in vehicle (10% acetonitrile in 0.9% saline) and studies were performed in satiated rats (n = 10-12) or mice (n = 9-10) in the early light phase (0900 - 1000 h) unless otherwise stated. Following injection, animals were returned to their home cage with pre-weighed chow. Food intake was measured at 1, 2, 4, 8, and 24 hours post injection. IntraPVN cannula position was verified histologically at the end of the study (Wren et al). Immediately following decapitation, 1 μl Indian ink was injected into the cannula. The brains were removed and fixed in 4% paraformaldehyde, dehydrated in 40% sucrose, frozen and stored at -7O⁰C. Brains were sliced on a cryostat (Bright, Huntingdon, UK) into 15 μm coronal sections and correct PVN placement determined by microscopy according to the position of the Indian ink. ICV cannula position was verified by a positive dipsogenic response to angiotensin II (150 ng/rat). Only those animals with correct cannula placement were included in the data analysis.

Behavioral response following iPVN administration ofrelaxin-3

Behavioral responses were monitored following iPVN administration of vehicle, 18 pmol or 180 pmol relaxin-3 (H3). Animals were immediately returned to their home cages and observed for an hour following injection by an investigator blinded to the treatment. Behavior was classified into one of nine categories. Each rat was observed for three 3 sec periods every 6 min and the behavior in each period scored as previously described (Kong WM, Martin NM, Smith KL, Gardiner JV, Connoley IP, Stephens DA, Dhillo WS, Ghatei MA, Small CJ, Bloom SR 2004 Triiodothyronine Stimulates Food Intake via the Hypothalamic Ventromedial Nucleus Independent of Changes in Energy Expenditure. Endocrinology 145:5252-5258; hereafter Kong et al).

Hypothalamic neuropeptide expression following relaxin-3 administration Hypothalamic neuropeptide mRNA expression was assessed following ICV administration of vehicle or relaxin-3 (H3) (180pmol). Food was removed immediately following injection and at four hours animals were killed, hypothalami dissected and snap frozen. Hypothalamic neuropeptide Y (NPY), agouti related peptide (AgRP) and proopiomelanocortin (POMC) mRNA expression were determined by ribonuclease protection assay (RPA). Briefly, RNA was extracted using Tri-Reagent (Helena Biosciences, Sunderland) according to the manufacturer's protocol. Rat β-actin (Ambion Inc.) was used to correct for RNA loading. RNA was hybridized overnight at 42⁰C with 1.3 x 10³ Bq Of³²P[CTP] labelled riboprobe. Reaction mixtures were digested with RNase A/Tl, the protected fragments precipitated and separated on a 4% polyacrylamide gel. The dried gel was exposed to a phosphorimager screen overnight and bands quantified by image densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) (Kong et al). Acute effects of high dose iPVN relaxin-3 administration on food intake Male Wistar rats (n = 10-12) received an iPVN injection of vehicle or increasing doses of H3 (180 pmol, 540 pmol and 1620 pmol). The lowest dose of H3 used was 10 fold higher than that used in previous iPVN injection studies (B.M.McGowan, S .A.Stanley, K.L.Smith, N.E. White, M.M.Connolly, E.L.Thompson, J.V.Gardiner, K.GMurphy, M.A.Ghatei, S.R.Bloom, Central relaxin-3 administration causes hyperphagia in male Wistar rats Endocrinology 2005; 146:3295-3300). For comparison, one group received the potent orexigen, NPY (500 pmol/animal iPVN). Following injection, animals were returned to their home cage with pre-weighed chow. Food intake was measured at 1, 2, 4, 8, and 24 hours post-injection.

Acute effects of high dose iPVN relaxin-3 administration on plasma TSHs Male Wistar rats (n = 9-11) received a single injection of vehicle or H3 (540 pmol). Fifteen or 30 minutes following administration, animals were killed by decapitation and plasma was collected into plastic lithium heparin tubes containing 4200 KIU aprotinin (Bayer, Haywards Heath, UK). Plasma was separated by centrifugation, frozen and stored at -70°C until radioimmunoassay (RIA) for thyroid stimulating hormone (TSH).

Acute effects of iPVN relaxin-3 administration on measurements of energy expenditure Metabolic parameters were measured following a single iPVN injection of vehicle or H3 (180 pmol) (n = 12 per group) in the early light phase (0900 h - 1000 h) by indirect calorimetry using an open-circuit Oxymax system of the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus instruments, Columbus, OH). Animals were individually housed in plexiglass cages through which air was passed at a flow rate of 2.5L/min. All rats were acclimatized to the cages for three days before the injection of vehicle or H3. Body weight was measured during the three day acclimatization period and the morning of the injection. Animals were maintained at 24⁰C with a 12:12hr light- dark cycle (light period 0700-1900). Food (ground RMl chow) and water were freely available and food was automatically weighed at 30 minute intervals. Exhaust air from each air tight chamber was sampled for one minute at 30 minute intervals. O₂ consumption and CO₂ production values were normalised with respect to body weight. The ambulatory activity of each animal was assessed simultaneously using the optical beam technique by an Opto M3 (Columbus Instruments). Consecutive photo-beam breaks were scored as an ambulatory movement. Cumulative activity counts were recorded for each 30 minute interval. Heat production was calculated by deriving a calorific value (CV) based on the observed respiratory exchange ratio (RER). This calorific value was then used with the observed oxygen consumption (VO₂) to calculate heat, expressed per gram lean body weight (cal/h/g BW). The effects of vehicle or H3 on metabolic indices were analyzed by determining the increment from baseline for each parameter.

Effects of 7 day repeated iP VN adm inistration ofrelaxin-3 on food intake and body weight

Male Wistar rats were randomized to one of three experimental groups (n= 8-11): 1) vehicle - animals received twice daily injections of vehicle (0900 h and 1900 h) for 7 days with ad libitum access to food and water, 2) H3 ad libitum fed group - rats received twice daily injections of H3 (180 pmol) for 7 days with ad libitum access to food and water, 3) H3 pair-fed group - rats received twice daily injections of H3 (180 pmol) for 7 days and were pair-fed to the median food intake of the vehicle group in the equivalent period 24 hours previously.

The dose of 180 pmol H3 was chosen as the lowest dose which gave a highly significant feeding response when administered acutely in this study. The injection interval was chosen based on a significant increase in cumulative food intake up to 8 hours but absent at 24 hours following injection during the early light phase.

Body weight was measured daily at 0900 h. Food was weighed immediately before and 1 hour after each injection to allow calculation of cumulative food intake and food intake in the first hour in response to each injection. Animals that lost more than 10 g in body weight over the course of the study were excluded. A final food and body weight measurement was taken at 0900 h on day 8. Effects of 7 day repeated iPVN administration ofrelaxin-3 on fat mass, plasma hormones and UCP-I expression

Rats from example 9 were killed by decapitation on day 8 at 0900-1000 h, and plasma was collected into plastic lithium heparin tubes containing 4200 KIU aprotinin. Plasma was separated by centrifugation, frozen and stored at -70°C until RIA. Plasma was assayed for pituitary hormones including TSH, prolactin, luteinizing hormone (LH), growth hormone (GH), Cortisol and leptin. Weights of epidydimal fat pads (white adipose tissue or WAT), interscapular brown adipose tissue (BAT), adrenals and testes were determined. Expression of UCP-I mRNA in interscapular brown adipose tissue was assessed by northern blot. RNA was extracted from BAT using Tri-reagent (Helena Biosciences, Sunderland, UK) according to the manufacturer's protocol. Northern Blot analysis was performed as previously described (W.S.Dhillo, C.J.Small, J.V.Gardiner, GA.Bewick, EJ.Whitworth, P.H.Jethwa, L.J.Seal, MΛ.Ghatei, J.P.Hinson, S.R.Bloom, Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

Biochem.Biophys.Res.Commun. 2003;301:102-107). Five micrograms RNA was size- separated on a denaturing MOPS/formaldehyde gel (1% agarose) and transferred to a Hybond-N membrane (GE Healthcare, Slough, UK). The RNA was fixed by baking at 8O⁰C for 2 hours before probing with a riboprobe corresponding to nucleotides 351-658 of the rat UCP-I cDNA sequence (accession number Ml 1814). The [α-³²P] dCTP labelled probe was denatured and then hybridized overnight at 55⁰C in a mixture of 2.5 mM EDTA pH 8, 0.5% dried milk, 0.25 M sodium phosphate buffer (pH 7.2), 5% SDS, 25 μM aurin tricarboxylic acid. Non-specific hybridization was removed by increasingly stringent washes, the final one being 0.1 x SSC/0.1% (w/v) SDS at 60°C for 30 minutes. The filter was exposed to phosphoscreen overnight prior to quantification of UCP-I mRNA expression using ImageQuant software (GE Healthcare, Chalfont St Giles, UK). Blots were reprobed with oligo(dT)_12-18 to enable differences in RNA loading to be corrected. Effect ofrelaxin-B on in vitro release of hypothalamic neuropeptides The static incubation system was used as previously described (S.A.Stanley, CJ.Small, MS.Kim, M.M.Heath, LJ.Seal, S.H.Russell, M.A.Ghatei, S.R.Bloom, Agouti related peptide (Agrp) stimulates the hypothalamo pituitary gonadal axis in vivo & in vitro in male rats Endocrinology 1999; 140:5459-5462, hereafter Stanley et al). Briefly, male Wistar rats were killed by decapitation and the brain immediately removed. The brain was mounted with ventral surface uppermost and placed in a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A 1.7 mm slice to include the PVN was taken from the basal hypothalamus and incubated in individual chambers containing 1 ml artificial cerebrospinal fluid (aCSF) (20 niM NaHCO₃, 126 niM NaCl, 0.09 mM

Na₂HPO4, 6 mM KCL, 1.4 mM CaCl₂, 0.09 mM MgSO₄, 5 mM glucose, 0.18 mg/ml ascorbic acid and 100 μg/ml aprotinin), equilibrated with 95% O₂ and 5% CO₂. The tubes were placed on a shaking platform in a water bath maintained at 37⁰C. After an initial 2 h equilibration period, the hypothalami were incubated for 45 min in 600 μl aCSF (basal period) before being challenged with H3 (10 nM) for 45 minutes. Finally, the viability of the tissue was verified by a 45 min exposure to 56 mM KCl; isotonicity was maintained by substituting K⁺ for Na⁺. At the end of each period, the aCSF was removed and frozen at -20⁰C until measurement of hypothalamic hormones [Thyrotropin releasing hormone (TRH), Somatotropin release inhibitory factor (SRIF)] by RIA.

Radioimmunoassays

Plasma pituitary hormone concentrations were assayed using reagents and methods provided by the National Institute of Diabetes and Digestive Diseases and the National Hormone and Peptide Program (Dr. A. Parlow, Torrance, CA), as previously described (Stanley et al; M.Desai, C.D.Byrne, K.Meeran, N.D.Martenz, S.R.Bloom, C.N.Hales, Regulation of hepatic enzymes and insulin levels in offspring of rat dams fed a reduced- protein diet Am. J.Physiol 1997;273:G899-G904; J.F.Todd, CJ.Small, K.O.Akinsanya, S.A.Stanley, D.M. Smith, S.R.Bloom, Galanin is a paracrine inhibitor of gonadotroph function in the female rat Endocrinology 1998; 139:4222-4229; M.S.Kim, CJ.Small, S.A.Stanley, D.G.Morgan, LJ.Seal, W.M.Kong, C.M.Edwards, S.Abusnana, D.Sunter, M.A.Ghatei, S.R.Bloom, The central melanocortin system affects the hypothalamo- pituitary thyroid axis and may mediate the effect of leptin J.Clin.Invest 2000;105:1005- 1011). Hypothalamic hormones (SRIF, TRH) were assayed using in-house radioimmunoassays (RIAs) as previously described (K.O.Akinsanya, M.A.Ghatei, S.R.Bloom, Gonadal steroids regulate rat anterior pituitary levels of TSH-releasing hormone- and pyroglutamyl-glutamyl-proline amide-like immunoreactivity

Endocrinology 1995;136:734-740; A.M.Wren, C.J.Small, C.V.Fribbens, N.M.Neary, HX. Ward, L.J.Seal, M.A.Ghatei, S.R.Bloom, The hypothalamic mechanisms of the hypophysiotropic action of ghrelin Neuroendocrinology 2002;76:316-324). Leptin (Linco Research, Inc, St. Charles, MO), corticosterone (MP Biomedicals, Irvine, CA) and free T3 (EuroDPC Ltd, Gwynedd, UK) were measured using commercially available RIA kits.

Statistical analysis

Results are shown as mean ± S.E.M. Data from acute feeding studies, plasma hormone levels and neuropepride expression were compared by ANOVA with post-hoc Dunnett's test or with post-hoc LSD test (Systat, Evanston, IL). Neuropeptide expression data were compared by unpaired Student's t-test between control and treated groups. Behavioral data were non-parametric and are expressed as median number of occurrences of behavior (interquartile ranges are expressed in square brackets). Comparison between groups was made by Mann-Whitney U test. Cumulative food intake and body weight data from the repeated injection study was analyzed using marginal models with exchangeable correlation matrix and robust standard errors (Stata 8, Statacorp LP, TX). Energy expenditure parameters were analyzed by unpaired Student /-test between control and treated group at each time interval. Hypothalamic explant data was compared by paired Student t-tβst between control and treated groups. In all cases, p < 0.05 was considered to be statistically significant.

Examples

Feeding studies

To investigate the hypothesis that relaxin-3 is involved in regulation of appetite, food intake was determined following central or peripheral relaxin-3 administration. Example 1: Effect of ICV relaxin-3 on food intake in satiated rats Animals received an ICV injection of vehicle or relaxin-3 (18, 54 or 180 pmol H3). Doses used were based on previously reported effects of porcine relaxin-1 on water intake (Thornton SM, Fitzsimons JT 1995 The effects of centrally administered porcine relaxin on drinking behaviour in male and female rats. J Neuroendocrinol 7:165-169). ICV relaxin-3 significantly increased food intake in the first hour at both 54 pmol and 180 pmol [0.96 ± 0.16 g (vehicle) vs 1.80 ± 0.27 g (54 pmol H3) and 1.81 ± 0.21 g (180 pmol H3), p< 0.05] (Fig IA). There was no significant difference in interval food intake between control and treated groups at later time points. However, cumulative food intake was significantly increased at all doses of relaxin-3 at 2 h and 4 h following ICV administration [1.38 ± 0.29 g (vehicle) vs 2.39 ± 0.29 g (18 pmol H3), 2.37 ± 0.31 g (54 pmol H3) and 2.28 ± 0.25 g (180 pmol H3) at 2 h and 1.76 g ± 0.29 g (vehicle) vs 2.73 ± 0.35 g (18 pmol H3), 2.95 ± 0.30 g (54 pmol H3) and 2.67 ± 0.29 g (180pmol H3) at 4 h, p< 0.05] (Fig IB). There was no effect on water intake following ICV H3 relaxin administration (data not shown).

Example 2: Effect of iP VN relaxin-3 on food intake in satiated rats

Animals received an iPVN injection of either vehicle or relaxin-3 (1.8, 5.4 or 18 pmol H3). Doses used were ten-fold less than those eliciting a feeding response following ICV administration (Wren et al). Intra-PVN relaxin-3 administration significantly increased food intake in the first hour at 18 pmol [0.34 ± 0.16 g (vehicle) vs 1.23 ± 0.30 g, p< 0.05] (Fig 2A). There was no significant difference in interval food intake but cumulative food intake was significantly increased at 2, 4, 8 and 24 hours following iPVN administration of 18 pmol relaxin-3 [0.38 ± 0.18 g (vehicle) vs 1.49 ± 0.31 g at 2 h, 0.63 ± 0.27 g

(vehicle) vs 1.61 ± 0.35 g at 4h, 1.24 ± 0.35 g (vehicle) vs 2.84 ± 0.61 g at 8 h and 25.9 ± 1.18 g (vehicle) vs 30.0 ± 1.1 g at 24 h, p< 0.05].

Example 3: Effect of iPVN relaxin-3 on dark phase food intake Rats received an iPVN injection of either vehicle or relaxin-3 (18 pmol H3) at the beginning of the dark phase. Nocturnal food intake was significantly increased in the first hour following relaxin-3 administration [4.43 ± 0.32 g (vehicle) vs 6.57 ± 0.42 g, p< 0.05] (Fig 2B). There was no significant effect on interval food intake at later time points but cumulative food intake was significantly increased in relaxin-3 -treated animals for 4 hours following administration in the early dark phase [9.68 ± 0.60 g (vehicle) vs 12.28 ± 0.76 g, p< 0.05].

Example 4: Effect of IP relaxin-3 on food intake

Satiated mice received an IP injection of either vehicle or increasing doses of relaxin-3 (0.03, 0.01 or 0.3 nmol/g H3). Doses of relaxin-3 used were equivalent to effective doses of the orexigenic peptide ghrelin following peripheral administration. Peripheral administration of relaxin-3 did not alter food intake in satiated mice at any time point following injection [0.06 ± 0.01 g (vehicle) vs 0.034 ± 0.02 g (0.3nmol/g H3)] (Fig 3).

Example 5: Effect of iPVN administration ofrelaxin-2 on food intake in satiated rats To differentiate the receptor mediating the effects of relaxin-3 on food intake, the feeding response to relaxin-3, which binds both LGR7 and GPCRl 35 receptors, was compared to that following administration ofrelaxin-2 (H2), which binds LGR7 but not GPCR135.

Satiated rats received an iPVN injection of either 1.8-18 pmol H3 or 1.8-18 pmol H2.

Following iPVN administration of equimolar doses, relaxin-3 stimulated one-hour food intake as previously shown in Example 2 [0.27 ± 0.11 g (vehicle) vs 1.52 ± 0.51 g (18 pmol H3), p< 0.05]. In contrast, relaxin-2 had no significant effect on food intake at any time point following administration [0.27 ± 0.11 g (vehicle) vs 0.14 ± 0.04 g (18 pmol

H2), p< 0.05] (Fig 4).

Example 6: Behavioral response following acute iPVN administration of relaxin-3

Feeding behavior was significantly increased following iPVN administration of relaxin-3 (180 pmol H3). There were no significant differences in other behaviors and there were no abnormal behaviors following an injection of relaxin-3 (Table 1). Table 1: Effect of iPVN administration of relaxin-3 (18 pmol or 180 pmol) on behavior in the first hour following injection. * = p < 0.05 vs vehicle

Relaxin-3 Relaxin-3

Behavior Vehicle (18pmol H3) (180pmol H3)

Feeding 2 [0-4] 2 [2-5] 6 [4-7] *

Drinking 0 [0] 0 [0] 0 [0]

Grooming 7 [1-8] 6.5 [4.25 -8] 6 [5-6]

Burrowing 0 [0] 0 [0] 0 [0]

Rearing 8 [ 3-10] 5 [4-9.75] 6 [4-8]

Locomotion 4 [2-4] 3 [3-4] 3 [1-5]

Sleep 0 [0-3] 0 [0-2.25] 0 [0-8]

Head down 10 [5-19] 14.5 [6.5-16] 9 [6-9]

Tremor 0 [0] 0 [0-1] 0 [0]

Example 7: Hypothalamic neuropeptide mRNA expression

Following an ICV injection of 180 pmol H3, there was no difference in hypothalamic NPY, AgRP or POMC mRNA expression 4 hours post injection compared to vehicle treated animals [NPY: 26.8 ± 1.26 (vehicle) vs 27.8 ± 2.90 (H3). AgRP: 13.1 ± 1.35 (vehicle) vs 13.0 ± 0.78 (H3). POMC: 1.90 ± 0.17 (vehicle) vs 1.85 ± 0.24 (H3), units are arbitrary, p< 0.05].

Example 8: Acute effects of high dose iPVN relaxin-3 administration on food intake A single iPVN injection of high dose human relaxin-3 (H3) to satiated male Wistar rats significantly increased food intake in the first hour post-administration at all doses [0-1 hour food intake: 0.4 ± 0.1 g (vehicle) vs 1.6 ± 0.5 g (180 pmol H3), 2.4 ± 0.5 g (540 pmol H3), and 2.2 ± 0.5 g (1620 pmol H3), p< 0.05 for all doses vs vehicle] (Figure 5). The effect was also significant at these doses in the second hour post-injection [1-2 hour food intake: 0.2 ± 0.1 g (vehicle) vs 2.2 ± 0.7 g (180 pmol H3), 2.6 ± 0.4 g (540 pmol H3), and 2.1 ± 0.5 g (1620 pmol H3), p< 0.05 for all doses vs vehicle]. There was no significant difference in interval food intake between control and treated groups at later time points. Cumulative food intake was significantly increased 2, 4 and 8 hours post iPVN administration of 180, 540 and 1620 pmol H3 [0-8 hour food intake: 2.7 ± 0.7 g (vehicle) vs 5.6 ± 1.0 g (180 pmol H3), 6.3 ± 0.6 g (540 pmol H3), and 6.9 ± 0.6 g (1620 pmol H3), p< 0.05 for all doses vs vehicle].

Although the maximum orexigenic effect of H3 (540 pmol) increased food intake almost seven-fold in the first hour, this response was equivalent to approximately 50% of the increase in food intake achieved by iPVN injection of NPY (500 pmol). However, by the end of the second hour, the increase in food intake with H3 was approximately 90% of that induced by NPY [0-1 hour food intake: 2.4 ± O.g (540 pmol H3) vs 5.2 ± 1.2 g (500 pmol NPY) and 0-2 hour food intake: 5.0 ± 0.7 g (540 pmol H3) vs 5.8 ± 1.3 g (500 pmol NPY) 2 hours post-injection].

Example 9: Acute effects of high dose iPVN relaxin-3 administration on thyroid stimulating hormone

IntraPVN administration of H3 (540 pmol) significantly reduced plasma thyroid stimulating hormone (TSH) at both 15 and 30 minutes following injection compared to vehicle [2.46 ± 0.34 ng/ml (vehicle) vs 1.58 ± 0.18 ng/ml (H3) at 15 min, p< 0.05 vs vehicle and 3.88 ± 0.44 ng/ml (vehicle) vs 2.48 ± 0.26 ng/ml (H3) at 30 min, p< 0.05 vs vehicle] (Figure 6).

Example 10: Acute effects of iP VN relaxin-3 administration on measurements of energy expenditure

A single iPVN injection of H3 (180 pmol) did not have a significant effect on the increment in VO2 although the trend was slightly higher for H3 treated animals [mean

VO2 consumption for the first 8 hours post-injection: 1296.6 ± 66.4 ml/kg/min (vehicle) vs 1348.2 ± 59.7 ml/kg/min (H3)]. VCO2, RER and physical activity did not differ when compared to vehicle. As previously observed, a significant increase in food intake was seen in the first hour after H3 administration (data not shown). Example 11: Effects of 7 day repeated iPVN administration ofrelaxin-3 on food intake and body weight

Three groups were studied: 1) vehicle treated, ad libitum fed, 2) H3 treated, ad libitum fed and 3) H3 treated pair-fed to the median food intake of the vehicle treated animals. At the onset of the study, there was no significant difference in body weight between the three experimental groups (day 1 body weight: 342 ± 9 g, [vehicle] vs 365 ± 7 g, [H3 ad libitum fed] vs 352 ± 1O g [H3 pair- fed]). Twenty-four hour food intake in the H3 ad libitum fed group was significantly higher compared to vehicle at the beginning and end of the study [day 1 : 23 ± 3 g (vehicle) vs 34 ± 4 g (H3), p< 0.05, and day 7: 33 ± 1 g (vehicle) vs 38 ± 1 g (H3), p< 0.05] (Figure 7A). IntraPVN injection of 180 pmol H3 stimulated feeding during the first hour following injection in both the early light phase and early dark phase on each study day. There was no attenuation in the acute orexigenic response in the early light phase on repeated administration up to 7 days [0-1 hour food intake: 0.3 ± 0.2 g (vehicle) and 2.3 ± 0.6 g (H3) (day 1) vs 0.7 ± 0.3 g (vehicle) and 3.0 ± 0.7 g (H3) (day 7), p< 0.05 vs vehicle] (Figure 7B). Trend analysis of cumulative food intake revealed that over the period of the study the H3-treated ad libitum fed group ate significantly more than vehicle-treated ad libitum fed controls [211.8 ± 7.1 g (vehicle) vs 261.6 ± 6.7 g (H3), p< 0.05 vs vehicle at the beginning of day 7] (Figure 8). There was also a trend towards greater cumulative body weight change in the H3-treated ad libitum fed group compared to vehicle-treated animals [8 ± 4 g (vehicle) vs 13 ± 4 g (H3)]. There was no difference in cumulative body weight change between the H3 pair-fed group compared to vehicle at day 7 [8 ± 4g (vehicle) vs 2 ± 2 g (H3 pair-fed)], but body weight change was significantly reduced in the H3 pair-fed compared to IB-treated ad libitum fed animals [2 ± 2 g (H3 pair-fed) vs 13 ± 4g (H3 ad libitum fed), p <0.05].

Example 12: Effects of 7 day repeated iPVN administration ofrelaxin-3 on fat mass, UCP-I expression and plasma hormones

Following an iPVN injection of H3 (180 pmol) or vehicle twice a day for 7 days, there was no significant change in interscapular BAT weight [0.54 ± 0.03 g (vehicle) vs 0.61 ± 0.04 g (H3 ad libitum fed) vs 0.56 ± 0.04 g (H3 pair-fed)]. There was no difference observed in interscapular BAT UCP-I mRNA expression between the experimental groups [12.83 ± 1.80 (vehicle) vs 14.28 ± 2.36 (H3 ad libitum fed) vs 12.42 ± 2.65 (H3 pair-fed), units are arbitrary] (Table 2).

There was no significant difference in epididymal fat mass between the 3 groups [3.35 ± 0.27 g (vehicle) vs 4.05 ± 0.32 g (H3 ad libitum fed) vs 3.31 ± 0.23 g (H3 pair-fed)]. However, plasma Ieptin was significantly elevated in the H3 ad libitum fed group compared to vehicle, [2.24 ± 0.40 ng/ml (vehicle) vs 3.67 ± 0.55 ng/ml (H3 ad libitum fed) vs 2.05 ± 0.49 (H3 pair-fed), p< 0.05 H3 ad libitum fed vs vehicle] (Table 2).

Table 2: The effect of 7-day repeated iPVN administration of vehicle or H3 (180pmol) on epididymal fat pad weight (WAT), BAT weight, UCP-I mRNA expression, plasma Ieptin, plasma TSH and free T3.

Vehicle - vehicle-treated, ad libitum fed; H3 - H3-treated, ad libitum, fed; H3 PF - H3- treated, pair-fed to median food intake of vehicle-treated animals. Data shown as mean ± SEM. * = p < 0.05 vs vehicle

A significant suppression of plasma TSH was observed in both the H3 ad libitum fed group and H3 pair-fed group when compared to vehicle-treated animals [3.93 ± 0.53 ng/ml (vehicle) vs 2.44 ± 0.30 ng/ml (H3 ad libitum fed), p<0.05, vs 2.59 ± 0.36 ng/ml (H3 pair-fed)] (Figure 9). There was no significant difference in plasma free T3 levels between the groups [1.24 ± 0.14 pg/ml (vehicle) vs 1.53 ± 0.14 pg/ml (H3 ad libitum fed) vs 1.76 ± 0.22 pg/ml (H3 pair-fed)].

There were no significant differences in adrenal and testicular weight between the treatment groups and no differences in plasma prolactin, corticosterone, LH and GH plasma levels amongst the three groups (data not shown).

Example 13: Effect ofrelaxin-3 on in vitro release of hypothalamic neuropeptides To examine the possible central mediators of the effects of H3 on plasma TSH, hypothalamic release of neuropeptides known to regulate thyroid function (TRH and

SRIF) was determined in response to H3 relaxin in vitro. TRH was significantly reduced by 10 nM H3 (100 ± 7.5 % basal vs 77.2 ± 7.2 % basal release, p< 0.05) whilst SRIF release increased significantly in response to 10 nM H3 (100 ± 8.1 % basal vs 163.6 ± 21.1 % basal release, p< 0.05).

Claims

Claims

1. A method for identifying compounds which will be useful as agents for the control of appetite and/or food intake in a mammal, comprising contacting a candidate molecule with a GPCRl 35 and monitoring the interaction of the candidate molecule and the GPCR 135.

2. A method for identifying compounds which will be useful in the treatment of obesity, comprising contacting a candidate molecule with GPCR135 and monitoring the interaction of the candidate molecule and the GPCR135.

3. A method for identifying compounds which will be useful as agents for the control of appetite and/or food intake, comprising ascertaining the binding affinity of a candidate molecule to GPCRl 35 receptor.

4. A method according to claim 3, which comprises ascertaining the GPCR135 agonist activity of the candidate molecule.

5. A method according to claim 4, which comprises determining whether the GPCR 135 agonist activity of the candidate molecule is greater than or equal to the agonist activity of human relaxin-3 under essentially the same conditions.

6. A method according to claim 3, which comprises ascertaining the GPCR135 antagonist activity of the candidate molecule.

7. A method according to any one of claims 1 to 6, in which the GPCR135 is human GPCRl 35.

8. A method according to any one of claims 1 to 6, in which the GPCRl 35 is rat GPCR135.

9. A method according to any one of claims 1 to 6, in which the GPCRl 35 is mouse GPCR135.

10. A method according to any one of claims 1 to 6, in which the GPCR135 has at least 75% homology with human GPCRl 35.

11. A method according to claim 10, in which the GPCR135 has at least 95% homology with human GPCRl 35.

12. A compound identified by a method according to any one of the preceding claims.

13. A compound identified by a method according to any one of the preceding claims, for use in a method of treatment of the human or animal body.

14. Relaxin-3, for use in a method of treatment of the human or animal body for an eating disorder.

15. Relaxin-3, for use in a method of treatment of anorexia nervosa or cachexia.

16. A pharmaceutical composition comprising a compound according to claim 13 or a physiologically acceptable salt, solvate or derivative thereof, together with one or more pharmaceutically acceptable carriers.

17. A pharmaceutical composition according to claim 16, in which the compound is a GPCRl 35 antagonist and is present in a therapeutically effective amount for the treatment of obesity.

18. The use of a compound which is an antagonist of GPCRl 35, or a physiologically acceptable salt, solvate or derivative thereof, for the manufacture of a medicament for suppression of appetite and/or food uptake.

19. The use of a compound which is an agonist of GPCR135, or a physiologically acceptable salt, solvate or derivative thereof, for the manufacture of a medicament for stimulation of appetite and/or food uptake.

20. A method for the treatment of an eating disorder in a mammal, including a human, comprising administration of a compound which agonises GPCR135.

21. A method as claimed in claim 20 or a use as claimed in claim 19 in which the disorder is cachexia.

22. A method according to claim 20, a use as claimed in claim 19 or a method or a use as claimed in claim 21 in which the compound is relaxin-3.

23. A method for the treatment of obesity in a mammal, including a human, comprising administration of a compound which antagonises GPCR 135.

24. A method for cosmetic weight loss in a mammal, the method comprising administering a composition comprising a GPCRl 35 antagonist to a mammal.