WO2009080608A1 - Y2 receptor agonists - Google Patents

Abstract

A polypeptide agonist of the Y2 receptor selective for the Y2 receptor over the Y1 and Y4 receptors, which is a functional mimic of the 3-36 truncated variant of the polypeptide PYYl- 36 and has the general formula (1) in which variant said PYY 1-36 sequence is modified to include a branch point (BP) at an amino acid position between position 1 and position 14 provided by a replacement or additional natural or artificial amino acid residue BP having a first amino group, a second amino acid group, which is not an α amino group, and a carboxylic acid group, the said first amino group having bonded thereto an amino acid sequence of from 0 to 15 amino acids, said second amino group being bonded to a PYY partial sequence, and said carboxylic acid group being bonded to an alpha helical amino acid sequence.

Description

Y2 Receptor Agonists

FIELD OF THE INVENTION

The present invention relates to the field of appetite regulation for cosmetic purposes or therapy and therapy of diseases associated with appetite regulation. In particular, the present invention relates to novel enhanced analogues of peptide YY (3-36) and the use of these analogues in prevention and treatment of diseases associated with appetite regulation, such as obesity and obesity related metabolic disorders such as insulin resistance, dyslipidaemia, glucose intolerance and diabetes.

BACKGROUND OF THE INVENTION

Peptide YY (SEQ ID NO: 1) is a 36 amino-acid peptide belonging to the pancreatic polypeptide (PP) family of peptides also known as the PP-fold peptides because they share a common hairpin-like three-dimensional structure (Fuhlendorff et al . , 1990, J Biol Chem 265:11706-12) . Pancreatic polypeptide was the first of the PP-fold peptides to be discovered and received its name because it was isolated from insulin extracts (Kimmel et al., 1968,

Endocrinology 83:1323-30) . Peptide YY (PYY) and neuropeptide Y (NPY) were discovered later from intestinal and brain extracts respectively (Tatemoto et al., 1982, Nature 296:659- 60, Tatemoto, 1982, Proc Natl Acad Sci U S A 79:2514-8) . There are two main forms of endogenous PYY: PYY1-36 and PYY3- 36 both of which can be found in the circulation (Grandt et al., 1994, Regul Pept 51:151-9, Grandt et al., 1994, Peptides 15:815-20) . The sequences of the human forms of PYY1-36 and PYY3-36 are as follows:

PYY1-36: YPIKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO: 1) PYY3-36: IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO: 2)

By way of comparison, the sequences of the human forms of NPY and PP are:

NPY: YPSKPDNPGE DAPAEDMARY YSALRHYINL ITRQRY (SEQ ID NO:3)

PP: APLEPVYPGD NATPEQMAQY AADLRRYINM LTRPRY (SEQ

ID NO:4)

Thus following this nomenclature it follows that PYYl -12 is YPIKPEAPGE DA (SEQ ID NO: 5) PYY3-12 is IKPEAPGE DA (SEQ ID NO: 6)

PYY13-36 is SPEELNRY YASLRHYLNL VTRQRY (SEQ ID

NO: 7)

Points in the sequence at which NPY differs from PYY in the first 14 amino acids have been marked in bold type.

The enzyme dipeptidyl peptidase-IV hydrolyses PYY1-36 at the Pro2-Ile3 bond yielding PYY3-36 (SEQ ID NO: 2) (Medeiros and Turner, 1994, Endocrinology 134:2088-94) . Peptide YY is synthesized by endocrine L-cells lining the gut and is released postprandially particularly following ingestion of fat (Adrian et al . , 1985, Gastroenterology 89:1070-7) . Plasma PYY levels increase within 15 minutes, are maximal at 90 minutes and are elevated for up to 6 hours following the ingestion of a meal (Adrian, et al., 1985, Gastroenterology 89:1070-7) . In the fasted state PYY1-36 has been found to be the predominant form, whereas PYY3-36 predominates following a meal (Grandt, et al . , 1994, Regul Pept 51:151-9, Grandt, et al., 1994, Peptides 15:815-20) . In addition to intestinal L- cells PYY expression has also been demonstrated in a small population of neurons in the brainstem, suggesting that PYY could function as a neurotransmitter (Broome et al., 1985, Acta Physiol Scand 125:349-52) .

To date five PP-fold receptors have been cloned and designated the Yl, Y2, Y4, Y5 and y6 receptors (Berglund et al., 2003, Exp Biol Med (Maywood) 228:217-44) . The existence of a Y3 NPY-preferring receptor has been suggested based on pharmacological studies, but the receptor remains to be cloned (Lee and Miller, 1998, Regul Pept 75-76:71-8) . The lower case designation of the y6 receptor is based on the fact that it encodes a truncated and presumably nonfunctional receptor in most mammals including humans (Michel et al., 1998, Pharmacol Rev 50:143-50) . The functional Y- receptors are G-protein coupled receptors all coupling to inhibitory G-proteins (Gi) therefore inhibiting cAMP production (Berglund, et al., 2003, Exp Biol Med (Maywood) 228:217-44, Michel, et al., 1998, Pharmacol Rev 50:143-50) .

The three PP-fold peptides, PYY, NPY and PP show different affinities to the Y-receptors. Whereas full length NPY and PYY show high affinity binding to Yl, Y2 and Y5 receptors, PYY3-36 and NPY 3-36 show high selectivity for Y2 over Yl receptors demonstrating the importance of the aminoterminal part of PP-fold peptides for Yl receptor activation (Grandt et al., 1996, Regul Pept 67:33-7, Grandt et al., 1992, Biochem Biophys Res Commun 186:1299-306) . In contrast, Y2 receptors are less strictly dependent on the amino-terminal portion, therefore permitting C-terminal truncated forms of PYY and NPY bind with almost equal affinity as the untruncated forms (Fuhlendorff, et al . , 1990, J Biol Chem 265:11706-12) . The Y4 subtype preferentially binds PP (Michel et al., 1998, Pharmacol Rev 50:143-50) .

Peripheral administration of PYY produces a variety of primarily inhibitory effects on digestion. It has been shown that PYY injected into the systemic circulation inhibits gastric emptying and acid secretion, reduce stimulated pancreatic exocrine secretion and increase intestinal transit time (Pappas et al., 1985, Gastroenterology 89:1387-92, Pappas et al., 1986, Gastroenterology 91:1386-9, Adrian et al., 1985, Gastroenterology 89:494-9, Allen et al., 1984, Digestion 30:255-62) . Inhibitory effects on digestive functions can also be elicited by injections of PYY into the hindbrain. Injection of PYY or PYY13-36 directly into the dorsal motor nucleus of the vagus can also inhibit gastric emptying (Martinez et al., 1998, Am J Physiol 274:G965-70, Chen and Rogers, 1997, Am J Physiol 273:R213-8, Browning and Travagli, 2003, J Physiol) . These effects are presumably mediated by the Y2 receptor as PYY and PYY13-36 (the latter a Y2 selective agonist) equally effectively elicits the effects (Chen and Rogers, 1997, Am J Physiol 273:R213-8) . When PYY, NPY or PP are injected into the cerebral ventricles or into the hypothalamus (notably the paraventricular nucleus or lateral hypothalamic area) they all increase food intake (Campbell et al., 2003, J Neurosci 23:1487-97, Stanley et al., 1985, Peptides 6:1205-11) . The stimulatory effects of NPY and PYY on food intake are believed to be mediated via activation of central Yl and Y5 receptors (Berglund, et al . , 2003, Exp Biol Med (Maywood) 228:217-44) whereas the orexigenic effects of PP presumably are caused by activation of Y4 receptors on neurons in the lateral hypothalamic area (Campbell et al., 2003, J Neurosci 23:1487-97) . In contrast to the postsynaptic Yl, Y2 and Y5 receptors the prototypical response for the Y2 receptor is the presynaptic inhibition of neurotransmitter release (Wahlestedt et al., 1986, Regul Pept 13:307-18) . This is consistent with the aforementioned predominantly inhibitory effects on vagal efferents. The Y2 agonist PYY13-36 applied onto vagal motor neurons inhibited the firing rate of approximately 50%, whereas only approximately 5% were activated (Chen and Rogers, 1997, Am J Physiol 273:R213-8) .

Recently, an inhibitory role for post-prandially released PYY3-36 in appetite regulation was proposed (Batterham et al., 2002, Nature 418:650-4) . It was shown that acute intraperitoneal (i.p.) injections of peptide YY (PYY3-36) dose-dependently (30, 300 and 3000μg/kg bw) inhibits 4 hour food intake and that chronic treatment (twice daily injections of 50μg/kg bw of PYY3-36) suppresses weight gain in rats (Batterham, et al., 2002, Nature 418:650-4) . In the same study 90 min intravenous infusion of PYY3-36 to healthy human volunteers lead to a reduction in appetite and a reduced caloric intake for the following 12 hours (Batterham et al., 2002, Nature 418:650-4) . The food inhibitory effect of peripherally administered PYY3-36 was recently shown to be present also in obese individuals (Batterham et al., 2003, N Engl J Med 349:941-8) . The food inhibitory effect of PYY3-36 is presumably mediated by Y2 receptors, as mice lacking this receptor fail to reduce caloric intake when injected with PYY3-36 (30, 300 and 3000μg/kg bw) (Batterham et al . , 2002, Nature 418: 650-4) .

It has been suggested that peripherally administered PYY3-36 inhibits food via activation of presynaptic Y2 receptors on NPY neurons in the hypothalamic arcuate nucleus (Batterham et al., 2002, Nature 418:650-4) . However, peripherally administered PP-fold peptides such as NPY and PYY gain access to the dorsal vagal complex (Whitcomb and Taylor, 1992, American Journal of the Medical Sciences 304:334-8), and vagal afferents terminating in the nucleus of the solitary tract are sensitive to several postprandially released gastrointestinal hormones (GLP-I, CCK) . Thus, it is equally possible that Y2 receptors expressed in neurons of the dorsal vagal complex mediate the anorectic actions of peripheral PYY3-36.

Obesity, defined as an excess of body fat relative to lean body mass, is highly associated with important psychological and medical morbidities. Of these the most severe include Type II or non-insulin-dependent diabetes mellitus (NIDDM) , hypertension, elevated blood lipids and coronary heart disease. Obesity, and especially upper body obesity, is the most common nutritional disorder of the world. Numerous studies indicate that lowering body weight dramatically reduces risk for chronic diseases, such as diabetes, hypertension, hyperlipidaemia, coronary heart disease, and musculo-skeletal diseases. For example, various measures of obesity, including, simple body weight, waist-to-hip ratios, and mesenteric fat depots, are strongly correlated with risk for non-insulin dependent diabetes (NIDDM) , also known as type II diabetes. Obesity is also a risk factor for the group of metabolic derangements collectively named the metabolic syndrome or "Syndrome X".

Current methods for promoting weight loss are not satisfactory. It is estimated that in the US alone approximately 33 billion USD is spent annually on weight reducing treatments, but considering that the prevalence of obesity continues to rise, the money spent appears largely futile.

The chronic nature of obesity, the worldwide epidemiological rise in the prevalence of obesity and the large number of associated diseases call for new methods and compositions such as pharmaceutical agents reducing caloric intake and hence promoting weight-loss.

There have been numerous publications of analogues of PYY intended to provide superior properties compared to PYY3-36. Amongst these, WO2005/080424 disclosed the concept of providing analogues of the PYY polypeptide in which by the use of an lysine residue substitution at the 29 position a short second amino acid chain was provided bonded to the ε amino acid group of the lysine, so as to provide the molecule with a second N-terminus. This was exemplified by molecules like IKPE-h-PYY (13-36) -K29 (H-IKPEA) -NH₂. OBJECT OF THE INVENTION

It is an object of the present invention to provide improvements in the treatment of appetite regulating diseases, such as obesity to provide agents effective in the treatment of conditions characterized by deposition of excess body fat. It is a further object of the invention to provide PYY analogues that produce after cessation of administration a longer lasting weight loss effect than does PYY3-36.

SUMMARY OF THE INVENTION

The present invention provides a polypeptide agonist of the Y2 receptor selective for the Y2 receptor over the Yl and Y4 receptors, which is a functional mimic of the 3-36 truncated variant of the polypeptide PYY1-36 and has the general formula 1

R¹

^R"

Formula 1

in which each Aaa is an amino acid chain and each of n, m, o and s is an integer representing the number of amino acids in a respective said chain,

said polypeptide of formula 1 being a variant of the polypeptide of SEQ ID NO: 1 (i.e. human PYY1-36)

YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY SEQ I D NO : 1

in which variant said SEQ ID NO:1 is modified to include a branch point (BP) at an amino acid position x between position 1 and position 14 of said sequence (the N-terminal Y being position 1 and the C-terminal Y being position 36) , said branch point being provided by a replacement or additional natural or artificial amino acid residue BP having a first amino group, a second amino acid group, which is not an α amino group, and a carboxylic acid group, the said first amino group being free (m = 0) or having bonded thereto an amino acid sequence (Aaa_m) of from 1 to 15 amino acids, said second amino group being bonded to an amino acid sequence (Aaa_n) containing a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence, and said carboxylic acid group being bonded to an amino acid sequence (Aaa₀) - (Aaa_s) , (Aaa₀) being an alpha helical amino acid sequence of from 12-19 amino acids and (Aaa_s) being the sequence -T-R-X' aa-R-Y;

or a function retaining variant of such a polypeptide in which in said partial PYY amino acid sequence in sequence Aaa_n one or more amino acids are deleted or substituted subject to there being maintained therein a motif of the form P-Xaa-Xaa-P,

wherein each residue Xaa or X' aa is any amino acid and each amino acid Xaa or X' aa is independently a natural or unnatural amino acid bonded to form an amide linkage via an α or other amino group, and wherein, and R¹ and R³ each independently are H, or an acylating group, e.g. acetyl, propanoyl, butanoyl or benzoyl, and R² is H, or an amidating group, e.g. NH2, NHR or NHRR' (where R and R' are alkyl or aromatic substituents) .

The amino acid sequence Aaa_n may contain as a variant of the PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence, a function retaining variant in which in said partial PYY amino acid sequence in sequence Aaa_n one or more amino acids are deleted or substituted subject to there being maintained therein a motif of the form P-Xaa-Xaa-P- Xaa-Xaa- P, or P-Xaa-Xaa-P-Xaa-Xaa-Xaa-P.

The amino acid sequence Aaa_n may contain a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 50% of non-proline amino acids therein and of up to one proline residue therein. For instance, the amino acid sequence Aaa_n may contain a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 3 non-proline amino acids therein or may contain a PYY partial sequence of from 6 to 9 amino acids of the PYY 1-9 sequence with substitutions of up to 3 non-proline amino acids therein.

The amino acid sequence Aaa_n preferably contains a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence .

Preferred polypeptides of the invention may be of the general formula 2

Formula 2 wherein each of Aaa_p is an amino acid sequence of from 4-8 amino acids containing the motif -Pro-Xaa-Xaa-Pro-, and wherein (Aaa_n' ) is an amino acid sequence consisting of a sequence of from 6 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 50% of non-proline amino acids therein, Aaa_q is an amino acid sequence of from 1-8 amino acids, Aaa_s is as defined in claim 1, and (Aaa_m' ) is absent (m = 0) or is an amino acid sequence consisting of a sequence of from 1 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 50% of non-proline amino acids therein .

In Formula 2, the amino acid sequence Aaa_n' may consist of a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence, and the amino acid sequence Aaa_m' is absent or consists of a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence.

Said amino acid sequence Aaa_p is preferably PRRP or GPRRP and said amino acid sequence Aaa_q is preferably PRRP or GPRRP.

Said amino acid sequence Aaa_q may be I (K) _r , where r representing the number of lysine residues is from 0 to 3. Said amino acid sequence Aaa_m may be I (K) _r , where r is from 0 to 3. Optionally, said amino acid sequence Aaa₀ matches in sequence an alpha helical portion of the peptide PYY, the peptide NPY or the peptide PP, but a match to PYY is preferred. The alpha helical portion of PYY is generally considered to run from residue 17 to residue 31 of the PYY1-36 sequence.

Said branch point amino acid may be an α amino acid. It may be lysine, or may be diaminopropionic acid. Whilst tri- functional branch point residues are preferred (two amino groups and a carboxylic acid group) it may be for instance tetra-, or pentafunctional . Furthermore, the branch point may be constructed from an amino acid having two carboxylic acid groups combined with an at least difunctional linker reacted with the second carboxylic acid such as to provide a free second amine group. For instance, the branch point may be glutamic acid or aspartic acid reacted on its second carboxylic acid group with a diamine linker, e.g. it may have the structure:

In the amino acid sequence Aaa_n, said PYY 1-14 partial sequence with optional amino acid substitutions may form an N-terminal end of the molecule. Optionally, said amino acid sequence Aaa_n comprises at its N- terminal end or consists of the sequence PYY 1-12, PYY 1-9, PYY 2-12, PYY 2-9, PYY 3-12, or PYY 3-9.

Alternatively, said amino acid sequence Aaa_n comprises at its N-terminal end or consists of the sequence NPY 1-12, NPY 1-9, NPY 2-12, NPY 2-9, NPY 3-12, or NPY 3-9.

Alternatively, said amino acid sequence Aaa_n comprises at its N-terminal end or consists of the sequence PP 1-12, PP 1-9, PP 2-12, PP 2-9, PP 3-12, or PP 3-9.

Each amino acid Xaa is preferably a natural amino acid forming a peptide bond via an α amino group. For instance, the amino acid sequence Aaa_s may be TRQRY or TRPRY.

Preferably, said branch point is immediately in the N- terminal direction from the residue S of SEQ ID NO: 1.

The polypeptide analogues of the present invention may contain variants of the relevant part of the PYY sequence and these are preferably conservative amino acid substitutions (defined further below) . Thus, in the sequence Aaa_n, which is permitted to contain substitutions of up to 50% of its non- proline amino acids and of one proline residue, the substitutions are preferably conservative substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . Substitution of one amino acid within a said family by another constitutes a conservative substitution.

Variants of the relevant part of the PYY sequence for use in this invention include those having an amino acid sequence sufficiently similar to the amino acid sequence of the peptides of this invention or a domain thereof. The term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain that is at least about 50%, about 75% through 98%, or identical are defined herein as sufficiently similar.

Substitutions are preferably of amino acids of the PYY sequence by corresponding amino acids from another PP fold polypeptide, e.g. by corresponding amino acids of NPY or PP.

In an alternative independent aspect, the invention provides a polypeptide agonist of the Y2 receptor selective for the Y2 receptor over the Yl and Y4 receptors, which is a functional mimic of the 3-36 truncated variant of the polypeptide PYYl- 36 and has the general formula 1

R¹ (Aaa)_n

(BP)- (Aaa- -(Aaa_s)- ^■R*

^R (Aaa) Formula 1

in which each Aaa is an amino acid chain and each of n, m, o and s is an integer representing the number of amino acids in a respective said chain,

(Aaa)_n being or comprising at its N-terminal end the sequence

PYY X-9 wherein X is from 1 to 3; preferably being no more than 12 amino acids and for instance being the sequence PYY 1-9 or the sequence PYY 3-9 or the sequence PYY 1-12 or the sequence PYY 3-12; (Aaa)_m being hydrogen or the amino acid residue I or being or comprising at its N-terminal end the sequence PYY X-9; preferably being no more than 12 amino acids; (Aaa) _o— (Aaa) _s being as defined above but preferably being the sequence PYY13-36, and BP, R]_, R2, and R3 being as previously defined.

Formulation of Peptide YY analogues

Route of administration The peptides of the present invention may serve as medicaments in their pure form or as pharmaceutical compositions and they may be administered via any of the usual and acceptable methods known in the art, either singly or in combination. Such compositions may be formulated for oral administration (including buccal cavity or sublingually) or for parenteral administration (including intravenous (i.v.), subcutaneous (s.c), intramuscular (i.m.), and intraperitoneal (i.p.)) administration. Other administration routes include epidural, rectal, intranasal or dermal administration or by pulmonary inhalation.

Types of Formulations The present invention contemplates a pharmaceutical composition comprising, as an active principle, a peptide of the invention in admixture with a pharmaceutically acceptable carrier, diluent, vehicle or excipient. Typically, such a pharmaceutical composition will be a dose form selected from the group consisting of an oral dosage form, a buccal dosage form, a sublingual dosage form, an anal dosage form, and a parenteral dosage form such as an intraveneous, an intraarterial, an intraperitoneal, a subdermal, an intradermal or an intracranial dosage form. Especially preferred formulations provide sustained release of the peptide of the invention.

The compositions may preferably be formulated to subcutaneous or oral administration, and such compositions may be prepared in a manner well known to the field. The compositions are preferably in the form of solid or liquid formulations and methods for their preparation are generally described in "Remington's Pharmaceutical Sciences", 17th Ed., Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985. Solid formulations are particularly suitable for oral administration, while solutions are most useful for injection or infusion (i.v., s.c, i.m., or i.p.) or intranasal administration .

Such compositions will contain an effective amount of the one or more active peptides of this invention together with a suitable carrier in order to provide the dosage in a form compatible with the route of administration selected. The compositions comprising at least one of the peptides of this invention together with a physiologically acceptable carrier in the form of a vehicle, a diluent, a buffering agent, a tonicity adjusting agent, a preservative and stabilizers. The excipients constituting the carrier must be compatible with the active pharmaceutical ingredient (s) and preferably capable of stabilizing the peptides without being deleterious to the subject being treated.

Solid compositions may appear in conventional form such as tablets, pills, capsules, suppositories, powders or enterically coated peptides. Liquid compositions may be in the form of solutions, suspensions, dispersions, emulsions, elixirs, as well as sustained release formulations, and the like. Topical compositions may be in the form of plasters or pastes and inhalation compositions may be contained in spray delivery systems.

Depot (sustained release) formulations

In a preferred embodiment of the invention depot formulations that include at least one of the present peptides are envisioned. A form of repository or depot formulation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or deposition. Formulations suitable for sustained release formulations include biodegradable polymers and may consist of appropriate biodegradable polymers, such as L-lactic acid, D-lactic acid, DL-lactic acid, glycolide, glycolic acid, and any isomers thereof. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax .

Other depot formulations may include, but are not limited to, formulations that include at least one of the present peptides disclosed herein combined with liposomes, microspheres, emulsions or micelles and liquid stabilizers.

Aqueous Formulation

Aqueous formulations of the peptides of this invention may be prepared for parenteral administration by injection or infusion (i.v., s.c, i.m. or i.p.) . Since the peptides of the invention are amphoteric, they may be utilized as free acids or bases, or as salts. The salts must, of course, be pharmaceutically acceptable, and these will include alkali and metal salts of acidic peptides, e,g., potassium, sodium or magnesium salts. The salts of basic peptides will include salts of halides and inorganic and organic acids, e.g. chloride, phosphate or acetate. Salts of the peptides are readily prepared by procedures well known to those skilled in the art .

The peptides of this invention may be provided as liquid or semi-liquid compositions for parenteral administration (e.g. injection, infusion or deposition of slow release depot formulations) . The peptides may be suspended or dissolved in an aqueous carrier, for example, in a suitably buffered solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate/citric acid, sodium phosphate/phosphoric acid, sodium acetate/acetic acid, or combinations thereof.

Such aqueous solutions may be rendered isotonic by adjusting the osmotic pressure with a buffering agent, by the inclusion of saline, aqueous dextrose, glycols or by the use of sugars such as lactose, glucose or mannitol and the like. The compositions may be other pharmaceutically acceptable excipients such as preservatives, stabilizing agents, and wetting or emulsifying agents as described in "Handbook of Pharmaceutical Excipients", 3^rd Ed., Arthur H. Kibbe (Ed.), Pharmaceutical Press, London, UK (2000) . The preservatives may include sodium benzoate, sodium sorbic acid, phenol or cresols and parabens . Stabilizing agents may include carboxymethyl- cellulose, cyclodextrins or detergents. The preparation may be produced immediately before use from active drug substance and sterile carrier solution. Alternatively, the compositions may be filled into sealed glass vials or ampoules, and if necessary purged with an inert gas, under aseptic conditions and stored until needed. This allows for continued multi-dose therapy but also demands the highest degree of stability of the compound.

Oleaginous Formulations Oleaginous formulations of the peptides of this invention may be prepared for parenteral administration by injection (s.c, i.m. or i.p.) or topically. The carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. The compositions may be in the form of solutions or suspensions. Solutions of the peptides may be prepared with the use of detergents and emulsifiers and suspensions may be prepared using powder or crystalline salts. The compositions may be stabilized with preservatives (e.g. butylated hydroxianisole or butylated hydroxytoluene) .

Nasal administration For nasal administration by pulmonary inhalation, the formulation may contain one or more peptides of the present invention, dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier may contain auxiliary additives such as solubilizing agents, e.g., propylene glycol, surfactants such as polyoxyethylene, higher alcohol ethers, and absorption enhancers such as lecithin or cyclodextrin and preservatives such as sorbic acid, cresols or parabens .

Topical Formulations

Topical administration for local application and action of the peptides of this invention may be in the form of pastes prepared by dispersing the active compound in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil or the like. Alternatively, the peptides may be incorporated into patches for dermal administration. Patches may be prepared in a form for iontophoretic application .

Suppositories Suppositories for transmucosal administration may be in the form of pellets containing an effective amount of a compound of the present invention can be prepared by admixing a compound of the present invention with a diluent such as carbowax, carnuba wax, and the like, and a lubricant, such as magnesium or calcium stearate.

Oral Formulations

Solid compositions are preferred for oral administration in the form of tablets, pills, capsules, powders, and the like. Tablets may contain stabilizing buffering agents (e.g. sodium citrate, calcium carbonate and calcium phosphate) , disintegrants (e.g. potato or tapioca starch, and complex silicates) binding agents (e.g. polyvinylpyrrolidone, lactose, mannitol, sucrose, gelatin, agar, pectin and acacia) and lubricating agents (e.g. magnesium stearate, stearic acid or sodium lauryl sulfate) as well as other fillers (e.g. cellulose or polyethylene glycols) . Liquid formulations for oral administration may be combined with various sweetening agents, flavoring agents, coloring agents, in addition to diluents such as water, ethanol, propylene glycol, glycerin.

Doses

The doses the peptides and compositions of the present invention required for the desired therapeutical effects will depend upon on the potency of the compound, the particular composition used and the route of administration selected. The peptides will typically be administrated in the range of about 0.001 mg to 100 mg per human adult patient per day, preferably from about 0.01 to about 10 mg per patient per day, more preferably from about 0.1 to about 1 mg per patient per day, about 0.5 mg per patient per day. Dosages for certain routes, for example oral and other non-parenteral administration routes, should be increased to account for any decreased bioavailability, for example, by about 5-100 fold.

Dosing Regimen

The most suitable dosing regimen may best be determined by a medical practitioner for each patient individually. The optimal dosing regimen with the peptides and pharmaceutical compositions of this invention depends on factors such as the particular disease or disorder being treated, the desired effect, and the age, weight or body mass index, and general physical conditions of the patient. The administration may be conducted in a single unit dosage form to alleviate acute symptoms or as a continuous therapy in the form of multiple doses over time. Alternatively, continuous infusion systems or slow release depot formulations may be employed. Two or more peptides or pharmaceutical compositions of this invention may be co-administered simultaneously or sequentially in any order. In addition, the peptides and compositions may be administered in a similar manner for prophylactic purposes.

The following non-limiting examples are presented merely in order to illustrate the invention. The skilled person in the area will understand that there are numerous equivalents and variations not exemplified but still forming part of the present invention.

Use of the PYY analogues in disease treatment The present invention contemplates in one embodiment a method for reducing body weight in a subject, the method comprising administering, to the subject, an effective amount of the peptide or pharmaceutical composition of the invention.

As will be appreciated from the above, administration of the inventive analogues is expected to in one embodiment providing effective means for reducing excess body fat in individuals in need thereof. It is contemplated that the presently suggested therapeutic treatment of humans should be accompanied by a controlled diet in order to ensure that the person undergoing treatment ingests necessary nutrients. At the same time the rate of weight loss or weight gain should be carefully monitored in order to avoid too drastic reductions in body weight over time and it should be ensured that the treated subject exerts a physical behaviour that aims at preserving muscle mass etc.

Overweight and obese individuals (BMI of 25 and above) are at increased risk for physical ailments such as: high blood pressure, hypertension; high blood cholesterol, dyslipidemia; Type 2 (non-insulin dependent) Diabetes; insulin resistance, glucose intolerance; hyperinsulinemia; coronary heart disease; angina pectoris; congestive heart failure; stroke; gallstones; cholescystitis and cholelithiasis; gout; osteoarthritis; obstructive sleep apnoea and respiratory problems; musculo-skeletal diseases; some types of cancer (such as endometrial, breast, prostate, and colon) ; complications of pregnancy; poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation) ; bladder control problems (such as stress inconti- nence) ; uric acid nephrolithiasis; psychological disorders (such as depression, eating disorders, distorted body image, and low self esteem) . Obesity is also a risk factor for the group of metabolic derangements collectively named the metabolic syndrome or "Syndrome X". The health consequences of obesity range from increased risk of premature death to serious chronic conditions that reduce the overall quality of life. Furthermore, severe obesity is associated with a 12 fold increase in mortality in 25-35 year olds when compared to lean individuals. Negative attitudes towards the obese can lead to discrimination in many areas of their life including health care and employment.

Since the present invention in one aspect provides means for reducing body fat deposits, any one of the above-listed syndromes, diseases and conditions are targets for the aspect of the invention that relates to therapy and prophylaxis and the inventive peptides are useful against any disease or condition characterized by excess body fat deposition.

The effective amount will be determined by the skilled person taking into account such factors as potency of the drug, age and constitution of the patient, body weight, pharmacokinetic profile of the drug, and in general the drug will be prescribed for each patient or group of patients. However, the effective amount of the peptide is preferably at least about 0.01 μg/kg body weight/day, such as at least 0.1 μg/kg body weight/day, at least 1 μg/body weight/day, and at least 10 μg/kg body weight/day. On the other hand, the effective amount of the peptide or dimer is preferably at most about 0.1 mg/kg body weight/day, such as at most 0.5 mg/kg body weight/day and at most 1 mg/kg body weight/day. It is expected that the effective amount of the peptide will be about 0.1 μg/kg body weight/day, about 0.3 μg/kg body weight/day or about 1 μg/kg body weight/day.

Compound illustrative of the invention are as follows (PYYl- 36 and PYY3-36 being shown for comparison) :

Table 1:

Ac-I-K[IKPEAPGEDA] - PYY 3-12 SPEELNRYYASLRHYLNLVTRQRY-NH2 Ac- I KI PYY13-3G I NH₂

IK- [PIKPEAPGEDA] - I PYY 2-12 SPEELNRYYASLRHYLNLVTRQRY-NH2 I K I PYY 13-36 INH₇

I-K[IKPEAPGEDA] - I PYY 3-12 I SPEELNRYYASLRHYLNLVTRQRY-NH2 I K l PYY 13-3R I NH₂

IK-K [ IKPEAPGEDA] - PYY 3-12 I SPEELNRYYASLRHYLNLVTRQRY-NH2 IK K I PYY 13-36 I NH₂

K-12K [IKPEAPGEDA] - SPEELNRYYASLRHYLNLVTRQRY-NH2

NH₂

IK [SKPDNPGEDA] SPEELNRYYASLRHYLN NPY 3-12 I LVTRQRY-NH2 KL PYY 13-3G J NH₂

IK [LEPVYPGDNA] SPEELNRYYASLRHYLN I PP 3-12 LVTRQRY-NH2 I K I PYY mR D NH₂

IK [APLEPVYPGDNA} SPEELNRYYASLRHY I PP 1-12 LNLVTRQRY-NH2 I K PYY 13-36 J NH₃ I PYY 1-12 1

12K [YPIKPEAPGEDA] - SPEELNRYYASLRHYLNLVTRQRY-NH2 K PYV 13-3G ~INH;

12K[IKPEAPGEDA] - PYY3-12 SPEELNRYYASLRHYLNLVTRQRY-NH2 PYY 13-3R NH,

PYY 1-12

Ac-12K [YPIKPEAPGEDA] - SPEELNRYYASLRHYLNLVTRQRY-NH2

Ac- K l PYY 1ΩE

Ac-12K[IKPEAPGEDA] - PYY 3-12 SPEELNRYYASLRHYLNLVTRQRY-NH2 Ac-K I PYY 13-3K NH₂

Ac-12K [Ac-YPIKPEAPGEDA] - Ac-I PYY 1-12 SPEELNRYYASLRHYLNLVTRQRY-NH2

Ac- K l PYY 13-3G NH,

Ac-12K [Ac-IKPEAPGEDA] - Ac-I PYY 3-12^" SPEELNRYYASLRHYLNLVTRQRY-NH2 Ac-K I PYY 13-36 INH,

IKPEAPGEDAK [YPIKPEAPGEDA] - I PYY 1-12 SPEELNRYYASLRHYLNLVTRQRY-NH2 I PYY 3-12 IK I PYY 13-3G ^NH₂

IKPEAPGEDA-K [ IKPEAPGEDA] - I PYY3-12 SPEELNRYYASLRHYLNLVTRQRY-NH2 I PYY 3-12 I R l PYY 13-3R I MH^

We describe below non-limiting general procedures for peptide synthesis. The scheme is illustrated in Figure 1. Other methods known in the art may of course be used.

Peptide synthesis

By way of example the resins used for the peptide synthesis are Tentagel S RAM, (bead size 90 μm, 0.24 mmoL/g loading) . The Fmoc-protected amino acids are purchased either from Fluka, Chem-Impex or Advanced ChemTech. Other chemicals such as ninhydrin, scavengers (thioanisole, trifluoroacetic acid, NMP and piperidine) and W-Hydroxybenzotrizaole (HOBt) are purchased from Spectrochem. N, Λ/'-diisopropylcarbodiimide, triisopropylsilane and 1, 2-ethanedithiol are purchased from Aldrich. Anhydrous solvents are used for reactions. All other reagents are obtained from local commercial sources and are used without further purification. The reactions on solid phase are carried out manually using a polyethylene vessel equipped with a polypropylene filter for filtration. The synthesis is also carried out automatically, for example using an automated peptide synthesizer, Symphony parallel synthesizer (PTI) using the synthesis programs constructed by the users themselves.

Removal of Fmoc group of Tenta gel resin:

The Fmoc group of the Tenta gel resin is deprotected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min. The resin is then filtered and washed with DMF. The deprotection of Fmoc group is determined by positive ninhydrin test.

Coupling of Fmoc-amino acid on Tenta gel resin

The coupling of amino acid to Fmoc-deprotected Tentagel resin is carried out by using the standard protocols DIC/HOBt or

HBTU as the coupling reagents or Fmoc AA/HBTU/NMM as coupling reagents with 2 h coupling time. The resin is filtered and washed with DMF, DCM and DMF. The completion of amide bond formation is confirmed by negative ninhydrin test. After the first amino acid attachment, the unreacted amino group in the resin is capped used acetic anhydride/pyridine/DCM (1:8:8) for 20 minutes to avoid any deletion of the sequence. After capping, resin is washed with DCM and DMF. The deprotection and coupling steps are repeated until the desired sequence of peptide is synthesized.

Final cleavage of peptide from the resin The cleavage of the peptides from the solid support is achieved by treating the peptide-resin with reagent K (82.5% TFA / 5% phenol / 5% thioanisole / 2.5% 1, 2-ethanedithiol / 5% water) at room temperature for 2.5 h. Cleavage mixture is collected by filtration and the resin is washed with TFA and DCM. The excess TFA and DCM is concentrated to small volume under nitrogen and a small amount of DCM is added to the residue and evaporated under nitrogen. The process is repeated 3-4 times to remove most of the volatile impurities. To the cooled residue chilled anhydrous ether is added to precipitate the peptide. The precipitated peptide is centrifuged and the supernatant ether is removed and fresh ether is added to the peptide and recentrifuged. The process is repeated several times to remove all the organics (washings continued till the traces of EDT are removed) . Finally the residue dissolved in millipore water and lyophilized to obtain the crude peptide.

Protocol using Symphony Parallel Synthesizer

Synthesis of branched peptides using Lysine

Fmoc-Lys- (Alloc) -OH or Fmoc-Lys- (Dde) -OH is used for branching the peptides from the ε-amino group of the lysine after the completion of the linear sequences.

Removal of Dde group of the ε-amino group of the lysine

The Dde protection of the ε-amino group of the lysine is orthogonally deprotected by treating the resin with 2.5% (v/v) Hydrazine hydrate solution in DMF for 5 and 15 minutes. The resin is then filtered and washed with DMF, DCM, and DMF. Deprotection is confirmed by positive ninhydrin test.

Removal of Alloc group of the ε-amino group of the lysine

Peptidyl resin is swelled in DCM for 1 h under argon atmosphere. To the swelled resin 20 equivalent excess of phenyl silane is added followed 10 equivalent excess of Tetrakis (triphenylphosphine) palladium (0) in DCM. Argon is continued to purge through the resin for 10 minutes at room temperature. The solution is filtered and washed with DCM. The procedure is repeated once again. Deprotection is confirmed by positive ninhydrin test.

Synthesis of branched peptides using Diaminopropionic acid

Fmoc-Dap (ivDde) -OH is used for branching the helix peptide from the β-amino group of the Dap after the completion of the linear sequences.

Removal of Dde group of the β-amino group of the Dap The Dde protection of the β-amino group of the Dap is orthogonally deprotected by treating the resin with 2.5% (v/v) Hydrazine hydrate solution in DMF for 5 and 15 minutes. The resin is then filtered and washed with DMF, DCM, and DMF. Deprotection is confirmed by positive ninhydrin test.

Synthesis of branched peptides using glutamic acid/ aspartic acid

Fmoc-Glu (ODmab)-OH or Fmoc-Glu (OAIl)-OH is used for the synthesis for branching the helix peptide initiating from glutamic acid and Fmoc-Asp (ODmab) -OH or Fmoc-Asp (OAIl) -OH for branching the helix peptide (sequence ID 4, 5, 6, 7, 8 or 9) initiating from aspartic acid.

Removal of Dmab protection group of δ-carboxy group of glutamic acid and γ-carboxy group of aspartic acid

The GIu (ODmab) protected resin is swelled in DMF. Dmab group is deprotected by the addition of 5 % hydrazine hydrate in DMF to the pre-swelled resin for 10 minute with occasional stirring. The process is repeated thrice. After 30 minute the resin is washed with DMF, DCM and DMF.

Removal of Allyl protection group of δ-carboxy group of glutamic acid and γ-carboxy group of aspartic acid

Peptidyl resin is swelled in chloroform for 1 h under argon atmosphere. To the swelled resin 20 equivalent excess of phenyl silane is added followed 10 equivalent excess of Tetrakis (triphenylphosphine) palladium (0) in 10 % NMP in chloroform. Argon is continued to purge through the resin for 7 hours at room temperature. The solution is filtered and washed with 0.5 % DIPEA in DMF and DCM.

Purification and Characterization of Peptides

Analytical HPLC Instrument: Agilent 1100 series liquid chromatograph

Column: Zorbax Eclipse XDB-Ci₈ (4.6mm X 150, 5μm) with 80 A° pore size

Temp: 25 ⁰C

Solvent A: 0.1% TFA/Water, Solvent B: 0.1%TFA/ CH₃CN Flow rate; lml/min

Wavelength monitored: 220nm (Diode array detector)

Gradient: start 100 % A, 0-1.5 minute 100 % A, 1.5-25 minute

0-50 % B

Crude peptide is injected by dissolving in the same buffer (1:1) used for the mobile phase.

Preparative HPLC (Agilent 1100 series)

Instrument: Agilent 1200 series liquid chromatograph

Column: Zorbax-Eclipse XDB-Ci₈ (21.2 mm X 250mm, 7μm)

Temp: 25 ⁰C Solvent A: 0.1% TFA/Water, Solvent B: 0.1%TFA/ CH₃CN

Flow rate 20 ml/min

Wavelength monitored: 220nm (Diode array detector)

Gradient: start 95 % A, 2-5 minute 5-25 % B, 5-50 minute 25-

60 % B Mass spectrometry

The peptide solutions are analyzed in positive polarity mode by Agilent Technologies LC/MSD VL.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows the general scheme of peptide synthesis used for synthesizing the branched peptides as set forth in the present application

Figure 2 is an outline of the experimental procedure used in the 14-day chronic subcutaneous PYY-analogue infusion study as set forth in Example 3

Figure 3 demonstrates the body-weight reduction (expressed as percent of starting body-weight (=day 0) caused by infusion of PYY3-36 and with an exemplary compound of the invention in the DIO-mouse study as set forth in Example 3

Figure 4 shows the reductions in Triglycerides (TG) , non- esterified free fatty acids (FFA) and LDL-cholesterol (LDL- Chol) following 14 days of treatment with PYY3-36 and with an exemplary compound of the invention in the DIO-mouse study as set forth in Example 3

Figure 5 shows the reduction in leptin and increase in adiponectin following 14 days of treatment with PYY3-36 and with an exemplary compound of the invention in the DIO-mouse study as set forth in Example 3

Examples

The present invention is described in more detail with reference to the following non-limiting examples, which are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof. The examples illustrate the preparation of the polypeptides and the testing of the polypeptides of the invention in vitro and/or in vivo.

Example 1

Peptide synthesis of IK- [PIKPEAPGEDA] -SPEELNRYYASLRHYLNLVTRQRY-NH2

The resins used for the peptide synthesis was Tentagel S RAM, Fluka, GA 10462; Sigma-Aldrich, Germany, bead size 90 μm, 0.24 mmoL/g loading) . The Fmoc-protected amino acids were purchased either from Fluka, Chem-Impex or Advanced ChemTech. Other chemicals such as ninhydrin, scavengers (thioanisole, trifluoroacetic acid, NMP and piperidine) and N- Hydroxybenzotrizaole (HOBt) were purchased from spectrochem. N, N'-diisopropylcarbodiimide, triisopropylsilane and 1,2 ethanedithiol were purchased from Aldrich. Anhydrous solvents were used for reactions. All other reagents were obtained from local commercial sources and were used without further purification.

Dry Tentagel S RAM resin (0.24 mmol/g, Ig) was placed in a polyethylene vessel equipped with a polypropylene filter.

Resin was swelled in DCM (10 m L) for 1 hr and DMF (10 m L) for 2 h. The Fmoc protecting group of the resin was removed by treatment with 20% piperidine in DMF for 5 and 15 min (10 mL) . The resin was washed with DMF (6 X 10 m L), DCM (6 X 10 m L) and DMF (6 X 10 m L) . Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive. The C-terminal amino acid, Fmoc-Tyr (tBu) -OH (6 equiv. 1.44 mmol) in DMF were added to the deprotected resin and coupling was initiated with DIC (6 Equiv.) and HOBT (6 equiv.) in DMF. The concentration of each reactant in the reaction mixture was approx. 0.4 M. The mixture was rotated on a rotator at room temperature for 2 h. Resin was filtered and washed with DMF (6 X 10 m L), DCM (6 X 10 m L) and DMF (6 X 10 m L) . Kaiser test on peptide resin aliquot upon the completion of the coupling was negative. After N^α-Fmoc-removal each coupling in the linear peptide sequence were initiated by the addition of N^α-Fmoc amino acid (6 equiv.), HOBt (6 equiv.) and DIC (6 equiv.) in DMF for 2 h at room temperature. The concentration of each reactant in the reaction mixture was approx. 0.4 M.

The guanidine group of Arginine was protected by Pbf, ε- amino group of lysine was protected by Dde group. The β and γ- carboxamide function of asparagine and glutamine and imidazole nitrogen of histidine were protected as trityl derivatives. β and γ-carboxyl function of aspartic acid and glutamic acid were protected as tert-butyl esters. The hydroxyl side chain of serine, threonine and phenolic hydroxyl group of tyrosine were protected as tert-butyl ethers.

The N-terminal amino acid isoleucine in the linear chain of the peptide was coupled as N^α-Boc amino acid in the same procedure as employed for N^α-Fmoc amino acid. The ε- amino group of lysine used as branching point amino acid was protected with Dde group. After the completion of the linear peptide chain Dde protection of the ε-amino group of the lysine was orthogonally deprotected by treating the resin with 2.5% (v/v) Hydrazine hydrate solution in DMF for 5 and 15 minutes. The resin was then filtered and washed with DMF (6 X 10 m L), DCM (6 X 10 m L) and DMF (6 X 10 m L) .

Deprotection of the Dde group was confirmed by positive ninhydrin test. The coupling of amino acid in the branch was carried out by the addition of N^α-Fmoc amino acid (6 equiv.), HOBt (6 equiv.) and DIC (6 equiv.) in DMF at room temperature for 2 h. The concentration of each reactant in the reaction mixture was approx. 0.4 M. Kaiser tests on peptide resin aliquots upon the completion of the couplings were negative.

The cleavage of the peptides from the solid support was achieved by treating the target peptidyl-resin with 15 mL reagent K (82.5% TFA / 5% phenol / 5% thioanisole / 2.5% 1,2 ethanedithiol / 5% water) at room temperature for 2.5 h. Cleavage mixture was collected by filtration and the resin was washed with TFA and DCM. The excess TFA and DCM was concentrated to small volume under nitrogen and a small amount of DCM was added to the residue and evaporated under nitrogen. The process was repeated 3-4 times to remove most of the volatile impurities. To the cooled residue chilled anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged and the supernatant ether removed and fresh ether was added to the peptide and recentrifuged. The process was repeated several times to remove all the organics (washings continued till the traces of EDT removed) . Finally the residue was dissolved in millipore water and lyophilized to obtain the crude peptide. The crude material was purified by preparative HPLC on Zorbax Eclipse XDB-C18 column (21.2 X 250 mm, 7μm particle size) and eluted with a linear gradient of 5- 40 % B (buffer A: 0.1 % TFA/H₂O; buffer B: 0.1%TFA/90% CH3CN/IO % H₂O) . The purified peptide was found to be homogeneous and purity was found to better than 95 %. The identity of the peptide was confirmed by LCMS (calculated 4388.01; observed m/3 = 1463.7)

Example 2

Yl, Y2 and Y4 BINDING ASSAYS

Yl Binding assay:

Materials: SKNMC cells were obtained from ATCC and propagated in minimum essential medium (MEM) with 1% penicillin- streptomycin (Sigma, Cat. No. P0781, Lot.35K2380) and 10% fetal bovine serum (Sigma, Cat. No. F7524 (Non-US)), Cell culture flasks, phosphate buffer saline (PBS), Homogenization buffer (5OmM Tris pH-7.5, 2.5mM MgCl₂, 1.OmM CaCl₂, Protease inhibitors (lμg/ ml Leupeptin, lμg /ml Pepstatin, 1 mM PMSF)), Binding buffer (5OmM Tris pH-7.5, 2.5mM MgCl₂, 1.OmM CaCl₂, 0.2% BSA, protease inhibitor cocktail), 0.6M Sucrose, ¹²⁵I-PYY, Cold ^Leu3iPro34 _{Npγ ^} pγγ3-36, other novel peptides, Optiphase supermix (Perkin elmer Cat. No. 1200-439), Scintillation counter.

Experimental procedure:

SKNMC cells were grown in a 75cm² flask to confluence using MEM with 10% FBS and 1% penicillin-streptomycin. The cells were washed with PBS and the cells were scraped into a 50 ml falcon tube. The suspension was centrifuged at 1,000 x g for 10 min at 4°C and the pellet was homogenized in a homogenizer for 15 to 30 seconds in 2 ml of ice cold homogenization buffer. An equal volume of 0.6M sucrose was added and the homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The pellet was washed with the binding buffer and re- centrifuged at 10,000 x g for 10 min at 4°C. The resulting pellet (membrane) was re-suspended in 20% glycerol made using the binding buffer, the protein concentration was estimated, aliquoted and frozen. For the binding assay, 40μg of the membrane protein was incubated with ¹²⁵I PYY (4OpM) in the presence or absence of test compound (eg. ^Leu3iPr _O ³⁴ _Npγ^ pγ_Y3_ 36) for lhr at room temperature (RT) . The reaction was stopped simply by centrifuging the samples at 10,000 rpm for 15 min. The pellet was washed with ice cold binding buffer two times and the counts were measured in a scintillation counter. The concentration of the unlabeled peptide required to compete for 50% of specific binding to the radioligand (IC50 value) was calculated using GraphPad Prism software. Example data are shown in Table 2 below.

Y2 BINDING ASSAY

Materials: Human embryonic kidney (HEK-293) cells stably transfected with Y2 receptor cDNA, Dulbecco ' s Modified Eagle's Medium (DMEM) (Sigma Cat. No. D5648-10L, Lot.084K8300) with 1% penicillin-streptomycin (Sigma, Cat. No. P0781, Lot.35K2380) and 10% fetal bovine serum (Sigma, Cat. No. F7524 (Non-US)), Cell culture flasks, phosphate buffer saline (PBS) , Homogenization buffer (2OmM Tris-HCL, 5mM EDTA) and Binding buffer (10% sucrose, 0.02M HEPES-NaOH (pH7.4), ImM MgCl₂, 2.5mM CaCl₂, 2mg/ml BSA, protease inhibitor cocktail), ¹²⁵I PYY, PYY3-36, scintillation fluid, scintillation counter.

Experimental procedure:

Human embryonic kidney (HEK-293) cells stably transfected with Y2 receptor cDNA were grown in a 75cm² flask to confluence using Dulbecco ' s Modified Eagle's Medium (DMEM) with 10% FBS and 1% penicillin-streptomycin. The cells were washed with PBS and the cells were scraped into a 50 ml falcon tube. The suspension was centrifuged at 1,000 x g for 10 min at 4°C and the pellet was homogenized in a homogenizer for 15 to 30 seconds in 2 ml of ice cold homogenization buffer. An equal volume of 0.6M sucrose was added and the homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The pellet was washed with the binding buffer and re- centrifuged at 10,000 x g for 10 min at 4°C. The resulting pellet (membrane) was re-suspended in 20% glycerol made using the binding buffer, the protein concentration was estimated, the aliquoted and frozen. For the binding assay, 40μg of the membrane proteins were incubated with ¹²⁵I PYY (4OpM) in the presence or absence of test compound (eg. PYY3-36) for lhr at room temperature (RT) . The reaction was stopped simply by centrifuging the samples at 10,000 rpm for 15 min. The pellet was washed with ice cold binding buffer two times and the counts were measured in a scintillation counter. The concentration of the unlabeled peptide required to compete for 50% of specific binding to the radioligand (IC₅O value) was calculated using GraphPad Prism software. Example data are shown in Table 2 below. Y4 BINDING ASSAY

Materials: Human embryonic kidney (HEK-293) cells stably transfected with Y4 receptor cDNA, Dulbecco ' s Modified Eagle's Medium (DMEM) (Sigma Cat. No. D5648-10L, Lot .084K8300) with 1% penicillin-streptomycin (Sigma, Cat. No. P0781, Lot.35K2380) and 10% fetal bovine serum (Sigma, Cat. No. F7524 (Non-US)), Cell culture flasks, phosphate buffer saline (PBS) , Homogenization buffer (2OmM Tris-HCL, 5mM EDTA) and Binding buffer (10% sucrose, 0.02M HEPES-NaOH (pH7.4), ImM MgCl₂, 2.5mM CaCl₂, 2mg/ml BSA, protease inhibitor cocktail), ¹²⁵I PP, PP, scintillation fluid, scintillation counter.

Experimental procedure:

Human embryonic kidney (HEK-293) cells stably transfected with Y4 receptor cDNA were grown in a 75cm² flask to confluence using Dulbecco ' s Modified Eagle's Medium (DMEM) with 10% FBS and 1% penicillin-streptomycin. The cells were washed with PBS and the cells were scraped into a 50 ml falcon tube. The suspension was centrifuged at 1,000 x g for 10 min at 4°C and the pellet was homogenized in a homogenizer for 15 to 30 seconds in 2 ml of ice cold homogenization buffer. An equal volume of 0.6M sucrose was added and the homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The pellet was washed with the binding buffer and re- centrifuged at 10,000 x g for 10 min at 4°C. The resulting pellet (membrane) was re-suspended in 20% glycerol made using the binding buffer, the protein concentration was estimated, the aliquoted and frozen. For the binding assay, 40μg of the membrane proteins were incubated with ¹²⁵I PP (4OpM) in the presence or absence of test compound (eg. PP) for lhr at room temperature (RT) . The reaction was stopped simply by centrifuging the samples at 10,000 rpm for 15 min. The pellet was washed with ice cold binding buffer two times and the counts were measured in a scintillation counter. The concentration of the unlabeled peptide required to compete for 50% of specific binding to the radioligand (IC₅O value) was calculated using GraphPad Prism software. Example data are shown in Table 2 below.

Table 2: Compound numbering is as in Table 1

Example 3

Y2 functional assay

Principle of test

The intracellular loops of NPY receptors are associated with the G proteins containing three subunits CC, β and γ. In an unstimulated state, the CC subunit of the receptor binds to GDP. Agonist binding causes the G protein to associate with the agonist bound receptor and exchange GDP for GTP. The GTP bound CC subunit remains active until GTP is hydrolyzed to GDP. It is possible to assess the activation of G protein linked to receptors by evaluating the exchange of GDP for GTP using stable radio labeled GTP analogue (³⁵SGTPγS) . Agonist activation exchanges the ³⁵SGTPyS instead of endogenous GTP and the receptor activation could be quantitated by measuring the radioactivity bound to the G protein.

Materials: Human embryonic kidney (HEK-293) FT cells, Y2 cDNA expression plasmid, Dulbecco ' s Modified Eagle's Medium (DMEM) (Sigma Cat. No. D5648-10L, Lot .084K8300) with 1% penicillin- streptomycin (Sigma, Cat. No. P0781, Lot .35K2380) and 10% FBS (Sigma, Cat. No. F7524 (Non-US)), Cell culture flasks, phosphate buffer saline (PBS), Homogenization buffer (2OmM Tris-HCL, 5mM EDTA) and assay buffer (2OmM Hepes, 100 mM

Nacl, 5 mM MgC12, pH 7.4/ 50 mM Tris HCl, 100 mM Nacl, 5 mM MgC12, pH 7.4), scintillation fluid, scintillation counter, LIPOFECTAMINE (Invitrogen) .

Experimental procedure:

The HEK293-FT cells were grown in a 75cm² flasks to confluence using DMEM with 10% FBS and 1% penicillin- streptomycin. The cells were split into four 75cm² flasks to have -60% confluency. The cells were washed with PBS and each plate was transfected with 2μg of Y2 cDNA expression plasmid using LIPOFECTAMINE transfection reagent. The cells were recovered for 36 hours and then scraped from all four flasks into 50 ml falcon tubes. The suspension was centrifuged and the pellet homogenized in a homogenizer for 15 to 30 seconds in 2 ml of homogenization buffer. The homogenate was centrifuged at ~2000 rpm for 5 mm. The supernatant was collected and re-centrifuged at 25,00Og for 25min and the resulting pellet (membrane) was resuspended in the assay buffer (pH7.4) . The protein concentration was estimated, and the membranes aliquoted and frozen at -80°C.

For the assay, the membranes (10μg) were thawed on ice and the assay mixtures (500 μl) in the assay buffer were preincubated with compounds for 30 min at room temperature. Then, 50 pM of [³⁵S] GTPyS was added. The assay mixtures were further incubated for 1 h at room temperature. Reactions were terminated by rapid filtration thought GF/C filters. Filters were washed twice with ice-cold 50 mM Tris-HCl, pH 7.4, containing 10 mM MgCl₂. Basal [³⁵S] GTPyS was measured in the absence of compounds. Stimulation of [^3oS]GTPγS is presented as percentage over basal. A control reaction in the presence of unlabelled GTPγS (100 μM) was performed to determine the non-specific binding (total binding - binding in the presence of unlabelled GTPγS = specific [³⁵S]GTPyS binding) . Agonist concentration-response curves for increases in [^3DS]GTPVS binding were analyzed by nonlinear regression using GraphPad Prism software (GraphPad Software Inc.) . The results are shown in Figure 3.

Example 4 The effect of Y2 receptor selective polypeptides on body- weight, lipids, leptin and adiponectin

Animals

Seventy male C57/bl6J mice (Aurigene, Bangalore, India) aged 6-8 weeks at the time of High-energy diet feeding were used. The mice were housed 5-6 per cage for 16-20 weeks. Mice were fed a high energy diet (HF diet 60% fat (5,24 kcal/g - Energy %: Carbohydrate 20,1 kcal %, Fat 59,9 kcal %, Protein 20 kcal %; Diet # D12492; Research Diets, New Jersey, USA) . Tap water was also provided ad libitum. Mice were kept under a

12:12 hour LD cycle with lights on at 07:00 AM, lights out at 19:00 PM. At least two weeks prior to pump implantation the animals were transferred to individual cages. From one week before experiment start (day -7) and until day 28, body- weight (BW) and food intake (FI) was recorded daily at 9.00 AM.

Compounds :

All test-compounds were dissolved in vehicle (0.9% NaCl, = physiological saline, pH=7.4) . Compounds were administered via Alzet osmotic minipumps (model 2002; 200μl; 0.5μl/h, 14 days of delivery) . The final concentration was calculated according to the following formulas (the calculation was based on the average body-weight "group average BW" of each group)

E . g : Group X : 0 . lμmole/kg/day Compound :

Concentration (μg/ml ) = ( 0 . lμmole/kg/day * (group average BW) kg * 14 days ) / 0 . 2ml Pumps were filled on day -1 and "primed" overnight according to the manufacturer' s recommendation (pump filled and kept in 0.9% normal saline at 37 °C overnight, approximately 19 hours) .

Experimen t :

The experimental schedule followed is illustrated schematically in Figure 2. On Experimental day -1 mice were weighed and randomized according to body-weight into the 6 treatment groups (n=8) . On day 0 animals were anaesthetized using gas anaesthesia (Halothane) and Alzet osmotic pumps prefilled with vehicle or compounds were implanted subcutaneously in the lower back and surgical wound closed with surgical staples. Povidone Iodine solution was applied topically on surgical site. Following the operation, mice were allowed to recover, and then transferred back to their cages. Pentazocin (5 mg/kg SQ) was administered once as analgesic .

On day 14 of the experiment, at 9 AM blood was sampled from all mice for the following analyses: plasma Triglycerides, Non Esterified fatty acids (NEFA) , LDL cholesterol, leptin and adiponectin. After blood sampling pumps were removed under halothane anesthesia, and the wound was closed with surgical staples. Povidone Iodine solution was applied topically on surgical site. Following the operation, mice were allowed to recover, and then transferred back to their cages. Pentazocin (5 mg/kg SQ) was administered once as analgesic . Triglycerides (Labkit, Chemelex S. A, Barcelona, Spain), Non Esterified fatty acids (NEFA, Randox Laboratories, Antrim, United Kingdom), and LDL cholesterol (Direct LDL cholesterol, Randox Laboratories, Antrim, United Kingdom) , were measured according to the manufacturer' s instructions using Vitalab Selectra E biochemistry analyser (Vital Scientific B. V., Netherlands) . Leptin and adiponectin levels were measured using Linco Research, Missouri, USA ELISA kits according to manufacturer's instructions. Fluorescence readout was measured using SpectraMax Gemini Spectrofluorometer (Molecular Devices Corporation, USA)

As is seen in Figure 3, administration of PYY3-36 over a 14 day period produces a reduction in body weight of treated mice and administration of compound 4 (Table 1) provides a similar weight loss. Upon cessation of treatment, the mice regain part of the lost weight. It can be seen that the mice receiving 0.25μmol/kg/day dose of the compound regained weight significantly more slowly than those receiving the same dose of PYY3-36. Those receiving a 0. lμmol/kg/day dose of the compound regained weight sufficiently slowly that at day 33 they weighed the same as the mice that received the higher dosage (0.25μmol/kg/day) of PYY1-36. As can be seen from Figure 4 mice treated with Compound 4 (0.25μmole/kg/day) showed greater reductions in Triglycerides, FFA and LDL- cholesterol at day 14 than mice treated with a similar (0.25μmole/kg/day) dose of PYY3-36. In Figure 5 it can be seen that Compound 4 (0.25μmole/kg/day) increased the circulating total adiponectin levels to a greater extent than did PYY3-36. Low circulating levels of Adiponectin have been linked to insulin resistance, upper body fat distribution and dyslipidemia (Hulthe et al, 2003, Metabolism 52:1612-1614) . Furthermore Adiponectin levels have been positively associated with insulin sensitization, glucose use and cardiovascular protection (Scherer, 2006, Diabetes 55:1537- 1545) . Increasing circulating Adiponectin levels is therefore believed to be one way by which the metabolic and cardiovascular complications associated with obesity can be reduced.

In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of . All prior teachings acknowledged above are hereby incorporated by reference in their entirety. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

Claims

1. A polypeptide agonist of the Y2 receptor selective for the Y2 receptor over the Yl and Y4 receptors, which is a functional mimic of the 3-36 truncated variant of the polypeptide PYY1-36 and has the general formula 1

Formula 1

in which each Aaa is an amino acid chain and each of n, m, o and s is an integer representing the number of amino acids in a respective said chain,

said polypeptide of formula 1 being a variant of the polypeptide of SEQ ID NO: 1 (i.e. human PYY1-36)

YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY SEQ ID NO: 1

in which variant said SEQ ID NO:1 is modified to include a branch point (BP) at an amino acid position x between position 1 and position 14 of said sequence (the N-terminal Y being position 1 and the C-terminal Y being position 36) , said branch point being provided by a replacement or additional natural or artificial amino acid residue BP having a first amino group, a second amino acid group, which is not an α amino group, and a carboxylic acid group, the said first amino group being free (m = 0) or having bonded thereto an amino acid sequence (Aaa_m) of from 1 to 15 amino acids, said second amino group being bonded to an amino acid sequence (Aaa_n) containing a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence, and said carboxylic acid group being bonded to an amino acid sequence (Aaa₀) - (Aaa_s) , (Aaa₀) being an alpha helical amino acid sequence of from 12-19 amino acids and (Aaa_s) being the sequence -T-R-X' aa-R-Y;

or a function retaining variant of such a polypeptide in which in said partial PYY amino acid sequence in sequence Aaa_n one or more amino acids are deleted or substituted subject to there being maintained therein a motif of the form P-Xaa-Xaa-P,

wherein each residue Xaa or X' aa is any amino acid and each amino acid Xaa or X' aa is independently a natural or unnatural amino acid bonded to form an amide linkage via an α or other amino group, and

wherein, and R¹ and R³ each independently are H, or an acylating group, and R² is H, or an amidating group.

A polypeptide as claimed in claim 1, wherein the amino acid sequence Aaa_n contains a variant of the PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence, which is a function retaining variant in which in said partial PYY amino acid sequence in sequence Aaa_n one or more amino acids are deleted or substituted subject to there being maintained therein a motif of the form P-Xaa-Xaa-P- Xaa-Xaa-P, or P-Xaa-Xaa- P-Xaa-Xaa-Xaa-P.

3. A polypeptide as claimed in claim 2, wherein the amino acid sequence Aaa_n contains a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 50% of non-proline amino acid residues therein and of up to one proline residue therein .

4. A polypeptide as claimed in claim 3, wherein the amino acid sequence Aaa_n contains a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 3 non-proline amino acids therein.

5. A polypeptide as claimed in claim 3, wherein the amino acid sequence Aaa_n contains a PYY partial sequence of from 6 to 9 amino acids of the PYY 1-9 sequence with substitutions of up to 3 non-proline amino acids therein .

6. A polypeptide as claimed in claim 2, wherein the amino acid sequence Aaa_n contains a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence.

7. A polypeptide as claimed in claim 1, of the general formula 2 :

Formula 2 wherein each of Aaa_p is an amino acid sequence of from 4-8 amino acids containing the motif -Pro-Xaa-Xaa-Pro-, and wherein (Aaa_n' ) is amino acid sequence consisting of a sequence of from 6 to 14 amino acids of the PYY 1- 14 sequence with substitutions of up to 50% of non- proline amino acids therein, Aaa_q is an amino acid sequence of from 1-8 amino acids, Aaa_s is as defined in claim 1, and (Aaa_m' ) is absent (m = 0) or is an amino acid sequence consisting of a sequence of from 1 to 14 amino acids of the PYY 1-14 sequence with substitutions of up to 50% of non-proline amino acid residues therein .

8. A polypeptide as claimed in claim 7, wherein the amino acid sequence Aaa_n' consists of a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence, and the amino acid sequence Aaa_m' is absent or consists of a PYY partial sequence of from 6 to 14 amino acids of the PYY 1-14 sequence.

9. A polypeptide as claimed in claim 7 or claim 8, wherein said amino acid sequence Aaa_p is PRRP or GPRRP.

10. A polypeptide as claimed in any one of claims 7 to 9, wherein said amino acid sequence Aaa_q is PRRP or GPRRP.

11. A polypeptide as claimed in any one of claims 7 to 10, wherein said amino acid sequence Aaa_q is I (K) _r , where r is from 0 to 3.

12. A polypeptide as claimed in any preceding claim, wherein said amino acid sequence Aaa_m is I (K) _r , where r is from 0 to 3.

13. A polypeptide as claimed in any preceding claim, wherein said amino acid sequence Aaa₀ matches in sequence an alpha helical portion of the peptide PYY, the peptide NPY or the peptide PP.

14. A polypeptide as claimed in any preceding claim, wherein said branch point amino acid is an α amino acid.

15. A polypeptide as claimed in any preceding claim, wherein said branch point is lysine, or diaminopropionic acid.

16. A polypeptide as claimed in any preceding claim, wherein in the amino acid sequence Aaa_n, said PYY 1-14 partial sequence with optional amino acid substitutions forms an N-terminal end of the molecule.

17. A polypeptide as claimed in any claim 16, wherein said amino acid sequence Aaa_n comprises at its N-terminal end or consists of the sequence PYY 1-12, PYY 1-9, PYY 2-12, PYY 2-9, PYY 3-12, or PYY 3-9.

18. A polypeptide as claimed in claim 16, wherein said amino acid sequence Aaa_n comprises at its N-terminal end or consists of the sequence NPY 1-12, NPY 1-9, NPY 2-12, NPY 2-9, NPY 3-12, or NPY 3-9.

19. A polypeptide as claimed in any preceding claim, wherein each amino acid Xaa is a natural amino acid forming a peptide bond via an α amino group.

20. A polypeptide as claimed in any preceding claim, wherein the amino acid sequence Aaa_s is TRQRY or TRPRY.

21. A polypeptide as claimed in any preceding claim, wherein said branch point is immediately in the N- terminal direction from the residue S of SEQ ID NO: 1.

22. A polypeptide having a sequence as given in any entry in Table 1.

23. A polypeptide as claimed in any preceding claim, formulated for pharmaceutical or veterinary administration .

24. A pharmaceutical or veterinary composition, comprising a polypeptide as claimed in any one of claims 1-23 and a pharmaceutically or veterinarily acceptable carrier.

25. A polypeptide as claimed in any one of claims 1-23, for use as a medicine.