WO2014178018A1 - Therapeutic peptides - Google Patents

Abstract

The present Invention relates to novel analogs of PYY that have an improved therapeutic profile when compared to native human PYY. These novel PYY analogs useful in the treatment of obesity, diabetes, and other disorders.

Description

THERAPEUTIC PEPTIDES

FIELD OF THE INVENTION

This invention relates to therapeutic peptides useful in the treatment of obesity and metabolic disorders. More specifically, the invention relates to novel analogs of Peptide YY (PYY) and their use.

BACKGROUND OF THE INVENTION

The prevalence of obesity in the United States is increasing, with 35.7% of adults considered obese (Blvll > 30) and 68.8% considered overweight (BMI≥ 25) in 2009-2010. See, for example, Flegal et al. (2012) JAMA 307(5):491-7. Worldwide, over 300 million people are considered obese. Obesity-related diseases, including Type 2 Diabetes Mellitus, hypertension, heart disease, joint disease, and some types of cancer have increased in prevalence as the population has grown heavier.

Prevention of obesity through diet and exercise is of critical importance to control these trends, but once patients become obese, the body's resistance to weight loss can be considerable. Diet and exercise alone may be insufficient to bring about significant weight change in severely obese patients, and both pharmacologic therapy and surgery have proven to be effective as additional aids to weight loss. Prevention and treatment of obesity are areas of high unmet medical need, with few medications currently available for chronic weight loss therapy.

Peptide YY (PYY) belongs to the PP-fold family of peptides together with pancreatic polypeptide and neuropeptide Y, which have a role in controlling appetite. See, for example, Schwartz et al. (2002) Nature :418(6698): 595-7. PYY is secreted as a 36 amino acid, straight chain polypeptide and then cleaved by dipeptidyl peptidase IV to produce PYY(3-36). Fasting and post-prandial concentrations of PYY in morbidly obese individuals after gastric bypass surgery are suggested as playing a role in their dramatic weight loss. See, for example, le Roux (2006) Ann Si/rg.243(1):108-14. Peripheral infusion of PYY(3-36) has been shown to increase energy expenditure and fat oxidation rates in obese and lean subjects. See, for example, Batterham et al. (2003) N Engl J Med. 349(10):941-8, and Sloth ef al. (2007) Am J Physiol Endocrinol Metab.: 293(2):E604-9. Administration of a PYY(3- 36) nasal spray reduced daily caloric intake of obese individuals by 2713 kJ, resulting in a weight loss of 0.6 kg over a six-day stud period. See, for example, Gantz ef al. (2007) J Clin Emiocrinot Metab. 92(5): 1 54-7. These results demonstrate that obese subjects retain sensitivity to PYY(3-36), in contrast to lepiin, where resistance limits its therapeutic usefulness in obesity. Accordingly, there remains a need in the art for improved PYY compositions for use in the treatment of obesity and obesity-related disorders. BRIEF SUMMARY OF INVENTION

The present invention relates to novel analogs of PYY that have an improved therapeutic profile when compared to native human PYY. These novei PYY analogs are useful in the treatment of obesity, diabetes, and other disorders.

Briefly, in one aspect, the invention provides a polypeptide comprising the amino acid sequence:

ProLysPiOGIuXaaiProGlyXaajAspAiaSerXaajGiuGiuXaa^XaasXaasTyrTyrAia

XaarLeuArgXaaaTyrXaas AsnTrpXaa_T0ThrArgGlnArgTyr (SEG ID NO:1)

or a salt thereof, wherein:

Xaa j is Ala, His, or Ser;

Xaa₂ is Glu or Lys;

Xaa₃ is Pro or Ala;

Xaa,_t is Leu or Trp;

Xaa_s is Asn, Ala, or Thr;

Xaa₆ is Arg or Lys;

Xaa₇ is Ser, Asp, or Ala;

aas is Leu or He; and

In another aspect, the invention provides a polypeptide selected from

NG:45);

and salts thereof.

In another aspect, the invention provides a nucleic acid molecule encoding a polypeptide of the invention In yet another aspect, the invention includes an expression vector comprising a nucleic acid molecule encoding a polypeptide of the invention.

In a further aspect, the invention encompasses a host cell containing an expression vector comprising a nucleic acid molecule encoding a poiypeptide of the invention. in another aspect, the invention provides a pharmaceutical combination comprising a novel PYY polypeptide of the invention and exendin-4.

in yet another aspect, the invention provides a pharmaceutical combination comprising a novel PYY polypeptide of the invention and GLP-1.

In an additional aspect the invention provides a pharmaceutical composition comprising a novel PYY polypeptide of the invention and one or more

pharmaceutically acceptable excipients.

in a further aspect, the invention encompasses a method of treating a metabolic disorder or obesity, the method comprising administering a novel PYY polypeptide or pharmaceutical combination of the invention to a subject in need thereof.

The invention also provides the use of a polypeptide or pharmaceutical combination of the invention in the preparation of a medicament for use in the treatment of obesity.

In addition, the invention provides a polypeptide or pharmaceutical combination of the invention for use in the treatment of a metabolic disorder or obesity.

BRE!F DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effects of the peptide shown in Example 5 (Analog # 5), PYY{3- 36}NH₂ (PYY3-36), and exendin-4 singly and in combination on changes in body weight in diet-induced obese (D!O) Long Evans (LE) rats.

Figure 2 shows the effects of the peptide shown in Example 5 (Analog # 5), PYY(3- 36)NH₂ (PYY3-36), and exendin-4 singly and in combination on body composition changes in DIO LE rats.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel analogs of PYY that have an improved therapeutic profile when compared to native human PYY. The novel PYY analogs of the invention show improved effects on food intake when compared with the native PYY sequence.

In one aspect, the novel PYY analogs comprise the amino acid sequence: ProLysProGluXaaiProGlyXaasAspAlaSerXaasGluGiuXaa^XaasXaaisTyrTyrAia Xaa_?LeuArgXaa_sTyrXaa_s AsnTrpXaa_1c.ThrArgGlnArgTyr (SEG ID NO; 1 )

or a salt thereof, wherein:

Xaa-; is Ala, His, or Ser; Xaa_£ is Gin or Lys;

Xaa_?. is Pro or Ala;

Xaa₅ is Asn, Aia, or Thr;

Xaa_e is Arg or Lys;

Xaa₇ is Ser, Asp, or Aia;

Xaa₈ is His o Lys;

Xaa_s is Leu or lie; and

Xaa-io is Vai or Leu.

The novel polypeptides of the invention show a statistically significant increase in the reduction of food intake in either a lean and/or diet-induced obesity animal model when compared with human PYY(3-36). Preferably the poiypeptides of the invention reduce the intake of food in a lean and/or diet-induced obesity animal mode! by at least 20%, at least 30%, or at least 40%. More preferably, the polypeptides reduce the intake of food in a lean and/or diet-induced obesity animal model by at least 50%.

in another aspect, the invention provides a polypeptide selected from the group consisting of:

NO:40);

NO'45);

and salts thereof.

Unless otherwise indicated, the polypeptides of the invention may have either a carboxamide or carboxylic acid at the end of the amino acid chain.

The invention encompasses salts of the recited polypeptides, including pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, including inorganic and organic acids and bases, including but not limited to, sulfuric, citric, maieic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfite, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oieate, tannate, pantothenate, b (tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, giutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1 , 1 '-methylene-bis-(2- hydroxy-3-naphthoate)) salts. Also included are salts formed with free amino groups such as, for example, hydrochloric, phosphoric, acetic, trifluoroacetic, oxalic, and tartaric acids. Also included are salts that ma form with free carboxy groups such as, for example sodium, potassium, ammonium, sodium, lithium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethano!, histidine, and procaine salts.

The polypeptides of the invention may be prepared using standard recombinant expression or chemical peptide synthesis techniques known in the art. See, for example, Ghan, Weng C, and Peter D. White, eds. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, New York: Oxford UP, 2000, and Howl, John, ed. Peptide Synthesis and Applications (Methods in Molecular Biology).

Totowa, J; Humana, 2005.

The compositions and pharmaceutical combinations of the invention are useful for the treatment of metabolic disorders including, for example, hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (including type 1 diabetes, type 2 diabetes, and gestational diabetes), non-alcoholic steatotic liver disease, steatosis of the liver, polycystic ovarian syndrome, hyper!ipidemia, and Metabolic Syndrome, The compositions and pharmaceutical combinations may be used for treating obesity or diseases characterized by overeating and for the suppression of appetite. The methods comprise administering to a subject a therapeuticaiiy effective amount of a composition of the invention to a subject in need thereof, preferably a human subject.

Other disorders that may be treated with the compositions and combinations of the invention include, but are not limited to, insulin resistance, insulin deficiency, hyperinsulinemia, hyperglycemia, dyslipidemia, hyperlipidemia, hyperketonemia, hyperg!ucagonemia, pancreatitis, pancreatic neoplasms, cardiovascular disease, hypertension, coronary artery disease, atherosclerosis, renal failure, neuropathy (e.g., autonomic neuropathy, parasympathetic neuropathy, and polyneuropathy), diabetic retinopathy, cataracts, endocrine disorders, and sleep apnea, polycystic ovarian syndrome, neoplasms of the breast, colon, prostate, rectum and ovarian, osteoarthritis steatosis of the liver.

The invention further encompasses methods of regulating insulin

responsiveness in a patient, as well as methods of increasing glucose uptake by a cell, and methods of regulating insulin sensitivit of a cell, using the conjugates or fusions of the invention. Also provided are methods of stimulating insulin synthesis and release, enhancing adipose, muscle or liver tissue sensitivity towards insulin uptake, stimulating glucose uptake, slowing digestive process, slowing of gastric emptying, inhibition of gastric acid secretion, inhibition of pancreatic enzyme secretion, reducing appetite, inhibition of food intake, modifying energy expenditure, or blocking the secretion of glucagon in a patient, comprising administering to said patient a composition of the invention e.g. comprising administering at least one dose of a composition e.g. a pharmaceutical composition or pharmaceutical combination of the present invention.

The invention also provides for use of a composition of the Invention in the manufacture of a medicament for treatment of a metabolic disease such as those described herein. The invention also relates to use of any of the compositions described herein for use i therapy.

The polypeptides of the present invention and their salts may be employed alone or in combination with other therapeutic agents (a "pharmaceutical

combination") for the treatment of the above-mentioned conditions. In some embodiments, the polypeptide of the present invention and the additional therapeutic agent or agents are administered together, whil in other embodiments, the polypeptide of the invention and the additional therapeutic agent or agents are administered separately. When administered separately, administration may occur simultaneously or sequentially, in any order. The amounts of the polypeptides(s) of the present invention and the other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. The administration in combination of a compound of the present invention with other treatment agents may be in combination by administration concomitantly in: (1) a unitary pharmaceutical composition including both therapeutic agents; or (2) separate pharmaceutical compositions each including one of the therapeutic agents. Alternatively, the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration ma be close in time or remote in time.

In one embodiment, the pharmaceutical combinations of the invention include a polypeptide according to the invention and an exendin-4 peptide (see, for example, US patent no. 5,424,286) or a fragment or analog thereof. Exendin-4 (Ex -4} and analogs thereof that are useful for the present invention include Byetta® and

Bydureon® (exenatide), Victoza® (iiraglutide), lixisenatide, LY2189265 (duiaglutide), PF-4856883, ZYD-I , and H1VI11260C (LAPS exendin) as well as those described in PCT patent publications WO 99/25728 (Beeley et a/.), WO 99/25727 (Beeley et a/.), WO 98/05351 (Young et a/.), WO 99/40788 (Young et a/.), WO 99/07404 (Beeley et ^■at.), and WO 99/43708 (Knudsen et a/.).

in another embodiment, the pharmaceutical combinations of the invention include a polypeptide according to the invention and GLP-1 (see, for example, Gutniak, M., et ai (1992) N. Engl. J Bled. 326: 316-22), or a fragment or analog thereof, for example, GLP-1 (7-37), GLP-1 (7-36) , GLP-1 (7-35), GLP-1 (7-38), GLP- 1 (7-39), GLP-1 (7-40), GLP-1 (7-41).

Further GLP-1 analogues are described in International Patent Application No. 90/11299, which relates to peptide fragments which comprise GLP-1 (7-36) and functional derivatives thereof and have an insulinotropic activity which exceeds the insulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37) and to their use as insulinotropic agents (incorporated herein by reference, particularly by way of examples of drugs for use in the present invention).

International Patent Application No. WO 91/1 1457 (Buckley et ai.) discloses analogues of the active GLP-1 peptides GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), and GLP-1 (7-37) which can also be useful as GLP-1 drugs according to the present invention (incorporated herein by reference, particularly by way of examples of drugs or agents for use in the present invention). The pharmaceutical combinations of the invention also include a polypeptide according to the invention and albigiutide.

in another embodiment, the pharmaceutical combinations include a polypeptide according to the invention and an enhancer of GLP-1 action such as a DPP-!V inhibitor (e.g. sitagliptin and/or saxagiiptin).

In other embodiments, the pharmaceutical combination comprises a PYY analog of the present invention and one or more therapeutic agents that are direct or indirect stimulators of GLP-1 secretion such as metformin, bile acid sequestrants {e.g. colestipol, cholestryramine, and/or colesevelam}, ilea! bile acid transport (iBAT) Inhibitors (e.g. ALBI-3309, AZD-7806, S-8921 , SAR-583G4, or those described in US20130029938), and SGLT-1 inhibitors (e.g. DSP-3235 and/or LX-4211 ).

The invention provides for methods of treatment where a "therapeutically effective amount" of a polypeptide of the invention is administered to a subject in need of such treatment. The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. As will be recognized by those in the field, an effective amount of therapeutic agent will vary with many factors including the age and weight of the patient, the patient's physical condition, the blood sugar level, the weight level to be obtained, and other factors

in one embodiment, a therapeutically effective amount of a polypeptide of the present invention is the amount required to suppress appetite in the subject to a desired degree. The effective daily appetite-suppressing dose of the compounds will typically be in the range of about 0.01 pg to about 500 pg /day, preferably about 0.05 pg to about 100 pg/day and more preferably about 1 pg to about 50 pg /day, most preferably about 5 pg to about 25 pg/day, for a 70 kg patient, administered in a single or divided doses.

In one aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the invention, and a pharmaceutically acceptable carrier, exclpient or diluent.

The pharmaceutical compositions and pharmaceutical combinations of the invention can be administered by any route, including intravenously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, transmucosa!iy, or by pulmonary inhaiation. For example, polypeptides of the inventio can be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous), nasal or oral administration. Methods for formulating and delivering polypeptides for various routes of administration are known in the art. See, for example, Swain et a/. (2013) Recent Pat. Btotechnol. 1 Feb 2013 Epub ahead of print, Hovgaard, Lars, Sven Frokiaer, and Marco Van De VVeert, eds. Pharmaceutical Formulation Development of Peptides and Proteins. 2^nd ed. Boca Raton: CRC Press, 2012, and Van Der Walle, Chris, ed. Peptide and Protein Delivery. London: Academic, 201 1.

In one embodiment, the invention encompasses a slow release formulation. Such formulations allow for therapeutically effective amounts of the therapeutic poiypepiide or polypeptides to be delivered into the bloodstream over many hours or days following injection or delivery to the subcutaneous space.

Slow release formulations of the invention may include one or more polymers useful in delaying the release of the therapeutic polypeptide. Non-limiting examples of suc polymers include poly(lactic-cc -glycolic acid) PLGA, poiycaprolactone, polydioxanone, poiytrimethylene carbonate, polyanhydrides, PEG-PLGA, polyg!utamic acid, polyethylene glycol terpbthalate/polybutylene

ferphthalate/polybutylehe ferphthalate, poly(aminoacid)-Leu/Glu copolymer, poSytyrosine carbonates, polyesteramides, poly (alpha aminoacid) based polymeric micelles, polyhydroxypropylmethacrylamide, polyalkyicyanoacrylate, collagen, hyaluronic acide, albumin, carboxymethylcelluiose, fleximer, chitosan, maltodextrin, dextran, or dextran sulfate,

In one aspect, the polypeptides of the invention may be delivered via a miniature device such as an implantable infusion pump which is designed to provide long-term continuous or intermittent drug infusion. Such devices can be used to administer a therapeutic polypeptide of the invention via intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal, epidural, or intraventricular routes. Such devices may be erodible, non-erodible and/or durable. Non-limiting examples of such devices include the Durasert™ device (pSivida), the DUROS® osmotic delivery system (Intarcia Therapeutics), MedLaunch™ Polymer Technology (Endo Health).

Other devices that could be used according to the present invention include the SnychroMed® pump (Medtronic), and the Codman® 3000 infusion

pump(Johnson & Johnson), the V-Go® delivery system (Valeritas), the OmniPod® pump (Insuiet), and the JeweiPump™ (Debiotech).

The poiypeptides of the invention may be administered in an in situ gel formulation. Such formulations typically are administered as liquids which form a gel either by dissipation of the water miscibie organic solvent or by aggregation of hydrophobic domains present in the matrix. Non-limiting examples include the FLUID CRYSTAL technology (Camurus) and the SABER technology (Durect), and the formulations described in US Patent serial nos. 5612051 , 5714159, 6413539, 6004573, and 61 17949.

The therapeutic polypeptides of the invention may also be encapsulated into a microsphere-based pharmaceutical formulation suitable for subcutaneous injection. Non-limiting examples of microsphere-based formulations for the delivery of peptides include Chroniject™ (Oakwood Labs), Medusa® (Flamel's), Q-Sphera (Q-CHIP), as well as those described in US patent serial nos. 4675189, 6669961 , and Amin et ai. (2001 ) J of Controlled Release 73: 49-57.

The formulation ma contain antibacterial or antifungal agents such as meta- cresol, benzyl alcohol, parabens (methyl, propyl, butyl), chlorobutano!, phenol, pheny!mercuric salts such as acetate, borate, or nitrate, or sorbic acid.

The compositions of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use> Any suitable lyophilization method {e.g., spray drying, cake drying) and/or reconstitution techniques can be employed. in a particular embodiment, the invention provides a composition comprising a lyophilized (freeze dried) polypeptide as described herein.

in certain aspects, the invention provides a nucleic acid encoding a polypeptide of the invention and recombinant expression vectors containing such nucleic acids. Recombinant expression vectors of the invention include a nucleic acid encoding a polypeptide of the invention operabiy linked to one more expression control elements such as, e.g., a promoter.

Host cells containing a nucleic acid or recombinant expression vector encoding a polypeptide of the invention are also included. Suitable host cells according to the invention include both prokaryotic host cells and eukaryotic hosts cells. Possible host cells include, but are not limited to, mammalian host cells, bacterial host cells (e.g. E. colt), yeast host cells, and plant host cells.

Nucleic acids and recombinant expression vectors encoding a polypeptide of the invention can be introduced into a suitable host cell to create a recombinant host cell using any method appropriate to the host cell selected, e.g., transformation, transfection, electroporation, or infection. In some embodiments, the nucleic acid or recombinant expression vector is integrated into the host ceil genome. The resulting recombinant host cell can be maintained under conditions suitable for expression (e.g. , in the presence of an inducer, in a suitable animal, i suitable culture media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), whereby the encoded polypeptide is produced. If desired, the encoded peptide or polypeptide can be isolated or recovered. The invention further provides a method for producing a polypeptide of the present invention where the method comprises maintaining a host cell such as those described above that comprises a nucleic acid or recombinant expression vector that encodes a polypeptide of the invention under conditions suitable for expression of said nucleic acid or recombinant expression vector. Methods for recombinant expression of polypeptides in host cells are well known in the art. See, for example, Rosalyn . Bill, ed. Recombinant Protei Production in Yeast: Methods and

Protocols (Methods in Molecular Biology, Vol. 866), Humana Press 2012; James L. Hartley, ed. Protein Expression in Mammalian Cells: Methods and Protocols

(Methods in Molecular Biology), Humana Press 2012, Loic Faye and Veronique

Gomord, eds.. Recombinant Proteins From Plants: Methods an Protocols (Methods in Molecular Biology), Humana Press 2008; and Argeiia Lorence, ed. Recombinant Gene Expression (Methods in Molecular Biology), Humana press 201 1.

In certain embodiments, the nucleic acids of the invention are "isolated.'^'' Nucleic acids referred to herein as "isolated" are nucleic acids which have been separated away from other material (e.g., other nucleic acids such as genomic DNA, cDNA and/or RNA) in its original environment (e.g., in cells or in a mixture of nucleic acids such as a library). An isolated nucleic acid can be isolated as part of a recombinant expression vector.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.

EXAMPLES

The examples make use of the following abbreviations;

amu atomic mass unit

Fmoc 9-f I uo re ny I m eth oxyca rbo n y [

HBTU 2-(1 H-benzotriazoie-1 -yl)~1 , ,3,3-tetramethyl uranium

hexafluorophosphate

HCTU 2-(6-ch ro-1-H-benzotriazoie-1-yi)-1 ,3.3-tetramethylaminiurn

hexafluorophosphate

DMF N,N-dimethylformamide

N.M.M N-methylmorpholine

D!PEA Ν,Ν-diisopropylethylamine

TFA trifluoroacetic acid

Trt trityl

f-Bu tert-buty!

Boc tert-butylcarbonyi Pbf 2,2,4,6₁7-pentamethyldihydro-benzofuran-5-sulfonyi AL maleimide

PBS phosphate buffered saline

ivDde 1-(4,4-dimethyl~2,6-dio ocyclohexylidene)-3-methy!butyl

MALDI matrix assisted laser desorption/ionizaticn

B PS N-p-maieimidopropyioxysuccinimide ester

DODT 2,2'-(ethylened!Oxy)diethanethio!

TIPS triisopropylsi!ane

PA mercaptopropionic acid

rt retention time

RP revolutions per minute

Peptide Synthesis

The peptides shown in the following examples were synthesized by solid- phase methods using Fmoc strategy with 2-{ H~benzotriazole-1-yl)-1 , 1 ,3,3- tetramethyl uranium hexaf!uorophosphate (HBTU) or 2-(6-chioro-1-H-benzotrtazoie- 1-yi)- , ,3,3-tetramethy!aminium hexafiuorophosphate (HCTU) activation (5 fold molar excess) in Ν,Ν-dimethyiformamide (DMF), and N-methyimorpholine (NMM) as base, 20% piperidine/D!vlF for Fmoc deprotection, on an automated peptide synthesizer (model Prelude or Overture; Protein Technologies, Tucson, AZ). The resin was Rink Amide IV1BHA LL (Novabiochem) or Rink Amide AM LL

(Novabiochern) with a loading of 0.29 - 0.38 mmol/g on a 20 - 400 μηιοΙ scale. The side chain protection groups used were Trt for Asn, Gin, Cys and His; ί-Bu for Ser, Thr, and Tyr; Boc for Lys and Trp; Or-Bu for Asp and Glu; and Pbf for Arg. Cieavage of peptide-resin was completed with a mixture of trifluoroacetic acid

(TFA):anisoie:vvater:triisopropyisilane (88:5:5:2). The crude peptide was precipitated in cold diethyl ether, the diethyl ether was decanted and the solids triturated again with cold diethyl ether. The crude solids were then purified by reverse phase HPLC on a Waters XBridge™ BEH 130, CI 8, 10 jjm, 130A, 30 X 250 mm ID column, using a gradient within the ranges of 5 - 75% acetonitriie/water with 0,1 % TFA over 3D - 45 minutes at a flow rate of 30 mL/min, λ -^■ 215 nm.

LC/MS Conditions

Method A:Performed using a Phenomenex UPLC Aeris™ Peptide XB C 8 column, 1.7 μιττ, 2.1 X 100 mm or ACQUiTY BEH300 or BEH130 CI 8 column, 1.77 m, 2.1 X 100 mm using 5 - 65% acetonltrile/water with 0.05% TFA over 30 minutes with a flow rate 0.5 mL/min, A - 215 nm, 280 nm. C18 HPLC Conditions:

Method A; Performed using a Waters XBridge™ BEH130 C18 column, 5 μηι, 4.6 X 250 mm. with 5 - 70% acetonitriie/wafer with 0.1 % TFA over 15 minutes with a flow rate 1.5 mL/min, 40° C, A - 215 nm, 280 nm.

Method B: Performed using a Waters XBridge™ BEH130 C18 column, 5 μιη, 4.6 X 250 mm, 5 - 75% acetonitrile/water with 0.1 % TFA over 20 minutes with a flow rate 1.5 mL/min, λ - 215 nm, 280 nm.

Method C: Performed using a Waters XBridge™ BEH130 C18 column, 5 μιη, 4,6 X 250 mm, 20 - 37.5% acetonitrile/water with 0, 1 % TFA over 15 minutes with a flow rate 1.0 mL/min, 60° C, K- 215 nm, 280 nm.

Method D: Performed using a Waters XBridge™ BEH300 C 8 column, 5 μιη, 4.6 X 250 mm, 5 - 70% acetonitrile/water with 0.1 % TFA over 15 minutes with a flow rate 1 5 mL/min, λ - 215 nm, 280 nm.

Example 1, PKPEAPGKDASPEELNRYYASLRHYLNWVTRQRY-NH₂ (SEQ ID NO:3)

Example 1 was prepared on a 35 μηιο! scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1369 amu and (M+4)/4 - 1027 amu, which corresponds to a peptide with the parent molecular weight of 4105 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 8.90 min) for the isolated peptide (25 mg, as the 8 trifluoroacetic acid salt). Example 2. PKPEAPGKDASPEELNRYYASLRKYLNWLTRQRY-NH₂ (SEQ ID NO:4)

Example 2 was prepared on a 35 μιτιοΙ scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1371 amu and (M+4)/4— 1028 amu, which corresponds to a peptide with the parent molecular weight of 41 1 1 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C S HPLC Method A, rt = 9.46 min) for the isolated peptide (20 mg, as the 8 trifluoroacetic acid salt).

Example 3. PKPEAPGKDASPEELISiRYYASLRHYLNWLTRQRY-NH₂ (SEQ ID NO:5)

Example 3 was prepared on a 35 μηιοΐ scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions {M+3}/3 - 1374 amu and (M+4)/4 ~ 1031 amu, which corresponds to a peptide with the parent molecular weight of 4120 amu (ESi-MS, LC/MS Method A). A purity of 90% was determined by C18 HPLC (C18 HPLC Method A, rt - 9,42 min) for the isolated peptide ( 6 mg, as the 8 trif!uoroacetic acid salt).

Example 4 PKPEAPGKDASPEEWNRYYADLRKYLNWLTRQRY-NH₂ (SEQ ID

NO:6)

Example 4 was prepared on a 35 prno! scale as a white soii^'d using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1405 amu and (M+4)/4 - 054 amu, which corresponds to a peptide with the parent molecular weight of 4212 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.54 min) for the isolated peptide (18 mg, as the 8 trifiuoroacetic acid salt). Example 5. PKPEAPGKDASPEEWNRYYADLRHYLNWLTRQRY-NH₂ (SEQ ID NO:7)

Example 5 was prepared on a 6X50 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions ( +3}/3 ~- 1407 amu and (M+4)/4 - 1056 amu, which corresponds to a peptide with the parent molecular weight of 4221 amu (ESI -MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.27 min) for the isolated peptide (180 mg, as the 8 trifiuoroacetic acid sait).

Alternatively, Example 5 was prepared via manual synthesis using a 250 mL jacketed reactor that was cooied to 15°C, Rink Amide AM Resin LL (100-200 mesh, 3.8 g, 0.29 mmol/g loading) was swelled with DMF (50 mL) 3 times for 0 min each with nitrogen sparge. The Fmoc group was removed with 20% piperic!ine in DMF (200 mL) over 5 min with nitrogen sparge, followed by 20% piperidine in DMF (200 mL) over 2 min with nitrogen sparge. The resin was then washed with DMF (100 mL) and then twice with DMF ( 00 mL) for 1 min with nitrogen sparge. Following Fmoc deprotecion and DMF washing, the first amino acid (100 mL, 200 mM solution in DMF) was added, followed by DSPEA solution (50 mL, 800 mM solution in DMF). A solution of HCTU in DMF (50 mL, 400 mM) was added over a 12 min period via peristaltic pump. After a minimum of 15 min, a Kaiser test on an aliquot of resin was performed to ensure complete reaction. The resin was then washed with DMF (100 mL) and then twice with DMF (100 mL) for 1 min with nitrogen sparge. This sequence (Fmoc deprotection with 20% piperidine/DMF; washes; amino acid coupling; Kaiser test; washes) was performed for the remaining sequence of the peptide, except for the histidine at position 24. The amino acid (His) and DtPEA solutions were cooled to ~10°C and added to the reactor. The reactor's chiller was set to 5"C and the reaction mixture cooled to 6.3°C in ~15 min. A cooled (-*1 Q°C} solution of HCTU in DMF (50 mL) was added dropwise over a 25 min period via peristaltic pump at 2 mL/min, during which time the solution warmed to 7.8°C. After 15 min, a Kaiser test showed complete reaction. The remaining amino acids were coupled using the standard protocol. At the completion of the synthesis (proline 1 was coupled), the resin was then washed twice with DMF (100 mL) for 1 min with nitrogen sparge, then washed three time with DCM (200 mL) for 5 min with nitrogen sparge, and then finally three times with methanol (200 mL) for 5 min with nitrogen sparge. The resin was dried with nitrogen sparge for 30 min to give 37.5 g of dry resin. The resin was cleaved in portions. Ten grams of resin was swelled with 120 mL DMF for 45 min with nitrogen sparge. The DMF was drained off and the final N- terminal Fmoc was removed with 20% piperidine in DMF ( 50 mL) over 5 min with nitrogen sparge, followed by 20% piperidine in DMF (150 mL) over 12 min with nitrogen sparge. The resin was then washed with DMF (100 mL) and then twice with DMF (100 mL) for 1 min with nitrogen sparge, then washed three time with DCM (120 mL) for 5 min with nitrogen sparge, and then finally three times with methanol ( 20 mL) for 5 min with nitrogen sparge. The resin was dried with nitrogen sparge for 30 min. Cleavage of peptide from the resin was performed using 100 - 120 mL of cleavage cocktail: TFA:phenol:DODT:water:TIPS (90:2.5:2.5:2.5:2.5) for 2.5 ~ 3 h. . The filtrates were split into vessels and treated with cold diethyl ether. The vessels were centrifuged for 10 min at 3000 RPM and the supernatant was poured off. The material was treated with cold diethyl ether again, shaken and then centrifuged for another 10 min at 3000 RPM. The supernatant was poured off again. The solids from the vessels were combined using 0.1 % aqueous TFA and lyophilized to give batch 1. The resin was subjected to second cleavage using the same procedure to give batch 2. This process (Fmoc deprotection; washes; cleavage from resin, trituration/resuspension, and lyophiiization) was repeated three times with ~9 g of resin to afford a total of ~1 1 g of crude peptide after lyophiiization. This material was dissolved in 0.1 % aqueous TFA to give an approximate concentration of 75 mg/mL and the material was purified by reverse phase HPLC using multiple injections (between 2 and 3 mL each) using the following ste gradient: 5 - 41.25%

acetonitrile/water with 0.1 % TFA over 75 min,; XBridge™ Prep C18, SO x 250 mm, 10 pm, flow rate 50 mL/min. Fractions containing product with >93% purity (HPLC Method C) were combined. Impure fractions (purity of -88 - 93%) were also collected and resubjected to the purification conditions. All pure fractions (>93%) were then combined and freeze-dried to give desired peptide as a. white solid. A purity of > 93% was determined by C18 HPLC (CIS HPLC Method C, rt ~ 14.12 min) for the isolated peptide (2.8g, as the 8 trifiuoroacetic acid salt).

A salt exchange from TFA to HOAc using Example 5 prepared by peptide synthesizer and manual synthesis was performed using a 2 x 60 mL Agilent

Stratospheres™ PL-HC0₃ MP SPE column. The column was equilibrated by first treating with 50 ml of MeOH, foliowed by 50 mL of Dl water. The column was then treated with 2 x 50 mL 1 N HOAc and then with 2 x 50 mL 0.1 N HOAc, and the filtrate was monitored to ensure pH ~ 3 (pH paper). A solution of Example 5 (~3.5 g including 2.8 g prepared as described above and 0.7 g derived from previously- prepared batches) in 0.1 N HOAc was split equally between the SPE columns and then eiuted with 5 x 50 mL of 0,1 HOAc. The column was then washed with 5 x 50 mL of MeOH. The methanol fractions containing product (as determined by HPLC, Method C) were concentrated via rotary evaporator to -40 mL, which was added to the 0.1 N HOAc washes. The solution was freeze-dried over 3 d to afford the desired isolated peptide as a white solid. A purity of > 95% was determined b C18 HPLC (C18 HPLC Method C, rt^■= 14.14 min) for the isolated peptide (2.95g, as the 8 acetic acid salt). Example 6. PKPEAPGKDASPEEvVNRYYASLRKYLNWLTRQRY-NH₂ (SEQ ID NO:8)

Example 6 was prepared on a 35 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1395 amu and (M+4)/4 - 1047 amu, which corresponds to a peptide with the parent molecular weight of 4184 amu {ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C 8 HPLC Method A, rt = 9.45 min) for the isolated peptide (18 mg, as the 8 trifiuoroacetic acid salt).

Example 7. PKPEAPGKDASPEEWNRYYASLRHYLNWLTRQRY-NH₂ (SEQ ID NO:9)

Example 7 was prepared on a 35 prnol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1398 amu and ( +4)/4 - 1049 amu, which corresponds to a peptide with the parent molecular weight of 4193 amu (ESS-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.47 min) for the isolated peptide (27 mg. as the 8 trifiuoroacetic acid salt). Example 8. PKPEAPGKDASPEEWNRYYADLRKYLNWVTRQRY-NH₂ (SEQ ID NO: 10)

Exarnpie 8 was prepared on a 35 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions ( +3)^'/3 - 1400 amu and ( +4)/4 - 1050 amu, which corresponds to a peptide with the parent molecular weight of 4198 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.35 min) for the isolated peptide (21 mg, as the 8 trifluoroacetic acid salt). Example s. PKPEAPGKDASPEEWNRYYAOLRHYLNV T QRY-NH₂ (SEQ ID NO:1 1 )

Example 9 was prepared on a 35 μηιοί scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1403 amu and (M+4)/4 - 1052 amu, which corresponds to a peptide with the parent molecular weight of 4207 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt - 9.36 min) for the isolated peptide (22 mg, as the 8 trifluoroacetic acid salt).

Example 10, PKPEAPGKDASPEEWNRYYASLRKYLNWVTRQRY-NH₂ (SEQ ID NO: 12)

Example 10 was prepared on a 35 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 391 amu and (M+4)/4 - 1043 amu, which corresponds to a peptide with the parent molecular weight of 4170 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt ~ 9.23 min) for the isolated peptide (23 mg, as the 8 trifluoroacetic acid salt).

Example 11, PKPEAPGKDASPEEVWRYYASLRHYLNWVTRQRY-NH₂ (SEQ ID NO: 13)

Example 11 was prepared on a 35 pmoi scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1393 amu and (M+4)/4 - 1045 amu, which corresponds to a peptide with the parent molecular weight of 4179 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.24 min) for the Isolated peptide (22 mg, as the 8 trifluoroacetic acid salt). Example 12. PKPEAPGEDASPEELNRYYASLRHYLNWVTRQRY-NH₂ (SEQ ID O: 14)

Exampie 12 was prepared on a 35 prnoi scaie as a white soiid using the genera! method. The moiecuiar mass of the isolated peptide was confirmed by fragment ions (M+3)/3 ~ 1389 amu and (M+4)/4 - 1027 amu, which corresponds to a peptide with the parent molecuiar weight of 4106 amu (ES^'i- S, LC/MS Method A). A purity of >90% was determined by C18 HPLC (CIS HPLC Method A, rt - 9.60 min) for the isolated peptide (22 mg, as the 7 trifiuoroacetic acid salt). Example 13. PKPEHPGKDASPEEWNRYYAALRKYLNWVTRQRY-NH? (SEQ ID NO: 15)

Example 13 was prepared on a 35 pmol scaie as a white soiid using the general method. The molecular mass of the isoiated peptide was confirmed by fragment ions (M+3)/3 - 1407 amu and (M+4)/4 ~ 1056 amu, which corresponds to a peptide with the parent moiecuiar weight of 4220 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.46 min) for the isoiated peptide (22 mg, as the 9 trifiuoroacetic acid salt).

Example 14. PKPEHPGKDASPEELNKYYAALRHYLNWVTRQRY-NHg (SEQ ID NO: 16)

Exampie 14 was prepared on a 35 pmoi scale as a white soiid using the general method. The molecuiar mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1377 amu and (M+4)/4 - 1033 amu, which corresponds to a peptide with the parent molecular weight of 4128 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by C 8 HPLC (C18 HPLC Method A, rt = 8.88 min) for the isoiated peptide (27 mg, as the 9 trifiuoroacetic acid sait).

Exampie 15. PKPEHPGKDASPEELNRYYASLRHY!N VTRGRY-NH₂ (SEQ !D NO: 17)

Example 6 was prepared on a 35 μη ο! scale as a white soiid using the general method. The molecuiar mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1391 amu and (M+4)/4 - 044 amu, which corresponds to a peptide with the parent moiecuiar weight of 4172 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt ^« 8.77 min) for the isoiated peptide (29 mg, as the 9 trifiuoroacetic acid salt). Example 18. PKPEHPGKDASPEELARYYASLRHYLNWVTRQRY-NH- (SEQ ID NO: 18)

Example 16 was prepared on a 35 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment Ions (M+3)/3 - 1377 amu and (,M+4)/4 - 1033 amu, which corresponds to a peptide with the parent molecular weight of 4129 amu (ESi-MS, LC/MS Method A), A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 8.89 min) for the isolated peptide (30 mg, as the 9 trifluoroacetic acid salt). Example 17. PKPEHPGKDASPEEWNRYYASLRHYINWVTRQRY-NH₂ (SEQ ID NO; 19)

Example 17 was prepared on a 35 μηιοΙ scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mL of 25% acetic acid to minimize the tryptophan C0₂ adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment ions (M^'+3) 3 ~ 1416 amu and (M+4)/4 - 1062 amu, which corresponds to a peptide with the parent molecular weight of 4245 amu (ESI-MS, LC/MS Method A). A purity of >9Q% was determined by C18 HPLC (C18 HPLC Method A, rt = 8.88 min) for the isolated peptide (35 mg, as the 9 trifluoroacetic acid salt).

Example 18. PKPEHPGKDASP£EWNRWADLRHYINVWTRQRY-NH₂ (SEQ ID NO:20)

Example 18 was prepared on a 35 pmol scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 rnL of 25% acetic acid to minimize the tryptophan CO?, adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment Ions (M+3)/3 - 1425 amu and (M÷4)/4 - 1069 amu, which corresponds to a peptide with the parent molecular weight of 4273 amu (ESi-MS, LC/MS Method A). A purity of 90% was determined by C S HPLC (C18 HPLC Method A, rt = 8.98 min) for the isolated peptide (17 mg, as the 9 trifluoroacetic acid salt).

Example 19. PKPEHPGKDASPE£WNRYYADLRHYLNWVTRGRY-NH₂ (SEQ ID NO:21)

Example 19 was prepared on a 35 p ol scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mL of 25% acetic acid to minimize the tryptophan C0₂ adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1425 amu and ( +4)/4 - 1069 amu, which corresponds to a peptide with the parent moiecuiar weight of 4273 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by CIS HPLC (C18 HPLC Method A, rt = 8.98 min) for the isolated peptide (34 mg, as the 9 trifluoroacetic acid salt).

Example 20, PKPESPGKDASPEEWNRYYADLRHY!NWVTRQRY-NH₂ (SEQ ID NO: 22}

Example 20 was prepared on a 35 pmoS scaie as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1408 amu and { +4)/4 - 1057 amu, which corresponds to a peptide with the parent moiecuiar weight of 4223 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLG (C18 HPLC Method A, rt « 8.30 min) for the isolated peptide (28 mg, as the 8 trifluoroacetic acid sa!t). Example 21. PKPESPGKDASPEEWNRYYADLRHYLNWVTRQRY-NH,, (SEQ ID NO:23)

Example 21 was prepared on a 35 prnol scale as a white solid using the general method. The moiecuiar mass of the isolated peptide was confirmed by fragment ions (M+3J/3 - 1408 amu and (M+4)/4 - 1057 amu, which corresponds to a peptid with the parent moiecuiar weight of 4223 amu (ESS-MS, LC/MS Method A). A purity of >90% was determined by CT8 HPLC (C18 HPLC Method A, rt 8.68 min) for the isolated peptid (28 mg, as the 8 trifluoroacetic acid salt).

Example 22. PKPEHPGKDASPEEWNRYYADLRHYLNWLTRQRY-NH₂ (SEQ ID NO-24)

Example 22 was prepared on a 40 pmol scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mi of 25% acetic acid to minimize the tryptophan C0₂ adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment ions (Svl+3)/3 - 1 30 amu and (M+4)/4 - 1073 amu, which corresponds to a peptide with the parent molecular weight of 4287 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by CI 8 HPLC (C18 HPLC Method A, rt = 9.85 min) for the isolated peptide (24 mg, as the 9 trifluoroacetic acid salt). Example 23. PKPEHPGKDASPEEWAKYYAALRHYINvWTRQRY-NH₂ (SEQ ID NO:25) Example 23 was prepared on a 40 μιηοί scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mL of 25% acetic acid to minimize the tryptophan C0₂ adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment ions ( +3)/3 - 1386 amu and (M+4)/4 - 1040 amu, which corresponds to a peptide with the parent molecular weight of 4158 amu (ESi-!vlS, LC/MS Method A). A purity of >90% was determined by CIS HPLC (C1S HPLC Method A, rt = 9.28 min) for the isolated peptide (20 mg, as the 9 trifluoroacetic acid salt). Example 24. PKPEAPGKDASPEEWNRYYADLRHY!NWVTRQRY-NH₂ (SEQ ID NO:26)

Example 24 was prepared on a 40 μϋΐοΙ scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mL of 25% acetic acid to minimize the tryptophan CG₂ adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1403 amu and (M+4)/4 - 1053 amu, which corresponds to a peptide with the parent molecular weight of 4207 amu (ES!-MS, LC/MS Method A). A purity of >90% was determined by CI 8 HPLC (€18 HPLC Method A, rt = 9,22 min) for the isolated peptide (28 mg, as the 8 trifluoroacetic acid salt).

Example 25. PKPEHPGKDASPEEWNRYYASLRKYLNWVTRQRY~NH₂ (SEQ ID NO:27)

Example 25 was prepared on a 40 pmo! scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mL of 25% acetic acid to minimize the tryptophan C0₂ adduct formed during cleavage from the resin. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 ~ 1412 amu and (M+4)/4 - 1060 amu, which corresponds to a peptide with the parent molecular weight of 4236 amu (E.SI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (CI 8 HPLC Method A, rt = 9.09 min) for the isolated peptide (21 mg, as the 9 trifluoroacetic acid salt).

Example 26. PKPEHPG DASAEEWA YYAALRHYINWVTRQRY-NH₂ (SEQ ID NO-28)

Example 26 was prepared on a 20 Mrnol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M÷3}/3 - 1378 amu and (M+4)/4 - 1034 amu, which corresponds to a peptide with the parent molecular weight of 4132 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by CIS HPLC (C18 HPLC Method A, rt = 9,44 min) for the isolated peptide (19 mg, as the 9 tnf!uoroacetic acid salt).

Example 27. PKPEAPGKDASAEEWNRYYASLRHYLNVWTRQRY-NH^ (SEQ !D NO:29)

Example 27 was prepared on a 20 pmoi scale as a white solid using the general method. The molecuiar mass of the isolated peptide was confirmed by fragment ions ( +3)/3 ~ 1385 amu and ( +4)/4 - 1039 amu, which corresponds to a peptide with the parent molecular weight of 4 53 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.31 min) for the isolated peptide ( 6 mg, as the 8 tn^'fiuoroacetic acid salt).

Example 28. PKPEHPGKDASAEELARYYASLRHYLNWVTRQRY-NH₂ (SEQ ID NO:30)

Example 28 was prepared on a 20 μιτιοΙ scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3}/3 - 1368 amu and (M+4)/4 - 1027 amu, which corresponds to a peptide with the parent molecular weight of 4103 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by CI 8 HPLC (C18 HPLC Method A, rt = 9.27 min) for the isolated peptide (14 mg, as the 9 tn^'fiuoroacetic acid salt).

Example 29. PKPEAPGKDASAEEWNRYYASLRKYLNVWTRQRY-NB_?. (SEQ ID NO:31)

Example 29 was prepared on a 20 rnol scale as a white solid using th general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1382 amu and (M+4) 4 - 037 amu, which corresponds to a peptide with the parent molecular weight of 4144 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 9.31 min) for the isolated peptide (27 mg, as the 8 trifluoroaceiic acid salt).

Example 30. PKPESPGKDASAEEWTKYYAALRHYINWVTRQRY-NH₂ (SEQ ID NO:32)

Example 30 was prepared on a 20 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3J/3 - 1371 amu and (M+4}/4 - 029 amu, which corresponds to a peptide with the parent molecular weight of 4112 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (CI S HPLC Method A, rt = 9.59 min) for the isolated peptide (33 mg, as the 8 trifiuoroacetic acid salt).

Example 31 PKPEAPGKDASPEELNRYYASLRKYLNWWRQRY-NH₂ (SEQ ID NO:33)

Example 31 was prepared on a 35 pmol scale as a white solid using the general method. The isolated crude solid was stirred for several hours in 8 mL of 25% acetic acid to minimize the tryptophan C0₂ adciuct formed during cleavage from the resin. The molecular mass of the Isolated peptide was confirmed by fragment ions (M+3)/3 - 1366 amu and (M+4)/4 - 1025 amu, which corresponds to a peptide with the parent molecular weight of 4097 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 8.93 min) for the isolated peptide (21 mg, as the 8 trifiuoroacetic acid salt). Example 32. PKPEHPGEDASPEEWAKYYAALRHY!NWVTRQRY-NH₂ (SEQ ID NO: 34)

Example 32 was prepared on a 20 pmoi scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 387 amu and ( +4)/4 - 1041 amu, which corresponds to a peptide with the parent molecular weight of 4 59 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by LC/MS (LC/MS Method A, rt ~ 13.58 min) for the isolated peptide (9.4 mg, as the 8 trifiuoroacetic acid salt).

Example 33, PKPEAPGEDASAEEWNRYYASLRHYLNWVTRQRY-NH, (SEQ ID NO:35)

Example 33 was prepared on a 20 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1385 amu and (M+4)/4 - 1039 amu, which corresponds to a peptide with the parent molecular weight of 4154 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by LC/MS (LC/MS Method A, rt = 13.35 min) for the isolated peptide (7.7 mg_s as the 7 trifiuoroacetic acid salt).

Example 34. PKPESPGEDASPEEWTKYYAALRHYINWVTRQRY-NH₂ (SEQ ID NO:35)

Example 34 was prepared on a 20 pmol scaie as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 ~ 1381 amu and (M+4)/4 - 036 amu, which corresponds to a peptide with the parent molecular weight of 413Θ amu. (ESI-MS, LC/MS Method A). A purity of >90% was determined by LC/MS (LC MS Method A, rt = 13.98 min) for the isolated peptide (8.2 mg, as the 7 trifluoroacetic acid salt). Example 3S. PKPEAPGEDASPEEWNRYYADLRHYLNWLTRQ Y-NH₂ (SEG ID NO:37)

Example 35 was prepared on a 20 pmo! scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1408 amu and {M+4)/4 ~ 1056 amu, which corresponds to a peptide with the parent molecular weight Of 4222 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by LC/MS (LC/MS Method A, rt - 3.84 min) for the isolated peptide (7.5 mg, as the 7 trifluoroacetic acid salt).

Examples 36-43 make reference to the following intermediates:

Intermediate 1

(SEQ ID O:39)

Intermediate 2

a-[3-(3-malejmido-1-oxopropyl)amino]propy!~w-methOxy, poiyoxyethylen (available from NOP Corporation or JenKEM Technology USA Inc.) intermediate 3

(SEQ !D NO:40)

intermediate 4

(SEQ !D NO:41)

intermediate 5

N-succinimidyl-S-maleinimidopropionate intermediate 6 HS' or

n ~ 950

-SH-40K, available from JenKem Technology USA Inc.

Intermediate 7

(SEQ ID- NO:42)

intermediate 8

(SEQ ID N0.43)

intermediate 9

(SEQ ID NO;44)

intermediate 10

Intermediate 11

(SEQ ID NO:45) Exam pie 36

Intermediate 1 was prepared on a 400 pmol scale as a white soiid using the general method. The moiecular mass of the isolated peptide was confirmed by fragment ions (fvH3)/3 - 1377 amu and ( +4)/4 - 1033 amu, which corresponds to a peptide with the parent moiecular weight of 4128 amu (ESI- S, LC/MS Method A). A purity of >9Q% was determined by C18 HPLC (C18 HPLC Method B, rt . = 10.98 min) for the isolated peptide (76 mg, as the 7 tnf!uoroacetic acid salt). A mixture of intermediate 1 (24.1 mg, 4.89 moi) and Intermediate 2 (NOF Corporation, ME-400 A, 226 mg, 5,14 pmol) i 3.5 mL of 1X PBS buffer at pH 7,4 was shaken for 45 minutes, during which time the reaction became homogenous. The reaction was then diluted with a solution of 20% MeOH in 0.1 M aqueous HCl and purified by ion exchange chromatography (Sepharose FF Media, 5-50% 1 NaCI in 20% methanol/10 mM aqueous HCl and over 5 column volumes, flow rate SmL/min, λ - 254 nm). The purified conjugate was desalted using size exclusion chromatography (GE HiPrep 26/10 Desalting column, 0.1 M acetic acid-, λ - 254 nm) to afford a white solid after lyophilization. The molecuiar mass of the isolated peptide was confirmed by positive fragment ion distribution with the apex at 47379 amu

(MALDl), Example 36 (107 mg, as the 7 acetic acid salt) gave a retention time equal to 9.95 min using size exclusion HPLC (Phenomenex BioSep-SEC~S3000 column, 7.8 x 300 mm, 5 μηΐ, 50% acetonitrile/water with 0.5 TFA over 20 min, flow rate 0.75 mL/rnin, A— 220 nm),

Example 37

NO:40)

intermediate 3 was prepared on a 40 prool scale as a white solid using the general method, except the lysine at position 8 of the peptide was protected with an ivDde group, and proline at position 1 was protected with a Boc, After the coupling of the last amino acid (proline 1 ), the IvDde was removed with repeated treatments of 4% hydrazine in D F, and Fmoc-Cys(Tri)-OH was coupled. The molecular mass of the isolated peptide was confirmed by fragment ions (!VR3)/3 - 1464 amu and ( +4)/4 - 1098 amu, which corresponds to a peptide ith the parent molecular

Weight of 4390 amu (ESi-!vlS, LC/MS Method A), A purity of >90% was determined by C18 HPLC (C 8 HPLC Method D, rt = 9,61 min) for the isolated peptide (32 mg, as the 9 trifiuoroaeetic acid salt). A mixture of intermediate 3 (10 mg, 1.85 |.imoi) and intermediate 2 (JenKem Technology USA inc., 74 mg, 1.85 pmo!) in 5 mL of IX PBS buffer at pH 7.4 was shaken overnight, during which time the reaction became homogenous. The reaction was then diluted wit 5 mL of 20% MeOH in 0 mM aqueous HCi and purified by ion exchange chromatography (Sepharose FF Media, 0 - 60% 1 IVI NaCi in 20% methanol/10 mM aqueous HCI over 7 column volumes, flow rate 5 mL/min, λ - 254 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine Desalting column, 0.1 acetic acid, λ - 254 nm) to afford a white solid after !yophilization. The molecular mass of the isolated peptide was confirmed by positive fragment ion distribution with the apex at 44588 amu (MALDI). Example 37 (35 mg, as the 9 acetic acid salt) gave a retention time equal to 11.58 min using size exclusion HPLC (Phenomenex BioSep-SEC-S3000 column, 7.8 x 300 mm, 5 prn, 0.15 mM NaCI in 30 mM PBS over 20 min, pH 8,8, flow rate 0.75 mL/min, λ - 215 nm).

Example 38

intermediate 4 was prepared on a 40 moi scale as a white solid using the general method, except the lysine at position 8 of the peptide was protected with an ivDde group, and proline at position 1 was protected with a Boc. After the coupling of the last amino acid (proline 1 ), the ivDde was removed with repeated treatments of 4% hydrazine in DMF, and the linker was coupled using the activated succinimide ester reagent intermediate 5, ^-maleimidoprGpyioxysuccinimide ester, without the use of activator (HCTU) or base (NMM). The molecular mass of the isolated peptide was confirmed by fragment ions (M^'+3)/3 - 1442 amu and (M+4)/^'4 - 1082 amu, which corresponds to a peptide with the parent molecular weight of 4323 amu (ESI- MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 8.79 min) for the isolated peptide (29 mg, as the 8 trifluoroacetic acid salt}. A mixture of intermediated (10 mg, 1.91 pmo!) and intermediate 6 (JenKem Technology USA inc., 76 mg, 1 ,91 μηιοΙ) in 5 mi of 1X PBS buffer at pH 7.4 was stirred overnight, during which time the reaction became homogenous. The reaction was then diluted with 5 ml of 20% MeOH in 0 mM aqueous HCI and purified by ion exchange chromatography (Sepharose FF Media , 0 - 60% 1 M NaCI in 20% methanol/10 mM aqueous HCI over 7 column volumes, flow rate 5 mL/min, A - 254 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine Desalting column, 0.1 M acetic acid, λ - 254 nm) to afford a white solid after iyophiiization. The molecular mass of the isolated peptide was confirmed fay positive fragment ion distribution with the apex at 44346 amu (MALDI). Example 38 (27 mg, as the 8 acetic acid salt) gave a retention time equal to 12.19 min using size exclusion HPLC (Phenomenex BioSep-SEC-S3Q00 column, 7.8 x 300 mm, 5 μηι, 0.15 mM NaCI in 30 mM PBS over 20 min, pH 6.8, flow rate 0.75 mL/min, λ - 215 nm).

Example 39

NO:42) Intermediate 7 was prepared on a 40 μ;ηοΙ scale as a white solid using the general method, except the lysine at position 8 of the peptide was protected with an ivDde group, while proline at position 1 was protected with a Boc. After the coupling of the last amino acid (proline 1 ), the ivDde was removed with repeated treatments of 4% aqueous hydrazine in DMF and Fmoc-Gly-OH and Fmoc-Cys(Trt)-OH were coupled. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1433 amu and (M+4)/4 - 1075 amu, which corresponds to a peptide with the parent molecular weight of 4298 amu (ESI-MS, LC/MS Method A). A purify of >90% was determined by LC/MS (LC/MS Method A, rt = 13.68 min) for the isolated peptide (28.4 mg, as the 8 tiifluoroacetic acid salt).

A mixture of Intermediate 7 (10.1 mg, 1.94 μιτιοΙ) and intermediate 2 (JenKem

Technology USA Inc., 78 mg, 1.94 mol) in 10 ml of 1X PBS buffer at pH 7.4 was stirred overnight. The reaction was then diluted with 10 ml of a solution of 20% MeOH in 10 mM aqueous HCI and purified by ion exchange chromatography (Sepharose FF Media, 0 - 80% 1 M NaCI in 20% methanol/ϊθ mM aqueous HCI over 7 column volumes, flow rate 5 mL/min, A - 215 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine, 50 x 130 mm column, 0.1 Macetic acid, λ - 254 nm) to afford a white solid after lyophilization. The molecular mass of the isolated peptide was confirmed by positive fragment io distribution with the apex at 44384 arau (MALDI). Example 39 (26.7 mg, as the 8 acetic acid salt) gave a retention time equal to 12.30 min using size exclusion HPLC (Phenomenex BioSep-SEOSSQOQ column, 7.8 x 300 mm, 5 pm, 0.15 mM NaCI in 30 mM PBS over 20 min, pH 6.8, flow rate 0.75 mL/rnin, λ - 215 nm), and a retention time equal to 12.31 min by G18 HPLC (C18 HPLC Method A).

Example 40

(SEQ ID

NO:43V, intermediate 8 was prepared on a 40 pmol scale as a white solid using the general method, except the lysine at position 8 of the peptide was protected with an ivDde group, while proiine at position 1 was protected with a Boc. After the coupling of the last amino acid (proline 1), the tvOde was removed with repeated treatments of 4% aqueous hydrazine in DMF and Fmoc-Giy-OH and Fmoc-Cys(Trt)-OH were coupled. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 ~ 1440 amu and (M+4)/4 - 1080 amu, which corresponds to a peptide with the parent molecular weight of 4318 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by LC/MS (LC/MS Method A, rt ~ 3.16 min) for the isolated peptide (39 mg, as the 9 trifiuoroacetic acid salt).

A mixture of Intermediate 8 ( 0.43 mg, 1.95 prnoi) and Intermediate 2 (JenKem Technology USA inc., 86 mg, 2.15 pmol) in 10 mL of 1X PBS buffer at pH 7.4 was stirred for 2 h. The reaction was then diluted with 10 mL of a solution of 20% MeOH in 0 mM aqueous HCI and purified by ion exchange chromatography (Sepharose FF Media, 0 - 60% 1 M NaCI in 20% methanoi/10 mM aqueous HCI over 7 column volumes, flow rate 5 mL/min, λ - 215 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine, 50 x 130 mm column, 0,1 M acetic acid, λ - 25 nm) to afford a white solid after lyophilization. The molecular mass of the isolated peptide was confirmed by positive fragment ion distribution with the apex at 44514 amu (MALDI). Example 40 (35 mg, as the 9 acetic acid salt) gave a retention time equal to 14.90 min using size exclusion HPLC (Phenomenex BioSep-SEC-S3000 column, 7.8 x 300 mm, 5 μιτι, 0.15 m NaCI in 30 mM PBS over 20 min, pH 6.8, flow rate 0,75 mL/min, λ - 215 nm), and a retention time equal to 12.08 min by C18 HPLC (C18 HPLC Method A). Example 41.

ID NO:44);

Intermediate 9 was prepared on a 40 μητοί scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1407 amu and (M+4)/4 - 056 amu, which corresponds to a peptide with the parent molecular weight of 4220 amu (ESI-MS, LC/MS Method A). A purity of >90% was determined by C18 HPLC (C18 HPLC Method A, rt = 8.98 min) for the isolated peptide (30 mg, as the 8 trifluoroacetic acid salt).

A mixture of intermediate 9 (13.2 mg, 2,57 mol) and Intermediate 2 (JenKem Technology USA Inc., 3 mg, 2.83 μιηοΙ) in 5 mL of ΊΧ PBS buffer at pH 7.4 was shaken for 1 h, during which time the reaction became homogenous. The reaction was then diluted with 5 mL of 20% MeOH in 10 mM aqueous HCi and purified by ion exchange chromatography (Sepharose FF Media, 0 - 80% 1 M NaCI in 20% methanol/10 mM aqueous HCI over ? column volumes, flow rate 5 mL/min, λ - 254 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine Desalting column, 0,1 M acetic acid, A - 254 nm) to afford a white solid after lyophjiization. The molecular mass of the isolated peptide was confirmed by positive fragment ion distribution with the apex at 44239 amu (MALDI). Example 41 (41 mg, as the 8 acetic acid salt) gave a retention time equal to 9.20 min using size exclusion HPLC (Phenomenex BioSep-SEC-S3GGQ column, 7,8 x 300 mm, 5 prn, 0.15 mM NaCI in 30 mM PBS over 20 min, pH 6,8, flow rate 0.75 mL/min, λ - 215 nm). Example 42.

intermediate 10 was prepared on a 40 prnoi scale as a white solid using the general method, except the iysine at position 8 of the peptide was protected with an ivDde group, while proline 1 was protected with a Boc. After the coupling of the last amino acid (proline 1 ), the ivDde was removed with repeated treatments of 4% aqueous hydrazine in DMF and Trt-mercaptopropionic acid (MPA) was coupled. The molecular mass of the isolated peptide was confirmed by fragment ions (M+3)/3 - 1416 amu and (M+4)/4 - 1062 amu, which corresponds to a peptide with the parent molecular weight of 4246 amu (ESi-MS, LC/MS Method A). A purity of >90% was determined by LC/MS (LC/MS Method A, rt = 3.88 min) for the isolated peptide (22.2 mg, as the 8 trifluoroacetic acid salt).

A mixture of Intermediate 10 (10.2 mg, 1.98 pmoi) and Intermediate 2 (JenKem Technology USA Inc., 87 mg, 2.18 pmol) in 10 ml of 1X PBS buffer at pH 7.4 was stirred overnight. The reaction was then diluted with 10 mL of a solution of 20% MeOH in 10 mM aqueous HCi and purified by ion exchange chromatography (Sepharose FF Media, 0 - 60% 1 M NaCI in 20% methanol/10 mM aqueous HCi over 7 coiumn volumes, flow rate 5 mL/min, λ ~ 215 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine, 50 x 130 mm column, 0.1 M acetic acid.A - 254 nm) to afford a white solid after lyopiiilization. The molecular mass of the isolated peptide was confirmed by positive fragment ion distribution with the apex at 44392 amu ( ALDI). Example 42 (32,2 mg, as the 8 acetic acid salt) gave a retention time equal to 13,83 min using size exclusion HPLC (Phenomene BioSep-SEC-S3000 column, 7,8 x 300 mm, 5 Mm, 0.15 mM NaCI in 30 mM PBS over 20 min, pH 6.8, f!ow rate 0.75 mL/min, λ - 215 nm), and a retention time equal to 12.06 min by CI 8 HPLC (C18 HPLC Method A). xample 43.

(SEQ ID

NO:45)

Intermediate 1 1 was prepared on a 35 pmol scale as a white solid using the general method. The molecular mass of the isolated peptide was confirmed by fragment ions (SVl+3)/3 - 1378 amu and ( +4)/4 - 1034 amu, which corresponds to a peptide with the parent molecular weight of 4133 amu (ESI-MS, LC/MS Method A), A purity of >90% was determined by LC/MS (LC/MS Method A, rt = 13.77 min) for the isolated peptide (13 mg, as the 8 trif!uoroacetie acid salt).

A mixture of Intermediate 11 (9.54 mg, 1.89 mol) and intermediate 2

(JenKem Technology USA Inc., 83 mg, 2.08 umol) in 10 mL of a solution of IX PBS buffer at pH 7.4 was stirred at ambient temperature overnight. The reaction was then diluted with 10 mL of a solution of 20% MeOH in 10 m aqueous HCi and purified by ion exchange chromatography (Sepharose FF Media, 0 - 60% 1 M NaCI in 20% methanol/10 mM aqueous HCI over 7 column volumes, flow rate 5 mL/min, λ - 2 5 nm). The purified conjugate was desalted using size exclusion chromatography (Sephadex G 25 Fine, 50 x 130 mm column, 0. acetic acid, λ - 254 nm) to afford a white solid after fyophilization. The molecular mass of the isolated peptide was confirmed by positive fragment ion distribution with the apex at 44117 amu (MALD!). Example 43 (32 mg, as the 8 acetic acid sa!t) gave a retention time equal to 10.65 min using size exclusion HPLC (Phenomenex BioSep-SEC~S3000 column, 7.8 x 300 mm, 5 |.im, 0.15 mM NaCI in 30 mM PBS over 20 min, pH 6.8, flow rate 0.75 mL/min, λ - 215 nm), and a retention time equal to 12.10 min by C18 HPLC (C18 HPLC Method A).

BIOLOGICAL EXAMPLES

Potency of PYY analogs at the human neuropeptide Y receptor type 2

The relative potency of PYY analogs at the human Neuropeptide Y receptor type 2 was determined using a melanophore assay essentially as described in Jayawickreme et at, (2005) Current Protocols in Pharmacology 12.9.1-12.

Effects of PYY analogs on food intake Cumulative food intake after 6 h was determined for the PYY analogs in eithe iean {Mode! A) or diet-induced obese (DiO) (Model B) C57BL/6 mice in a BioDaQ system for continuous monitoring of food intake (Research Diets Inc., New

Brunswick, N J). Model A utilized 10 week old male C57BL/6 mice (laconic, Germantown, New York) fed a normal chow (Purina P i 5001 ) whereas Model B utilized 25 week old male C57BL/6 mice fed a 45% high fat chow for 20 weeks (Research Diets D12451 ). Mice were placed singly into the BioDaQ cages and acclimatized for a minimum of 6 days and were ailowed ad libitum access to food and water. Approximately 1 hour prior to lights-out, animals were dosed subcutaneously with either vehicle (20 mM Acetat buffer, pH 4.9 or 20% DMSO in water) or analogs dissolved in vehicle (1 mg/kg) (8 animals per group). Once all animals have been dosed, feeder gates were opened providing ad-libitum access to food. Continuous food intake was monitored and collected for 15 hours. Hourly food intake, as well as 6 and 15-hour cumulative food intake, was summarized as % inhibition relative to vehicle controls. The data were analyzed in JMP 6.0.0 (SAS institute, Gary, NC) using a pooled variance t-test ^'vs. groups treated with human PYY(3-36)NH₂. P~ values < 0.05 were considered to indicate a significant difference between treatment groups. Table 1 shows potency at the human Neuropeptide Y receptor and food intake reduction for the PYY analogs shown in Examples 1.-35.

Example 10.1 -91 <0.0001

Exam le 8 10.7 -74 <0.0001

Example 9 10.7 -86 O.00Q1

Example 10 10.4 -88 <Q.0001

Example 1 1 10.3 -86 <O.0Q01

Example 12 10.2 -88 <0.0001

Example 13 9.9 -85 <0.0001

Example 14 10.5 -89 <0.0001

Example 15 10 -90 <0.0001

Example 16 10.4 -90 <G.0001

Example 17 9.9 -90 <0.0001

Example 18 10.5 -92 <0.0001

Example 19 10.2 -89 <0.0001

Example 20 10.5 -86 <0.0001

Example 21 10.5 -90 <0.0001

Example 22 10.2 -80 <0,0001

Example 23 9.8 -86 <0.0001

Example 24 10.6 J5B 0.0035

Example 25 9.6 -68 0.0003

Example 26 9,7 -81 <0.0001

Example 27 9,8 -69 <0,0001

Example 28 10 -65 <0.0001

Example 29 9.7 -54 0.0006

Example 30 9.7 -73 <0.0001

Example 31 9.8 -59 0.0004

Example 32 10.1 -7 <0,0001

Example 33 10 -67 <0.0001

Example 34 10 -69 <Q.Q001

Example 35 10.3 -70 <0.0001

Table 2 shows examples of PYY anaiogs which have potency at the human Neuropeptide Y receptor but do not show food intake reduction greater than human PYY(3^36)NH₂ at the 6 h time point. Table 2

SEQ. ID NO hNPY reduction

Peptide Peptide Sequence Y2 in food

EC50 intake,

modei A

46

Peptide 1 P PEAPGCDASPEE NRYYASLR YLNWVT QNY- Hj 7.9 5

47

Peptide 2 i PEAPLSKQLEEEAVRYYASLRHYLNLVTRQRY-NHj 8.6 -12

48

Peptide 3 P PEAPGEDASP EWNRYYASLRKYLNVVVTRQRY-NH, 9.2 -30

49

Peptide 4 P PEHPGEDA$PEEL RYHAALRAYLNLVTRQRY-NH₂ 11.1 -26

50

Peptide 5 PKPEHPGEDASPEELNRYYAALRAYLNLVTRQKY-NH₂ 8.5 -7

51

Peptide 6 P PEHPGEDASPEELNRYYAALRAYLNLVTKQ.RY-N H₂ 9.7 -14

Peptide 7 PQPESPGC ASP£ELAI<YHAALRHYVNLiTRQRY~NH _: 10.2 -25 52

53

Peptide 8 i PPYPGCDASPEEQN YYASLRAYWNLVTRQRY-i\iH₂ 9.3 -19

54

Peptide 9 PKP ES PGS N AS P E D W A !< YQ.A AV RHYVM UTRQ.R Y~ N H ₂ 10.6 -24

Peptide 10 PEPEHPGCDASPEDQNKYHASLRKYLNWVTRQRY-NHj 9.5 -21 55

56

Peptide 11 iKPPEPGCDASPEEQN YYASLRHYWNLVTRQRY-NH₂ 9.5 -5

57

Peptide 12 iEPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH₂ 9.8 -15

Peptide 13 PKPESPGSDASPEDLAi<YHAAVRHYV UTRQRY-N¾ 10.9 -23 58

Peptide 14 P PEAPGCDASPEE NRYYASLRKYL WVTRQHY-iMH₂ 8.2 2 59

Peptide 15 P PVAPGCDASPAELNRQYSDLRNYWNLVTRQRY-NH, 8.9 -17 60

61

Peptide 16 iQPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NHj 10 -32

Peptide 17 PKPESPGKDASPEDLA YHAAVRHYVNLITRQRY-NHj 11 -36 62

Peptide IS PQPESPEGNASPEDWACYHAAVRHYVNLiTRQRY-NH₂ 9.7 63

-17

Peptide 19 iHPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH₂ 10 -26 64

65

Peptide 20 ! PEAPGEDASPEQLMAQYASLRHYL!MLVTRQ.RY-NH₂ 9.9 -16

Peptide 21 PKPEAPLSKQLEEEAVRYYASLRHYLNLVTRQRY-NH 8.7 -2 66

67

Peptide 22 PKPEAPGCDASPEELNRYQASLRHYLi\!LVTRQRY-NH₂ 10.3 -16

Effects of Example 5 in combination with Exendin-4 on body weight, body

composition and food intake reduction

A chronic (41 days) in vivo efficacy study was conducted in a rodent model for

obesity (diet- induced obese (DIO) Long Evans rat) to investigate the efficacy and durability of Example 5 sing!y and in combination with exendin-4 as anti-obesity

agents.

Male Diet-Induced Obese (DiO) Long Evans (LE) rats were used (Harlan

Laboratories, Inc., Indianapolis, IN) and beginning at weaning (about 3 weeks of age), the rats were fed a high fat chow (Tekiad TD 95217, 40% kcal from fat, Harlan Laboratories, Madison, Wl). Rats were 17 weeks old at the start of the study. The rats were housed 1 per cage and given ad libitum access to TD.95217 chow and water, maintained on a 12 h light/dark cycle from 5:00 AM to 5:00 P at 21 °C and 50% relative humidity and allowed to acclimate for at least 7 days prior to baseline measurements. Baseline fat mass and non-fat mass measurements were taken 3 days before the start of peptide infusion and on day 40 of treatment using a OMR instrument (Echo Medical Systems, Houston, TX). Rats were randomized according to their percent body fat mass Into 6 groups: (1) vehicle (sterile water, n=8), {2} Exendin-4 (ED₅₀=0.15 mg/kg/day, n=8), (3) Example 5 (EDso-Q.OSmg/kg/day, n~8), (4) PYY(3-36)NH₂ (1.5 mg/kg/day, n=8), (5) Exendin--4+ Example 5 (h=8) and (6) Exendin-4+ PYY(3-36)NH₂ (n=8). AIZET© mini-osmotic pumps (6 week; Model 2006, Durect Corporation, Cupertino, CA) were filled under sterile condition with either vehicle or peptide one day prior to the surgery. Each rat was implanted with two osmotic pumps subcutaneous!y In the scapula region containing vehicle or peptide according to their treatment group. Body weight and food intake were measured twice per week beginning three days before the 41 -day treatment period. On day 41 of treatment, whole biood was collected by cardiac stick under isoflurane anesthesia. Plasma and serum were then prepared from the whole blood for serum chemistry analysis. All the data are presented as mean ± SEM. The data were analyzed in either Prism (GraphPad Software, Inc., La Joiia, CA) or Excel using a Student's T- test to compare each group to the appropriate control group. P-values < 0.05 were considered to indicate a significant difference between treatment groups.

Ail procedures were performed in compliance with the Animal Welfare Act, USDA regulations and approved by the GiaxoSmithKline Institutional Anima! Care and Use Committee.

In DIG rats, administration of Example 5 at the EDso for weight loss for 40 days resulted in -6.1% (p<0.05) weight loss whereas native PYY(3-36)NH₂ at the EDso resulted in -1.3% (p~0.46) weight loss vs, vehicle (Figure 1). The combination of Example 5 and exendin-4 at combo ED₅₀ doses for 40 days resulted in sustained and significant weight loss of -30.9% vs. vehicle (p<0.05), which far exceeded the expected additive effect based on weight loss of exendin-4 and Example 5 when administered alone (-11.3% and -6.1 %, respectively, with a projected additive weight loss of -17.4%). Whereas, native PYY{3-36)NH₂ in combination with exendin-4 resulted in -10.2% weight loss vs. vehicle which was sub-additive based on weight loss of exendin-4 and PYY(3-36)NH₂when administered alone (-1 1.3% and -1.3%, with a projected additive weight loss of -12.6%). Changes in body composition were primarily driven by loss of body fat mass, with some changes in non-fat mass and mirrored the body weight changes in all treatment groups (Figure 2). Specifically, the animals treated with Exampie 5 lost - 34.2 grams of fat mass from vehicle control (p<0.G5), the PYY(3~36)iMH₂ animals lost -10.9 grams fat mass (p=0.12 vs. vehicle control) and animals treated with exendin-4 lost -55.8 grams fat mass (p<0.05 vs. vehicle control) during the treatment period. The Exampie 5 + exendin-4 combination had a more than additive effect on fat mass where the combination lost -110.1 grams (p<0.05 vs. vehicle control), which was significantly greater than the predicted additivity value of -90 grams (p<0.05) (Figure 3). i contrast, PYY(3~38)NH₂ in combination with exendin-4 resulted in -54.0 grams fat mass loss vs. vehicle (p<0.0^'5) which was less than the predicted additivity value of -86.7 grams.

In addition, a -57.1% inhibition of cumulative food intake (p<0.05 vs. vehicle control) was observed when Example 5 was co-administered with exendin-4 compared with -18.8% inhibition (p-0.87 vs. vehicle control) with the PYY(3-36)NH₂ + exendin-4 combination. There appears to be a more than additive efficacy with the Example 5 + exendin-4 combination based upon the food intake inhibition of each peptide administered alone (- 1 ,5% and -20.1 %, respectively, with a projected additive food intake inhibition of -3 .6%). in contrast, the native PYY(3-36)NH₂ ⁺ exendin-4 combination resulted in sub-additive food intake inhibition based upon the food intake inhibition of each peptide administered alone (-0.7% for PYY(3-36)NH₂ and -20.1 % for exendin-4, with a projected additive food intake inhibition of -20.8%).

Example 23 in Combination with exendin-4 causes more than additive effects on glucose parameters In Diabetic ZDF rats

A chronic (26 days) in vivo efficacy study was conducted in a rodent model for diabetes (Zueker Diabetic Fatty (ZDF) rat) to investigate the efficacy and durability of Exampie 23 singly and in combination with exendin-4 as anti-diabetes agents.

Male ZDF rats were 12 weeks old at the start of the study (Charles River, Inc., Boston, MA). The ZDF rats were housed 1 per cage and given ad libitum access to diet (Purina PMl 5008) and water, maintained on a 1 hr light/dark cycle from 5:00 AM to 5:00 PM at 2 Γ C and 50% relative humidity and allowed to acclimate for at least 6 days prior to baseline measurements and 10 days prior to the surgeries, Baseline fat mass and non-fat mass measurements were taken 3 days before the start of peptide infusion and on day 26 of treatment using a OMR instrument (Echo Medical Systems, Houston, TX). Blood samples were taken via tail snip to measure feci glucose values and % HbA1c values two days before the start of drug dosing; this data was used to randomize the animals into 7 groups: (1 ) Lean vehicle control (sterile phosphate buffered saline (PBS), pH 4,9, n=8), (2) ZDF vehicle control (sterile PBS, pH 4,9, n=8), (3) Exendin-4 {ED₂₀=Q.0Q55 mg/kg/day, n=8), (4) Example 23 (ED₂o=0.02mg/kg/day, n-8), (5) PYY(3-36)NH₂ (0.02 mg/kg/day, n=8), (6) Exendin-4+Example 23 (n=4) and (7) Exendin-4+ PYY(3-36)NH₂ (n=8), ALZET® mini-osmotic pumps (4-week; Model 2006, Durect Corporation, Cupertino, CA) were filled under sterile condition with either vehicle or peptide one day prior to the surgery. Similar surgical implantation of the mini-pumps was performed as described for the DIG rats above (except animals were injected ID with !idocaine (0.1 m.L of 0, 125% !idocaine). Body weight and food intake were measured twice per week beginning 3 days before the 26-day treatment period. On day 26 of treatment, whole blood was collected by cardiac stick under isoflurane anesthesia. The whole blood was used to determine the % HbA1 c and the serum was used to measure giucose. The data were analyzed in either Prism (GraphPad Software, inc., La Jolla, CA) or Excel using a Student's T-test to compare each group to the appropriate control group. P-values < 0.05 were considered to indicate a significant difference between treatment groups.

Ail procedures were performed in compliance with the Animal Welfare Act, USDA regulations and approved by the GlaxoSmithKline Institutional Animal Care and Use Committee.

Table 3 shows the glucose and glycosylated HbA1 c changes from baseline and from vehicle control ZDF animals (ΔΔ) following chronic treatment (26 days) with Example 23, PYY(3-36)NH₂,or exendin-4 singly or in combination. Singly, only the exendin-4 and Example 23 achieved statistically significant glucose lowering from vehicle control (ΔΑ -53.9 and -54.5 mg/dL, respectively; p<0.05) compared to PYY(3- 36)NH₂ (ΔΔ-33.1 ; p=0.1 1). Treatment with the combination of Example 23 and exendin-4 combo at ED₂₀ doses for HbA1 c lowering for 26 days resulted in significant glucose lowering AA - 52.3 mg/dL (pO.05 vs. vehicle control), which exceeded the expected additive effect based on glucose lowering of exendin-4 and Example 23 when administered alone (ΔΔ -53.9 and -54.5 mg/dL, with a projected additive giucose lowering of -108,4 mg/dL vs. vehicle control). The AA%HbA1 c levels closely mirrored the glucose changes in all treatment groups, however, none of the groups were deemed statistically significant. Table 3. Changes in glucose and HbA c after 26 days of treatment with Example 23 and/or exendin-4 singly and in combination in ZDF rats Treatment G rou p AAGlucose ΔΔ %HbA1c

Exendin-4 -53.9 ± 18.3 -0.4 ± 0.2

Example 23 -54.5 ± 13.7 -0.4 + 0.2

PYY(3~36)NH₂ -33.1 ± 17 0.01 ± 0.4

Ex-4 + Exampie 23 -152.3 ± 91.5 -1.3 + 0.8

Ex-4 + PYY(3-36)NH₂ -17.6 + 24.4 -0,4 ± 0.3

ΔΔ-Cbanqe in parameter from baseline and vehicle control Bold- p 0.05 from vehicle control

Claims

That which is claimed:

1 , A polypeptide comprising the amino acid sequence:

ProLysProGiuXaa₁PfoeiyXaa₂AspA!aSerXaa₃GiuG[uXaa₄Xaa_sXaa_sTyrTyrAla

Xaa₇LeuArgXaa₈TyrXaa₉ AsnTrpXaa₁₀ThrArgGlnArgTyr (SEQ ID NO.:1)

or a salt thereof, wherein:

Xaa_! is Ala, His, or Ser;

Xaa₂ is Glu or Lys;

Xaa₃ is Pro or Ala;

Xaa is Leu or Trp;

Xaa_s is Asn, Ala, or Thr;

Xaa_i3 is Arg or Lys;

Xaa₇ is Ser, Asp, or Aia;

Xaa₈ is His or Lys;

Xaa₈ is Leu or lie; and

2. A polypeptide consisting of the amino acid sequence;

ProLysProGlLiXaa-i ProGiyXaazAspAiaSerXaaaGSuGlu Xaa₄Xaa₅

Xaa_sTyrTyrAlaXaa₇LeuArg Xaajyr Xaa₉ AsnTrp Xaa^.ThrArgGinArgTyr -HH₂ (SEQ ID NQ:2) or a salt thereof, wherein:

Xaa is Ala, His, or Ser;

Xaa₂ is Giu or Lys;

Xaa₃ is Pro or Ala;

Xaa« is Leu or Trp;

Xaa_s is Asn, Ala, or Thr;

Xaa_s is Arg or Lys;

Xaa-_/ is Ser, Asp, or Ala;

Xaa₈ is His or Lys;

Xaa_s is Leu or lie; and

Xaaio is Val or Leu.

3. The polypeptide of claim 2 which is selected from the group consisting of. ProLysProGiuAiaProGlyLysAspAlaSerProGluGluLeuAsnArgTyrTyrAla

SerLeuArgHisTyrLeuAsnTrpVa!ThrArgG!nArgTyr-NHz (SEQ ID NO:3),

ProLysProGiuAlaPi GIyLysAspAiaSerProGluGluLeuAsnArgTyrTyrAla

SerLeuArgLysTyrLeuAsnTrpLeuThrArgGlnArgTyr-NH₂ (SEQ iD NO:4),

ProLysProGluAlaProGiyLysAspAlaSerProGluG!uLeuAsnArgTyrTyrA!a

SerLeuArgHisTyrLeuAsnTrpLeuThrArgGinArgTyr-NH₂ (SEQ ID NO:5),

ProLysProGluAlaProGiyLysAspAiaSerProGiuGiuTrpAsnArgTyrTyrAla

AspLeuArgLysTyrLeuAsnTrpLeuThrArgGinArgTyr--NH₂ (SEQ ID NO:6), ProLysProGluAlaProGlyLysAspAlaSerProG!uG!uTrpAsnArgTyrTyrAia

AspLeuArgHisTyrLeuAsnTrpLeuThrArgGinArgTyr-NH₂ (SEQ iD NO:7),

ProLysProGiuA!aProGlyLysAspAiaSerProGluGiuTrpAsnArgTyrTyrAla

SerLeuArgLysTyrLeuAsnTrpLeuThrArgGinArgTyr-NH₂ (SEQ I DNO;8),

ProLysProGluA!aProGlyLysAspAlaSerProG!uGluTrpAsnArgTyrTyrAla

SerLeuArgHisTyrLeuAsnTrpLeuThrArgGlnArgTyr-NH₂ (SEQ ID NO:9),

ProLysProGluAlaProGiyLysAspAiaSerProGluGiuTrpAsnArgTyrTyrAla

AspLeuArgLysTyrLeuAsnTrpVaiThrArgGlnArgTyr-NH₂ (SEQ iD NO: 10), ProLysProG!uAiaProG!yLysAspAlaSerProGluGluTrpAsnArgTyrTyfAla

AspLeuArgHisTyrLeuAsnTrpValThrArgGlnArgTyr-NH₂ (SEQ ID NO:11), ProLysProGluAlaProGlyLysAspA!aSerProGluGiuTrpAsnArgTyrTyrAla

SerLeuArgLysTyrLeuAsnTrpVaiThrArgGinArgTyr--NH₂ (SEQ ID NO:12)_> ProLysProGluAlaProGlyLysAspAiaSerProGiuGiuTrpAsnArgTyrTyrAla

SerLeuArgHisTyrLeuAsriTrpVa[ThrArgGinArgTyr-NH₂ (SEQ ID NO:13), ProLysProGiuAfaProGiyGluAspAiaSerProG!uGluLeuAsnArgTyrTyrAla

SerLeuArgHisTyrLeuAsnTrpValThrArgGlnArgTyr-NH₂ (SEQ ID NO: 14), ProLysProGiuHisProGlyLysAspAlaSerProGluGluTrpAsnArgTyrTyrA!a

AiaLeuArgLysTyrLeuAsnTrpYalThrArgGinArgTyr-NH₂ (SEQ ID NO: 15), ProLysProGluHisProGiyLysAspAlaSerProGluGiuLeuAsnLysTyrTyrAla

AlaLeuArgHisTyrLeuAsnTrpValThrArgGlnArgTyr-NHz (SEQ ID NO: 16),

ProLysProGiuHisProGlyLysAspAlaSerProGiuGiuLeuAsnArgTyrTyrAla

SerLeuArgHisTyrl!eAsnTrpValThrAi-gGlnArgTyr- Hs (SEQ !D NO: 17),

ProLysProGluHisProGlyLysAspAiaSerProGiuGiuLeuAiaArgTyrTyrAla

SerLeuArgHisTyrLeuAsnTrpVa!ThrArgGinArgTyr-NH₂ (SEQ ID NO: 18), ProLysPt G!uHisProGiyLysAspAlaSerProGluGiuTrpAsnArgTyrTyrAla SerLeuArgH!sTyfi!eAsnTrpVaIThrArgGlnArgTyr-NH₂ (SEQ ID NO: 19), ProLysProGluHisProGSyLysAspAiaSerProGiuGluTrpAsnArgTyrTyfAia AspLeuArgHisTyr!leAsnTrpVaiThrArgGlnArgTyr-NH₂ (SEQ ID NQ:20), ProLysProGluHisProGlyLysAspAiaSerProGluGluTrpAsnArgTyrTyrAla AspLeuArgHisTyrLeuAsnTrpValThrArgGlnArgTyr-~NH₂ (SEQ ID MQ:21), ProLysPiOGiuSerProGlyLysAspA!aSerProGSuG!uTrpAsnArgTyrTyrAia AspLeuArgHisTyrSSeAsnTrpVa!T rArgGinArgTyr~NH₂ (SEQ ID NQ:22), ProLysProGluSerProGlyLysAspAlaSerProGluGiuTrpAsnArgTyrTyrA!a AspLeyArgHisTyrLeuAsnTrpVaiThrArgGlnArgTyr-NH₂ (SEQ ID NO:23), ProLysProGiuHfeProGlyLysAspAlaSerProGluGluTrpAsnArgTyrTyrAia AspLeuAi-gHisTyrLeuAsnTrpLeuThrArgGSnArgTyr~NH₂ (SEQ ID NG:24}, ProLysProGiuHisProGlyLysAspAiaSerProGluGluTrpAiaLysTy^'rTyrAia AlaLeuArgHfsTyrlleAsnTrpValThrAigGlnArgTyr-NH₂ (SEQ ID N0.25), ProLysProGiuA!aProG!yLysAspAlaSerProGluGluTrpAsnArgTyrTyrAla AspLeuArgHisTyrlieAsriTrpVaSThrArgGinArgTyr~NH₂ (SEQ ID NO:26), ProLysProGluHisProGlyLysAspAlaSerProGluGluTrpAsnArgTyrTyrAla SerLeuArgLysTyrLeuAsnTrpVaiThrArgG!nArgTyr~NH₂ (SEQ !D NO:27), PiOLysProGIuHssProGlyLysAspAlaSerAlaGiuGluTrpAlaLysTyrTyrAia AlaLeuArgHisTyrSleAsnTrpVaiT rArgGinArgTyr-NH₂ (SEQ ID NO;28), ProLysProGluAlaProGlyLysAspAlaSerAlaGluGSuTrpAsnArgTyrTyrAla SerLeuArgHisTyrLeuAsnTrpVaiThrArgGlnArgTyr~NH₂ (SEQ ID NO:29), PioLysProGluHisProGlyLysAspAlaSerAlaGiuGluLeuAlaArgTyrTyrAla SerLeuArgH^'!sTyrLeuAsnTrpValThrArgGlnArgTyr-NH₂ (SEQ ID WO:30), ProLysProGiuAlaProGlyLysAspAlaSerAlaGluGluTrpAsnArgTyrTyrASa SerleuArgLysTyrLeuAsnTrpValThrArgGlnArgTyr-NHs (SEQ ID NG:31), ProLysProGluSerProGlyLysAspAlaSerA!aGiuGluTrpThrLysTyrTyrAla AlaLeuArgHisTyrlleAsnTrpVaiT rArgGinArgTyr~NH₂ (SEQ ID NO:32), ProLysProGiuAiaProGiyLysAspAlaSerProG!yGSuLeuAsnArgTyrTyrAia SerLeuArgLysTyrLeuAsnTrpValThrArgGlnArgTyr--NH₂ (SEQ ID NG:33), ProLysProGluHisProGlyGluAspAlaSerProGluGluTrpAlaLysTyrTyrASa AlaLeuArgHisTyrlleAsnTrpValThrArgG!nArgTyi-NH₂ (SEQ ID NO:34), ProLysProGiuAlaProGiyGluAspA!aSerAlaGluGluTrpAsnArgTyrTyi-Aia SerLeuArgHisTyrLeuAsnTrpValThrArgGSnArgTyr-NH₂ (SEQ ID NG:35), ProLysProGluSerProGlyGluAspAlaSerProGluGiuTrpThrLysTyrTyrAla AlaLeuArgHisTyr!ieAshTrpVaiThrArgGinArgTyr-NH2 (SEQ ID NO; 36), and

ProLysProGluAlaProG!yGluAspAlaSerProGluGluTrpAsnArgTyrTyrAla

AspLeuArgHisTyrLeuAsnTrpLeuThrArgGlnArgTyr-NH₂ (SEQ ID NO;37).

4. The polypeptide of claim 1 which comprises the amino acid sequence:

ProLysProGiuA!aProGlyLysAspAiaSerPf GluGluTrpAsnArgTyrTyrAla

AspLeuArgHisTyrLeuAsnTrpLeuThrArgGlnArgTyr (SEQ !DNO:38).

5. The polypeptide of claim 1 which consists of the amino acid sequence:

ProLysProGiuAlaProGlyLysAspAiaSerProGluGluTrpAsnArgTyrTyrAla

AspLeuArgHisTyrLeuAsnTrpLeuThrArgGlnArgTyr (SEQ IDNO:38).

6. The polypeptide of claim 2 which consists of the amino acid sequence:

ProLysProGiuAiaProGlyLysAspAlaSerProG!uGiuTrpAsnArgTyrTyrAia

AspLeuArgHisTyrLeuAsnTrpLeuThrArgGlnArgTyr--NH2 (SEQ ID NO:7).

7. A pharmaceutical combination comprising a polypeptide according to any one of cialms 1-6 and exendin-4.

8. A pharmaceutical combination comprising; a polypeptide according to any one of claims 1-6 and GLP-1 ,

9. A pharmaceutical composition comprising a polypeptide according to any one of claims 1-6 and a pharmaceutically acceptable carrier,

10. A method of treating a metabolic disorder in a human subject, said method comprising administering a polypeptide or pharmaceutica! combination of any one of claims 1-8 to a subject in need thereof.

11. A method of treating obesity in a human subject, said method comprising administering a polypeptide or pharmaceutica! combination of any one of claims 1-8 to a su ject in need thereof.

12. Use of a polypeptide or pharmaceutical combination of any one of ciaims 1-8 in the preparation of a medicament for use in the treatment of a metabolic disorder.

13. Use of a polypeptide or pharmaceutical combination of any one of claims 1-8 in the preparation of a medicament for use in the treatment of obesity

14. A polypeptide or pharmaceutical combination according to any one of claims 1-8 for use in the treatment of a metabolic disorder. 5. A polypeptide or pharmaceutical combination according to any one of ciaims 1-8 for use in the treatment of obesity. 6. The method or use of any one of claims 10, 12, or 14 where the metabolic disorder is Type 2 Diabetes Meilitus.

17. A nucleic acid molecule encoding a polypeptide sequence according to any one of claims 1-8.

18. An expression vector comprising a nucleic acid molecule according to claim 17. 9. A host cell containing an expression vector according to claim 18.