(12) Patent Application Publication (10) Pub. No.: US 2009/0036364 A1 Levy Et Al

US 20090036364A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0036364 A1 Levy et al. (43) Pub. Date: Feb. 5, 2009

(54) GIPANALOG AND HYBRID POLYPEPTIDES (86). PCT No.: PCT/US2O06/OOSO2O WITH SELECTABLE PROPERTIES S371 (c)(1), (75) Inventors: Odile Esther Levy, San Diego, CA (2), (4) Date: May 21, 2008 (US); Alain D. Baron, San Diego, Related U.S. Application Data CA (US); Lawrence J. D’Souza, San Diego, CA (US); Mary (60) Provisional application No. 60/652,662, filed on Feb. Erickson, San Diego, CA (US); 11, 2005, provisional application No. 60/651,647, Soumitra S. Ghosh, San Diego, CA filed on Feb. 11, 2005, provisional application No. (US); Michael R. Hanley, San 60/653,433, filed on Feb. 15, 2005, provisional appli Diego, CA (US); Samuel Janssen, cation No. 60/707,244, filed on Aug. 11, 2005, provi San Diego, CA (US); Carolyn M. sional application No. 60/707,369, filed on Aug. 11, Jodka, San Diego, CA (US); Diana 2005, provisional application No. 60/709,320, filed on Y. Lewis, San Diego, CA (US); Aug. 17, 2005, provisional application No. 60/709, Christine M. Mack, San Diego, 316, filed on Aug. 17, 2005. CA (US); David G. Parkes, San Diego, CA (US); Richard A. Publication Classification Pittner, San Diego, CA (US); (51) Int. Cl. Christopher J. Soares, San Diego, A638/22 (2006.01) CA (US); Ved Srivastava, San C07K I4/47 (2006.01) Diego, CA (US); Andrew A. A6IP3/10 (2006.01) Young, San Diego, CA (US) (52) U.S. Cl...... 514/12:530/324 Correspondence Address: (57) ABSTRACT Intellectual Property Department The present invention relates generally to novel GIP analogs Amylin Pharmaceuticals, Inc. and GIP hybrid polypeptides with selectable properties, use 9360 Towne Centre Drive ful as agents for the treatment and prevention of metabolic San Diego, CA 92121 (US) diseases and disorders, for example those which can be alle viated by control plasma glucose levels, insulin levels, and/or (73) Assignee: Amylin Pharmaceuticals, Inc., San insulin secretion, positive inotropic effects, reduction of cata Diego, CA (US) bolic effects, slowing of gastric emptying. Such conditions and disorders include, but are not limited to, hypertension, (21) Appl. No.: 11/816,095 dyslipidemia, cardiovascular disease, eating disorders, criti cal care, insulin-resistance, obesity, and diabetes mellitus of (22) PCT Filed: Feb. 10, 2006 any kind, including type 1, type 2, and gestational diabetes.

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Fig. 13A Effect of Compounds of the Invention in Food Intake Assay

Food intake Assay: Gumulative food intake - E. Vehicle -3 Cpl. 25mmol/kg

25rmcl.g

25rmolikg

-- Cmpd 10 25mmotg

Patent Application Publication Feb. 5, 2009 Sheet 63 of 78 US 2009/0036364 A1

Fig. 13B Effect of Compounds of the Invention on Blood Glucose (Oral Glucose Tolerance Test)

Glucos e OGTT 25 Wehicle n-1 Cmpd4 0.1mg/kg n=5

2) H. Cmpdf 35 nmolkg n=8 III). Cmpd2 350mmol/kg n=8 Cmptl3 35 nmol/kg n=8 5 ; 3.% O) 2.3. 8 3 % 2 5 3.3E. %

O B Y

Patent Application Publication Feb. 5, 2009 Sheet 64 of 78 US 2009/0036364 A1

Fig. 14 Effect of Compounds of the Invention in Food Intake Assay Food Intake Assay: Cumulative food intake

O 60 120 18O minutes

Figure Legend: Vehicle A Cmpd 2 3 nmol/kg Cmpd 2 10 nmol/kg V Cmpd 2 30 nmol/kg V Cmpd 2 100 nmol/kg O Cmpd 2 300 nmol/kg Patent Application Publication Feb. 5, 2009 Sheet 65 of 78 US 2009/0036364 A1

Fig. 15 Effect of Compounds of the Invention in Food Intake Assay Food intake Assay: Cumulative food intake

-T-T-T O 60 120 180 minutes

Figure Legend: Vehicle O Cmpd3 3nmol/kg A Cmpd 3 10nmol/kg sk Cmpd 3 30nmol/kg 0 Cmpd3 100nmol/kg - Cmpd 3 300nmol/kg Patent Application Publication Feb. 5, 2009 Sheet 66 of 78 US 2009/0036364A1

Fig. 16A

Effect of Compound 10 in Food Intake Assay Food Intake Assay: Cumulative food intake 9

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O 120 180 minutes Figure Legend: Vehicle O Compound 10 25nmol/kg Patent Application Publication Feb. 5, 2009 Sheet 67 of 78 US 2009/0036364 A1

Fig. 16B

Effect of sGT (amide form) in Food Intake Assay Food intake Assay: cumulative food intake

O 4. O O 3 S. 2

O 6 120 18O minutes Figure Legend: Vehicle O scT 25nmol/kg Patent Application Publication Feb. 5, 2009 Sheet 68 of 78 US 2009/0036364 A1

Fig. 17

Effect of Compounds of the Invention on Blood Glucose Glucose Assay: Change in blood glucose 150

125

100- C

| 75

mean pre-treatment glucose 125 mg/dL O 60 120 180 240 minutes

Figure Legend: Vehicle O Compound 10 160 nmol/kg A Compound 3 160 nmol/kg Compound 2 160 nmol/kg A Compound 1 160 nmol/kg Patent Application Publication Feb. 5, 2009 Sheet 69 of 78 US 2009/0036364 A1

Fig. 18A

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Fig. 18B EC50=11.7462(M) GIP Hybrid

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O. O O. 1. 1OOO Concentration?nM) Patent Application Publication Feb. 5, 2009 Sheet 70 of 78 US 2009/0036364A1

Fig. 19A

4) lea Aterial Pressure

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20 --vehicle r=11 - GIP 8Ohmokg n=5 -C- Compound G 80nmol/kg = 9 -4 maal t Sl -----Mrm-- O O 3. SO inutes after flous Patent Application Publication Feb. 5, 2009 Sheet 73 of 78 US 2009/0036364 A1

Fig. 19D

Systolic Pressure 40

AA R O 5uititR :S

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Fig. 19E

Diastolic Pressure

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Fig. 20A Food Intake Assays

--Wehicle - A - GP 1000pg/kg 5 -o-ramylin 100g/kg

-The O 6 20 minutes

Fig. 20B

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3 2 -W-Exendin-440-o-Exendin-4400g/kg g/kg

O 30 60 90 120 Minutes Patent Application Publication Feb. 5, 2009 Sheet 76 of 78 US 2009/0036364 A1

Fig. 21 Weight Loss in DIO Mice

s d5 s S 1 E 9. S e O 0 8 S HF Saline OLF Saline GP 75nmol/kg/d Ex-42.5nmol/kg/d -1 1 2 Week of treatment Patent Application Publication Feb. 5, 2009 Sheet 77 of 78 US 2009/0036364 A1

Fig. 22 Mammalian and Non-Mammalian GIPAlignment

(1 SEQ ID NO: BOVINE GIBO (1 13 COW XP 588333 (1 420 MOUSE NP O32145 (1 10 PIG 2 GIP AY609506 (1 12 CHICKEN XP 418111 (1 300 Xenopus Trop GIP (1 301 ZEBRAFISH POTENTIAL GIP (1 302 MONKEY GIP (1 303 Opussum GIP peptide (1 304 HUMAN NP OO4114 (1 2 HAMSTER GDIP TRANSLATION (1 305 NP 062604 RAT GIP (1) : V 11 Consensus (1) YAEGTFTSDYSIAMDKIRQQDFVN WLLAQKGKKS DWKHNITO 306 sy sy y: 'sN 's' & & S.

Positions. Y1, E3, D9, S11, D15, F22, V23, L26, L27, and K32 are conserved across all species. Patent Application Publication Feb. 5, 2009 Sheet 78 of 78 US 2009/0036364 A1

Fig. 23A

E inhibition of G as tric E in pty ing 88Wehicle 8 Compd 3 60nmokg Ed halP 1 (Onimo'kg S III Cmpd to 6nmokg

Fig. 23B

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GPANALOG AND HYBRD POLYPEPTDES incretin mimetics). GIP and GLP-1 both belong to the gluca WITH SELECTABLE PROPERTIES gon peptide Superfamily and thus share amino acid sequence homology. GIP and GLP-1 are secreted by specialized cells in RELATED APPLICATIONS the gastrointestinal tract and have receptors located on islet 0001. The present application claims priority to com cells as well as other tissues. As incretins, both are secreted monly-owned and pending U.S. provisional applications: from the intestine in response to ingestion of nutrients, which U.S. Provisional Application No. 60/652,662 filed Feb. 11, results in enhanced insulin secretion. The insulinotropic 2005; U.S. Provisional Application No. 60/653,433 filed Feb. effect of GIP and GLP-1 is dependent on elevations in ambi 15, 2005: U.S. Provisional Application No. 60/651,647 filed ent glucose. Both are rapidly inactivated by the ubiquitous Feb. 11, 2005: U.S. Provisional Application No. 60/707,244 enzyme dipeptidyl peptidase IV (DPP-IV). filed Aug. 11, 2005: U.S. Provisional Application No. 60/707, 0006 Native human GIP is a single 42-amino acid peptide 369 filed Aug. 11, 2005: U.S. Provisional Application No. synthesized in and secreted by specialized enteroendocrine 60/709.320 filed Aug. 17, 2005; and U.S. Provisional Appli K-cells. These cells are concentrated primarily in the duode cation No. 60/709,316 filed Aug. 17, 2005, each of which are num and proximal jejunum, although they also can be found hereby incorporated by reference in their entirety. The present throughout the intestine. The main stimulant for GIP secre application hereby incorporates by reference in their entirety tion is ingestion of carbohydrate- and lipid-rich meals. Fol commonly-owned: PCT/US05/04178 filed Feb. 11, 2005 and lowing ingestion, circulating plasma GIP levels increase 10 published as WO2005/077072; PCT/US2005/004351 filed to 20-fold. The half-life of intact GIP is estimated to be Feb. 11, 2005; PCT/US2005/004631 filed Feb. 11, 2005; approximately 7.3 minutes in healthy Subjects and 5.2 min PCT/US2005/045471 filed Dec. 15, 2005; U.S. patent appli utes in diabetic Subjects. cation Ser. No. 11/055,093: U.S. patent application Ser. No. 0007. The physiologic effects of GIPhave been elucidated 11/201,664 filed Aug. 10, 2005; and U.S. Ser. No. 206,903 using GIP receptor antagonists, GIP peptide antagonists, and filed Aug. 17, 2005. The applications teach compounds and GIP receptor knockout mice, in addition to GIP infusion uses related to and useful for the present invention. protocols. Blocking GIP binding to its receptor results in attenuated glucose-dependent insulin secretion following FIELD OF THE INVENTION oral glucose load in rats and mice. Similarly, administration of GIP antagonists or GIP antisera markedly reduces the 0002 The present invention relates to peptide chemistry postprandial insulin release in rats. GIP receptor knockout and pharmaceuticals, and more particularly to novel gastric mice demonstrate normal fasting glucose levels but mild glu inhibitory peptide (“GIP) analog and GIP-containing hybrid cose intolerance following oral glucose loads. Interestingly, polypeptides with selectable properties. It further relates to they also exhibit resistance to diet-induced obesity following the use of these and other GIP compounds either alone or in months of high-fat feeding. Additionally, in the leptin-defi adjunct with other compounds such as glucagon-like peptide cient ob?ob mouse, the GIP receptor knockout genotype 1 (“GLP1), exendin-4, or amylin polypeptides, to treat meta appears to decrease the extent of obesity that develops. bolic disorders and conditions. 0008 GIP infusion has consistently demonstrated stimu lation of insulin secretion in isolated rat islets, isolated per BACKGROUND OF THE INVENTION fused rat pancreas, dogs, and humans. During stepwise eug 0003 Incretin peptides are hormones and peptide mimet lycemic, mild hyperglycemic (54 mg/dL above basal), and ics that cause an increase in the amount of insulin released moderate hyperglycemic (143 mg/dL above basal) clamps, it when glucose levels are normal or particularly when they are has been demonstrated that GIP infusion results in insulin elevated. These incretin peptides have other actions beyond secretion only in the presence of elevated glucose concentra the initial incretin action defined by insulin secretion. For tions. Furthermore, it has been demonstrated that GIP is not instance, they may also have actions to reduce glucagon pro glucagonotropic in normal humans during either euglycemic duction and delay gastric emptying. In addition, they may or hyperglycemic conditions. Thus, the effect of endog have actions to improve insulin sensitivity, and they may enously released GIP appears to be an important mechanism increase islet cell neogenesis—the formation of new islets. of postprandial insulin secretion and does not appear to play 0004. The concept of the incretin effect developed from a role in the fasting state. the observation that insulin responses to oral glucose 0009 GIP has many non-incretin effects as well. Unlike exceeded those measured after intravenous administration of other insulin secretagogues, GIP stimulates beta-cell prolif equivalent amounts of glucose. It was concluded that gut eration and cell survival in INS-1 islet cell-line studies. Fur derived factors, or incretins, influenced postprandial insulin thermore, animal studies have suggested a role for GIP in release. Nutrient entry into the stomach and proximal gas lipid metabolism by stimulating lipoprotein lipase activity, trointestinal tract causes release of incretin hormones, which inducing fatty acid incorporation into adipose tissue and then stimulate insulin secretion. This insulinotropism, orabil stimulating fatty acid synthesis. However, in humans, there is ity to stimulate insulin secretion, can be quantified by com no clear evidence for an effect of GIP on lipid metabolism. paring insulin or C-peptide responses to oral vs. intravenous GIP also appears to stimulate glucagon Secretion from the glucose loads. In this way, it has been shown that the incretin isolated perfused rat pancreas, although human studies have effect is responsible for about 50% to 70% of the insulin not demonstrated any significant influence on glucagon response to oral glucose in healthy individuals. secretion. Furthermore, unlike GLP-1. GIP appears to act by 0005. Although many postprandial hormones have incre accelerating emptying of the stomach rather than by inhibit tin-like activity, predominant incretin peptides include glu ing gastrointestinal motility. cose-dependent insulinotropic polypeptide, also known as 0010. The GIP-receptor, a member of the G-protein gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 coupled receptor family has a high specificity for GIP and (GLP-1), and exendin peptides (which are non-endogenous does not bind other peptides of the glucagon family. For this US 2009/0036364 A1 Feb. 5, 2009 reason, GLP-1/GIP chimeric peptides show nearly no affinity increased GIP receptor binding, increased receptor selectiv for the GIP-receptor. From such studies it has been concluded ity, and/or increased resistance to degradation by chemical that the GIP(1-30) sequence of the GIP(1-42) is sufficient for and/or proteolytic means. In another embodiment the GIP recognizing the receptor. GIP(6-30)-amide contains the high analog further has least 50% sequence identity to native GIP affinity binding region of GIP(1-42) but exhibits antagonist (1-30), native GIP(1-14), native GIP(19-30) or native GIP(1- activity. 42) over the entire length of each molecule, and at least one 0.011 Despite potent glucoregulatory actions through glu biological property of a GIP. In certain embodiments, novel cose-dependant stimulation of insulin secretion, the insulino GIP analog polypeptides include unnatural amino acids. Such tropic effect of GIP is significantly reduced in diabetic sub as a Damino acid. In a further embodiment a GIP analog or jects compared to normal individuals (16-18). Consequently, hybrid is modified to have reduced renal clearance, such as by clinical use of GIP has not been significantly advanced. Fur fatty acyl derivitization. ther, there remains a need to develop additional diabetic treat 0015. In another embodiment are provided novel GIP ment modalities as well as treatments for metabolic diseases, containing hybrid polypeptides with selectable properties. conditions, and disorders. Accordingly, it is an object of the The hybrid polypeptides exhibit at least one hormonal activ present invention to provide GIP analog and GIP-containing ity. In one embodiment the GIP-hybrid polypeptides com hybrid polypeptides and methods for their use to treat or prise at least two biologically active (“bio-active') peptide prevent metabolic diseases, conditions, and disorders. hormone modules covalently linked together, wherein at least 0012 All documents referred to herein are incorporated one of the bio-active peptide hormone modules comprises a by reference into the present application as though fully set GIP polypeptide. The other bio-active peptide hormone mod forth herein. ule can be: (a) a native component peptide hormone, (b) an analog or derivative of a native component peptide hormone SUMMARY OF THE INVENTION that retains hormonal activity, (c) a fragment of a native 0013 Provided are methods for treating or preventing component peptide hormone that retains hormonal activity, metabolic diseases and disorders including those which can (d) afragment of analogs or derivatives of a native component be alleviated by control of plasma glucose levels, insulin peptide hormone that retains hormonal activity, (e) a struc levels, and/or insulin secretion, such as diabetes and diabetes tural motif of a native component peptide hormone that related conditions, and conditions and disorders including, imparts a desired chemical stability, conformational stability, but not limited to, hypertension, dyslipidemia, cardiovascular metabolic Stability, bioavailability, organ/tissue targeting, disease, eating disorders, insulin-resistance, obesity, and dia receptorinteraction, protease inhibition, plasma proteinbind betes mellitus of any kind, including type 1, type 2, and ing, and/or other pharmacokinetic characteristic to the hybrid gestational diabetes. The methods comprise administering a polypeptide; or (f) a structural motif of analogs orderivatives therapeutically or prophylactically effective amount of a GIP of a native component peptide hormone that imparts a desired or GIP analog, fragment orderivative thereof, or a GIPhybrid chemical stability, conformational stability, metabolic stabil or derivatives thereof as described herein, alone (mono ity, bioavailability, organ/tissue targeting, receptor interac therapy) or in combination with another agent or therapy tion, protease inhibition, plasma protein binding, and/or other (adjunct therapy), for example a glucose lowering agent (e.g., pharmacokinetic characteristic to the hybrid polypeptide. antidiabetic) or agents or methods that inhibit or reduce gas The structural motifs of (e) and (f) will collectively be tric emptying (examples of such agents are presented herein), referred to herein as “peptidic enhancers’. An example of a to a subject in need thereof. By providing a GIP bioactivity as peptidic enhancer is a Trp cage sequence, particularly one part of a GIP hybrid having one or more other hormonal derived from exendin-4. bio-activities, e.g., pramlintide, exendin, BNP, compounds 0016. In a further embodiment a GIP-hybrid polypeptide with one or more selectable functions will have the additional comprises at least two bio-active peptide hormone modules benefit of a property of a GIP bio-activity such as lowering covalently linked together, wherein at least one of the bio plasma glucose, increasing insulin secretion, without the side active peptide hormone modules is comprised of a GIP effects associated with other incretin molecules. For example, polypeptide, and the second exhibits at least one hormonal a GIP-SCT hybrid compound of the invention can have a activity of a component peptide hormone. The bio-active selectable property of a salmon calcitonin, Such as decreasing peptide hormone modules are independently selected from: bone loss and bone resorption or reducing cartilage turnover component peptide hormones, fragments of component pep (chondroprotection), and a property of a GIP. Such as plasma tide hormones that exhibit at least one hormonal activity of glucose lowering (concomitant with an anti-catabolic aspect the component peptide hormones, analogs and derivatives of as described herein) and/or inhibiting bone resorption and component peptide hormones that exhibit at least one hor maintaining or increasing bone density. AGIP hybrid with monal activity of the component peptide hormones, and frag Such selectable properties can enhance treatment of ments of analogs and derivatives of component peptide hor osteoporosis or conditions of high cartilage turnover, particu mones that exhibit at least one hormonal activity of the larly in those who can also benefit from glycemic control, component peptide hormones. Such as Subjects with diabetes or undergoing critical care. 0017. In one embodiment the GIP-hybrid polypeptides 0014. In one embodiment are provided novel GIP analogs comprise a novel GIP analog polypeptide of the invention having one or more enhanced properties. The GIP analogs covalently linked to at least one additional bio-active peptide have increased resistance to DPP-IV or to other proteases, hormone module. In a further embodiment the bio-active Such as those found in human plasma and/or on kidney brush peptide hormone module is a peptidic enhancer. In one border membranes, which increases in vivo half-life. In a embodiment the GIPhybrid polypeptide of the invention will further embodiment the GIP analogs have at least one substi exhibit at least 50% sequence identity to a native GIP(1-30), tution, modification, insertion or deletion in amino acids native GIP(1-14), native GIP(19-30) or native GIP(1-42) over 4-30. These changes can provide enhanced properties such as the entire length of the GIP portion. In certain embodiments US 2009/0036364 A1 Feb. 5, 2009

the GIP portion can comprise a novel GIP analog further neuromedins, urocortin, calcitonin, or salmon calcitonin, a comprising a peptidic enhancer, Such as a trp-cage motif. natriuretic peptide or analog, derivative or fragment thereof. Accordingly, a GIPhybrid can comprise a GIP portion that is 0021. In other embodiments the peptidic enhancer is a tail a GIP analog, fragment or derivative thereof with a peptidic or terminal extension derived from a second hormone, such as enhancer, such as a trp-cage motif, and having enhanced exendin, human GLP-1, or frog GLP-1, or is empirically properties, that is linked covalently to an additional bio-active determined. In one embodiment the peptidic enhancer is a peptide hormone module, such as another hormone or growth heterologous C-terminal tail or terminal extension to the GIP factor or fragment thereof. portion. As with the other GIP hybrids described herein, in 0018 Component peptide hormones for use in a GIP one embodiment of the peptidic-enhancer containing hybrid, hybrid polypeptide include: amylin, adrenomedulin (ADM), calcitonin (CT), calcitonin gene related peptide (CGRP), the GIP portion can be native GIP, an active fragment thereof, intermedin, cholecystokinin (“CCK), leptin, peptide YY or their analogs or derivatives. In another aspect the GIP (PYY), glucagon-like peptide-1 (GLP-1), glucagon-like pep portion of the hybrid comprises at least one modification, tide 2 (GLP-2), oxyntomodulin (OXM), natriuretic peptides substitution, deletion or addition that provides one or more (e.g. ANP, BNP. CNP or urodilatin), urocortin family peptide, enhanced properties, e.g. increased resistance to proteolytic e.g., Ucn-2 and Ucn-3, neuromedin family peptide, e.g. neu digestion (thus prolonging half-life), fatty acyl derivitization romedin U25 or splice variants, exendin-3 and exendin-4. that reduces renal clearance. In one embodiment the tail com 0019. In other GIPhybrid embodiments the GIP portion is prises a Trp-cage motif sequence. In another embodiment the combined with a gastrin/CCK receptor ligand; an amylin GIP analog polypeptide portion includes unnatural amino receptor ligand; a calcitonin receptor ligand; a CGRP recep acids, such as a Damino acid, e.g. that inhibits to reduces the tor ligand, a PYY receptor ligand, an EGF receptor ligand; a rate of proteolysis by DPP-IV. Glucagon-like peptide 1 receptor ligand; a Glucagon-like 0022. The present invention also provides for the treat peptide 2 receptor ligand; a gastric inhibitory polypeptide ment and prevention of metabolic diseases and disorders, (GIP) receptor ligand; a keratinocyte growth factor (KGF) particularly those which can be alleviated by control of receptor 1 ligand; a dipeptidyl peptidase IV inhibitor; a REG plasma glucose levels, insulin levels, and/or insulin secretion, protein receptor ligand; a Growth Hormone receptor ligand; a Such as diabetes and diabetes-related conditions. Such con Prolactin (PRL) receptor ligand; an Insulin-like Growth Fac ditions and disorders include, but are not limited to, hyper tor (IGF) receptor ligand; PTH-related protein (PTHrP) tension, dyslipidemia, cardiovascular disease, eating disor receptor ligand; hepatocyte growth factor (HGF) receptor ders, insulin-resistance, obesity, and diabetes mellitus of any ligand; a bone morphogenetic protein (BMP) receptor ligand, kind, including type 1, type 2, and gestational diabetes, dia a transforming growth factor (TGF receptor ligand; a laminin betes complications (e.g. neuropathy (treating with a GIP receptor ligand; a vasoactive intestinal peptide (VIP) receptor hybrid containing an exendin family hormone module for ligand; a fibroblast growth factor (FGF) receptor ligand; a example), neuropathic pain (treating with GIP hybrids com nerve growth factor (NGF) receptor ligand; an islet neogen prising an amylin family hormone module for example), ret esis associated protein (INGAP) receptor ligand; an inopathy, nephropathy, conditions of insufficient pancreatic Activin-A receptor ligand; a vascular endothelial growth fac beta cell mass (based on, e.g., islet neogenesis actions of tor (VEGF) receptor ligand; an erythropoietin (EPO) receptor exendin-4 and GLP-1). Accordingly, provided are methods ligand; a pituitary adenylate cyclase activating polypeptide for treating or preventing Such conditions, wherein the (PACAP) receptor ligand; a granulocyte colony stimulating method comprises administering atherapeutically or prophy factor (G-CSF) receptor ligand; a granulocyte-macrophage lactically effective amount of a GIP or an analog or derivative colony stimulating factor (GM-CSF); a platelet-derived thereof, including a novel GIP analog of the invention, or a growth factor (PDGF) receptor ligand, a cannabinoid CB1 GIP-hybrid of the invention, including one having a peptidic receptor antagonist, and a secretin receptor ligand. enhancer, to a Subject in need thereof. In one embodiment the 0020. In one embodiment desirable GIP hybrid polypep polypeptides of the invention can be provided as mono tides include an N-terminal GIP or novel GIP analog frag therapy. In another embodiment for treating diabetes or con ment in combination with a C-terminal polypeptide or frag ditions associated with elevated glucose levels, the GIP com ment thereof having a glucose lowering activity (e.g., pound can be administered in adjunct therapy with a glucose antidiabetics, exendin) or the ability to inhibit or reduce gas lowering agents (e.g., antidiabetics) or agents or methods that tric emptying. Such desirable GIP hybrids include an N-ter inhibit or reduce gastric emptying. Examples of Such agents minal GIP fragment or novel GIP analog or derivative frag are presented herein. For example, in one embodiment is ment in combination with a C-terminal exendin, GLP1, provided an adjunct therapy method for reducing blood glu symlin (pramlintide), amylin, CCK, gastrin, PYY, secretin, cose levels of a subject, e.g., one having type 1, type 2 or GRP neuromedins, urocortin, calcitonin, or salmon calcito gestational diabetes mellitus, comprising administering to the nin, a natriuretic peptide (e.g., ANP BNP. CNP, urodilatin) or Subject atherapeutically effective amount of an exendin oran analog (e.g. amylin-SCT-amylin chimera), derivative or frag exendin agonist, such as wherein said exendin agonist is a ment thereof. In other embodiments desirable GIP hybrids peptide, in adjunct therapy with a GIP or novel GIP analog of include a C-terminal GIP or novel GIP analog fragment in the invention, oran effective amount of a GIP-exendin hybrid. combination with an N-terminal polypeptide or fragment 0023 Compounds of the invention, alone or in combina thereofhaving a glucose lowering activity (e.g., antidiabetics, tion with a glucose lowering agent (e.g., antidiabetics) or with exendin) or the ability to inhibitor reduce gastric emptying. In agents or methods that inhibitor reduce gastric emptying, can Such embodiments, the chimeric polypeptides can include a also be useful for potentiating, inducing, enhancing or restor C-terminal GIP, a novel GIP analog, or fragment thereof, in ing glucose responsivity in pancreatic islets or cells. These combination with a N-terminal exendin, GLP1, symlin actions may also be used to treat or prevent conditions asso (pramlintide), amylin, CCK, gastrin, PYY, secretin, GRP, ciated with metabolic disorders such as those described above US 2009/0036364 A1 Feb. 5, 2009

and in U.S. Patent Application No. US20040228846, incor 0027. In another aspect GIP analogs and hybrids are useful porated herein by reference in its entirety. for decreasing or inhibiting bone resorption and maintaining 0024. In another aspect methods for treating or preventing or increasing bone density. When combined with an appro obesity are provided, wherein the method comprises admin priate second hormonal module, GIP hybrids are useful to istering a therapeutically or prophylactically effective treat these conditions as well as decreasing plasma calcium, amount of a GIP or an analog or derivative thereof, including and/or inducing an analgesic effect, particularly to treat bone a novel GIP analog of the invention, or a GIP-hybrid of the disorders such as osteopenia and osteoporosis, and treating invention, including those having a peptidic enhancer, to a painful neuropathy. In one embodiment Such hybrids contain subject in need thereof. In one embodiment, the subject is an an exendin, GLP1, amylin and/or SCT portion. For example, obese or overweight subject. While “obesity' is generally a GIP-SCT or GIP-amylin/sCT hybrid compound of the invention can have a selectable property of a salmon calcito defined as a body mass index over 30, for purposes of this nin or amylin/SCT/Amylin chimera, Such as decreasing bone disclosure, any subject, including those with a body mass loss and bone resorption or reducing cartilage turnover (chon index of less than 30, who needs or wishes to reduce body droprotection), and a property of a GIP. Such as plasma glu weight is included in the scope of “obese.” Subjects who are cose lowering (concomitant with an anabolic aspect as insulin resistant, glucose intolerant, or have any form of dia described herein) and/or inhibiting bone resorption and main betes mellitus (e.g., type 1, 2 or gestational diabetes) can taining or increasing bone density. A GIP hybrid with such benefit from this method. Compounds of the invention can selectable properties can enhance treatment of osteoporosis also be useful in treating or preventing other conditions asso or conditions of high cartilage turnover, particularly in those ciated with obesity including stroke, cancer (e.g., endome who can also benefit from glycemic control. Such as Subjects trial, breast, prostate, and colon cancer), gallbladder disease, with diabetes or undergoing critical care. sleep apnea, reduced fertility, and osteoarthritis, (see 0028. GIP compounds, particularly GIP analogs, Lyznicki et al, Am. Fam. Phys. 63:2185, 2001). Where con extended half-life GIP hybrids (e.g. DPP-IV cleavage resis ditions are associated with elevated glucose or hyperglyce tant (such as a D-Ala2, N-Acetyl or N-pyroglutamyl analogs) mia, the method comprises administering atherapeutically or optionally further comprising a peptidic enhancer Such as a prophylactically effective amount of a GIP compound, alone heterologous C-terminal tail, and GIP hybrids comprising or in combination with a glucose lowering agent (e.g., other hormone modules known to provide beneficial cardio antidiabetic) or agent or method that inhibits or reduces gas vascular effects, are useful to treat cardiovascular disease and tric emptying. related conditions. As demonstrated herein GIP compounds 0025. In yet another aspect, GIP compounds, particularly increase cardiac contractility (dp/dt), decrease blood pressure GIP hybrids of the invention, can be used in methods of (for example by acute vasodilatation), decrease systolic pres reducing food intake, reducing appetite, inducing Satiety, Sure, decrease diastolic pressure, and can provide a direct reducing nutrient availability, reducing caloric efficiency, beneficial action on cardiac cells. GIP compounds also causing weight loss, affecting body composition, altering improve cardiac function via metabolic actions, e.g. glucose body energy content or energy expenditure, and improving lowering, insulin secretion, beta cell proliferation. However, lipid profile (including reducing LDL cholesterol and triglyc by also providing direct effects on cardiovascular system, the eride levels and/or changing HDL cholesterol levels) wherein GIP compounds are Surprisingly even more beneficial. the methods comprise administering to a Subject an effective 0029 Compounds of the invention can also be useful in amount of a GIP compound, particularly a GIP hybrid com the treatment or prevention of any number of gastrointestinal pound, of the invention. In one embodiment, the methods of disorders that are associated with excess gastric secretion, the invention are used to treat or prevent conditions or disor excess intestinal electrolytes and water secretion as well as ders which can be alleviated by reducing nutrient availability decreased absorption, e.g., infectious (e.g., viral or bacterial) in a Subject in need thereof, comprising administering to said diarrhea, inflammatory diarrhea, short bowel syndrome, or Subject atherapeutically or prophylactically effective amount the diarrhea which typically occurs following Surgical proce of a GIP compound of the invention. Conditions and disorders dure, e.g., ileostomy (see e.g., Harrison's principles of Inter include, but are not limited to, hypertension, dyslipidemia, nal Medicine, McGraw Hill Inc., New York, 12th ed.). cardiovascular disease, eating disorders, insulin-resistance, Examples of infectious diarrhea include, without limitation, obesity, and diabetes mellitus of any kind, including type 1, acute viral diarrhea, acute bacterial diarrhea (e.g., salmo type 2, and gestational diabetes, diabetes complications (neu nella, Campylobacter, and clostridium) or diarrhea due to ropathy (based on, e.g., neurotrophic actions of exendin-4), protozoal infections, or travellers' diarrhea (e.g., Norwalk neuropathic pain (based on, e.g., amylin action), retinopathy, virus or rotavirus). Examples of inflammatory diarrhea nephropathy, conditions of insufficient pancreatic beta cell include, without limitation, malabsorption syndrome, tropi mass (based on, e.g., islet neogenesis actions of exendin-4 cal spue, chronic pancreatitis, Crohn's disease, diarrhea, and and GLP-1). Where conditions are associated with elevated irritable bowel syndrome. GIP and GIP compounds of the glucose or hyperglycemia, the method comprises administer invention can be used to treat or prevent an emergency or ing a therapeutically or prophylactically effective amount of life-threatening situation involving a gastrointestinal disor a GIP compound, alone or in combination with a glucose der, e.g., after Surgery or due to cholera. Furthermore, the lowering agent (e.g., antidiabetic) or agent or method that compounds can be used to treat intestinal dysfunction in inhibits or reduces gastric emptying. patients with Acquired Immune Deficiency Syndrome 0026. In addition to the amelioration of hypertension in (AIDS), especially during cachexia. The compounds may subjects in need thereof as a result of reduced food intake, also be useful for inhibiting small intestinal fluid and electro weight loss, and/or treating obesity, compounds of the inven lyte secretion, and augmenting nutrient transport, as well as tion may be used to treat or prevent hypotension and condi increasing cell proliferation in the gastrointestinal tract, regu tions associated therewith. lating lipolysis in, e.g., adipase tissue and regulating blood US 2009/0036364 A1 Feb. 5, 2009 flow in a mammal. GIP compounds of the invention may also g/kg) was given at t=0, Sample was taken at t=30 min. Blood be useful for treating or preventing the above conditions by glucose was measured with a OneTouch.R. Ultra R. (LifeScan, their gastrointestinal protective activity (e.g., inhibition of Inc., a Johnson & Johnson Company, Milpitas, Calif.) p<0. gastric secretion). Accordingly, a GIP compound of the 05 vs. vehicle control: ANOVA, Dunnett's test. In FIG. 3B, invention may be used to treat gastrointestinal or muscosal points represent meantisem, n=8-15. Peptide was injected IP damage. Exemplary types of damage include, but are not at t=0 immediately following baseline sample into 2-hour limited to, inflammatory bowel disease, bowel atrophy, con fasted NIH/Swiss mice. Samples were taken at t=60, 120, and ditions characterized by loss of bowel mucosa or bowel 180 minutes. Blood glucose was measured with a One mucosal function, and other conditions of the gastrointestinal Touch(R) Ultra(R) (LifeScan, Inc., a Johnson & Johnson Com tract, including those which may be brought about by expo pany, Milpitas, Calif.). p-0.05 vs. vehicle control: ANOVA, Sure to cytotoxic agents, radiation, toxicity, infection and/or Dunnett's test. Compound No. Gis (D-Ala2)GIP(1-30)-PSS injury. Moreover, these compounds of the invention may be GAPPPS amideform: sequenceY(D-Ala)EGTFISDYSIAM combined with analgesics, anti-inflammatory agents, growth DKIHQQDFVNWLLAQKPSSGAPPPS-NH2. Compound hormone, heparin, or any other therapies that may be used to G has both an N-terminal modification providing DPP-IV treat inflammatory bowel disease or other conditions listed resistance and a C-terminal Trp-cage shield. above. 0036 FIG. 4 presents a comparison of the glucose lower 0030. In another embodiment GIP compounds can be use ing action of Compound G with full length GIP and exendin-4 ful for treating or preventing gastritis, pancreatitis, Barrett's in diabetic db/db mice. Points represent meanisem. n=6-10. esophagus, Gastroesophageal Reflux Disease (GERD) and Peptide was injected IP at t=0 immediately following baseline conditions associated therewith. Such conditions can include, sample into 2-hour fasted db/db mice. Samples were taken at but are not limited to, heartburn, heartburn accompanied by 60, 120, and 180 min. Blood glucose was measured with a regurgitation of gastric/intestinal contents into the mouth or OneTouch.R. Ultra R. (LifeScan, Inc., a Johnson & Johnson the lungs, difficulty in Swallowing, coughing, intermittent Company, Milpitas, Calif.) *p-0.05 vs. vehicle control; wheezing and Vocal cord inflammation (conditions associ ANOVA, Dunnett's test. ated with GERD), esophageal erosion, esophageal ulcer, 0037 FIGS. 5A-5D present examples of GIP hybrids esophageal stricture, Barrett's metaplasia (replacement of comprising GIP and amylin/calcitonin-like analog peptides. normal esophageal epithelium with abnormal epithelium), The compounds would have both GIP activity (e.g., glucose and pulmonary aspiration. GIP compounds can have anti lowering) and reduction of gastric emptying. secretory properties. Such as inhibition of gastric acids, inhi bition of bile acids, and inhibition of pancreatic enzymes. 0038 FIG. 6 depicts the structures of various chemical Moreover, GIP compounds can also have gastroprotective groups mentioned herein. effects. Accordingly, GIP compounds of the invention may be 0039 FIG. 7 depicts the structures of various chemical particularly useful in the treatment or prevention of gastritis, Fmoc derivatives mentioned herein. pancreatitis, Barrett's esophagus, and/or GERD and related 0040 FIGS. 8A and 8B depict glucose lowering effect of or associated conditions. novel GIP analogs. The figures demonstrate that a D-alanine 0031. The present invention also relates to pharmaceutical Substitution at position 2 in the analogs herein improves glu compositions comprising a therapeutically or prophylacti cose lowering ability. Points represent meanisem. Peptide cally effective amount of at least one novel GIP analog or GIP was injected IP at t=0 immediately following baseline sample hybrid polypeptide of the invention, or a pharmaceutically into 2-hour fasted NIH/Swiss mice. Samples were taken at acceptable salt thereof, together with pharmaceutically t=60, 120, 180 and 240 minutes. Blood glucose was measured acceptable diluents, preservatives, Solubilizers, emulsifiers, with a OneTouch(R Ultra R. (LifeScan, Inc., a Johnson & adjuvants and/or carriers useful in the delivery of the GIP Johnson Company, Milpitas, Calif.). p-0.05 vs. vehicle con compound. trol: ANOVA, Dunnett's test. 0032. These and other aspects of the invention will be 004.1 FIG.9 depicts glucose lowering effect of novel GIP more clearly understood with reference to the following analogs, particularly the effect of a Trp-cage. Points represent embodiments and detailed description. meanisem. Peptide was injected IP at t=0 immediately fol lowing baseline sample into 2-hour fasted NIH/Swiss mice. BRIEF DESCRIPTION OF THE DRAWINGS Samples were taken at t=60, 120, 180 and 240 min. Blood 0033 FIGS. 1A and 1B present views of the exendin-4 glucose was measured with a OneTouch.R. Ultra R. (LifeScan, Trp-cage. FIG. 1A shows an NMR-derived ensemble struc Inc., a Johnson & Johnson Company, Milpitas, Calif.). p-0. ture of exendin-4 in 30% aqueous trifluoroethanol. The Trp 05 vs. vehicle control: ANOVA, Dunnett's test. cage can be seen folding back towards the central region of 0042 FIGS. 10A and 10B depict glucose lowering effect exendin. FIG. 1B shows a CPK view of the “Trp-cage” motif of various analogs. In this example, Ac modification and a (residues 21-38) of a representative structure from the solu Pro3 Substitution did not significantly enhance glucose low tion-state NMR structure ensemble of exendin-4. ering ability. Points represent meanisem. Peptide was 0034 FIG. 2 presents sequences and receptor binding and injected IP at t=0 immediately following baseline sample into glucose lowering (oral glucose tolerance test (OGTT) in non 2-hour fasted NIH/Swiss mice. Samples were taken at t=60, diabetic NIH/Swiss mice) activities of reference sequences 120, 180 and 240 min. Blood glucose was measured with a and novel GIP analog sequences of the invention. OneTouch.R. Ultra R. (LifeScan, Inc., a Johnson & Johnson 0035 FIGS. 3A and 3B present suppression of glucose Company, Milpitas, Calif.). p-0.05 vs. Vehicle control; excursion following an OGTT, and basal glucose lowering ANOVA, Dunnett's test. activity of GIP and Compound G in NIH/Swiss mice. In FIG. 0043 FIG. 11 demonstrates superior protease resistance 3A, bars represent meantsd, n=6-10. Peptide was injected IP of an exemplary GIP hybrid, 0601GIP3794 versus a native at t=-5 into overnight-fasted NIH/Swiss mice. Gavage (1.5 GIP. US 2009/0036364 A1 Feb. 5, 2009

0044 FIGS. 12A-12AT depict further exemplary analogs tin effects in diabetic subjects (19). Despite the reduced and reference peptides of the invention. It is intended that the insulinotropic response to GIP in subjects with type 2 diabe various modifications and variants shown are to be used in the tes, it is possible that administration of elevated pharmaco present invention, and may be combined as discussed herein. logical doses of GIP or analogues could have therapeutic For example, the terms exendin tail or exendin trp-cage motif utility. Of note, GIP lacks the gastrointestinal effects of includes any of the exendin tail variants depicted, which are GLP-1 (20) that has limited the latter peptide's therapeutic useful as shield sequences (peptidic enhancers). Of further window, thus permitting the possibility of higher dosing regi interest are the frog GLP1 C-terminal extensions as shown in mens (21). the figures, which are yet another example of a shield 0057. One of the major hurdles in the therapeutic devel sequence that can be used in place of an exendin tail. opment of these incretin hormones is their short duration of 004.5 FIGS. 13A and 13B demonstrate food intake inhi action due to enzymatic degradation in vivo. The enzyme bition and lowering of blood glucose by GIP hybrids. dipeptidyl peptidase IV (DPP-IV) plays a key role in the 0046 FIG. 14. Effect of GIP hybrids in food intake assay. N-terminal cleavage of the peptides in Vivo, and more 0047 FIG. 15. Effect of GIP hybrid in food intake assay. recently, neutral endopeptidase 24.11 (NEP) has also be 0048 FIGS. 16A and 16B demonstrate effect of Com implicated in their degradation (22-26). Several studies have pound 10 (FIG. 16A) and sGT (FIG. 16B) in food intake reported greater in vivo efficacy of DPP-1 V resistant GIP assay. analogues in rodent diabetic models (27-28). 0049 FIG. 17 demonstrates effect of GIPhybrids on low 0058 Provided herein are novel GIP analogs and GIP ering of blood glucose. containing hybrid polypeptides, or derivatives thereof, which 0050 FIGS. 18A and 18B depict cAMP production in have enhanced or novel properties, including enhanced DPP whole cardiomyocytes from receptor activation in response to IV resistance, dual hormonal activity, and improved plasma varying doses of test compound, human GIP(1-42) free acid half-life. Also provided are methods for treating or preventing form (FIG. 18A) and GIP hybrid compound G (FIG. 18B). metabolic diseases and disorders including those which can 0051 FIGS. 19A-E depict the response of mean arterial be alleviated by control of plasma glucose levels, insulin pressure (FIG. 19A), heart rate (FIG. 19B), and rate of change levels, and/or insulin secretion, Such as diabetes and diabetes in blood pressure (dP/dt, FIG. 19C), systolic pressure (FIG. related conditions, and conditions and disorders including, 19D) and diastolic pressure (FIG. 19E) as determined by but not limited to, hypertension, dyslipidemia, cardiovascular telemetry in conscious rats to administration of GIP com disease, eating disorders, insulin-resistance, obesity, and dia pounds. Mean arterial pressure is presented as % of predose betes mellitus of any kind, including type 1, type 2, and values measured over the 30 minutes prior to drug adminis gestational diabetes. The methods comprise administering a tration. FIG. 19C reflects the inotropic response to GIP com therapeutically or prophylactically effective amount of a GIP pounds. The rate of change of blood pressure (dP/dt) is indica or GIP analog, fragment or derivative thereof or a novel GIP tive of cardiac contractility. analog or GIP hybrid or derivatives thereof as described 0052 FIGS. 20A, 20B and 20O depict the lack an acute herein, alone (monotherapy) or in combination with another effect of GIP(1-42) and a GIP DPP-IV-resistant-analog/ex agent or therapy (adjunct therapy), for example a glucose endin-tail hybrid on food intake in contrast to exendin-4. The lowering agent (e.g., antidiabetic) or agents or methods that pancreatic hormone amylin produced a significant effect as inhibit or reduce gastric emptying (examples of Such agents expected. are presented herein), to a Subject in need thereof. 0053 FIG. 21 depicts the lack of effect on weight loss in 0059. In addition to novel GIP analogs and derivatives, the diet-induced obesity mice, in contrast to the effect of exendin present invention relates to novel, GIP-containing selectable 4. hybrid polypeptides useful as agents for the treatment and 0054 FIG. 22 provides an alignment of mammalian and prevention of metabolic diseases and disorders which can be non-mammalian GIP. Positions Y1, E3, D9, S11, D15, F22, alleviated by control of plasma glucose levels, insulin levels, V23, L26, L27 and K32 are conserved across all species. and/or insulin Secretion, such as diabetes and diabetes-related 0055 FIGS. 23A and B depict beneficial activity of a conditions. Such conditions and disorders include, but are not GIP-amylin/sCT/amylin hybrid in slowing of gastric empty limited to, hypertension, dyslipidemia, cardiovascular dis ing and reducing intracellular calcium levels. ease, eating disorders, insulin-resistance, obesity, and diabe tes mellitus of any kind, including type 1, type 2, and gesta DETAILED DESCRIPTION OF THE INVENTION tional diabetes. 0056 Gastric inhibitory polypeptide (GIP) and glucagon 0060. In one aspect, the invention involves the modular like peptide 1 (GLP-1) are gut peptide hormones that exert assembly of physiologically, metabolically, and/or pharma potent glucoregulatory action through their glucose-depen cokinetically active peptidic modules that may be selectable dant stimulation of insulin secretion. Consequently, these based on “bio-activities, e.g., therapeutic efficacy, scope of incretin hormones have attracted great interest as potential function, duration of action, physicochemical properties, anti-diabetic agents with reduced risk for hypoglycemia. and/or other pharmacokinetic properties. Whereas GLP-1, GLP-1 analogs and mimetics have been 0061. Without intending to be limited by theory, the shown to be efficacious in controlling glucose levels in type 2 present invention relates at least in part to a “toolbox” diabetic patients, the insulinotropic effect of GIP is reportedly approach, wherein bio-active peptide hormone modules are significantly reduced in diabetic Subjects, compared to nor linked in binary, tertiary or higher order combinations to mal individuals (16-18). The preservation of insulinotropic create novel, efficacious therapeutic agents with selectable action of GLP-1 but not of GIP in the same diabetic subjects properties. The “bio-active peptide hormone modules' may Suggests that GIP signal transduction is impaired in type 2 be peptide hormones, peptide fragments with hormonal activ diabetes. Reduced GIP receptor expression in pancreatic beta ity, or structural motifs of peptide hormones that impart cells has been proposed to contribute to overall reduced incre chemical, metabolic, and/or other pharmacokinetic stability. US 2009/0036364 A1 Feb. 5, 2009

The peptide hormones can include native peptide hormones, 0065. Useful in the therapies disclosed herein and in the as well as peptide hormone analogs and derivatives, as known novel GIP hybrids disclosed herein, are native GIP peptide in the art and described herein. hormones, and functional peptide analogs and derivatives 0062. In one aspect of the invention, it has been found that thereof. Certain exemplary native peptides, peptide analogs the combination of certain physicochemical characteristics of and derivatives are described herein, however it should be two or more peptide hormones into a single modality can recognized that any known GIP peptide that exhibits hor facilitate intervention at several points in a dysfunctional monal activity known in the art may be used either as a metabolic circuit. As such, in one aspect of the invention, component of a novel GIP analog or hybrid herein or in the rationally-designed hybrid polypeptides are provided that novel adjunct therapies disclosed herein. In one embodiment, integrate selectable bio-activities into a single polypeptide the GIP peptide analogs and derivatives have at least one agent. In one embodiment, the selectable hybrid polypeptides hormonal activity of a native GIP peptide. In certain embodi of the invention may involve the use of chemically stable ments, the GIP peptide analogs are agonists of a receptor that linkers to covalently attach the bio-active modules. In another a native GIP peptide is capable of specifically binding. Exem embodiment, the selectable hybrid polypeptides of the inven plary GIP peptide analogs and derivatives include those tion may involve the use of cleavable linkers, which them described in the references herein, which are hereby incorpo selves may be or form part of a bio-active module. rated by reference. While the present application describes 0063. Again, without intending to be limited by theory, GIP polypeptide compounds as GIP analogs, novel GIPana design of the hybrid polypeptides of the present invention logs, and novel GIP hybrids for use in the methods and thera may generally involve: (1) the identification, selection and pies described herein, it is further intended that any suitable pairing of bio-active peptide hormone modules for desired GIPagonist can be administered in place of a GIP compound, efficacy and therapeutic use, and (2) the covalent linking of Such agonists include agonist antibodies and antibody frag the bio-active modules (e.g. native peptide hormones, peptide ments and derivatives, and Small molecule GIP receptor ago hormone analogs or derivatives with hormonal activity, pep nists. Accordingly, when a GIP compound or polypeptide is tide hormone fragments with hormonal activity, stabilizing indicated for use in a particular therapeutic method, in motifs, etc.) either directly or via a linker without loss of another embodiment it is intended that a GIPagonist be used, bio-activity of the component modules. In certain embodi particularly an agonist antibody or fragment or derivative ments, module selection criteria may include, but not be thereof. limited to: (a) desired in vivo efficacy for desired therapeutic 0066. In serum, GIP is degraded by dipeptidyl peptidase or prophylactic indication, such as an additive or a synergistic IV (DPP-IV). The resulting short biological half-life (about 2 effect; (b) optional Synergism or dual action of the linked minutes in vivo) limits the therapeutic use of GIP. modules for multiple therapeutic or prophylactic indications; 0067. The following references relate to various GIPana and/or (c) a desired chemical stability, conformational stabil logs that are useful to provide as components for the novel ity, metabolic stability, bioavailability, organ/tissue targeting, GIP analogs and GIPhybrids of the present invention and find receptor interaction, protease inhibition, plasma protein bind use in the novel therapies of the present invention based on ing, and/or other pharmacokinetic characteristic. their function on various target organs. 0064. GIP, GIP Analogs and Novel GIP Analogs. (The 0068 German Patent Application 1992 1537 discloses a section headings are used herein for organizational purposes method for extending the Survival of insulin producing beta only, and are not to be construed as in any way limiting the cells by stimulation of their proliferation and prevention of Subject matter described.) Reference sequences include their programmed cell death. The specific goal is to increase human GIP truncated GIP, human GLP-1, exendin-4, an the endogenous insulin content and insulin response to exemplary Trp-cage, and exemplary 'shield” sequences (e.g. elevated blood glucose levels. An important component of a short and a long “exendin tail'): this invention is the activation of protein kinase B/Akt in

SEQ ID NO Description Sequence

2 GIP (1 - 42) YAEGTFISDYSIAMDKIHOODFWNWLLAOKGKKNDWKHNITO-OH

293 GIP (1-30) YAEGTFISDYSIAMDKIHQQDFVNWLLAQK-NH

4 GLP-1 HAEGTFTSDVSSYLEGOAAKEFIAWLVKGR-NH

5 Exedin-4 HGEGTFTSDLSKOMEEEAVRLFIEWLKNGGPSSGAPPPS-NH

6 Ex 4...... KNGGPSSGAPPPS tail/long

1. Ex-4 ...... PSSGAPPPS tail/short

7 Trp - Cage ...... FIEWLKNGGPSSGAPPPS US 2009/0036364 A1 Feb. 5, 2009

insulin producing beta-cells in response to the administration (0075 O'Harte et al. and Ghault et al. have reported GIP of effectors such as GLP-1. GIP Exendin-4 or GLP-1 recep (1-30) analogs Tyr1-glucitol-GIP and (Pro3)GIP-display toragonists or GIP-receptor agonists. ing DPP-IV resistance and enhanced bioactivity. (O'Harte et 0069 European Patent Application 0479210 discloses al., NH2-terminally modified gastric inhibitory polypeptide GIP analogs of the formula GIP(1-13)-X-GIP(15-30)-Y. exhibits amino-peptidase resistance and enhanced antihyper wherein X is an amino acid residue other than Met, and Y is selected from homoserine (inclusive homoserine-lactone) glycemic activity, Diabetes 48, 758-765 (1999)); and see (referred to as “Hse'), homoserine amide (Hse-NH2), H-Gly Gault et al. “Characterization of the cellular and metabolic Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln-Hse O effects of a novel enzyme-resistant antagonist of Glucose H-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln dependent insulinotropic polypeptide. Biochemical and HSe-NH2. Biophysical Research Communications 290, 1420-1426 0070 United States Patent Application 20030232761 by (2002)). Hinke et al., published Dec. 18, 2003, reports C-terminal 0076 Specific active GIP and GIP analogs known in the truncated fragments and N-terminal modified analogs of GIP art include:

SEQ ID No : Description Sequence 2 hCGIP (1-42) YAEGTFISDYSIAMDKIHOODFWNWLLAOKGKKNDWKHNITO

3 hCGIP (1-3O) YAEGTFISDYSIAMDKIHOODFWNWLLAOK

1O Mouse YAEGTFISDYSIAMDKIROODFWNWLLAORGKKSDWKHNITO

11 Rat YAEGTFISDYSIAMDKIROODFWNWLLAOKGKKNDWKHNLTO

12 Pig YAEGTFISDYSIAMDKIROODFWNWLLAOKGKKSDWKHNITO

13 Bowline YAEGTFISDYSIAMDKIROODFWNWLLAOKGKKSDWIHNITO

14 GIP (1-14) YAEGTFISDYSIAM

15 GIP (19-30) QODFWNWLLAOK

16 GIP (3-42) EGTFISDYSIAMDKIHOODFVNWLLAOKGKKNDWKHNITO antagonist as well as various GIP analogs with a reduced peptide bond or 0077. Of particular interest are analogs modified at or near alterations of the amino acids close to the dipeptidyl pepti the dipeptidyl peptidase IV (DPP-IV) specific cleavage site, dase IV (DPP-IV)-specific cleavage site providing DPP-IV which improve DPP-IV-resistance and consequently prolong resistance and prolonged half-life. Also reported are analogs half-life. Amino acid alterations include modifications of the with different linkers between potential receptor binding sites first 3 or 4 residues of GIP and/or the bond between residues of GIP. 2 and 3, which is cleaved by DPP-IV. Modifications and 0071 WO98/24464 discloses an antagonist of glucose Substitutions include N-terminal modifications, L-amino dependent insulinotropic polypeptide (GIP) consisting essen acids, D-amino acids, proteinogenic and non-proteinogenic tially of a 24 amino acid polypeptide corresponding to posi amino acids. Proteinogenic amino acids are defined as natural tions 7-30 of the sequence of GIP, a method of treating non protein-derived alpha-amino acids. Non-proteinogenic insulin dependent diabetes mellitus and a method of improving glucose tolerance in a non-insulin dependent dia amino acids are defined as all otheramino acids, which are not betes mellitus patient. building blocks of common natural proteins. 0072 WO 00/58360 and EP1171465 disclose peptides, 0078. Of further interest are novel GIP analogs having one which stimulate the release of insulin. This disclosure pro or more modifications as described herein and that exhibit at vides a process of N terminally-modifying GIP and the use of least 50%, at least 60%, at least 65%, at least 70%, at least the peptide analogues for treatment of diabetes. The specific 75%, at least 80%, at least 85%, at least 90%, at least 95% or peptide analog, which is disclosed in this invention, com at least 98% sequence identity to a native GIP(1-30), native prises at least 15 amino acid residues from the N terminal end GIP(1-26), native GIP(1-14), native GIP(1-39), native GIP of GIP (1-42). In another embodiment, Tyr1-glucitol GIP (19-30), native GIP(19-26), native GIP(19-39), native GIP (1-42) is disclosed. (19-42) or native GIP(1-42) over the entire length of the GIP 0073 WO 00/20592 discloses GIP or anti-idiotypic anti portion. bodies of GIP or fragments thereofas GIP-analogs for main (0079. Of particular interest are those GIP compounds taining or increasing bone density or bone formation. comprising at least 11, 12, 13, 14 or -15 amino acid residues 0074 Kuhn-Wache et al. (2000) discloses analogs of GIP from the N-terminal end of a GIP, e.g., GIP(1-42); having a with increased dipeptidyl peptidase IV resistance (Kuhn least one amino acid Substitution or modification at position Wache et al., in Langner & Ansorge, Cellular peptidases in 1-3 and being biologically active. This includes modification Immune Functions and Diseases 2. Kluwer Academic/Ple by fatty acid addition at an epsilon amino group of at least one num Publishers, 187-195.) lysine residue, either present in or introduced into the mol US 2009/0036364 A1 Feb. 5, 2009

ecule. Particular modifications include Tyr1-glucitol of a GIP. or Ser; Met14 to Leu; His 18 to Ala, Arg, or Lys: Asp21 to Glu; for example Tyr1-Glucitol GIP(1-42) or Tyr1-glucitol GIP(1- Lys30 to Arg or His; and/or an N-terminus as Gly(Oct). 30). GIP analogs of interest include those comprising a sub I0084. Further exemplary GIP modifications and combina stitution or modification selected from the group comprising tions are shown in the following compounds: D-amino acid substitutions in 1, 2 and/or 3 positions and/or N-terminal glycation, alkylation, acetylation or acylation, or other N-terminal modifications described herein. Of further SEO ID interest are analogs wherein the amino acid in the 2 or 3 NO: Sequence position is Substituted by lysine, serine, 4-amino butyric amino acid, Aib, D-alanine, Sarcosine or proline. Further 3 YAEGTFISDYSIAMDKIHOODFVNWLLAOK exemplary Substitutions in the 1, 2, or 3 position, and more YaFGTFISDYSIALDKIAQOEFWNWLLAOR particularly in the 2 position of GIP are dAla, Val, dinorVal, dSer, Abu, dAbu, homo-Ser, d-homoSer, dPro, cyclopropyl YaBEGT S DYSIALDKIROQEFVNWLLAQR Ala, d-cyclopropyl Ala, cycloHexyl Ala, d-cyclohexyl Ala, YaBEGT S DYSIALDKIKQQEFVNWLLAQR A(NMe), Aib, and cyclpropCly. 0080 Further exemplary GIP analog compounds have a YaBEGT S DYSIALDKIAQQEFVNWLLAQH modification at the N-terminus, retaining their GIP Receptor YaBEGT S DYSIALDKIROQEFVNWLLAQH binding, in which the N-terminus modification includes H. isocap, isoBuOCO, octylglycine, Y(Me) and succinoyl. Fur YaBEGT S DYSIALDKIKQQEFVNWLLAQH ther exemplary GIP compounds include those with fatty acid YaBEGT S DYSIAMDKIHOVKFVNWLLAOK modifications or combinations of modifications as described herein, while retaining their GIP Receptor binding and recep YaBEGT S DYSIALDKIROQEFVNWLLAOK tor activation activity, For example, an N-terminus modifica tion can be combined with a substitution or modification at YaBEGT S DYSIALDKIKQQEFVNWLLAOK positions 1, 2 or 3 (which imparts resistance to DPP-IV as YaBEGT S DYSIALDKIAQQEFVNWLLAOK described herein) or with a fatty acyl derivative modification (which can reduce renal clearance). In another example, a YaBEGT S DYSIALDKIROQEFVNWLLAQH substitution or modification at positions 1, 2 or 3 is combined with a fatty acyl derivative. For example an N-terminus YaEGTFT A. DYSKALDKIHOODFWNWLLAOK octylglycine is combined with a d-amino acid at 1, 2, or, YaGTFT S DYSKALDKIHOODFWNWLLAOK particularly a D-Ala at position 2. In one embodiment a fatty acyl Substitution is a octylglycine for lysine at position 16 or YaBEGT S DYSKAMDKIROQEFVNWLLAOK methionine at position 14. In other embodiments the methion YaBEGT S DYSIALEKIROOKFVNWLLAOK ine at position 14 is deleted, and which, for example, can be further combined with a octylglycine for lysine at position 16 YaBEGT S DYSIALDKIRQODFWEWLLAOK or 30. Another substitution is acylation of the lysine at posi tion 16 or 30, for example with an octyl or palmitoyl group. YaBEGT S DYSIALDKIROQEFVNWLLAOK Other embodiments include a fatty acyl substitution where an YaBEGT S DYSIALDKIROQEFVNWLLAOK octylglycine is substituted for lysine at position 16 or 30 or for methionine at position 14. To eliminate or reduce oxidation, YaBEGT S DYSIAMDKIHOOL FIEWLKNGG the methionine at position 14 is deleted or substituted, for YaBEGT S DYSIAMDKIROQEFVNWLLAOK example with a lecince or other Small hydrophobic amino acid, and/or the tryptophan at position 25 is deleted or sub YaBEGT S DYSIAMDKIHOODFWNFLLAOK stituted, for example with a phenylalanine. YAEGT S DYSIAMDKIHOODFWNFLLAOK 0081. In one embodiment are analogs having at least one or two amino acid deletions in amino acids 1-30 of GIP, those having one or two deletions in amino acids 4 to 30, and those I0085. Accordingly, it is intended that the modifications having one or more deletions in amino acids 4-15, and those described herein can be combined with at least one other having one amino acid deletion in amino acids 1-30, 4-30 or change as described herein, Such as a change that imparts 4-15 of a GIP. Ofcourse it is intended that such a modification DPP-IV resistance, reduces or eliminates oxidation, reduces can be combined with at least one other change as described renal clearance, improves receptor binding, or improves herein, such as a change that imparts DPP-IV resistance, receptor activation. For example, intended are specific ana reduces or eliminates oxidation, reduces renal clearance, logs that have one or more replacements or modifications as improves receptor binding, or improves receptor activation. described herein, such as a GIPD-Ala2 or L-Ala2 analog that 0082 Further exemplary substitutions are those derived also has a Phe for Trp replacement at position 25. from GIP of other (non-human) species, for example the methionine 14 replaced by leucine, the D at position 9 or 21 GIP Hybrid Polypeptides replaced by E, the histidine 18 replaced by alanine, arginine I0086) Bio-Active Peptide Hormone Modules. As dis or lysine, the lysine at position 30 replaced by alanine, argi cussed herein the GIP hybrid polypeptides of the present nine or histidine, the alanine at position 13 replaced by leu invention (also referred to as “phybrids), generally comprise cine, and the alanine at position 28 replaced by serine. at least two bio-active peptide hormone modules covalently 0083. In one embodiment the GIP analogs have one or linked together, with a GIP peptideas one of the modules. The more of the following modifications: dAla2 to Abu, Ala, Gly, bio-active peptide hormone modules may be: (a) native com US 2009/0036364 A1 Feb. 5, 2009 ponent peptide hormones, (b) analogs or derivatives of native component peptide hormones that retain hormonal activity, - Continued (c) fragments of native component peptide hormones that retain hormonal activity, (d) fragments of analogs or deriva AFP - 6: TOAOLLRVGCGNLSTCOWONLSHRLWOLMGPAGRODSAPWDP tives of native component peptide hormones that retain hor SSPHSY, monal activity, (e) structural motifs of native component pep (SEQ ID NO: 18) tide hormones that impart a desired chemical stability, TOAOLLRVGCDTATCOWONLSHRLWOLMGPAGRODSAPWDPS SPHSY, conformational stability, metabolic stability, bioavailability, (SEQ ID NO: 19) organ/tissue targeting, receptor interaction, protease inhibi TOAOLLRVGMWLGTMOWONLSHRLWOLMGPAGRODSAPWDPS tion, plasma protein binding, and/or other pharmacokinetic SPHSY, characteristic to the hybrid polypeptide; or (f) structural (SEQ ID NO: 2O) TOAOLLRVGCVLGTCOWONLSHRLWOLMGPAGROESAPWEPS motifs of analogs or derivatives of native component peptide SPHSY, hormones that impart a desired chemical stability, conforma (SEQ ID NO: 21) tional stability, metabolic stability, bioavailability, organ/tis TOAOLLRVGCVLGTCOWONLSHRLWOLMGPAGROESAPWEPS Sue targeting, receptor interaction, protease inhibition, SPHSY, plasma protein binding, and/or other pharmacokinetic char (SEQ ID NO: 22) acteristic to the hybrid polypeptide. The structural motifs of CCK : DY (OSO3H) MGWMDF (e) and (f) will collectively be referred to herein as "peptidic (SEQ ID NO: 23) enhancers'. An example of a peptidic enhancer is a Trp cage DYMGWMDF (SEQ ID NO: 24) sequence, particularly one derived from exendin-4. Such as MGWMDF the Ex-4 short or long tails. (SEQ ID NO: 25) 0087 Exemplary bio-active peptide hormone modules GWMDF (SEQ ID NO: 26) include native peptide hormones selected from: amylin, WMDF ADM, CT, CGRP intermedin, CCK(1-33), CCK-8, leptin, (SEO ID NO: 27) PYY(1-36), PYY(3-36), GLP-1(1-37), GLP-1 (7-37), GLP-1 KDY (OSO3H) MGWMDF (7-36), GLP-2, OXM, GIP exendin-3, exendin-4, natriuretic (SEQ ID NO: 28) KDYMGWMDF peptide hormones, urocortin family peptides, e.g., Ucn-2 and (SEQ ID NO: 29) Ucn-3, neuromedin family peptides, e.g. neuromedin U25 or KMGWMDF splice variants, and ANP, BNP. CNP or urodilatin. (SEQ ID NO: 3 O) KGWMDF 0088. Other exemplary bio-active peptide hormone mod (SEQ ID NO: 31) ules include analogs and derivatives of a component peptide KWMDF hormone selected from: amylin, ADM, CT, CGRP interme (SEQ ID NO: 32) din, CCK, leptin, PYY(1-36), PYY(3-36), GLP-1(1-37), Leptin: “Asp-leptin, “Glu-leptin, Ala-leptin, GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, a natriuretic pep Glu-leptin, Des-AA-leptin, "Ala tide hormone, a urocortin family peptide, e.g., Ucn-2 and leptin, Lieu-leptin, Asp-leptin, Glu Ucn-3, a neuromedin family peptide, e.g. neuromedin U25 or leptin, Ala-leptin, Lieu-leptin, splice variants, exendin-3, and exendin-4, wherein the analog or derivative exhibits at least one hormonal activity of the PYY: Leu-PYY, Val-PYY, Arg-PYY, Gln-PYY, component peptide hormone. The analog may comprise one “Asn-PYY, Lys-PYY, “Pro-PYY, “His-PYY, or more insertions, deletions, or Substitutions of the amino Tyr-PYY, Pro14Ala-PYY, Leu Pro acid sequence of the component peptide hormone, and the PYY, des-AA-4-PYY derivative may comprise one or more chemical modifications GLP-1 Gln-GLP-1 (7-37), D-Gln-GLP-1 (7-37), of an amino acid residue of an analog or component peptide 'Thr-Lys-GLP-1 (7-37), Lys - GLP hormone, as described more fully herein and known in the art. 1 (7-37), Gly-GLP-1 (7-36), Gln-GLP 1 (7-37), D-Gln-GLP-1 (7-37), acetyl-Lys 0089 More specifically, analogs and derivatives may be GLP-1 (7-37), Thr-GLP-1 (7-37), D-Thr-GLP selected from any described above and/or known in the art. 1 (7-37), Asn-GLP-1 (7-37), D-Asn-GLP Particularly exemplary analogs and derivatives that exhibit at 1 (7-37), Ser? Arg Arg-Gln-GLP 1 (7-37), Thr'Llys- GLP-1 (7-37), Lys least one hormonal activity useful as bio-active peptide hor GLP-1 (7-37), Arg-GLP-1 (7-37), Arg-GLP mone modules of the invention include the following: 1 (7-37)

GIP Y - dAla2 - GIP Amylin: Ala-h-amylin, "Ala-h-amylin, Pro-h- Exendin Leu, Phe-exendin-4, Leu, Phe amylin, Pro-h-amylin, ' ' ' 'Pro-h- exendin-4, Ala, '“Leu, Phe-exendin-4, amylin, Pro, Val, Pro-h-amylin, and Leu, * Ala, Phe-exendin-4. "Arg, Pro-h-amylin, Arg, ° 2 °Pro-h-amylin, Pro, 2-val, ? Pro-h-amylin, Arg, Leu?, ?, ?? Pro 0090. As known in the art, such peptide compounds may h-amylin, Arg'Leu, Pro-h-amylin, preferably be amidated, but within the context of the present and 2, 7- Cyclo- (“Asp, 7Lys) -h-amylin invention, may optionally be in the acid form unless other C T : Glu-SCT, Arg-SCT, ''''Arg-SCT, Glu, wise specified. Arg-SCT, Glu, ' ' 'Arg-SCT 0091 Still other exemplary bioactive peptide hormone CGRP : D-Ser- CGRP, D-Thr- CGRP, D-Asp- CGRP, modules include fragments of a component peptide hormone D-Asn- CGRP, Ser- CGRP, Hse-CGRP, selected from: amylin, ADM, CT, CGRP intermedin, CCK, Asp- CGRP, Thr- CGRP, Asn-CGRP leptin, PYY(1-36), PYY(3-36), GLP-1(1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, a natriuretic peptide, aurocortin US 2009/0036364 A1 Feb. 5, 2009 family peptide, e.g., Ucn-2 and Ucn-3, a neuromedin family may be combined with any of the analogs or derivatives peptide, e.g. neuromedin U25 or splice variant, exendin-3, discussed herein or known in the art. For example, exemplary and exendin-4, wherein the fragment exhibits at least one analog fragments may include Ala, '“Leu, Phe-exendin-4 hormonal activity of the component peptide hormone. (1-28), ''Leu, Phe-exendin-4(1-27), Ala, '“Leu, Phe 0092. Yet other exemplary bioactive peptide hormone exendin-4(1-28), ''Leu, Phe-exendin-4(1-27), or any other modules include fragments of analogs or derivatives of a combinations of the disclosed fragments, analogs, and deriva component peptide hormone selected from: amylin, ADM, tives. Further embodiments include NN2211 and ZP-10. CT, CGRP intermedin, CCK, leptin, PYY(1-36), PYY(3- 0.095 Yet other exemplary bio-active peptide modules 36), GLP-1(1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, include "peptidic enhancer, i.e., structural motifs of compo OXM, ANP, BNP, CNP, urodilatin, Ucn-2 and Ucn-3, neuro nent peptide hormones (including analogs and derivatives medin U25 or splice variant, neuromedin S, exendin-3 and thereof) that impart a desired chemical stability, conforma exendin-4, wherein the fragment exhibits at least one hor tional stability, metabolic stability, bioavailability, organ/tis monal activity of the component peptide hormone. Again, the Sue targeting, receptor interaction, protease inhibition, analog may comprise one or more insertions, deletions, or plasma protein binding, and/or other pharmacokinetic char Substitutions of the amino acid sequence of the component acteristic to the hybrid polypeptide. Exemplary peptidic peptide hormone, and the derivative may comprise one or enhancers include the following.

Amylin Family amylin (32-37), amylin? 33-37), amylin (34-37), amylin (35-37), amylin? 36 37), amylin37), ADM(47-52), ADM(48-52), ADM(49-52), ADM(50-52), ADM(51-52), ADM(52), CT(27-32), CT(27-32), CT(28-32), CT(29-32), CT(30-32), CT(31-32), CT(32), CGRP(32-37), CGRP(33-37), CGRP(34 37), CGRP(35-37), CGRP(36-37), CGRP(37), intermedin (42-47), intermedin (43-47), intermedin (44-47), intermedin (45-47), intermedin (46 47), intermedin (47) PYY(25-36), PYY(26-36), PYY(27-36), PYY(28-36), PYY(29-36), PYY(30-36), PYY(31-36), PYY(32-36), PYY(25-35), PYY(26-35), PYY(27-35), PYY(28-35), PYY(29-35), PYY(30-35), PYY(31-35), PYY(32-35) GLP-1 and 2 frog GLP-1(29-37); frog GLP-1 (30-37); frog GLP-2(24-31), frog GLP-2(25 31) GIP GIP(31-42), GIP(32–42), GIP(33–42), GIP(34–42), GIP(35–42), GIP(36–42), GIP(37-42), GIP(38-42), GIP(39–42), GIP(40-42), GIP(41-42), GIP(42) Exendin-4 exendin-4(31-39), exendin-4(32-39), exendin-4(33-39), exendin-4(34-39), exendin-4(35-39), exendin-4(36-39), exendin-4(37-39), exendin-4(38-39), exendin-4(39) more chemical modifications of an amino acid residue of an 0096. Again, it should be understood that combinations of analog or component peptide hormone, as described more the above-described GIP analogs and derivatives taken fully herein and known in the art. together with the bio-active peptide modules described herein 0093 Certain exemplary fragments that exhibit at least are contemplated. For example, the last six amino acid resi dues of amylin family peptide hormone analogs and deriva one hormonal activity include the following. However, it tives known in the art and/or described above are also con should be understood that combinations of the above-de templated as exemplary bio-active peptide modules. For scribed analogs and derivatives taken with fragments known example, as further discussed herein, the peptidic enhancer in the art, including the exemplary fragments described Ex-4 short tail, which is an exemplary Trp-Cage sequence, or herein, are contemplated. analog thereof, is added to the C-terminus of any GIP analog, and in further embodiments the peptidic enhancer is attached using a linker. Amylin: amylin? 1-36), amylin (1-35), amylin (1-20), amylin? 1-18), (0097. In one aspect, the novel GIP hybrid include a GIP amylin? 1-17), amylin (1-16), amylin? 1-15), amylin (1-7) portion exhibiting at least 50%, at least 60%, at least 65%, at CT: CT(8-32), CT(8-27), CT(8-26), CT(8-10), CT(18-26), CT(18-27) least 70%, at least 75%, at least 80%, at least 85%, at least AFP-6: AFP-6(18-27) 90%, at least 95% or at least 98% sequence identity to a native CCK: CCK-8, CCK-5, CCK-4 GIP(1-30), native GIP(1-26), native GIP(1-14), native GIP(1- Leptin: leptin (22-167), leptin (56-73) PYY: PYY(1-35), PYY(1-30), PYY(1-25), PYY(1-15), PYY(1-10), 39), native GIP(19-30), native GIP(19-26), native GIP(19 PYY(2-36), PYY(3-36), PYY(4-36), PYY(5-36) 39), native GIP(19-42) or native GIP(1-42) over the entire GLP-1 GLP-1 (7-37), GLP-1 (7-36), GLP-1 (7-35) length of that GIP portion. GIP GIP(1-14), GIP(1-30) or longer, GIP(1-39) or longer 0098. Accordingly, in certain embodiments the GIP por Exendin exendin-4(1-27), exendin-4(1-28), exendin-4(1-29), tion of a GIP hybrid can comprise a trp-cage motif. Such exendin-4(1-30) or longer desirable GIP hybrids include an N-terminal GIP or novel GIP analog fragment in combination with a C-terminal 0094. Again, as known in the art, Such peptide compounds polypeptide or fragment thereof having a glucose lowering may preferably be amidated, but within the context of the activity (e.g., antidiabetics, exendin) or the ability to inhibitor present invention, may optionally be in the acid form unless reduce gastric emptying. Such desirable GIP hybrids include otherwise specified. Further, the above exemplary fragments an N-terminal GIP fragment or novel GIP analog fragment in US 2009/0036364 A1 Feb. 5, 2009 12 combination with a C-terminal exendin, GLP1, symlin esis associated protein (INGAP) receptor ligand; an (pramlintide), amylin, CCK, gastrin, PYY, secretin, GRP, Activin-A receptor ligand; a vascular endothelial growth fac tor (VEGF) receptor ligand; an erythropoietin (EPO) receptor neuromedins, urocortin, calcitonin, or salmon calcitonin, or ligand; a pituitary adenylate cyclase activating polypeptide fragment thereof. In other embodiments desirable GIP (PACAP) receptor ligand; a granulocyte colony stimulating hybrids include a C-terminal GIP or novel GIP analog frag factor (G-CSF) receptor ligand; a granulocyte-macrophage ment in combination with an N-terminal polypeptide or frag colony stimulating factor (GM-CSF); a platelet-derived ment thereofhaving a glucose lowering activity (e.g., antidia growth factor (PDGF) receptor ligand, and a secretin receptor betics, exendin) or the ability to inhibit or reduce gastric ligand. emptying. In Such embodiments, the chimeric polypeptides 0100. The polypeptides of the present invention will pref can include a C-terminal GIP, a novel GIP analog (in which erably retain, at least in part, a biological activity of native case a Trp-cage forming sequence is present), or fragment human GIP, e.g., the polypeptides of the present invention thereof, in combination with a N-terminal exendin, GLP1, will generally be GIPagonists orantagonists. In one embodi symlin (pramlintide), amylin, CCK, gastrin, PYY, secretin, ment, the polypeptides of the present invention will exhibit GRP neuromedins, urocortin, calcitonin, or salmon calcito biological activity in the treatment and prevention of meta nin, or fragment thereof. bolic conditions and disorders. Further, the novel GIP analog 0099. In other embodiments the GIP or novel GIP analog polypeptides of the invention may include internal linker is combined with a gastrin/CCK receptor ligand; an amylin compounds, may include chemical modifications at internal receptor ligand; a calcitonin receptor ligand; an CGRP recep amino acid residues, or may be chemically modified at the N-terminal or C-terminal residue. In yet another embodi tor ligand, a PYY receptor ligand, an EGF receptor ligand; a ment, the polypeptides of the invention include only natural L Glucagon-like peptide 1 receptor ligand; a Glucagon-like amino acid residues and/or modified natural L amino acid peptide 2 receptor ligand; a gastric inhibitory polypeptide residues. Alternatively, in another embodiment, the polypep (GIP) receptor ligand; a keratinocyte growth factor (KGF) tides of the invention do not include unnatural amino acid receptor 1 ligand; a dipeptidyl peptidase IV inhibitor; a REG residues. protein receptor ligand; a Growth Hormone receptor ligand; a 0101. In exemplary GIP hybrid embodiments, the GIP Prolactin (PRL) receptor ligand; an Insulin-like Growth Fac portion comprises a GIPN-terminal region modified or sub tor (IGF) receptor ligand; PTH-related protein (PTHrP) stituted to provide DPP-IV resistance superior to that of receptor ligand; hepatocyte growth factor (HGF) receptor native GIP ligand; a bone morphogenetic protein (BMP) receptor ligand, a transforming growth factor (TGF receptor ligand; a laminin Exemplary Peptide Component Families. receptor ligand; a vasoactive intestinal peptide (VIP) receptor 0102 Native peptide hormones are known in the art, as are ligand; a fibroblast growth factor (FGF) receptor ligand; a their analogs and derivatives. For reference, the sequences of nerve growth factor (NGF) receptor ligand; an islet neogen several native peptide hormones are provided herein.

Exemplary Peptide Hormones

SEQ ID No. Description Sequence 33 Rat Amylin KCNTATCATORLANFLVRSSNNLGPVLPPTNVGSNTY 34 h-Amylin: KCNTATCATORLANFLVHSSNNFGAILSSTNVGSNTY

35 h-ADM: YROSMNNFOGLRSFGCRFGTCTVOKLAHOIYOFTDKDKDNVAPRSKISP

36 S- CT: CSNLSTCVLGKLSOELHKLOTYPRTNTGSGTP

37 h- CT: CGNLSTCMLGTYTODFNKFHTFPOTAIGVGAP

38 h-CGRP C. : ACDTATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF

39 h-CGRP B: ACNTATCWTHRLAGLLSRSGGMWKSNFWPTNWGSKAF

4 O h-AFP - 6 (1 - TOAOLLRVGCVLGTCOWONLSHRLWOLMGPAGRODSAPWDPSSPHSY 47)

41 h-AFP - 6 (8 - WGCVLGTCOWONLSHRLWOLMGPAGRODSAPWDPSSPHSY 47) :

42 Mouse AFP - 6 PHAOLLRVGCVLGTCOWONLSHRLWOLVRPAGRRDSAPWDPSSPHSY (1 - 47) :

43 Mouse AFP - 6 WGCVLGTCOVONLSHRLWOLVRPAGRRDSAPWDPSSPHSY (8 - 47) :

44 CCK-8 - DY (SO) MGWMDF sulfated: US 2009/0036364 A1 Feb. 5, 2009 13

- Continued Exemplary Peptide Hormones

SEO ID No. Description Sequence

45 h-Leptin: MHWGTLCGFLWLWPYLFYVOAVPIQKVODDTKTLIKTIVTRINDISHTQ SVSSKOKVTGLDFIPGLHPILTLSKMDOTLAVYOOILTSMPSRNVIOISND LENLRDLLHWLAFSKSCHIPWASGLETLDSLGGWLEASGYSTEWWALSR LQGSLODMLWOLDLSPGC

46 hPYY: YPIKIPEAPGEDASPEELNRYYASLRHYLNLVTRORY 47 hPYY (3-36.) IKIPEAPGEDASPEELNRYYASLRHYLNLVTRORY 48 hCGLP-1 (1 - HDEFERHAEGTFTSDWSSTLEGOAALEFIAWLWKGRG 37) :

49 Frog GLP-1: HAEGTYTNDVTEYLEEKAAKEFIEWLIKGKPKKIRYS-OH,

SO HAEGTFTSDWTOOLDEKAAKEFIDWLINGGPSKEIIS-OH 51 h- GLP-1 (7- HAEGTFTSDWSSYLEGOAALEFIAWLWKGR 36) :

52 h- GLP-2 HADGSFSDEMINTILDNLAARDFINWLIETKITD

53 Frog GLP-2 : HAEGTFTNDMTNYLEEKAAKEFVGWLIKGRP-OH

54 OXM: HSOGTFTSDYSKYLDSRRAODFWOWLMNTKRNRNNIA 55 Exendin-3 ; HSDGTFTSDLSKOMEEEAVR LFIEWLKNGG PSSGAPPPS

5 Exendin-4 HGEGTFTSDLSKOMEEEAVRLFIEWLKNGGPSSGAPPPS

0103) These peptides are generally C-terminally amidated mone. It was isolated, purified and chemically characterized when expressed physiologically, but need not be for the pur as the major component of amyloid deposits in the islets of poses of the instant invention. In other words, the C-terminus pancreases of human Type 2 diabetics (Cooper et al., Proc. of these peptides, as well as the GIP hybrid polypeptides of Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The amylin the present invention, may have a free —OH or NH2 group. molecule has two post-translational modifications: the C-ter These peptides may also have other post-translational modi minus is amidated, and the cysteines in positions 2 and 7 are fications. One skilled in the art will appreciate that the hybrid cross-linked to form an N-terminal loop. The sequence of the polypeptides of the present invention may also be constructed open reading frame of the human amylin gene shows the with an N-terminal methionine residue. presence of the Lys-Arg dibasic amino acid proteolytic cleav 0104. It is also intended that in addition to comprising a age signal, prior to the N-terminal codon for Lys, and the Gly GIP hybrid, in other embodiments the hormones described prior to the Lys-Arg proteolytic signal at the CLAIMS-termi herein can be used in adjunct therapy, co-administered with, nal position, a typical sequence for amidation by protein a GIP analog of the invention. amidating enzyme, PAM (Cooper et al., Biochem. Biophys. 0105. The Amylin Family. As discussed herein component Acta, 1014:247-258 (1989)). peptide hormones useful in the present invention with a GIP 0107 Amylin is believed to regulate gastric emptying, and or a novel GIP analog include amylin family peptide hor Suppress glucagon secretion and food intake, thus regulating mones including amylin, adrenomedulin (ADM), calcito the rate of glucose appearance in the circulation. It appears to nin (“CT), calcitonin gene related peptide (“CGRP”), inter complement the actions of insulin, which regulates the rate of medin (also known as 'AFP-6') and related peptides. Native glucose disappearance from the circulation and its uptake by amylin family peptide hormones are known in art, as are peripheral tissues. These actions are Supported by experimen functional peptide analogs and derivatives. Certain exem tal findings in rodents and humans, which indicate that amylin plary native peptides, peptide analogs and derivatives are complements the effects of insulin in postprandial glucose described herein, however it should be recognized that any control by at least three independent mechanisms, all of known amylin family peptides that exhibit hormonal activity which affect the rate of glucose appearance. First, amylin known in the art may be used in conjunction with the present Suppresses postprandial glucagon Secretion. Compared to invention. Any amylin analog or derivative known in the art healthy adults, patients with type 1 diabetes have no circulat may be used in conjunction with the present invention. ing amylin and patients with type 2 diabetes have diminished 010.6 Another family of peptide hormones implicated in postprandial amylin concentrations. Furthermore, infusion of metabolic diseases and disorders is the amylin family of an amylin specific monoclonal antibody, which bound circu peptide hormones, including amylin, calcitonin, calcitonin lating amylin, again resulted in greatly elevated glucagon gene related peptide, adrenomedulin, and intermedin (also concentrations relative to controls. Both of these results point known as 'AFP-6). Amylin is a 37-amino acid peptide hor to a physiological role of endogenous amylin in the regulation US 2009/0036364 A1 Feb. 5, 2009

of postprandial glucagon Secretion. Second, amylin slows 239). An important role of CGRP is to control blood flow to gastrointestinal motility and gastric emptying. Finally, intra various organs by its potent vasodilatory actions, as evi hypothalamic injections of rat amylin were shown to reduce denced by a decrease of mean arterial pressure following feeding in rats and alter neurotransmitter metabolism in the intravenous administration of C-CGRP. The vasodilatory hypothalamus. In certain studies, food intake was signifi actions are also supported by recent analysis of homozygous cantly reduced for up to eight hours following the intrahypo knockout CGRP mice, which demonstrated elevated periph thalamic injection of rat amylin and rat CGRP. In human eral vascular resistance and high blood pressure caused by trials, an amylin analog, pramlintide, has been shown to increased peripheral sympathetic activity (Kurihara H. et al., reduce weight or weight gain. Amylin may be beneficial in Targeted disruption of ADM and C.CGRP genes reveals their treating metabolic conditions such as diabetes and obesity. distinct biological roles. Hypertens Res. 2003 February; 26 Amylin may also be used to treat pain, bone disorders, gas Suppl:S105-8). Thus, CGRP appears to elicit vasodilatory tritis, to modulate lipids, in particular triglycerides, or to effects, hypotensive effects and an increase in heart rate affect body composition such as the preferential loss offat among other actions. and sparing of lean tissue. 0111 Prolonged infusion of CGRP into patients with con 0108. The hormone calcitonin (CT) was named for its gestive cardiac failure has shown a Sustained beneficial effect secretion in response to induced hypercalcemia and its rapid on hemodynamic functions without adverse effects, suggest hypocalcemic effect. It is produced in and secreted from ing a use in heart failure. Other indications of CGRP use neuroendocrine cells in the thyroid that have since been include renal failure, acute and chronic coronary artery termed C cells. The best-studied action of CT(1-32) is its ischemia, treatment of cardiac arrhythmia, other peripheral effect on the osteoclast. In vitro effects of CT include the vascular disease Such as Raynaud's phenomenon, Subarach rapid loss of ruffled borders and decreased release of lysoso noid hemorrhage, hypertension, and pulmonary hyperten mal enzymes. Ultimately, the inhibition of osteoclast func Sion. Preeclamptic toxemia of pregnancy and preterm labor tions by CT results in a decrease in bone resorption. However, are also potentially treatable. (Wimalawansa, 1997). Recent neither a chronic reduction of serum CT in the case of thy therapeutic uses include the use of CGRPantagonists for the roidectomy nor the increased serum CT found in medullary treatment of migraine headaches. thyroid cancer appears to be associated with changes in serum 0112 Adrenomedullin (ADM) is almost ubiquitously calcium or bone mass. It is thus most likely that a major expressed with many more tissues containing the peptide than function of CT(1-32) is to combat acute hypercalcemia in not. A published review of ADM, (Hinson, J. P. et al., Endo emergency situations and/or protect the skeleton during peri crine Reviews (2000) 21(2): 138-167) details its effects on the ods of "calcium stress' such as growth, pregnancy, and lac cardiovascular system, cellular growth, the central nervous tation. (Reviewed in Becker, JCEM, 89(4): 1512-1525 (2004) system and the endocrine system, with a range of biological and Sexton, Current Medicinal Chemistry 6: 1067-1093 actions including vasodilation, cell growth, regulation of hor (1999)). Consistent with this is recent data from the calcitonin mone secretion, and natriuresis. Studies in rat, cat, sheep, and gene knockout mouse, which removes both the calcitonin and man confirm that intravenous infusion of ADM results in the CGRP-I peptides, that revealed that the mouse had normal potent and Sustained hypotension, and is comparable to that levels of basal calcium-related values, but an increased cal of CGRP. However, the hypotensive effect of ADM on mean cemic response (Kurihara H. et al., Hypertens Res. 2003 arterial pressure in the anesthetized rat is not inhibited by the February; 26 Suppl:S105-8). CGRPantagonist CGRP8-37 suggesting that this effect is not 0109 CT has an effect on plasma calcium levels and inhib mediated via CGRP receptors. Acute or chronic administra its osteoclast function and is widely used for the treatment of tion of human ADM in rats, anesthetized, conscious or hyper osteoporosis. Therapeutically, salmon CT (SCT) appears to tensive, results in a significant decrease in total peripheral increase bone density and decrease fracture rates with mini resistance accompanied by a fall in blood pressure, with a mal adverse effects. CT has also been successfully used over concomitant rise in heart rate, cardiac output and stroke Vol the past 25 years as a therapy for Paget's disease of bone, le which is a chronic skeletal disorder that may result in 0113 ADM has also been proposed as an important factor enlarged or deformed bones in one or more regions of the in embryogenesis and differentiation and as an apoptosis skeleton. CT is also widely used for its analgesic effect on survival factor for rat endothelial cells. This is supported by bone pain experienced during osteoporosis, although the recent mouse ADM knockout studies, in which mice homozy mechanism for this effect is not clearly understood. gous for loss of the ADM gene demonstrated defective vas 0110 Calcitonin gene related peptide (CGRP) is a neu cular formation during embryogenesis and thus died mid ropeptide whose receptors are widely distributed in the body, gestation. It was reported that ADM--/-heterozygous mice including the nervous system and the cardiovascular system. had high blood pressure along with Susceptibility to tissue This peptide seems to modulate sensory neurotransmission injury (Kurihara H. et al., Hypertens Res. 2003 February; 26 and is one of the most potent endogenous vasodilatory pep Suppl:S105-8). tide discovered to date. Reported biological effects for CGRP 0114 ADMaffects such endocrine organs as the pituitary, include: modulation of substance Pin inflammation, nicotinic the adrenal gland, reproductive organs and the pancreas. The receptor activity at the neuromuscular junction, stimulation peptide appears to have a role in inhibiting ACTH release of pancreatic enzyme secretion, a reduction of gastric acid from the pituitary. In the adrenal gland, it appears to affect the secretion, peripheral vasodilation, cardiac acceleration, secretory activity of the adrenal cortex in both rat and human neuro-modulation, regulation of calcium metabolism, osteo and it increases adrenal blood flow, acting as a vasodilator in genic stimulation, insulin secretion, an increase in body tem the adrenal vascular bed in intact rats. ADM has been shown perature and a decrease in food intake. (Wimalawansa, Amy to be present throughout the female reproductive tract and lin, calcitonin gene-related peptide, calcitonin and ADM: a plasma levels are elevated in normal pregnancy. Studies in a peptide superfamily. Crit. Rev Neurobiol. 1997: 11(2-3):167 rat model of preeclampsia show that ADM can reverse hyper US 2009/0036364 A1 Feb. 5, 2009 tension and decrease pup mortality when given to rats during binding to two closely related type II G protein-coupled late gestation. Because it did not have a similar effect in receptors (GPCRs), the calcitonin receptor (CTR) and the animals in early gestation or non-pregnant rats in the preec calcitonin receptor like receptor (CRLR). Cloning and func lampsia model, this Suggests that ADM may play an impor tional studies have shown that CGRP. ADM, and amylin tant regulatory role in the utero-placental cardiovascular sys interact with different combinations of CTR or the CRLR and tem. In the pancreas, ADM most likely plays an inhibitory the receptor activity modifying protein (RAMP). Many cells role since it attenuated and delayed insulin response to an oral express multiple RAMPs. It is believed that co-expression of glucose challenge, resulting in initial elevated glucose levels. RAMPs and either the CTR or CRLR is required to generate ADM can also affect renal function. A bolus administered functional receptors for calcitonin, CGRP. ADM, and amylin. peripherally can significantly lower mean arterial pressure The RAMP family comprises three members (RAMP1, -2, and raise renal blood flow, glomerular filtration rate and urine and -3), which share less then 30% sequence identity, but have flow. In some cases, there is also an increase in Na+ excretion. a common topological organization. Co-expression of CRLR and RAMP1 leads to the formation of a receptor for CGRP. 0115 ADM also has other peripheral effects on bone and Co-expression of CRLR and RAMP2 leads to the formation on the lung. For bone, studies have Supported a role beyond of a receptor for ADM. Co-expression of CRLRandRAMP3 the cardiovascular system and fluid homeostasis and have leads to the formation of a receptor for ADM and CGRP. demonstrated that ADM acts on fetal and adult rodent osteo Co-expression of hCTR2 and RAMP1 leads to the formation blasts to increase cell growth comparable to those of known of a receptor for amylin and CGRP. Co-expression of hCTR2 osteoblast growth factors such as transforming growth factor and RAMP3 leads to the formation of a receptor for amylin. alpha. This is important clinically as one of the major chal 0119 Thus a GIP hybrid comprising an amylin family lenges in osteoporosis research is to develop a therapy that hormone module can provide the functions and uses associ increases bone mass via osteoblastic stimulation. In the lung, ated with the amylin family module, e.g. amylin, amylin/SCT/ ADM not only causes pulmonary vasodilation, but also inhib amylin, ADM, CGRP as discussed, in addition to a GIP its bronchoconstriction induced by histamine or acetylcho function. line. Recent studies using aerosolized ADM to treat pulmo nary hypertension in a rat model indicate that inhalation I0120 In one embodiment, the amylin analogs and deriva treatment of this condition is effective, as evidenced by the tives have at least one hormonal activity of native amylin. In fact that mean pulmonary arterial pressure and total pulmo certain embodiments, the amylin analogs are agonists of a nary resistance were markedly lower in rats treated with receptor which native amylin is capable of specifically bind ADM than in those given saline. This result was achieved ing. Exemplary amylin analogs and derivatives include those without an alteration in Systemic arterial pressure or heart rate described in US 2003/0026812 A1, which is hereby incorpo (Nagaya N et al., Am J Physiol Heart Circ Physiol. 2003: rated by reference. 285:H2125-31). I0121 Exemplary amylin analogs include: 0116. In healthy volunteers, i.v. infusion of ADM has been shown to reduce arterial pressure and to stimulate heart rate, ‘’’’Pro-h-amylin (pramlintide) cardiac output, plasma levels of cAMP prolactin, norepi des-Lys-h-amylin nephrine and rennin. In these patients, there was little or no 2Pro, 26Val, 28.29Pro-h-amylin increase in urine Volume or Sodium excretion observed. In Arg, ''Pro-h-amylin des-Lys, Arg, ''Pro-h-amylin patients with heart failure or chronic renal failure, i.v. ADM 18Arg, 25.28.29Pro-h-amylin had similar effects to those seen in normal Subjects, and also des-Lys, Arg, 2-28.2°Pro-h-amylin induced diuresis and natriuresis, depending on the dose des-', Lys’’’Pro-h-amylin 2Pro, 26Val, 28.2°Pro-h-amylin administered (Nicholls, MG et al. Peptides. 2001; 22:1745 °Pro-h-amylin, 2,7-Cyclo-Asp, "Lys-h-amylin 1752) Experimental ADM treatment has also been shown to 27h-amylin be beneficial in arterial and pulmonary hypertension, septic 'Ala-h-amylin shock and ischemia/reperfusion injury (Beltowski J., Pol J. ‘Ala-h-amylin °-Ala-h-amylin Pharmacol. 2004:56:5-27). Other indications for ADM treat Ser-h-amylin ment include: peripheral vascular disease, Subarachnoid ‘’Pro-h-amylin hemorrhage, hypertension, preeclamptic toxemia of preg 22 Pro-h-amylin nancy and preterm labor, and osteoporosis. des-Lys, 2-Pro-h-amylin 2Leu, 2 Pro, 26Val, 28-29Pro-h-amylin 0117 Expression of AFP-6 (i.e., intermedin) is primarily 2Leu’Pro?Val?Pro-h-amylin in the pituitary and gastrointestinal tract. A specific receptor des-Lys, Leu, Pro, Val, Pro-h-amylin for AFP-6 has not been reported; however, binding studies Arg, 2-Leu, 2 Pro, 2Val, 2 Pro-h-amylin 18Arg, 2-Leu, 25-28.29Pro-h-amylin indicate that AFP-6 binds to all the known receptors of the Arg’Leu, ''Pro-h-amylin Amylin Family. AFP-6 has been shown to increase cAMP 7Ile, 2Leu, 25-28.29Pro-h-amylin production in SK-N-MC and L6 cells expressing endogenous 7Ile, 25.28.29Pro-h-amylin CGRP receptors and competes with labeled CGRP for bind des-Lys, ''Ile, Leu, '''Pro-h-amylin ing to its receptors in these cells. In published in vivo studies, ''Ile, Arg, Leu-h-amylin "Ile, Arg, 2-Leu,2Val, 29Pro-h-amylin AFP-6 administration led to blood pressure reduction in both "Ile, Arg, 2-Leu, 2 Pro, 2Val, 28.2°Pro-h-amylin, normal and spontaneously hypertensive rats, most likely via Thr, His, Leu, Ala, Leu, 'Pro, Asp-h-amylin interactions with the CRLR/RAMP receptors. In vivo admin Thr, His, Leu, Ala, 'Pro, Asp-h-amylin istration in mice led to a Suppression of gastric emptying and des-Lys, 'Thr, His, Leu, Ala, Pro, Asp-h-amylin *Thr, Arg, 2 His, 2Leu, 26Ala, 29Pro, Asp-h-amylin food intake. (Roh et al. J. Biol. Chem. 2004 Feb. 20; 279(8): Thr, Arg, His, Leu, 'Pro, Asp-h-amylin 7264-74.) Thr, Arg, His, Leu, Pro, Ala, 'Pro, Asp-h-amylin 0118. It has been reported that the biological actions of amylin family peptide hormones are generally mediated via US 2009/0036364 A1 Feb. 5, 2009

0122 AS known in the art, such amylin analogs are pref 0.125. As known in the art, such CT analogs are preferably erably amidated, but within the context of the present inven amidated, but within the context of the present invention, may optionally be in the acid form unless otherwise specified. tion, may optionally be in the acid form unless otherwise 0.126 Any CGRP analog or derivative known in the art specified. may be used in conjunction with the present invention. In one 0123. Any ADM analog or derivative known in the art may embodiment, the CGRP analogs and derivatives have at least be used in conjunction with the present invention. In one one hormonal activity of native CGRP. In certain embodi embodiment, the ADM analogs and derivatives have at least ments, the CGRP analogs are agonists of a receptor which one hormonal activity of native ADM. In certain embodi native CGRP is capable of specifically binding. Exemplary ments, the ADM analogs are agonists of a receptor which CGRP analogs and derivatives include those described in native ADM is capable of specifically binding. U.S. Pat. Nos. 4,697.002; and 4,687,839, which are hereby incorporated by reference. 0.124. Any CT analog orderivative known in the art may be I0127 Exemplary CGRP analogs include: used in conjunction with the present invention. In one embodiment, the CT analogs and derivatives have at least one hormonal activity of native CT. In certain embodiments, the 3D-Ser-CGRP CT analogs are agonists of a receptor which native CT is 36D-Thr-CGRP capable of specifically binding. Exemplary CT analogs and D-Asp-CGRP derivatives include those described in U.S. Pat. Nos. 4,652, D-Asn-CGRP 3°Ser-CGRP 627; 4,606,856; 4,604,238; 4,597,900; 4,537,716; 4,497,731: Hse-CGRP 4,495,097: 4,444,981; 4,414,149; 4,401,593; and 4,397,780, Asp-CGRP which are hereby incorporated by reference. Thr-CGRP Asn-CGRP

Gly-CT I0128. Any AFP-6 analog or derivative known in the art 22Leu-CT may be used in conjunction with the present invention. In one °Gly, Ser, Gly, 'des-Tyr-CT embodiment, the AFP-6 analogs and derivatives have at least 'Glu-sCT, Arg-sCT, one hormonal activity of native AFP-6. In certain embodi 11.8Arg-sCT, ments, the AFP-6 analogs are agonists of a receptor which 'Glu, Arg-sCT, native AFP-6 is capable of specifically binding. Exemplary 'Glu, Arg-sCT AFP-6 analogs and derivatives include those described in WO 2003/022304, which is hereby incorporated by reference. I0129. Exemplary AFP-6 analogs include:

SEQ ID No.: Sequence 18 TOAOLLRVGCGNLSTCOWONLSHRLWOLMGPAGRODSAPWDPSSPHSY

19 TOAOLLRVGCDTATCOWONLSHRLWOLMGPAGRODSAPWDPSSPHSY

TOAOLLRVGMWLGTMOVONLSHRLWOLMGPAGRODSAPWDPSSPHSY

21 TOAOLLRVGCVLGTCOWONLSHRLWOLMGPAGRODSAPWEPSSPHSY

22 TOAOLLRVGCVLGTCOWONLSHRLWOLMGPAGROESAPWEPSSPHSY

56 TOAOLLRVGCVLGTCOWONLSHRLWOL - - - -RODSAPWDPSSPHSY

st TOAOLLRVGCVLGTCOWONLSHRLWOL - - - -DSAPWDPSSPHSY

58 RVGCVLGTCOVONLSHRLWOLMGPAGRODSAPWDPSSPHSY

59 VGCVLGTCOVONLSHRLWOLMGPAGRODSAPVEPSSPHSY

60 WGCVLGTCOWONLSHRLWOL- - - -RODSAPWEPSSPHSY

61 GCVLGTCOWONLSHRLWOLMGPAGRODSAPWDPSSPHSY

62 GCNTATCOWONLSHRLWOL - - - -RODSAPWDPSSPHSY

63 GCNTATCOWONLSHRLWOL - - - -RODSAPWEPSSPHSY

64 GCSNLSTCOWONLSHRLWOL - - - -RODSAPWEPSSPHSY

65 GCGNLSTCOWONLSHRLWOL - - - -RODSAPWEPSSPHSY

66 GCVLGTCOWONLSHRLWOL - - - -RQESAPWEPSSPHSY

67 CVLGTCOVONLSHRLWOLMGPAGRODSAPWDPSSPHSY US 2009/0036364 A1 Feb. 5, 2009 17

- Continued SEQ ID No.: Sequence

68 QVONLSHRLWOLMGPAGRODSAPWDPSSPHSY

69 WONLSHRLWOLMGPAGRODSAPWDPSSPHSY

70 WONLSHRL - - - - QLMGPAGRODSAPWDPSSPHSY

71. GTMOVONLSHRLWQL - - - -RODSAPWEPSSPHSY

0130. As known in the art, such AFP-6 analogs are pref 713:236-241 (1994). It has also been suggested that estradiol erably amidated, but within the context of the present inven and CCK can have a synergistic effect on Satiety. Dulawa et tion, may optionally be in the acid form unless otherwise al., Peptides 15:913–918 (1994); Smith and Gibbs, supra. It specified. has also been proposed that signals arising from the Small 0131 The CCK Family. CCKs, including hCCK (chole intestine in response to nutrients therein may interact syner cystokinin) and species variants, and various analogs thereof gistically with CCK to reduce food intake. Cox, Behav. Brain are known in the art. Generally, CCK has a 33-amino acid Res. 38:35-44 (1990). Additionally, it has been reported that sequence first identified in humans, and includes a 8-amino CCK induces Satiety in several species. For example, it has acid in vivo C-terminal fragment (“CCK-8) that has been been reported that feeding depression was caused by CCK reportedly demonstrated in pig, rat, chicken, chinchilla, dog injected intraperitoneally in rats, intraarterially in pigs, intra and humans. Other species variants include a 39-amino acid venously in cats and pigs, into the cerebral ventricles in mon sequence found in pig, dog and guinea pig, and a 58-amino keys, rats, dogs and sheep, and intravenously in obese and acid found in cat, dog and humans, and a 47-amino acid non-obese humans. See Lieverse et al., Supra. Studies from sequences homologous to both CCK and gastrin. The C-ter several laboratories have reportedly confirmed the behavioral minal tyrosine-Sulfated octapeptide sequence (CCK-8) is specificity of low doses of CCK on inhibition in feeding, by relatively conserved across species, and may be the minimum comparing responding for food to responding for nonfood sequence for biological activity in the periphery of rodents. reinforces in both monkeys and rats and by showing that CCK Thus, the term CCK-33 will generally refer to human CCK elicits the sequence of behaviors normally observed after (1-33), while CCK-8 (CCK(26-33)) will refer to the C-termi meal ingestion (i.e., the postprandial Satiety sequence). Addi nal octapeptide generically in both the Sulfated and unsul tionally, comparison of behavior after CCK to behavior after fated unless otherwise specified. Further, pentagastrin or food ingestion, alone or in combination with CCK has report CCK-5 will refer to the C-terminal peptide CCK(29-33), and edly revealed behavioral similarities between CCK and food the CCK-4 will refer to the C-terminal tetrapeptide CCK(30 ingestion. Crawley and Corwin, Supra. It has also been 33). reported that CCK in physiological plasma concentrations 0.132. CCK was reportedly identified in 1928 from prepa inhibits food intake and increases Satiety in both lean and rations of intestinal extracts by its ability to stimulate gall obese humans. See Lieverse et al., Supra. bladder contraction. Other biological actions of CCK have 0.134. CCK was characterized in 1966 as a 33-amino acid since been reported, including stimulation of pancreatic peptide. Crawley and Corwin, Supra. Species-specific secretion, delayed gastric emptying, stimulation of intestinal molecular variants of the amino acid sequence of CCK have motility and stimulation of insulin secretion. See Lieverse et been identified. The 33-amino acid sequence and a truncated al., Ann. N.Y. Acad. Sci. 713:268-272 (1994). The actions of peptide, its 8-amino acid C-terminal sequence (CCK-8) have CCK, also reportedly include effects on cardiovascular func been reportedly identified in pig, rat, chicken, chinchilla, dog tion, respiratory function, neurotoxicity and seizures, cancer and humans. A 39-amino acid sequence was reportedly found cell proliferation, analgesia, sleep, sexual and reproductive in pig, dog and guinea pig. A 58-amino acid sequence was behaviors, memory, anxiety and dopamine-mediated behav reported to have been found in cat, dog and humans. Frog and iors. Crawley and Corwin, Peptides 15: 731-755 (1994). turtle reportedly show 47-amino acid sequences homologous Other reported effects of CCK include stimulation of pancre to both CCK and gastrin. Very fresh human intestine has been atic growth, stimulation of gallbladder contraction, inhibition reported to contain Small amounts of an even larger molecule, of gastric acid secretion, pancreatic polypeptide release and a termed CCK-83. In the rat, a principal intermediate form has contractile component of peristalsis. Additional reported been reportedly identified, and is termed CCK-22. Walsh, effects of CCK include vasodilation. Walsh, “Gastrointestinal “Gastrointestinal Hormones. In Physiology of the Gas Hormones. In Physiology of the Gastrointestinal Tract (3d trointestinal Tract (3d ed. 1994; Raven Press, New York). A ed. 1994; Raven Press, New York). non-sulfated CCK-8 and a tetrapeptide (termed CCK-4 (CCK 0133. It has been reported that injections of combinations (30-33)) have been reported in rat brain. The C-terminal of glucagon, CCK and bombesin potentiated the inhibition of pentapeptide (termed CCK-4 (CCK(29-33)) conserves the intake of condensed milk test meals in nondeprived rats over structural homology of CCK, and also homology with the the inhibitions observed with individual compounds. Hinton neuropeptide, gastrin. The C-terminal Sulfated octapeptide et al., Brain Res. Bull. 17:615-619 (1986). It has also been sequence, CCK-8, is reportedly relatively conserved across reported that glucagon and CCK Synergistically inhibit sham species. Cloning and sequence analysis of a cDNA encoding feeding in rats. LeSauter and Geary, Am. J. Physiol. 253: preprocholecystokinin from rat thyroid carcinoma, porcine R217-225 (1987); Smith and Gibbs, Annals N.Y. Acad. Sci. brain, and porcine intestine reportedly revealed 345 nucle US 2009/0036364 A1 Feb. 5, 2009 otides coding for a precursor to CCK, which is 115 amino 0.139. The Leptin Family. Component peptide hormones acids and contains all of the CCK sequences previously useful in the present invention also include leptin family reported to have been isolated. Crawley and Corwin, supra. peptide hormones. Native leptin family peptide hormones are 0135 CCK is said to be distributed throughout the central known in art, as are functional peptide analogs and deriva nervous system and in endocrine cells and enteric nerves of tives. Certain exemplary native peptides, peptide analogs and the upper small intestine. CCK agonists include CCK itself derivatives are described herein, however it should be recog (also referred to as CCK-33), CCK-8 (CCK(26-33)), non nized that any known leptin family peptides that exhibit hor sulfated CCK-8, pentagastrin (CCK-5 or CCK(29-33)), and monal activity known in the art may be used in conjunction the tetrapeptide, CCK-4 (CCK(30-33)). At the pancreatic with the present invention. CCK receptor, CCK-8 reportedly displaced binding with a 1000-5000 greater potency than unsulfated CCK-8 or CCK 0140 Yet another peptide hormone family implicated in 4, and CCK-8 has been reported to be approximately 1000 metabolic diseases and disorders is the leptin family. The fold more potent than unsulfated CCK-8 or CCK-4 in stimu mature form of circulating leptin is a 146-amino acid protein lating pancreatic amylase secretion. Crawley and Corwin, that is normally excluded from the CNS by the blood-brain Supra. In homogenates from the cerebral cortex, CCK recep barrier (BBB) and the blood-CSF barrier. See, e.g., Weigle et tor binding was said to be displaced by unsulfated CCK-8 and al., 1995. J. Clin Invest 96: 2065-2070. Leptin is the afferent by CCK-4 at concentrations that were equimolar, 10-fold or signal in a negative feedback loop regulating food intake and 100-fold greater than sulfated CCK-8. Id. Receptors for CCK body weight. The leptin receptor is a member of the cytokine have been reportedly identified in a variety of tissues, and two receptor family. Leptin's anorexigenic effect is dependent on primary Subtypes have been described: type A receptors and binding to homodimer of the Ob-Rb isoform of this receptor type B receptors. Type A receptors have been reported in which encodes along intra-cytoplasmic domain that includes peripheral tissues including pancreas, gallbladder, pyloric several motifs for protein-protein interaction. Ob-Rb is sphincter and afferent Vagal fibers, and in discrete areas of the highly expressed in the hypothalamus Suggesting that this brain. The type A receptor subtype (CCKA) has been reported brain region is an important site of leptin action. Mutation of to be selective for the sulfated octapeptide. The Type B recep the mouse ob gene has been demonstrated to result in a tor subtype (CCKB) has been identified throughout the brain syndrome that exhibits-pathophysiology that includes: obe and in the stomach, and reportedly does not require Sulfation sity, increased body fat deposition, hyperglycemia, hyperin or all eight amino acids. See Reidelberger, J. Nutr. 124 (8 Sulinemia, hypothermia, and impaired thyroid and reproduc Suppl.) 1327S-1333S (1994); Crawley and Corwin, supra. tive functions in both male and female homozygous ob?ob 0.136 Various in vivo and in vitro screening methods for obese mice (see e.g., Ingalis, et al., 1950. J Hered 41: 317 CCK analogs are known in the art. Examples include in vivo 318. Therapeutic uses for leptin or leptin receptor include (i) assays involving the contraction of the dog or guinea pig diabetes (see, e.g., PCT Patent Applications WO 98/55139, gallbladder after rapid intravenous injection of the compound WO 98/12224, and WO 97/02004); (ii) hematopoiesis (see, to be tested for CCK-like activity, and in vitro assays using e.g., PCT Patent Applications WO 97/27286 and WO strips of rabbit gallbladder. See Walsh, “Gastrointestinal Hor 98/18486); (iii) infertility (see, e.g., PCT Patent Applications mones”. In Physiology of the Gastrointestinal Tract (3d ed. WO97/15322 and WO 98/36763); and (iv) tumor suppres 1994; Raven Press, New York). sion (see, e.g., PCT Patent Applications WO 98/48831), each 0.137 Certain exemplary CCKs and CCK analogs with of which are incorporated herein by reference in their entirety. CCK activity include: 0.141. The leptin receptor (OB-R) gene has been cloned (GenBank Accession No. AF098792) and mapped to the db locus (see, e.g., Tartaglia, et al., 1995. Cell 83: 1263-1271). Several transcripts of the OB-R, resulting from alternative SEQ ID splicing, have also been identified. Defects in OB-R produce NO : Sequence a syndrome in the mutant diabetic ob?ob mouse that is phe notypically identical to the ob?ob mouse (see, e.g., Ghilardi, 72 DY (SOH) MGWMDF et al., 1996. Proc. Natl. Acad. Sci. USA 93: 6231-6235). In 24 DYMGWMDF contrast to obfob mice, however, administration of recombi nant leptin to C57BLKS/J-mob/ob mice does not result in 25 MGWMDF reduced food intake and body weight (see, e.g., Roberts and 26 GWMDF Greengerg, 1996. Nutrition Rev. 54: 41-49). 0.142 Most leptin-related studies able to report weight loss 27 WMDF activity from administration of recombinant leptin, leptin 73 KDY (SOH) MGWMDF fragments and/or leptin receptor variants have administered said constructs directly into the ventricles of the brain. See 29 KDYMGWMDF e.g., Weigle, et al., 1995.JClin Invest 96: 2065-2070; Barash, 3O KMGWMDF et al., 1996. Endocrinology 137: 3144-3147. 0143. Other studies have shown significant weight loss 31 KGWMDF activity due to administration of leptin peptides through intra peritoneally (i.p.) administration to test Subjects. See, Grasso 32 KWMDF et al., 1997. Endocrinology 138: 1413-1418. Further, leptin fragments, and most particularly an 18 amino acid fragment 0.138. As known in the art, such CCK peptides are prefer comprising residues taken from full length human leptin, ably amidated, but within the context of the present invention, have been reported to function in weight loss, but only upon may optionally be in the acid form unless otherwise specified. direct administration through an implanted cannula to the US 2009/0036364 A1 Feb. 5, 2009

lateral brain ventricle of rats. See, e.g., PCT Patent Applica Acad. Sci. USA 79: 2514-8 (1982)). A third related peptide tions WO 97/46585, which is incorporated herein by refer was later found in extracts of brain and named Neuropeptide ence in its entirety. Y (“NPY) (Tatemoto, Proc. Natl. Acad. Sci. USA 79: 5485-9 0144. Thus a GIP hybrid comprising a leptin family hor (1982); Tatemoto et al., Nature 296: 659-60 (1982)). mone module can provide the functions and uses associated 0150. These three related peptides have been reported to with the leptin family module, e.g. leptin, leptin fragment, as exert various biological effects. Effects of PP include inhibi discussed, in addition to a GIP function. tion of pancreatic secretion and relaxation of the gallbladder. 0145 Any leptin analog or derivative known in the art may Centrally administered PP produces modest increases in be used in conjunction with the present invention. In one feeding that may be mediated by receptors localized to the embodiment, the leptin analogs and derivatives have at least hypothalamus and brainstem (reviewed in Gehlert, Proc. Soc. one hormonal activity of native leptin. In certain embodi Exp. Biol. Med. 218: 7-22 (1998)). ments, the leptin analogs are agonists of a receptor which 0151 Release of PYY occurs following a meal. An alter native leptin is capable of specifically binding. Exemplary nate molecular form of PYY is PYY(3-36) (Eberlein et al., leptin analogs and derivatives include those described in, e.g., Peptides 10: 797-803 (1989); Grandt et al., Regul. Pept. 51: WO 2004/039832, WO 98/55139, WO 98/12224, and WO 151-9 (1994)). This fragment constitutes approximately 40% 97/02004, all of which are hereby incorporated by reference. of total PYY-like immunoreactivity in human and canine 0146 Exemplary leptin analogs include those where the intestinal extracts and about 36% of total plasma PYY immu amino acid at position 43 is substituted with Asp or Glu; noreactivity in a fasting state to slightly over 50% following a position 48 is substituted Ala; position 49 is substituted with meal. It is apparently a dipeptidyl peptidase-IV (DPP4) cleav Glu, or absent; position 75 is substituted with Ala; position 89 age product of PYY. PYY(3-36) is reportedly a selective is substituted with Leu; position 93 is substituted with Asp or ligand at the Y2 and Y5 receptors, which appear pharmaco Glu; position 98 is substituted with Ala; position 117 is sub logically unique in preferring N-terminally truncated (i.e., stituted with Ser, position 139 is substituted with Leu, posi C-terminal fragments of) NPY analogs. Peripheral adminis tion 167 is substituted with Ser, and any combination thereof. tration of PYY reportedly reduces gastric acid secretion, gas 0147 Certain exemplary leptin and leptin analogs with tric motility, exocrine pancreatic secretion (Yoshinaga et al., leptin activity include: Am. J. Physiol. 263: G695-701 (1992); Guan et al., Endocri nology 128: 911-6 (1991); Pappas et al., Gastroenterology 91: 1386-9 (1986)), gallbladder contraction and intestinal motility (Savage et al., Gut 28:166-70 (1987)). The effects of 43Asp-leptin 43Glu-leptin central injection of PYY on gastric emptying, gastric motility 48Ala-leptin and gastric acid secretion, as seen after direct injection in or 49Glu-leptin around the hindbrain/brainstem (Chen and Rogers, Am. J. 49Des-AA-leptin Physiol. 269: R787-92 (1995); Chen et al., Regul. Pept. 61: 75Ala-leptin 89Leu-leptin 95-98 (1996); Yang and Tache, Am. J. Physiol. 268: G943-8 93Asp-leptin (1995); Chen et al., Neurogastroenterol. Motil. 9: 109-16 93Glu-leptin (1997)), may differ from those effects observed after periph 98Ala-leptin eral injection. For example, centrally administered PYY had 117 Ser-leptin 139Leu-leptin some effects opposite to those described herein for peripher 167Ser-leptin ally injected PYY(3-36) in that gastric acid secretion was 43Asp, 49Glu-leptin stimulated, not inhibited. Gastric motility was suppressed 43Asp, 75 Ala-leptin only in conjunction with TRH stimulation, but not when 89Leu, 117 Ser-leptin administered alone, and was indeed stimulatory at higher 93Glu, 167Ser-leptin doses through presumed interaction with PP receptors. PYY has been shown to stimulate food and water intake after 0148. The PPF or PYY Family. Component peptide hor central administration (Morley et al., Brain Res. 341: 200-3 mones useful in the present invention also include Pancreatic (1985); Corp et al., Am. J. Physiol. 259: R317-23 (1990)). Polypeptide Family (PPF) peptide hormones, including PP, 0152 Any PPF analog or derivative known in the art may -NPY and PYY. Native PPF peptide hormones are known in be used in conjunction with the present invention. In one art, as are functional peptide analogs and derivatives. Certain embodiment, the PPF analogs and derivatives have at least exemplary native peptides, peptide analogs and derivatives one hormonal activity of a native PPF polypeptide. In certain are described herein, however it should be recognized that embodiments, the PPF analogs are agonists of a receptor any known PYY family peptides that exhibit hormonal activ which native PPF polypeptide is capable of specifically bind ity known in the art may be used in conjunction with the ing. Exemplary PPF analogs and derivatives include those present invention. described in WO 03/026591 and WO 03/057235, which are 0149. Yet another family of peptide hormones implicated herein incorporated by reference in their entirety. In one in metabolic diseases and disorders is the pancreatic polypep embodiment, exemplary PPF analogs and derivatives that tide family (“PPF). Pancreatic polypeptide (“PP) was dis exhibit at least one PPF hormonal activity generally comprise covered as a contaminant of insulin extracts and was named at least two PYY motifs including a polyproline motif and by its organ of origin rather than functional importance (Kim C-terminal tail motif. Such analogs are generally described in mel et al., Endocrinology 83: 1323-30 (1968)). PP is a U.S. Provisional Application No. 60/543,406 filed Feb. 11, 36-amino acid peptide containing distinctive structural 2004, which is herein incorporated by reference. Other exem motifs. A related peptide was Subsequently discovered in plary PPF analogs are disclosed in PCT/US2005/004351, extracts of intestine and named PeptideYY (“PYY) because entitled “Pancreatic Polypeptide Family Motifs and Polypep of the N- and C-terminal tyrosines (Tatemoto, Proc. Natl. tides Comprising the Same'. Attorney Docket 18528.832, the US 2009/0036364 A1 Feb. 5, 2009 20 contents of which is hereby incorporated by reference. By ing effect on insulin Secretion. GLP-1 is processed from pro way of background, the receptors to which PYY family pep glucagon in the gut and enhances nutrient-induced insulin tides bind are generally referred to as Y receptors, and release (Kreymann B., et al., Lancet, 2:1300-1303 (1987)). research has suggested that the differences in Y receptor Various truncated forms of GLP-1, are known to stimulate binding affinities are correlated with secondary and tertiary insulin secretion (insulinotropic action) and cAMP formation structural differences. See, e.g., Keire et al., Biochemistry (see, e.g., Mojsov, S., Int. J. Pep. Pro. Res., 40:333-343 2000, 39,9935-9942. Native porcine PYY has been charac (1992)). A relationship between various in vitro laboratory terized as including two C-terminal helical segments from experiments and mammalian, especially human, insulinotro residues 17 to 22 and 25 to 33 separated by a kink at residues pic responses to exogenous administration of GLP-1, GLP-1 23, 24, and 25, a turn centered around residues 12-14, and the N-terminus folded near residues 30 and 31. Further, full (7-36) amide, and GLP-1 (7-37) acid has been established length porcine PYY has been characterized as including the (see, e.g., Nauck, M. A., et al., Diabetologia, 36:741-744 PP fold, stabilized by hydrophobic interactions among resi (1993); Gutniak, M., et al., New Eng. J. of Med., 326(20): dues in the N- and C-termini. Seeid. 1316-1322 (1992): Nauck, M. A., et al., J. Clin. Invest., 0153. A “PYY motif is generally a structural component, 91:301-307 (1993); and Thorens, B., et al., Diabetes, primary, secondary, or tertiary, of a native PP family polypep 42:1219-1225 (1993)). tide that is critical to biological activity, i.e., biological activ 0156 GLP-1 (7-36) amide exerts a pronounced antidiabe ity is substantially decreased in the absence or disturbance of togenic effect in insulin-dependent diabetics by stimulating the motif. Exemplary PYY motifs include the N-terminal insulin sensitivity and by enhancing glucose-induced insulin polyproline type II motif of a native PP family polypeptide, release at physiological concentrations (Gutniak M., et al., the type II beta-turn motif of native PP family polypeptide, New Eng. J. Med., 326:13.16-1322 (1992)). When adminis the C-helical motif at the C-terminal end of native PP family tered to non-insulin dependent diabetics, GLP-1 (7-36) amide polypeptide, and the C-terminal tail motif of native PP family stimulates insulin release, lowers glucagon secretion, inhibits polypeptide. More particularly, in the N-terminal polyproline gastric emptying and enhances glucose utilization (Nauck, region, amino acids corresponding to residues 5 and 8 of a 1993; Gutniak, 1992: Nauck, 1993). However, the use of native PP family polypeptide are generally conserved as a GLP-1 type molecules for prolonged therapy of diabetes has proline. The type II beta-turn motif will generally include been complicated because the serum half-life of such pep amino acids corresponding to residues 12-14 of a native PP tides is quite short. family polypeptide. The C-helical motif can generally extend (O157 Moreparticularly, GLP-1 is a 30-amino acid peptide from amino acids corresponding to approximately residue 14 derived from proglucagon, a 160-amino acid prohormone. of a native PP family polypeptide to any point up to and Actions of different prohormone convertases in the pancreas including the C-terminal end, so long as the C-helical motif and intestine result in the production of glucagon and other includes a sufficient number of amino acid residues such that ill-defined peptides, whereas cleavage of proglucagon results an O-helical turn is formed in solution. The C-helical motif in the production of GLP-1 and GLP-2 as well as two other can also includeamino acid Substitutions, insertions and dele peptides. The amino acid sequence of GLP-1 is 100% tions to the native PP family sequence, so long as the C-helical homologous in all mammals studied so far, implying a critical turn is still formed in solution. The C-terminal tail motif physiological role. GLP-1 (7-37) acid is C-terminally trun generally includes amino acids corresponding to approxi cated and amidated to form GLP-1 (7-36) NH2. The biologi mately the last 10 residues of a native PP family polypeptide, cal effects and metabolic turnover of the free acid GLP-1 more preferably the last 7, 6, or 5 residues of a native PP (7-37) OH, and the amide, GLP-1 (7-36) NH2, are indistin family polypeptide, and more preferably amino acid residues guishable. By convention, the numbering of the amino acids 32-35. Exemplary PYY analogs include those with internal is based on the processed GLP-1 (1-37) OH from progluca deletions, insertions, and substitutions in areas of the PYY gon. The biologically active GLP-1 is the result of further molecule not corresponding to the polyproline motif and/or processing: GLP-1 (7-36) NH2. Thus the first amino acid of the C-terminal tail motif. For instance, internal deletions at GLP-1 (7-37) OH or GLP-1 (7-36)NH2 is 7H is. positions 4, 6, 7, 9, or 10 are envisioned. 0158. In the gastrointestinal tract, GLP-1 is produced by 0154 Additional Incretins and Incretin Mimetics. Com L-cells of intestinal, colonic and rectal mucosa, in response to ponent peptide hormones useful in the present invention also stimulation by intraluminal glucose. The plasma half-life of include GLP-1 peptide hormones. Native GLP-1 peptide hor active GLP-1 is <5 minutes, and its metabolic clearance rate mones, including GLP-1(1-37), GLP-1 (7-37), and GLP-1 is around 12-13 minutes (Holst, Gastroenterology 107(6): (7-36)amide, are known in art, as are functional peptide ana 1848-55 (1994)). The major protease involved in the metabo logs and derivatives. As used herein, GLP-1 refers to all native lism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26) forms of GLP-1 peptide hormones. Certain exemplary native which cleaves the N-terminal His-Ala dipeptide, thus produc peptides, peptide analogs and derivatives are described ing metabolites, GLP-1 (9–37) OH or GLP-1 (9–36). NH2 herein, however it should be recognized that any known which are variously described as inactive, weak agonist or GLP-1 peptides that exhibit hormonal activity known in the antagonists of GLP-1 receptor. The GLP-1 receptor (GLP art may be used in conjunction with the present invention. 1R) is a G protein coupled receptor of 463 amino acid and is 0155 Central to many metabolic diseases and disorders is localized in pancreatic beta cells, in the lungs, and to a lesser the regulation of insulin levels and blood glucose levels. extent in the brain, adipose tissue and kidneys. The stimula Insulin secretion is modulated in part by Secretagogue hor tion of GLP-1R by GLP-1 (7-37) OH or GLP-1 (7-36)NH2 mones, termed as incretins, which are produced by enteroen results in adenylate cyclase activation, cAMP synthesis, docrine cells. The incretin hormone, glucagon-like peptide-1 membrane depolarization, rise in intracellular calcium and (“GLP-1) is a peptide hormone secreted by intestinal cells increase in glucose-induced insulin secretion (Holz et al., J. that has been shown in multiple studies to produce an enhanc Biol. Chem. 270(30): 17749-57 (1995)). US 2009/0036364 A1 Feb. 5, 2009

0159 GLP-1 is a potent insulin secretagogue that is 0.165 Any GLP-1 peptide analog or derivative known in secreted from the intestinal mucosa in response to food the art may be used in conjunction with the present invention. intake. The profound incretin effect of GLP-1 is underscored In one embodiment, the GLP-1 peptide analogs and deriva by the fact that GLP-1R knockout mice are glucose-intoler tives have at least one hormonal activity of a native GLP-1 ant. The incretin response of i.v. infused GLP-1 is preserved peptide. In certain embodiments, the GLP-1 peptide analogs in diabetic Subjects, though the incretin response to oral glu are agonists of a receptor which a native GLP-1 peptide is cose in these patients is compromised. GLP-1 administration capable of specifically binding. Exemplary GLP-1 peptide by infusion or Sc injections controls fasting glucose levels in analogs and derivatives include those described in, e.g., WO diabetic patients, and maintains the glucose threshold for 91/11457, which is hereby incorporated by reference. insulin secretion (Gutniak et al., N. Engl. J. Med. 326:1316– 0166 GLP-1 analogs known in the art include: 22 (1992): Naucket al., Diabet. Med. 13:(9 Suppl S):S39-S43 (1996): Naucket al., J. Clin. Endocrinol. Metab. 76:912-917 (1993)). GLP-1 has shown tremendous potential as a thera 'Gln-GLP-1 (7-37) peutic agent capable of augmenting insulin secretion in a D-9Gln-GLP-1 (7-37) 6Thr-18Lys GLP-1 (7-37) physiological manner, while avoiding hypoglycemia associ Lys-GLP-1 (7-37) ated with Sulfonylurea drugs. Gly-GLP-1 (7-36) 0160 Other important effects of GLP-1 on glucose 'Gln-GLP-1 (7-37) D-9Gln-GLP-1 (7-37) homeostasis are Suppression of glucagon secretion and inhi acetyl-Lys-GLP-1 (7-37) bition of gastric motility. GLP-1 inhibitory actions on pan 'Thr-GLP-1 (7-37) creatic alpha cell secretion of glucagon leads to decreases in D-9Thr-GLP-1 (7-37) hepatic glucose production via reduction in gluconeogenesis 'Asn-GLP-1 (7-37) D-'Asn-GLP-1 (7-37) and glycogenolysis. This antiglucagon effect of GLP-1 is ’Ser’Arg’Arg’Gln-GLP-1 (7-37) preserved in diabetic patients. 6ThrLys-GLP-1 (7-37) (0161 The so-called ileal brake effect of GLP-1, in which Lys-GLP-1 (7-37) gastric motility and gastric secretion are inhibited, is effected *Arg-GLP-1 (7-37) via Vagal efferent receptors or by direct action on intestinal *Arg-GLP-1 (7-37) smooth muscle. Reduction of gastric acid secretion by GLP-1 contributes to a lag phase in nutrient availability, thus obvi 0.167 As known in the art, such GLP-1 analogs may pref ating the need for rapid insulin response. In summary, the erably be amidated, but within the context of the present gastrointestinal effects of GLP-1 contribute significantly to invention, may optionally be in the acid form unless other delayed glucose and fatty acid absorption and modulate insu wise specified. lin secretion and glucose homeostasis. 0168 Other GLP-1 analogs and derivatives are disclosed in U.S. Pat. No. 5,545,618 which is incorporated herein by 0162 GLP-1 has also been shown to induce beta cell spe reference. A exemplary group of GLP-1 analogs and deriva cific genes, such as GLUT-1 transporter, insulin (via the inter tives include those disclosed in U.S. Pat. No. 6,747,006, action of PDX-1 with insulin gene promoter), and hexoki which is herein incorporated by reference in its entirety. The nase-1. Thus GLP-1 could potentially reverse glucose use in the present invention of a molecule described in U.S. intolerance normally associated with aging, as demonstrated Pat. No. 5,188,666, which is expressly incorporated by refer by rodent experiments. In addition, GLP-1 may contribute to ence, is also contemplated. Another group of molecules for beta cell neogenesis and increase beta cell mass, in addition to use in the present invention includes compounds described in restoring beta cell function during states of beta cell insuffi U.S. Pat. No. 5.512.549, which is expressly incorporated ciency. herein by reference. Another exemplary group of GLP-1 0163 Central effects of GLP-1 include increases in satiety compounds for use in the present invention is disclosed in coupled with decreases in food intake, effected via the action WO 91/11457, which is herein incorporated by reference. of hypothalamic GLP-1R. A 48 hour continuous SC infusion (0169. In another embodiment useful with GIP or novel of GLP-1 in type II diabetic subjects, decreased hunger and GIP analogs are GLP1 analogs with Trp-Cage tails (e.g. exen food intake and increased satiety. These anorectic effects din C-terminal sequence with or without the tryptophan (or were absent in GLP-1R knock out mice. similar residue) (which can be optionally provided as 0164. Thus a GIP hybrid comprising an incretin family described, e.g. by the presence of a tryptophan advanta hormone module can provide the functions and uses associ geously located in the hormone that is either naturally occur ated with the incretin family module, e.g. exendin-4, GLP1, ring, added as a Substitution or as part of a linker.). These GLP2, as discussed, in addition to having a GIP function. include:

GLP1 (7-26) Gly8Ex- 4 (21–39) : HGEGTFTSDWSSYLEGOAAKLFIEWLKNGG PSSGAPPPS-NH2, (SEO ID NO : 74)

GLP1 (7-33) (G8 E3 O, K33) Ex- 4 (28-39): HGEGTFTSDWSSYLEGOAAKEFIEWLKNGGPSSGAPPPS-NH2; (SEO ID NO : 75)

GLP1 (7-33) (G8, K33) EX4 (28-39): HGEGTFTSDWSSYLEGOAAKEFIAWLKNGGPSSGAPPPS-NH2; (SEO ID NO : 76)