US 20060293.232A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0293232 A1 Levy et al. (43) Pub. Date: Dec. 28, 2006

(54) HYBRID POLYPEPTIDES WITH Related U.S. Application Data SELECTABLE PROPERTIES (63) Continuation-in-part of application No. 11/201,664, filed on Aug. 11, 2005, now abandoned, which is a (75) Inventors: Odile Esther Levy, San Diego, CA continuation-in-part of application No. 11/055,093, (US); Michael R. Hanley, Corte filed on Feb. 11, 2005. Madera, CA (US); Carolyn M. Jodka, Encinitas, CA (US); Diana Y. Lewis, (60) Provisional application No. 60/543,407, filed on Feb. San Diego, CA (US); Christopher J. 11, 2004. Soares, La Jolla, CA (US); Soumitra S. Publication Classification Ghosh, San Diego, CA (US); Lawrence J. D’Souza, San Diego, CA (US); (51) Int. Cl. David G. Parkes, Del Mar, CA (US); A6II 38/22 (2006.01) Christine M. Mack, San Diego, CA C07K I4/575 (2006.01) (US) (52) U.S. Cl...... 514/12: 530/399 (57) ABSTRACT Correspondence Address: ARNOLD & PORTER LLP (18528) The present invention relates generally to novel, selectable 555 TWELFTH ST, NW hybrid polypeptides useful as agents for the treatment and WASHINGTON, DC 20004 (US) prevention of metabolic diseases and disorders which can be alleviated by control plasma glucose levels, levels, (73) Assignee: , Inc., San and/or insulin , Such as and diabetes Diego, CA related conditions. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardio (21) Appl. No.: 11/206,903 vascular disease, eating disorders, insulin-resistance, obe sity, and diabetes mellitus of any kind, including type 1, type (22) Filed: Aug. 17, 2005 2, and gestational diabetes. Patent Application Publication Dec. 28, 2006 Sheet 1 of 9 US 2006/02932.32 A1

Figure 1: Effect in DIO Mouse of Exendin/PYY Hybrids

Effect of treatment on body weight in fattened C57BL/6 mice. 8 --LF diet control - HF diet control A Compound 175nmol/kg/day 4 -v- Compound 275nmol/kg/day -C- Compound 375nmol/kg/day -O- Compound 475nmol/kg/day O S. -4 A A -8 KX C -12 tf O -16 O 1 2 3 4. 5 6 7 Day of treatment PCO.05; ANOVA vs HF Vehicle Patent Application Publication Dec. 28, 2006 Sheet 2 of 9 US 2006/02932.32 A1

Figure 2: Effect in DIO Mouse of Exendin/Amylin Hybrids

Effect of treatment on body weight in fattened C57BL/6 mice. 8 --LF diet Control - - HF diet Contro A Compound 175nmol/kg/day -O- Compound 5.75nmol/kg/day A Compound 675nmol/kg/day

-4

-12

-1 6 O 1 2 3 4 5 6 7 Day of treatment PCO.05; ANOVA vs HF Vehicle Patent Application Publication Dec. 28, 2006 Sheet 3 of 9 US 2006/02932.32 A1

Figure 3A: Effect in DIO of Exendin/CCk-8 Hybrids

Effect of treatment on body weight in fattened C57BL/6 mice. 4. HF diet control -- Compound 7 25 nmol/kg/day 2

-2

-4

8 O 1 2 3 4. 5 6 7 P-0.05; ANOVA vs HF ve pays of Treatment Patent Application Publication Dec. 28, 2006 Sheet 4 of 9 US 2006/02932.32 A1

Figure 3B: Effect in DIO of Exendin/CCk-8 Hybrids

Effect of treatment on body weight in 8 fattened C57BL/6 mice. -HF Vehicle O4. -e-Compound 9 25nmol/kg/d

-4

-8

1 2 O 1 2 3 4 5 6 7 Days of Treatment PCO.05; ANOVA vs HF Vehicle Patent Application Publication Dec. 28, 2006 Sheet 5 of 9 US 2006/02932.32 A1

Figure 3C: Effect in DIO of Exendin/CCk-8 Hybrids

Effect of treatment on body weight in fattened C57BL/6 mice. 6 -- HF vehicle E V- Cmpd 8 25mmol/kg/d as a - - g 4 - Cmpd 1 75nmol/kg/d G5 X 2 - O E 0 O 9 2 -4 8 SS -6 -8 O 1 2 3 4. 5 6 7 Days of treatment *P<0.05; ANOVA vs HF vehicle Patent Application Publication Dec. 28, 2006 Sheet 6 of 9 US 2006/02932.32 A1

Figure 4A. Effect of Compounds of the Invention in Food Intake Assay (Compared to Parent Compounds)

Food Intake Assay: cumulative food intake

-- Vehicle -- Compound 10 10nmol/kg -- Compound 11 10nmol/kg -O- Compound 510nmol/kg - A - Compound 6 10nmol/kg -O-Compounds 10 and 11 pooled 10nmol/kg each

90 12O minutes Patent Application Publication Dec. 28, 2006 Sheet 7 of 9 US 2006/02932.32 A1

Figure 4.B. Effect of Compounds of the Invention in Food Intake Assay (Compared to Parent Compounds)

Food Intake Assay: cumulative food intake

--Vehicle -V- Compound 23nmol/kg -O-Compound 33nmol/kg -O- Compound 43nmol/kg - A - Compound 13nmol/kg - V - Compound 123nmol/kg -O- Compounds 1 and 12 pooled 3nmol/kg each

30 60 90 120 minutes Patent Application Publication Dec. 28, 2006 Sheet 8 of 9 US 2006/02932.32 A1

Figure 5A. Effect of Compounds of the Invention on Blood Glucose

Glucose Assay: Change in blood glucose

125

w s 100 E w t O) t O) o SS 75

mean pre-treatment glucose 123 mg/dL O 60 120 180 240 minutes

Glucose Assay Legend: Vehicle A Compound 11 16 nmol/kg 0 Compound 10 16 nmol/kg O Compound 10 with 11 16 nmol/kg each V Compound 15 16 nmol/kg O Compound 14 16 nmol/kg A Compound 5 16 nmol/kg Patent Application Publication Dec. 28, 2006 Sheet 9 of 9 US 2006/02932.32 A1 Figure 5B. Effect of Compounds of the Invention in Food Intake Assay Food Intake Assay: Cumulative food intake

8

7

6 5 E 4. O (9 C 3 O 9 2

1

O --- O 60 120 minutes

Food Intake Assay Legend: Vehicle A Compound 11 3nmol/kg 0 Compound 10 3nmol/kg O Compounds 10 with 11 3nmol/kg each V Compound 15 3nmol/kg O Compound 14 3nmol/kg A Compound 5 3nmol/kg US 2006/0293.232 A1 Dec. 28, 2006

HYBRD POLYPEPTIDES WITH SELECTABLE The biological effects and metabolic turnover of the free acid PROPERTIES GLP-1 (7-37) OH, and the amide, GLP-1 (7-36) NH, are indistinguishable. By convention, the numbering of the RELATED APPLICATIONS amino acids is based on the processed GLP-1 (1-37) OH 0001. The present application claims priority to com from . The biologically active GLP-1 is the monly-owned U.S. Provisional Application No. 60/543,407, result of further processing: GLP-1 (7-36) NH. Thus the filed Feb. 11, 2004, to U.S. Ser. No. 11/055,093, filed Feb. first of GLP-1 (7-37) OH or GLP-1 (7-36)NH 11, 2005, and to U.S. application Ser. No. filed as is His. Attorney Docket No. 0701-CIP-0 entitled “Hybrid Polypep 0006. In the gastrointestinal tract, GLP-1 is produced by tides With Selectable Properties', which are hereby incor L-cells of intestinal, colonic and rectal mucosa, in response porated by reference in their entirety. to stimulation by intraluminal glucose. The plasma half-life of active GLP-1 is <5 minutes, and its metabolic clearance FIELD OF THE INVENTION rate is around 12-13 minutes (Holst, Gastroenterology 0002 The present invention relates to chemistry, 107(6):1848-55 (1994)). The major protease involved in the and more particularly to hybrid polypeptides with selectable metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV properties. (CD26) which cleaves the N-terminal His-Ala , thus producing metabolites, GLP-1 (9-37) OH or GLP-1 (9-36) NH which are variously described as inactive, weak BACKGROUND OF THE INVENTION or antagonists of GLP-1 receptor. The GLP-1 recep 0003 Central to many metabolic diseases and disorders is tor (GLP-1R) is a G protein coupled receptor of 463 amino the regulation of insulin levels and blood glucose levels. acid and is localized in pancreatic beta cells, in the lungs, Insulin secretion is modulated in part by secretagogue and to a lesser extent in the brain, and , termed as , which are produced by kidneys. The stimulation of GLP-1R by GLP-1 (7-37) OH or enteroendocrine cells. The , -like GLP-1 (7-36)NH results in adenylate cyclase activation, peptide-1 (“GLP-1) is a secreted by cAMP synthesis, membrane depolarization, rise in intracel intestinal cells that has been shown in multiple studies to lular calcium and increase in glucose-induced insulin secre produce an enhancing effect on insulin secretion. GLP-1 is tion (Holz et al., J. Biol. Chem. 270(30): 17749-57 (1995)). processed from proglucagon in the gut and enhances nutri 0007 GLP-1 is a potent insulin secretagogue that is ent-induced insulin release (Krcymann B., et al., Lancet, secreted from the intestinal mucosa in response to food 2:1300-1303 (1987)). Various truncated forms of GLP-1, are intake. The profound incretin effect of GLP-1 is underscored known to stimulate insulin secretion (insulinotropic action) by the fact that GLP-1R knockout mice are glucose-intol and cAMP formation (see, e.g., Mojsov, S., Int. J. Pep. Pro. erant. The incretin response of i.v. infused GLP-1 is pre Res., 40:333-343 (1992)). A relationship between various in served in diabetic Subjects, though the incretin response to vitro laboratory experiments and mammalian, especially oral glucose in these patients is compromised. GLP-1 human, insulinotropic responses to exogenous administra administration by infusion or Sc injections controls fasting tion of GLP-1, GLP-1 (7-36) amide, and GLP-1 (7-37) acid glucose levels in diabetic patients, and maintains the glucose has been established (see, e.g., Nauck, M. A., et al., Diabe threshold for insulin secretion (Gutniak et al., N. Engl. J. tologia, 36:741-744 (1993); Gutniak, M., et al., New Eng. J. Med. 326:13.16-22 (1992): Naucket al., Diabet. Med. 13:(9 of Med., 326(20): 1316-1322 (1992): Nauck, M.A., et al., J. Suppl 5): S39-S43 (1996): Naucket al., J. Clin. Endocrinol. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Metab. 76:912-917 (1993)). GLP-1 has shown tremendous Diabetes, 42:1219-1225 (1993)). potential as a therapeutic agent capable of augmenting 0004 GLP-1 (7-36) amide exerts a pronounced antidia insulin secretion in a physiological manner, while avoiding betogenic effect in insulin-dependent diabetics by stimulat hypoglycemia associated with drugs. ing insulin sensitivity and by enhancing glucose-induced 0008. Other important effects of GLP-1 on glucose insulin release at physiological concentrations (Gutniak M., homeostasis are Suppression of glucagon Secretion and inhi et al., New Eng. J. Med., 326:13.16-1322 (1992)). When bition of gastric motility. GLP-1 inhibitory actions on pan administered to non-insulin dependent diabetics, GLP-1 (7- creatic alpha cell secretion of glucagon leads to decreases in 36) amide stimulates insulin release, lowers glucagon secre hepatic glucose production via reduction in gluconeogenesis tion, inhibits gastric emptying and enhances glucose utili and glycogenolysis. This antiglucagon effect of GLP-1 is zation (Nauck, 1993; Gutniak, 1992: Nauck, 1993). preserved in diabetic patients. However, the use of GLP-1 type molecules for prolonged therapy of diabetes has been complicated because the serum 0009. The so-called ileal brake effect of GLP-1, in which half-life of such is quite short. gastric motility and gastric secretion are inhibited, is effected 0005 More particularly, GLP-1 is a 30-amino acid pep via Vagal efferent receptors or by direct action on intestinal tide derived from proglucagon, a 160-amino acid prohor Smooth muscle. Reduction of gastric acid secretion by mone. Actions of different prohormone convertases in the GLP-1 contributes to a lag phase in nutrient availability, thus and intestine result in the production of glucagon obviating the need for rapid insulin response. In Summary, and other ill-defined peptides, whereas cleavage of proglu the gastrointestinal effects of GLP-1 contribute significantly cagon results in the production of GLP-1 and GLP-2 as well to delayed glucose and absorption and modulate as two other peptides. The amino acid sequence of GLP-1 is insulin secretion and glucose homeostasis. 100% homologous in all mammals studied so far, implying 0010 GLP-1 has also been shown to induce a critical physiological role. GLP-1 (7-37) acid is C-termi specific genes, such as GLUT-1 transporter, insulin (via the nally truncated and amidated to form GLP-1 (7-36) NH. interaction of PDX-1 with insulin gene ), and US 2006/0293.232 A1 Dec. 28, 2006

hexokinase-1. Thus GLP-1 could potentially reverse glucose Monster (Heloderma suspectum), with a 53% amino acid intolerance normally associated with aging, as demonstrated sequence identity to the GLP-1 peptide sequence. See, e.g., by rodent experiments. In addition, GLP-1 may contribute to Eng, J., et al. “Isolation and Characterization of Exendin-4, beta cell neogenesis and increase beta cell mass, in addition and Exendin-3 Analogue from Heloderma Suspectum to restoring beta cell function during states of beta cell Venom.J. Bio. Chem., 267:11, p. 7402-7405 (1992), Young, insufficiency. A. A., et al., “Glucose-Lowering and Insulin-Sensitizing 0011 Central effects of GLP-1 include increases in sati Actions of Exendin-4. Diabetes, Vol. 48, p. 1026-1034, ety coupled with decreases in food intake, effected via the May, 1999. In terms of its activity, exendin-4 is a highly action of hypothalamic GLP-1 R. A 48 hour continuous SC specific agonist for the GLP-1 receptor, and, like GLP-1, is infusion of GLP-1 in type II diabetic subjects, decreased able to stimulate insulin secretion. Therefore, like GLP-1, hunger and food intake and increased Satiety. These anorec exendin-4 is regarded as an insulinotropic peptide. tic effects were absent in GLP-1R knock out mice. 0016. However, unlike GLP-1, exendin4 has a relatively long half-life in humans, because of its resistance to the 0012 Exendins are another family of peptides implicated dipeptidyl peptidase IV which rapidly degrades the GLP-1 in insulin secretion. Exendins are found in the saliva of the sequence in vivo. Furthermore, it has been shown that, as Gila-monster, a lizard endogenous to Arizona, and the compared to GLP-1, exendin4 has a stronger capability to Mexican Beaded Lizard. Exendin-3 is present in the saliva stimulate insulin secretion, and that a lower concentration of of Heloderma horridum, and exendin-4 is present in the exendin4 may be used to obtain Such stimulating activity. saliva of Heloderma Suspectum (Eng., J., et al., J. Biol. See, e.g., U.S. Pat. No. 5,424,286, herein incorporated by Chem., 265:20259-62, 1990; Eng., J., et al., J. Biol. Chem., reference. Therefore exendin4 peptides or derivatives 267: 7402-05 (1992)). The exendins have some sequence thereof (for examples of such derivatives, see, e.g., U.S. Pat. similarity to several members of the glucagon-like peptide No. 6,528,486, herein incorporated by reference, and its family, with the highest identity, 53%, being to GLP-1 corresponding international application WO 01/04156) have (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993)). a greater potential utility for the treatment of conditions 0013 Exendin4 binds the GLP-1 receptors on insulin involving the dysregulation of insulin levels (e.g., conditions secreting TC1 cells, at dispersed acinar cells from guinea pig such as diabetes) than either insulin or GLP-1. pancreas, and at parietal cells from ; the peptide also stimulates release and inhibits release in 0017 Another family of peptide hormones implicated in isolated stomachs (Goke, et al., J. Biol. Chem., 268:19650 metabolic diseases and disorders is the amylin family of 55 (1993); Schepp, et al., Eur: J. Pharmacol., 69:183-91 peptide hormones, including amylin, , calcitonin (1994); Eissele, et al., Life Sci., 55:629-34 (1994)). Exen gene related peptide, adrenomedulin, and intermedin (also din-3 and exendin-4 were found to bind the GLP-1 receptors known as “AFP-6'). Amylin is a 37-amino acid peptide on, to stimulating cAMP production in, and amylase release hormone. It was isolated, purified and chemically charac from, pancreatic acinar cells (Malhotra, R., et al., Relulatory terized as the major component of deposits in the Peptides, 41:149-56 (1992); Raufman, et al., J. Biol. Chem. islets of of human Type 2 diabetics (Cooper et 267:21432-37 (1992); Singh, et al., Regul. Pept., 53:47-59 al., Proc. Natl. Acad. Sci., USA, 84:8628-8632 (1987)). The (1994)). The use of the insulinotropic activities of exendin-3 amylin molecule has two post-translational modifications: and exendin-4 for the treatment of diabetes mellitus and the the C-terminus is amidated, and the in positions 2 and 7 are cross-linked to form an N-terminal loop. The prevention of hyperglycemia has been proposed (Eng., U.S. sequence of the open reading frame of the human amylin Pat. No. 5,424,286). gene shows the presence of the Lys-Arg dibasic amino acid 0014 Truncated exendin peptides such as exending-39). proteolytic cleavage signal, prior to the N-terminal codon a carboxyamidated molecule, and fragments 3-39 through for Lys, and the Gly prior to the Lys-Arg proteolytic signal 9-39 have been reported to be potent and selective antago at the CLAIMS-terminal position, a typical sequence for nists of GLP-1 (Goke, et al., J. Biol. Chem., 268: 19650-55 amidation by protein amidating enzyme, PAM (Cooper et (1993); Raufman, J. P. et al., J. Biol. Chem., 266:2897-902 al., Biochem. Biophys. Acta, 1014:247-258 (1989)). (1991); Schepp, W., et al., Eur: J. Pharm., 269:183-91 0018 Amylin is believed to regulate gastric emptying, (1994); Montrose-Rafizadeh, et al., Diabetes, 45(Suppl. and Suppress glucagon Secretion and food intake, thus regu 2): 152A (1996)). Exendin9-39 blocks endogenous GLP-1 lating the rate of glucose appearance in the circulation. It in vivo, resulting in reduced insulin secretion (Wang, et al., appears to complement the actions of insulin, which regu J. Clin. Invest., 95:417-21 (1995): D’Alessio, et al., J. Clin. lates the rate of glucose disappearance from the circulation Invest., 97:133-38 (1996)). The receptor apparently respon and its uptake by peripheral tissues. These actions are sible for the insulinotropic effect of GLP-1 has been cloned Supported by experimental findings in rodents and humans, from rat pancreatic islet cells (Thorens, B., Proc. Natl. Acad. which indicate that amylin complements the effects of Sci. USA 89:8641-8645 (1992)). Exendins and exendin9 insulin in postprandial glucose control by at least three 39 bind to the cloned GLP-1 receptor (rat pancreatic -cell independent mechanisms, all of which affect the rate of GLP-1 receptor: Fehmann H C, et al., Peptides, 15 (3): glucose appearance. First, amylin Suppresses postprandial 453-6 (1994); human GLP-1 receptor: Thorens B, et al., glucagon Secretion. Compared to healthy adults, patients Diabetes, 42 (11): 1678-82 (1993)). In cells transfected with with type I diabetes have no circulating amylin and patients the cloned GLP-1 receptor, exendin-4 is an agonist, i.e., it with have diminished postprandial amylin increases cAMP, while exendin9-39 is an antagonist, i.e., concentrations. Furthermore, infusion of an amylin specific it blocks the stimulatory actions of exendin4 and GLP-1. Id. monoclonal antibody, which bound circulating amylin, 0.015 More particularly, exendin4 is a 39 amino acid again resulted in greatly elevated glucagon concentrations C-terminal amidated peptide found in the saliva of the Gila relative to controls. Both of these results point to a physi US 2006/0293.232 A1 Dec. 28, 2006 ological role of endogenous amylin in the regulation of wansa, Amylin, calcitonin gene-related peptide, calcitonin postprandial glucagon secretion. Second, amylin slows gas and ADM: a peptide superfamily. Crit Rev Neurobiol. 1997: trointestinal motility and gastric emptying. Finally, intrahy 11(2-3):167-239). An important role of CGRP is to control pothalamic injections of rat amylin were shown to reduce blood flow to various organs by its potent vasodilatory feeding in rats and alter neurotransmitter metabolism in the actions, as evidenced by a decrease of mean arterial pressure . In certain studies, food intake was signifi following intravenous administration of C-CGRP. The cantly reduced for up to eight hours following the intrahy vasodilatory actions are also supported by recent analysis of pothalamic injection of rat amylin and rat CGRP. In human homozygous knockout CGRP mice, which demonstrated trials, an amylin analog, , has been shown to elevated peripheral vascular resistance and high blood pres reduce weight or weight gain. Amylin may be beneficial in Sure caused by increased peripheral sympathetic activity treating metabolic conditions such as diabetes and obesity. (Kurihara H. et al., Targeted disruption of ADM and C.CGRP Amylin may also be used to treat pain, bone disorders, genes reveals their distinct biological roles. Hypertens Res. gastritis, to modulate lipids, in particular triglycerides, or to February 2003; 26 Suppl:S105-8). Thus, CGRP appears to affect body composition such as the preferential loss of fat elicit vasodilatory effects, hypotensive effects and an and sparing of lean tissue. increase in rate among other actions. 0019. The hormone calcitonin (CT) was named for its 0022 Prolonged infusion of CGRP into patients with secretion in response to induced hypercalcemia and its rapid congestive cardiac failure has shown a Sustained beneficial hypocalcemic effect. It is produced in and secreted from effect on hemodynamic functions without adverse effects, neuroendocrine cells in the that have since been suggesting a use in heart failure. Other indications of CGRP termed C cells. The best-studied action of CT(1-32) is its use include renal failure, acute and chronic coronary artery effect on the osteoclast. In vitro effects of CT include the ischemia, treatment of cardiac arrhythmia, other peripheral rapid loss of ruffled borders and decreased release of lyso vascular disease Such as Raynaud's phenomenon, Subarach somal enzymes. Ultimately, the inhibition of osteoclast noid hemorrhage, hypertension, and pulmonary hyperten functions by CT results in a decrease in bone resorption. Sion. Preeclamptic toxemia of pregnancy and preterm labor However, neither a chronic reduction of serum CT in the are also potentially treatable. (Wimalawansa, 1997). Recent case of thyroidectomy nor the increased serum CT found in therapeutic uses include the use of CGRPantagonists for the appears to be associated with treatment of migraine headaches. changes in serum calcium or bone mass. It is thus most likely that a major function of CT(1-32) is to combat acute 0023) Adrenomedulin (ADM) is almost ubiquitously hypercalcemia in emergency situations and/or protect the expressed with many more tissues containing the peptide skeleton during periods of "calcium stress' such as growth, than not. A published review of ADM, (Hinson, J. P. et al., pregnancy, and lactation. (Reviewed in Becker, JCEM, Endocrine Reviews (2000) 21(2): 138-167) details its effects 89(4): 1512-1525 (2004) and Sexton, Current Medicinal on the cardiovascular system, cellular growth, the central Chemistry 6: 1067-1093 (1999)). Consistent with this is nervous system and the , with a range of recent data from the calcitonin gene knockout mouse, which biological actions including vasodilation, cell growth, regu removes both the calcitonin and the CGRP-I peptides, that lation of hormone secretion, and natriuresis. Studies in rat, revealed that the mouse had normal levels of basal calcium cat, sheep, and man confirm that intravenous infusion of related values, but an increased calcemic response (Kurihara ADM results in potent and Sustained hypotension, and is H, et al., Hypertens Res. February 2003; 26 Suppl:S105-8). comparable to that of CGRP. However, the hypotensive effect of ADM on mean arterial pressure in the anesthetized 0020 CT has an effect on plasma calcium levels and rat is not inhibited by the CGRP antagonist CGRPs, inhibits osteoclast function and is widely used for the suggesting that this effect is not mediated via CGRP recep treatment of osteoporosis. Therapeutically, salmon CT (SCT) tors. Acute or chronic administration of human ADM in rats, appears to increase bone density and decrease fracture rates anesthetized, conscious or hypertensive, results in a signifi with minimal adverse effects. CT has also been successfully cant decrease in total peripheral resistance accompanied by used over the past 25 years as a therapy for Paget’s disease a fall in blood pressure, with a concomitant rise in heart rate, of bone, which is a chronic skeletal disorder that may result cardiac output and stroke Volume. in enlarged or deformed bones in one or more regions of the skeleton. CT is also widely used for its analgesic effect on 0024 ADM has also been proposed as an important bone pain experienced during osteoporosis, although the factor in embryogenesis and differentiation and as an apo mechanism for this effect is not clearly understood. ptosis survival factor for rat endothelial cells. This is sup ported by recent mouse ADM knockout studies, in which 0021 Calcitonin gene related peptide (CGRP) is a neu mice homozygous for loss of the ADM gene demonstrated ropeptide whose receptors are widely distributed in the body, defective vascular formation during embryogenesis and thus including the nervous system and the cardiovascular system. died mid-gestation. It was reported that ADM + heterozy This peptide seems to modulate sensory neurotransmission gous mice had high blood pressure along with Susceptibility and is one of the most potent endogenous vasodilatory to tissue injury (Kurihara H. et al., Hypertens Res. February peptide discovered to date. Reported biological effects for 2003; 26 Suppl:S 105-8). CGRP include: modulation of substance P in inflammation, nicotinic receptor activity at the neuromuscular junction, 0025 ADM affects such endocrine organs as the pitu stimulation of pancreatic enzyme secretion, a reduction of itary, the , reproductive organs and the pan gastric acid secretion, peripheral vasodilation, cardiac accel creas. The peptide appears to have a role in inhibiting ACTH eration, neuro-modulation, regulation of calcium metabo release from the pituitary. In the adrenal gland, it appears to lism, osteogenic stimulation, insulin secretion, an increase in affect the secretory activity of the adrenal cortex in both rat body temperature and a decrease in food intake. (Wimala and human and it increases adrenal blood flow, acting as a US 2006/0293.232 A1 Dec. 28, 2006

vasodilator in the adrenal vascular bed in intact rats. ADM tion in both normal and spontaneously hypertensive rats, has been shown to be present throughout the female repro most likely via interactions with the CRLR/RAMP recep ductive tract and plasma levels are elevated in normal tors. In vivo administration in mice led to a Suppression of pregnancy. Studies in a rat model of preeclampsia show that gastric emptying and food intake. (Roh et al. J. Biol Chem. ADM can reverse hypertension and decrease pup mortality Feb. 20, 2004:279(8):7264-74.) when given to rats during late gestation. Because it did not 0029. It has been reported that the biological actions of have a similar effect in animals in early gestation or non amylin family peptide hormones are generally mediated via pregnant rats in the preeclampsia model, this Suggests that binding to two closely related type II G protein-coupled ADM may play an important regulatory role in the utero receptors (GPCRs), the (CTR) and the placental cardiovascular system. In the pancreas, ADM most calcitonin receptor like receptor (CRLR). Cloning and func likely plays an inhibitory role since it attenuated and delayed tional studies have shown that CGRP. ADM, and amylin insulin response to an oral glucose challenge, resulting in interact with different combinations of CTR or the CRLR initial elevated glucose levels. ADM can also affect renal and the receptor activity modifying protein (RAMP). Many function. Abolus administered peripherally can significantly cells express multiple RAMPs. It is believed that co-expres lower mean arterial pressure and raise renal blood flow, sion of RAMPs and either the CTR or CRLR is required to glomerular filtration rate and urine flow. In some cases, there generate functional receptors for calcitonin, CGRP. ADM, is also an increase in Na+ excretion. and amylin. The RAMP family comprises three members 0026 ADM also has other peripheral effects on bone and (RAMP1-2, and -3), which share less then 30% sequence on the lung. For bone, studies have Supported a role beyond identity, but have a common topological organization. Co the cardiovascular system and fluid homeostasis and have expression of CRLR and RAMP1 leads to the formation of demonstrated that ADM acts on fetal and adult rodent a receptor for CGRP. Co-expression of CRLR and RAMP2 osteoblasts to increase cell growth comparable to those of leads to the formation of a receptor for ADM. Co-expression known osteoblast growth factors such as transforming of CRLR and RAMP3 leads to the formation of a receptor growth factor-B. This is important clinically as one of the for ADM and CGRP. Co-expression of hCTR2 and RAMP1 major challenges in osteoporosis research is to develop a leads to the formation of a receptor for amylin and CGRP. therapy that increases bone mass via osteoblastic stimula Co-expression of hCTR2 and RAMP3 leads to the formation tion. In the lung, ADM not only causes pulmonary vasodi of a receptor for amylin. lation, but also inhibits bronchoconstriction induced by 0030 Yet another peptide hormone family implicated in histamine or acetylcholine. Recent studies using aerosolized metabolic diseases and disorders is the family. The ADM to treat pulmonary hypertension in a rat model indi mature form of circulating leptin is a 146-amino acid protein cate that inhalation treatment of this condition is effective, as that is normally excluded from the CNS by the blood-brain evidenced by the fact that mean pulmonary arterial pressure barrier (BBB) and the blood-CSF barrier. See, e.g., Weigle and total pulmonary resistance were markedly lower in rats et al., 1995. J Clin Invest 96 : 2065-2070. Leptin is the treated with ADM than in those given saline. This result was afferent signal in a negative feedback loop regulating food achieved without an alteration in systemic arterial pressure intake and body weight. The is a member of or heart rate (Nagaya N et al., Am J Physiol Heart Circ the family. Leptin's anorexigenic effect is Physiol. 2003:285:H2125-31). dependent on binding to homodimer of the Ob-Rb isoform 0027. In healthy volunteers, i.v. infusion of ADM has of this receptor which encodes a long intra-cytoplasmic been shown to reduce arterial pressure and to stimulate heart domain that includes several motifs for protein-protein inter rate, cardiac output, plasma levels of cAMP , action. Ob-Rb is highly expressed in the hypothalamus and rennin. In these patients, there was little Suggesting that this brain region is an important site of leptin or no increase in urine Volume or Sodium excretion action. Mutation of the mouse ob gene has been demon observed. In patients with heart failure or chronic renal strated to result in a syndrome that exhibits-pathophysiology failure, i.v. ADM had similar effects to those seen in normal that includes: obesity, increased body fat deposition, hyper Subjects, and also induced diuresis and natriuresis, depend glycemia, hyperinsulinemia, hypothermia, and impaired ing on the dose administered (Nicholls, MG et al. Peptides. thyroid and reproductive functions in both male and female 2001; 22:1745-1752) Experimental ADM treatment has also homozygous ob?ob obese mice (see e.g., Ingalis, et al., 1950. been shown to be beneficial in arterial and pulmonary J Hered 41: 317–318. Therapeutic uses for leptin or leptin hypertension, septic shock and ischemia/reperfusion injury receptor include (i) diabetes (see, e.g., PCT Patent Applica (Beltowski J., Pol J Pharmacol. 2004:56:5-27). Other indi tions WO 98/55139, WO 98/12224, and WO 97/02004); (ii) cations for ADM treatment include: peripheral vascular hematopoiesis (see, e.g., PCT Patent Applications WO disease, Subarachnoid hemorrhage, hypertension, preec 97/27286 and WO 98/18486); (iii) infertility (see, e.g., PCT lamptic toxemia of pregnancy and preterm labor, and Patent Applications WO 97/15322 and WO 98/36763); and osteoporosis. (iv) tumor Suppression (see, e.g., PCT Patent Applications 0028 Expression of AFP-6 (i.e., intermedin) is primarily WO 98/48831), each of which are incorporated herein by in the pituitary and gastrointestinal tract. A specific receptor reference in their entirety. for AFP-6 has not been reported; however, binding studies 0031. The leptin receptor (OB-R) gene has been cloned indicate that AFP-6 binds to all the known receptors of the (GenBank Accession No. AF098792) and mapped to the db Amylin Family. AFP-6 has been shown to increase cAMP locus (see, e.g., Tartaglia, et al., 1995. Cell 83: 1263-1271). production in SK-N-MC and L6 cells expressing endog Several transcripts of the OB-R, resulting from alternative enous CGRP receptors and competes with labeled CGRP for splicing, have also been identified. Defects in OB-R produce binding to its receptors in these cells. In published in vivo a syndrome in the mutant diabetic ob/ob mouse that is studies, AFP-6 administration led to blood pressure reduc phenotypically identical to the ob?ob mouse (see, e.g., US 2006/0293.232 A1 Dec. 28, 2006

Ghilardi, et al., 1996. Proc. Natl. Acad. Sci. USA 93: intraarterially in pigs, intravenously in cats and pigs, into the 6231-6235). In contrast to ob/ob mice, however, adminis cerebral ventricles in monkeys, rats, dogs and sheep, and tration of recombinant leptin to C57BLKS/J-mob/ob mice intravenously in obese and non-obese humans. See Lieverse does not result in reduced food intake and body weight (see, et al., supra. Studies from several laboratories have report e.g., Roberts and Greengerg, 1996. Nutrition Rev. 54: 4149). edly confirmed the behavioral specificity of low doses of CCK on inhibition in feeding, by comparing responding for 0032 Most leptin-related studies able to report weight food to responding for nonfood reinforcers in both monkeys loss activity from administration of recombinant leptin, and rats and by showing that CCK elicits the sequence of leptin fragments and/or leptin receptor variants have admin behaviors normally observed after meal ingestion (i.e., the istered said constructs directly into the ventricles of the postprandial satiety sequence). Additionally, comparison of brain. See e.g., Weigle, et al., 1995. J. Clin Invest 96: behavior after CCK to behavior after food ingestion, alone 2065-2070; Barash, et al., 1996. Endocrinology 137: 3144 or in combination with CCK has reportedly revealed behav 3.147. ioral similarities between CCK and food ingestion. Crawley 0033. Other studies have shown significant weight loss and Corwin, supra. It has also been reported that CCK in activity due to administration of leptin peptides through physiological plasma concentrations inhibits food intake and intraperitoneally (i.p.) administration to test Subjects. See, increases Satiety in both lean and obese humans. See Liev Grasso et al., 1997. Endocrinology 138: 1413-1418. Further, erse et al., Supra. leptin fragments, and most particularly an 18 amino acid 0036 CCK was characterized in 1966 as a 33-amino acid fragment comprising residues taken from full length human peptide. Crawley and Corwin, Supra. Species-specific leptin, have been reported to function in weight loss, but molecular variants of the amino acid sequence of CCK have only upon direct administration through an implanted can been identified. The 33-amino acid sequence and a truncated nula to the lateral brain ventricle of rats. See, e.g., PCT peptide, its 8-amino acid C-terminal sequence (CCK-8) have Patent Applications WO 97/46585, which is incorporated been reportedly identified in pig, rat, chicken, chinchilla, herein by reference in its entirety. dog and humans. A 39-amino acid sequence was reportedly 0034. Another peptide hormone implicated in metabolic found in pig, dog and guinea pig. A 58-amino acid sequence diseases and disorders is (CCK). CCK was was reported to have been found in cat, dog and humans. reportedly identified in 1928 from preparations of intestinal Frog and turtle reportedly show 47-amino acid sequences extracts by its ability to stimulate gallbladder contraction. homologous to both CCK and gastrin. Very fresh human Other biological actions of CCK have since been reported, intestine has been reported to contain small amounts of an including stimulation of pancreatic secretion, delayed gas even larger molecule, termed CCK-83. In the rat, a principal intermediate form has been reportedly identified, and is tric emptying, stimulation of intestinal motility and stimu termed CCK-22. Walsh, “Gastrointestinal Hormones. In lation of insulin secretion. See Lieverse et al., Ann. N.Y. Physiology of the Gastrointestinal Tract (3d ed. 1994; Raven Acad. Sci. 713: 268-272 (1994). The actions of CCK, also Press, New York). A non-sulfated CCK-8 and a reportedly include effects on cardiovascular function, respi (termed CCK-4 (CCK(30-33)) have been reported in rat ratory function, neurotoxicity and seizures, cancer cell pro brain. The C-terminal pentapeptide (termed CCK-4 liferation, analgesia, sleep, sexual and reproductive behav (CCK(29-33)) conserves the structural homology of CCK, iors, memory, anxiety and -mediated behaviors. and also homology with the , gastrin. The Crawley and Corwin, Peptides 15: 731-755 (1994). Other C-terminal Sulfated octapeptide sequence, CCK-8, is report reported effects of CCK include stimulation of pancreatic edly relatively conserved across species. Cloning and growth, stimulation of gallbladder contraction, inhibition of sequence analysis of a cDNA encoding preprocholecystoki gastric acid secretion, release and a nin from rat thyroid carcinoma, porcine brain, and porcine contractile component of peristalsis. Additional reported intestine reportedly revealed 345 nucleotides coding for a effects of CCK include vasodilation. Walsh, “Gastrointesti precursor to CCK, which is 115 amino acids and contains all nal Hormones. In Physiology of the Gastrointestinal Tract of the CCK sequences previously reported to have been (3d ed. 1994; Raven Press, New York). isolated. Crawley and Corwin, Supra. 0035) It has been reported that injections of combinations 0037 CCK is said to be distributed throughout the central of glucagon, CCK and potentiated the inhibition nervous system and in endocrine cells and enteric nerves of of intake of condensed milk test meals in nondeprived rats the upper small intestine. CCK include CCK itself over the inhibitions observed with individual compounds. (also referred to as CCK-33), CCK-8 (CCK(26-33)), non Hinton et al., Brain Res. Bull. 17:615-619 (1986). It has also sulfated CCK-8, (CCK-5 or CCK(29-33)), and been reported that glucagon and CCK Synergistically inhibit the tetrapeptide, CCK-4 (CCK(30-33)). At the pancreatic sham feeding in rats. LeSauter and Geary, Am. J. Physiol. CCK receptor, CCK-8 reportedly displaced binding with a 253:R217-225 (1987); Smith and Gibbs, Annals N.Y. Acad. Sci. 713:236-241 (1994). It has also been suggested that 1000-5000 greater potency than unsulfated CCK-8 or CCK and CCK can have a synergistic effect on Satiety. 4, and CCK-8 has been reported to be approximately 1000 Dulawa et al., Peptides 15:913–918 (1994); Smith and fold more potent than unsulfated CCK-8 or CCK-4 in Gibbs, Supra. It has also been proposed that signals arising stimulating pancreatic amylase secretion. Crawley and Cor from the Small intestine in response to nutrients therein may win, Supra. In homogenates from the cerebral cortex, CCK interact synergistically with CCK to reduce food intake. receptor binding was said to be displaced by unsulfated Cox, Behav. Brain Res. 38:35-44 (1990). Additionally, it has CCK-8 and by CCK-4 at concentrations that were equimo been reported that CCK induces satiety in several species. lar, 10-fold or 100-fold greater than sulfated CCK-8. Id. For example, it has been reported that feeding depression 0038 Receptors for CCK have been reportedly identified was caused by CCK injected intraperitoneally in rats, in a variety of tissues, and two primary Subtypes have been US 2006/0293.232 A1 Dec. 28, 2006 described: type A receptors and type B receptors. Type A and was indeed stimulatory at higher doses through pre receptors have been reported in peripheral tissues including sumed interaction with PP receptors. PYY has been shown pancreas, gallbladder, pyloric sphincter and afferent vagal to stimulate food and water intake after central administra fibers, and in discrete areas of the brain. The type A receptor tion (Morley et al., Brain Res. 341: 200-3 (1985); Corp et al., subtype (CCKA) has been reported to be selective for the Am. J. Physiol. 259: R317-23 (1990)). sulfated octapeptide. The Type B receptor subtype (CCK) 0042 Metabolic diseases and disorders take on many has been identified throughout the brain and in the stomach, forms, including obesity, diabetes, dyslipidemia, insulin and reportedly does not require Sulfation or all eight amino resistance, cellular , etc. Obesity and its associated acids. See Reidelberger, J. Nutr. 124 (8 Suppl.) 1327S disorders are common and very serious public health prob 1333S (1994); Crawley and Corwin, supra. lems in the United States and throughout the world. Upper 0039. Yet another family of peptide hormones implicated body obesity is the strongest risk factor known for type 2 in metabolic diseases and disorders is the pancreatic diabetes mellitus, and is a strong risk factor for cardiovas polypeptide family (“PPF). Pancreatic polypeptide (“PP) cular disease. Obesity is a recognized risk factor for hyper was discovered as a contaminant of insulin extracts and was tension, atherosclerosis, congestive heart failure, stroke, named by its organ of origin rather than functional impor gallbladder disease, osteoarthritis, sleep apnea, reproductive tance (Kimmel et al., Endocrinology 83: 1323-30 (1968)). disorders such as polycystic ovarian syndrome, cancers of PP is a 36-amino acid peptide containing distinctive struc the breast, prostate, and colon, and increased incidence of tural motifs. A related peptide was Subsequently discovered complications of general anesthesia (see, e.g., Kopelman, in extracts of intestine and named Peptide YY (“PYY) Nature 404: 635-43 (2000)). It reduces life-span and carries because of the N— and C-terminal tyrosines (Tatemoto, a serious risk of co-morbidities above, as well disorders such Proc. Natl. Acad. Sci. USA 79: 2514-8 (1982)). A third as infections, varicose veins, acanthosis nigricans, eczema, related peptide was later found in extracts of brain and exercise intolerance, , hypertension hyper named (“NPY) (Tatemoto, Proc. Natl. cholesterolemia, cholelithiasis, orthopedic injury, and Acad. Sci. USA 79: 5485-9 (1982); Tatemoto et al., Nature thromboembolic disease (Rissanen et al., Br: Med. J 301: 296: 659-60 (1982)). 835-7 (1990)). Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or "Syndrome 0040. These three related peptides have been reported to X. Recent estimate for the medical cost of obesity and exert various biological effects. Effects of PP include inhi associated disorders is S150 billion worldwide. The patho bition of pancreatic secretion and relaxation of the gallblad genesis of obesity is believed to be multifactorial but the der. Centrally administered PP produces modest increases in basic problem is that in obese subjects nutrient availability feeding that may be mediated by receptors localized to the and energy expenditure do not come into balance until there hypothalamus and brainstem (reviewed in Gehlert, Proc. is excess adipose tissue. Obesity is currently a poorly Soc. Exp. Biol. Med. 218: 7-22 (1998)). treatable, chronic, essentially intractable metabolic disorder. 0041 Release of PYY occurs following a meal. An A therapeutic drug useful in weight reduction of obese alternate molecular form of PYY is PYY(3-36) (Eberleinet persons could have a profound beneficial effect on their al., Peptides 10: 797-803 (1989); Grandt et al., Regul. Pept. health. 51: 151-9 (1994)). This fragment constitutes approximately 0043. Diabetes is a disorder of carbohydrate metabolism 40% of total PYY-like immunoreactivity in human and characterized by hyperglycemia and glucosuria resulting canine intestinal extracts and about 36% of total plasma PYY immunoreactivity in a fasting state to slightly over from insufficient production or utilization of insulin. Diabe 50% following a meal. It is apparently a dipeptidyl pepti tes severely affects the quality of life of large parts of the dase-IV (DPP4) cleavage product of PYY. PYY(3-36) is populations in developed countries. Insufficient production reportedly a selective ligand at the Y2 and Y5 receptors, of insulin is characterized as and insufficient which appear pharmacologically unique in preferring N-ter utilization of insulin is type 2 diabetes. However, it is now minally truncated (i.e., C-terminal fragments of) NPY ana widely recognized that there are many distinct diabetes logs. Peripheral administration of PYY reportedly reduces related diseases which have their onset long before patients gastric acid secretion, gastric motility, exocrine pancreatic are diagnosed as having overt diabetes. Also, the effects secretion (Yoshinaga et al., Am. J. Physiol. 263: G695-701 from the Suboptimal control of glucose metabolism in dia (1992); Guan et al., Endocrinology 128: 911-6 (1991); betes gives rise to a wide spectrum of related lipid and Pappas et al., Gastroenterology 91: 1386-9 (1986)), gall cardiovascular disorders. bladder contraction and intestinal motility (Savage et al., 0044) Dyslipidemia, or abnormal levels of lipoproteins in Gut 28: 166-70 (1987)). The effects of central injection of blood plasma, is a frequent occurrence among diabetics. PYY on gastric emptying, gastric motility and gastric acid Dyslipidemia is typically characterized by elevated plasma secretion, as seen after direct injection in or around the triglycerides, low HDL (High Density Lipoprotein) choles hindbrain/brainstem (Chen and Rogers, Am. J. Physiol. 269: terol, normal to elevated levels of LDL (Low Density R787-92 (1995); Chen et al., Regul. Pept. 61: 95-98 (1996): Lipoprotein) cholesterol and increased levels of Small dense, Yang and Tache, Am. J. Physiol. 268: G943-8 (1995); Chen LDL (Low Density Lipoprotein) particles in the blood. et al., Neurogastroenterol. Motil. 9; 109-16 (1997)), may Dyslipidemia is one of the main contributors to the increased differ from those effects observed after peripheral injection. incidence of coronary events and deaths among diabetic For example, centrally administered PYY had some effects Subjects. Epidemiological studies have confirmed this by opposite to those described herein for peripherally injected showing a several-fold increase in coronary deaths among PYY(3-36) in that gastric acid secretion was stimulated, not diabetic subjects when compared with non-diabetic subjects. inhibited. Gastric motility was suppressed only in conjunc Several lipoprotein abnormalities have been described tion with TRH stimulation, but not when administered alone, among diabetic Subjects. US 2006/0293.232 A1 Dec. 28, 2006

0045 Insulin resistance is the diminished ability of insu disorders include, but are not limited to, hypertension, lin to exert its biologically action across a broad range of dyslipidemia, cardiovascular disease, eating disorders, insu concentrations. In insulin resistance, the body secretes lin-resistance, obesity, and diabetes mellitus of any kind, abnormally high amounts of insulin to compensate for this including type 1, type 2, and gestational diabetes. defect and a state of impaired glucose tolerance develops. 0051. In one aspect of the invention, hybrid polypeptides Failing to compensate for the defective insulin action, the exhibiting at least one hormonal activity are provided. The plasma glucose concentration inevitable rises, resulting in hybrid polypeptides of the invention comprise at least two the clinical state of diabetes. It is being recognized that bio-active peptide hormone modules covalently linked insulin resistance and relative hyperinsulinemia have a con together, wherein at least one of the bio-active peptide tributory role in obesity, hypertension, atherosclerosis and hormone modules exhibits at least one hormonal activity of type 2 diabetes. The association of insulin resistance with a component peptide hormone. The bio-active peptide hor obesity, hypertension and angina has been described as a mone modules are independently selected from: component syndrome, Syndrome X, having insulin resistance as the peptide hormones, fragments of component peptide hor common pathogenic link. mones that exhibit at least one hormonal activity of the 0046 Apoptosis is an active process of cellular self component peptide hormones, analogs and derivatives of destruction that is regulated by extrinsic and intrinsic signals component peptide hormones that exhibit at least one hor occurring during normal development. It is well documented monal activity of the component peptide hormones, frag that apoptosis plays a key role in regulation of pancreatic ments of analogs and derivatives of component peptide endocrine beta cells. There is increasing evidence that in hormones that exhibit at least one hormonal activity of the adult mammals the beta-cell mass is subject to dynamic component peptide hormones, and peptidic enhancers. changes to adapt insulin production for maintaining eugly 0052. In one embodiment a hybrid polypeptide is exhib cemia in particular conditions, such as pregnancy and obe iting at least one hormonal activity, the hybrid polypeptide sity. The control of beta cell mass depends on a subtle containing at least a first bio-active peptide hormone module balance between cell proliferation, growth and programmed covalently linked to at least one additional bio-active peptide cell death (apoptosis). A disturbance of this balance may hormone module; wherein the bio-active peptide hormone lead to impairment of glucose homeostasis. For example, it modules are independently selected from the group consist is noteworthy that glucose intolerance develops with aging ing of component peptide hormones; fragments of compo when beta cell replication rates are reduced and human nent peptide hormones that exhibit at least one hormonal autopsy studies repeatedly showed a 40-60% reduction of activity of the component peptide hormones; analogs and beta cell mass in patients with non-insulin-dependent-dia derivatives of component peptide hormones that exhibit at betes mellitus compared with nondiabetic subjects. It is least one hormonal activity of the component peptide hor generally agreed that insulin resistance is an invariable mones; fragments of analogs and derivatives of component accompaniment of obesity but that normoglycemia is main peptide hormones that exhibit at least one hormonal activity tained by compensatory hyperinsulinemia until the beta cells of the component peptide hormones; and peptidic enhancers. become unable to meet the increased demand for insulin, at The component peptide hormones are typically indepen which point type 2 diabetes begins. dently selected from at least two of the group consisting of 0047. Attempts to treat the multiple abnormalities asso amylin, (ADM), calcitonin (CT), calcitonin ciated with diabetes have prompted for the administration of gene related peptide (CGRP), intermedin, cholecystokinin several anti-diabetic medicaments in order to address these (“CCK), leptin, peptide YY (PYY), glucagon-like pep abnormalities in the different patients. Examples of anti tide-1 (GLP-1), glucagon-like peptide 2 (GLP-2), oxynto diabetic medicaments are proteins such as insulin and insu modulin (OXM), a , and exendin-4. Typi lin analogues, and Small molecules Such as insulin sensitiz cally peptidic enhancers are independently selected from the ers, insulin Secretagogues and appetite regulating group consisting of structural motifs of component peptide compounds. hormones that impart a desired chemical stability, confor mational stability, metabolic stability, bioavailability, organ/ 0.048. There remains a need to develop polypeptides tissue targeting, receptor interaction, protease inhibition, useful in the above described metabolic diseases, conditions, plasma protein binding, or other pharmacokinetic character and disorders. Accordingly, it is an object of the present istic to the hybrid polypeptide, and structural motifs of invention to provide hybrid polypeptides and methods for analogs or derivatives of component peptide hormones that producing and using them. The compounds of the invention impart a desired chemical stability, conformational stability, find use in the metabolic diseases, conditions, and disorders metabolic stability, bioavailability, organ/tissue targeting, described above and herein. receptor interaction, protease inhibition, plasma protein 0049 All documents referred to herein are incorporated binding, or other pharmacokinetic characteristic to the by reference into the present application as though fully set hybrid polypeptide. In yet a further embodiment at least one of the bio-active peptide hormone modules exhibits at least forth herein. one hormonal activity of a component peptide hormone. In SUMMARY OF THE INVENTION yet further alternative embodiments when the at least one bio-active peptide hormone module that exhibits at least one 0050. The present invention relates generally to novel, hormonal activity of a component peptide hormone is amy selectable hybrid polypeptides useful as agents for the lin, a fragment of amylin that exhibits at least one hormonal treatment and prevention of metabolic diseases and disor activity, an analog or derivative of amylin that exhibits at ders which can be alleviated by control of plasma glucose least one hormonal activity, or a fragment of an analog or levels, insulin levels, and/or insulin secretion, Such as dia derivative of amylin that exhibits at least one hormonal betes and diabetes-related conditions. Such conditions and activity, and the at least one other bio-active peptide hor US 2006/0293.232 A1 Dec. 28, 2006

mone module is CCK, a fragment of CCK that exhibits at ment, the methods of the invention are used to treat or least one hormonal activity, an analog or derivative of CCK prevent conditions or disorders which can be alleviated by that exhibits at least one hormonal activity, a fragment of an reducing nutrient availability in a Subject in need thereof, analog or derivative of CCK that exhibits at least one comprising administering to said subject a therapeutically or hormonal activity, CT, a fragment of CT that exhibits at least prophylactically effective amount of a hybrid polypeptide of one hormonal activity, an analog or derivative of CT that the invention. In another embodiment, the methods of the exhibits at least one hormonal activity, or a fragment of an invention are used to treat or prevent conditions or disorders analog or derivative of CT that exhibits at least one hor which can be alleviated by control plasma glucose levels, monal activity, then the hybrid polypeptide can further insulin levels, and/or insulin secretion. In yet another contain at least three bio-active peptide hormone modules embodiment, the methods of the invention are used to treat selected from at least three different component peptide diabetes and/or diabetes-related conditions. Such conditions hormones. In yet a further alternative embodiment, when the and disorders include, but are not limited to, hypertension, at least one bio-active peptide hormone module that exhibits dyslipidemia, cardiovascular disease, eating disorders, insu at least one hormonal activity of a component peptide lin-resistance, obesity, and diabetes mellitus of any kind, hormone is GLP-1, a fragment of GLP-1 that exhibits at including Type I, Type II, and gestational diabetes, diabetes least one hormonal activity, an analog or derivative of complications (neuropathy (based on, e.g., neurotrophic GLP-1 that exhibits at least one hormonal activity, or a actions of exendin4), neuropathic pain (based on, e.g., fragment of an analog or derivative of GLP-1 that exhibits amylin action), retinopathy, nephropathy, conditions of at least one hormonal activity, and the at least one other insufficient pancreatic beta cell mass (based on, e.g., islet bio-active peptide hormone module is a peptidic enhancer neogenesis actions of exendin-4 and GLP-1). comprising an exendin fragment, then the hybrid polypep 0057 The present invention also relates to pharmaceuti tide can further contain at least three bio-active peptide cal compositions comprising a therapeutically or prophylac hormone modules. tically effective amount of at least one hybrid polypeptide of 0053 Component peptide hormones of the invention the invention, or a pharmaceutically acceptable salt thereof, include: amylin, adrenomedullin (ADM), calcitonin (CT), together with pharmaceutically acceptable diluents, preser calcitonin gene related peptide (CGRP), intermedin, chole Vatives, Solubilizers, emulsifiers, adjuvants and/or carriers cystokinin (“CCK), leptin, peptide YY (PYY), glucagon useful in the delivery of the hybrid polypeptides. like peptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2), 0058. These and other aspects of the invention will be (OXM), natriuretic peptides, and exendin-4: more clearly understood with reference to the following 0054 Peptidic enhancers of the invention include: struc preferred embodiments and detailed description. tural motifs of component peptide hormones that impart a desired chemical stability, conformational stability, meta BRIEF DESCRIPTION OF THE DRAWINGS bolic stability, bioavailability, organ/tissue targeting, recep 0059 FIG. 1 demonstrates the effect of exemplary com tor interaction, protease inhibition, plasma protein binding, pounds of the invention in DIO mouse assay. or other pharmacokinetic characteristic to the hybrid polypeptide, and structural motifs of analogs or derivatives 0060 FIG. 2 demonstrates the effect of exemplary com of component peptide hormones that impart a desired chemi pounds of the invention in DIO mouse assay. cal stability, conformational stability, metabolic stability, 0061 FIGS. 3A-3C demonstrate the effect of exemplary bioavailability, organ/tissue targeting, receptor interaction, compounds of the invention in DIO mouse assay. protease inhibition, plana protein binding, or other pharma cokinetic characteristic to the hybrid polypeptide. 0062 FIGS. 4A-4B demonstrate the effects of exemplary compounds of the invention in food intake assay, compared 0055. In another aspect of the invention, methods for to parent peptide compounds. treating or preventing obesity are provided, wherein the method comprises administering a therapeutically or pro 0063 FIGS.5A-5B demonstrate the effects of exemplary phylactically effective amount of a hybrid polypeptide of the compounds of the invention in blood glucose lowering assay invention to a subject in need thereof. In a preferred embodi and food intake assay, respectively. ment, the subject is an obese or overweight subject. While “obesity' is generally defined as a body mass index over 30, DETAILED DESCRIPTION OF THE for purposes of this disclosure, any subject, including those INVENTION with a body mass index of less than 30, who needs or wishes 0064. The present invention relates generally to novel, to reduce body weight is included in the scope of "obese.” selectable hybrid polypeptides useful as agents for the Subjects who are insulin resistant, glucose intolerant, or treatment and prevention of metabolic diseases and disor have any form of diabetes mellitus (e.g., type 1, 2 or ders which can be alleviated by control of plasma glucose gestational diabetes) can benefit from this method. levels, insulin levels, and/or insulin secretion, such as dia 0056. In yet another aspect of the invention, methods of betes and diabetes-related conditions. Such conditions and reducing food intake, reducing nutrient availability, causing disorders include, but are not limited to, hypertension, weight loss, treating diabetes mellitus or diabetes-associated dyslipidemia, cardiovascular disease, eating disorders, insu conditions, and improving lipid profile (including reducing lin-resistance, obesity, and diabetes mellitus of any kind, LDL cholesterol and triglyceride levels and/or changing including type 1, type 2, and gestational diabetes. HDL cholesterol levels) are provided, wherein the methods 0065. In one aspect, the invention involves the modular comprise administering to a subject an effective amount of assembly of physiologically, metabolically, and/or pharma a hybrid polypeptide of the invention. In a preferred embodi cokinetically active peptidic modules that may be selectable US 2006/0293.232 A1 Dec. 28, 2006 based on “bio-activities', e.g., therapeutic efficacy, scope of peptide hormone, and may preferably include at least one function, duration of action, physicochemical properties, additional bio-activity of a second component peptide hor and/or other pharmacokinetic properties. O. 0066. Without intending to be limited by theory, the 0071. In one embodiment, the hybrid polypeptides of the present invention relates at least in part to a “toolbox” invention may comprise at least two bio-active peptide approach, wherein bio-active peptide hormone modules are hormone modules, wherein each of said at least two bio linked in binary, tertiary or higher order combinations to active peptide hormone modules exhibits at least one hor create novel, efficacious therapeutic agents with selectable monal activity of a component peptide hormone. In another properties. The “bio-active peptide hormone modules' may embodiment, the hybrid polypeptides of the invention may be peptide hormones, peptide fragments with hormonal comprise at least two bio-active peptide hormone modules, activity, or structural motifs of peptide hormones that impart wherein at least one of said bio-active peptide hormone chemical, metabolic, and/or other pharmacokinetic stability. modules exhibits at least one hormonal activity of a com The peptide hormones can include native peptide hormones, ponent peptide hormone and at least one of said bio-active as well as peptide hormone analogs and derivatives, as peptide hormone modules imparts a desired chemical sta known in the art and described herein. bility, conformational stability, metabolic stability, bioavail ability, organ/tissue targeting, receptor interaction, protease 0067. In one aspect of the invention, it has been found that the combination of certain physicochemical character inhibition, plasma protein binding, and/or other pharmaco istics of two or more peptide hormones into a single modal kinetic characteristic to the hybrid polypeptide. ity can facilitate intervention at several points in a dysfunc 0072. In a preferred embodiment, the hybrid polypeptides tional metabolic circuit. As such, in one aspect of the of the invention may have comparable or higher potency in invention, rationally-designed hybrid polypeptides are pro the treatment and/or prevention of metabolic conditions and vided that integrate selectable bio-activities into a single disorders, as compared to the component peptide hormones. polypeptide agent. In one embodiment, the selectable hybrid In another embodiment, the hybrid polypeptides of the polypeptides of the invention may involve the use of chemi invention may have comparable or higher potency in the cally stable linkers to covalently attach the bio-active mod treatment and/or prevention of diabetes and/or diabetes ules. In another embodiment, the selectable hybrid polypep related disorders, as compared to the component peptide tides of the invention may involve the use of cleavable hormones. Alternatively, preferred hybrid polypeptides of linkers, which themselves may be or form part of a bio the invention may exhibit improved ease of manufacture, active module. stability, and/or ease of formulation, as compared to the 0068 Again, without intending to be limited by theory, component peptide hormones. design of the hybrid polypeptides of the present invention 0073 More particularly, the hybrid polypeptides of the may generally involve: (1) the identification, selection and present invention will generally comprise a first bio-active pairing of bio-active peptide hormone modules for desired peptide hormone module covalently linked to at least one efficacy and therapeutic use, and (2) the covalent linking of additional bio-active peptide hormone module. The bio the bio-active modules (e.g. native peptide hormones, pep active peptide hormone modules may be covalently linked tide hormone analogs or derivatives with hormonal activity, together in any manner known in the art, including but not peptide hormone fragments with hormonal activity, stabi limited to direct amide bonds or chemical linker groups, as lizing motifs, etc.) either directly or via a linker without loss described in further detail herein. In one embodiment, of bio-activity of the component modules. In certain chemical linker groups may include peptide mimetics which embodiments, module selection criteria may include, but not induce or stabilize polypeptide conformation. be limited to: (a) desired in vivo efficacy for desired thera peutic or prophylactic indication, Such as an additive or a 0074 The first bio-active peptide hormone module may synergistic effect; (b) optional synergism or dual action of be selected from a first component peptide hormone, and the linked modules for multiple therapeutic or prophylactic may be a peptide hormone (including native peptide hor indications; and/or (c) a desired chemical stability, confor mones as well as analogs and derivatives thereof), a peptide mational stability, metabolic stability, bioavailability, organ/ fragment with hormonal activity (including fragments of tissue targeting, receptor interaction, protease inhibition, native peptides hormones as well as analogs and derivatives plasma protein binding, and/or other pharmacokinetic char thereof), or a structural motif of a peptide hormone (includ acteristic. ing native peptide hormones as well as analogs and deriva tives thereof) that imparts a desired chemical stability, 0069. The section headings are used herein for organiza conformational stability, metabolic stability, bioavailability, tional purposes only, and are not to be construed as in any organ/tissue targeting, receptor interaction, protease inhibi way limiting the Subject matter described. tion, plasma protein binding, and/or other pharmacokinetic 1. Hybrid Polypeptides of the Invention characteristic to the hybrid polypeptide. Likewise, the addi tional bio-active peptide module(s) may be selected from 0070. As mentioned above, the present invention relates component peptide hormones, and may be a peptide hor in part to hybrid polypeptides comprising at least two mone (including native peptide hormones as well as analogs bio-active peptide hormone modules selectable from com and derivatives thereof), a peptide fragment with hormonal ponent peptide hormones described herein. The hybrid activity (including fragments of native peptides hormones as polypeptides of the present invention will generally be well as analogs and derivatives thereof), or a structural motif useful in the treatment and prevention of metabolic condi of a hormone peptide (including native peptide hormones as tions and disorders. The hybrid polypeptides of the invention well as analogs and derivatives thereof) that imparts a will exhibit at least one hormonal activity of a component desired chemical stability, conformational stability, meta US 2006/0293.232 A1 Dec. 28, 2006 bolic stability, bioavailability, organ/tissue targeting, recep (b) cholecystokinin (“CCK); (c) the leptin family, including tor interaction, protease inhibition, plasma protein binding, leptin and leptin-like peptides; (d) the pancreatic polypep and/or other pharmacokinetic characteristic to the hybrid tide family, including pancreatic polypeptide (“PP) and polypeptide. The first peptide hormone and the additional peptide YY (“PYY); (e) incretins and incretin mimetics, peptide hormone may be the same peptide hormone, may be including: peptide hormones derived from the proglucagon from the same family of peptide hormones, or may be gene Such as: glucagon, glucagon-like peptide-1 (“GLP-1), different peptide hormones, depending on the desired char glucagon-like peptide 2 (“GLP-2), and oxyntomodulin acteristics of the bio-active peptide hormone modules. (“OXM”); and exendins such as: exendin-3, and exendin4; 0075). As used herein, the term “bio-active” refers to (1) and (f) matriuretic peptides including ANP, BNP. CNP and biological activity in at least one in vivo hormonal pathway, , their precursor forms and peptides derived there or (2) modulation of the therapeutic efficacy, scope of from (g) family and the (h) neuromedin family, function, duration of action, physicochemical properties, and analogs, derivatives and fragments thereof. As discussed and/or other pharmacokinetic properties of Such biological herein component peptide hormones of the invention also activity. Biological activity may be evaluated through target include analogs and derivatives that retain hormonal activity hormone receptor binding assays, or through metabolic of these native peptide hormones. In one embodiment, Such studies that monitor a physiological indication, as known in analogs and derivatives are agonists of the target hormone the art and described herein. Modulation of the therapeutic receptor. efficacy, scope of function, duration of action, physico 0079. By “amylin' is meant the human peptide hormone chemical properties, and/or other pharmacokinetic proper referred to as amylin and secreted from the beta cells of the ties of such biological activity may be modified through pancreas, and species variations thereof, as described in U.S. changed in, e.g., chemical stability, conformational stability, Pat. No. 5,234,906, issued Aug. 10, 1993, for “Hyperglyce metabolic stability, bioavailability, organ/tissue targeting, mic Compositions, the contents of which are hereby incor receptor interaction, protease inhibition, plasma protein porated by reference. More particularly, amylin is a binding, and/or other pharmacokinetic characteristics. 37-amino acid polypeptide hormone normally co-secreted 0076. In one embodiment, the hybrid polypeptides of the with insulin by pancreatic beta cells in response to nutrient invention retain at least about 25%, preferably about 30%, intake (see, e.g., Koda et al., Lancet 339:1179-1180, 1992). 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% In this sense, "amylin,”“wild-type amylin, and “native percent of the biological activity of a component peptide amylin, i.e., unmodified amylin, are used interchangeably. hormone. Preferred hybrid polypeptides are those having a 0080) By “adrenomedullin' or "ADM is meant the potency in one of the metabolic-related assays known in the human peptide hormone and species variants thereof. More art or described herein (e.g., receptor binding, food intake, particularly, ADM is generated from a 185 amino acid gastric emptying, pancreatic secretion, insulin secretion, preprohormone through consecutive enzymatic cleavage blood glucose lowering, weight reduction, etc.) which is and amidation. This process culminates in the liberation of equal to or greater than the potency of component peptide a 52 amino acid bioactive peptide. hormone in that same assay. Alternatively, preferred hybrid polypeptides of the invention may exhibit improved ease of 0081. By "calcitonin' or “CT is meant the human pep manufacture, Stability, and/or ease of formulation, as com tide hormone and species variants thereof, including salmon pared to component peptide hormones. calcitonin (“sCT). More particularly, CT is a 32 amino acid peptide cleaved from a larger prohormone. It contains a 0077. In another embodiment, the hybrid polypeptides of single bond, which causes the amino terminus to the invention retain at least about 25%, preferably about assume the shape of a ring. Alternative splicing of the 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% calcitonin pre-mRNA can yield a mRNA encoding calcito percent of the biological activity of a native component nin gene-related peptide; that peptide appears to function in peptide hormone with regard to the reduction of nutrient the nervous and vascular systems. The calcitonin receptor availability, the reduction of food intake, the effect of body has been cloned and shown to be a member of the seven weight gain, and/or the treatment and prevention of meta transmembrane, G protein-coupled receptor family. bolic conditions and disorders. In yet another embodiment, the hybrid polypeptides of the invention exhibit at least 0082) By “calcitonin gene related peptide' or “CGRP” is about 110%, 125%, 130%, 140%, 150%, 200%, or more of meant the human peptide hormone and species variants the biological activity of a native peptide hormone with thereof, in any physiological form. regard to the reduction of nutrient availability the reduction of food intake, the effect of body weight gain, and/or the 0083. By “intermedin' or “AFP-6' is meant the human treatment and prevention of metabolic conditions and dis peptide hormone and species variants thereof, in any physi orders. In another embodiment, the hybrid polypeptides of ological form. the invention exhibit improved component peptide hormone 0084. By “cholecystokinin” or “CCK' is meant the receptor agonist activity. human peptide hormone and species variants thereof. More 2. Component Peptides Hormones, Analogs and Derivatives particularly, CCK is a 33-amino acid sequence first identi fied in humans, and includes a 8-amino acid in Vivo C-ter 0078 Component peptide hormones generally include minal fragment ("CCK-8) that has been reportedly dem peptide hormones useful in the treatment or prevention of onstrated in pig, rat, chicken, chinchilla, dog and humans. metabolic diseases and disorders including: (a) the amylin Thus, the term CCK-33 will generally refer to human family, including amylin, adrenomedulin (ADM'), calci CCK(1-33), while CCK-8 (CCK(26-33)) will refer to the tonin (“CT'), calcitonin gene related peptide (“CGRP”), C-terminal octapeptide generically in both the sulfated and intermedin (also known as “AFP-6') and related peptides: unsulfated unless otherwise specified. Further, pentagastrin US 2006/0293.232 A1 Dec. 28, 2006

or CCK-5 will refer to the C-terminal peptide CCK(29-33), Chem., 265:20259-62, 1990; Eng., J., et al., J. Biol. Chem., and the CCK-4 will refer to the C-terminal tetrapeptide 267:7402-05 (1992)). The exendins have some sequence CCK(30-33). However, as used herein, CCK will generally similarity to several members of the glucagon-like peptide refer to all naturally occurring variations of the hormone, family, with the highest identity, 53%, being to GLP-1 including CCK-33, CCK-8, CCK-5, and CCK-4, in the (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993)). In this sulfated and unsulfated form unless otherwise specified. sense, “exendin,”“wild-type exendin,” and “native exen 0085. By “leptin' is meant the naturally occurring leptin din, i.e., unmodified exendin, are used interchangeably. from any species, as well as biologically active D-isoforms, 0092. By “urocortin' is meant a human urocortin petide or fragments of naturally occurring leptin and variants hormone or species variants thereof in any physiological thereof, and combinations of the preceding. Leptin is the form. More particularly, there are three human urocortins: polypeptide product of the ob gene as described in the Ucn-1, Ucn-2 and Ucn-3. For example, human urocortin 1 International Patent Publication No. WO96/05309, which is has the formula: Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu incorporated herein by reference in its entirety. Putative Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu -Glu-Leu-Ala analogs and fragments of leptin are reported in U.S. Pat. No. Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg 5,521,283, U.S. Pat. No. 5,532,336, PCT/US96/22308 and Ile-Ile-Phe-Asp-Ser-Val-NH 2 (SEQ ID NO: ). Rat-derived PCT/US96/01471, each of which is incorporated herein by urocortin is identical but for 2 substitutions: Asp2 for Asn2 reference in its entirety. and Pro4 for Ser4. Human Ucn-2 has the sequence Ile Val Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu Leu 0.086 By “PP” is meant human pancreatic peptide Glu Gln Ala Arg Ala Arg Ala Ala Arg Glu Gln Ala Thr Thr polypeptide or species variants thereof, in any physiological Asn Ala Arg Ile Leu Ala Arg Val Gly His Cys. Human Ucn-3 form. Thus, the term “PP includes both the human full has the sequence Phe Thr Leu Ser Leu Asp Val Pro Thr Asn length, 36 amino acid peptide as set forth in SEQID NO: 1. Ile Met Asn Leu Leu Phe ASn Ile Ala Lys Ala Lys Asn Leu and species variations of PP, including, e.g., murine, ham Arg Ala Glin Ala Ala Ala Asn Ala His Leu Met Ala Glin Ile. ster, chicken, bovine, rat, and dog PP. In this sense, “PP, Ucn-3 is preferably in amide form. Further urocortins and “wild-type PP” and “native PP” i.e., unmodified PP, are analogs are described in the literature, for example in U.S. used interchangeably. Pat. No. 6,214,797. Urocortins Ucn-2 and Ucn-3, which 0087. By “PYY is meant human peptide YY polypeptide retain the food-intake Suppression and antihypertensive/ or species variants thereof, in any physiological form. Thus, cardioprotective/inotropic properties, find particular use in the term “PYY” includes both the human full length, 36 the hybrids of the ivention. Stresscopin (Ucn-3) and Stress amino acid peptide, and species variations of PYY, including copin-related peptide (Ucn 2), named for their ability to e.g., murine, hamster, chicken, bovine, rat, and dog PYY. In suppress the chronic HPA activation following a stressful this sense, “PYY and “wild-type PYY and “native PYY.” stimulus Such as dieting/fasting, are specific for the CRF i.e., unmodified PYY. are used interchangeably. In the type 2 receptor and do not activate CRF-R1 which mediates context of the present invention, all modifications discussed ACTH release. Hybrids comprisig aurocortin, e.g., Ucn-2 or with reference to the PYYanalog polypeptides of the present Ucn-3, are particularly useful for vasodilation and thus for invention are based on the 36 amino acid sequence of native cardiovascular uses as described herein, e.g. CHF. Urocortin human PYY. containing hybrids of the invention find particular use in treating or preventing conditions associated with stimulating 0088. By “GLP-1 is meant human glucagon like pep ACTH release, hypertension due to vasodilatory effects, tide-1 or species variants thereof, in any physiological form. inflammation mediated via other than ACTH elevation, The term “GLP-1” includes human GLP-1 (1-37), GLP-1 (7- hyperthermia, appetite disorder, congestive heart failure, 37), and GLP-1 (7-36)amide, with reference to the full length stress, anxiety, and psoriasis. Such compounds are also human GLP-1 (1-37), and species variations of GLP-1. useful for an antiproliferative effect, such as for treating or including, e.g., murine, hamster, chicken, bovine, rat, and preventing cancers or tumor growth. Of particular interest dog PP. In this sense, “GLP-1,”“wild-type GLP-1,” and are urocortin peptide hormone module combined with a “native GLP-1, i.e., unmodified GLP-1, are used inter natriuretic peptide module, amylin family, aan exendin changeably. family, or a GLP1 family module to provide an enhanced 0089. By “GLP-2 is meant human glucagon like pep cardiovascular benefit, e.g. treating CHF, as by providing a tide-2 or species variants thereof, in any physiological form. beneficial vasodilation effect. More particularly, GLP-2 is a 33 amino acid peptide, co 0093. By “neuromedin' is meant the neuromedin family secreted along with GLP-1 from intestinal endocrine cells in of peptides including and S peptides, more the Small and large intestine. particularly their active hormone sequences. For example, the native active human nueromedin U peptide hormone is 0090. By “OXM” is meant human oxyntomodulin or neuromedin-U25: Phe Arg Val Asp Glu Glu Phe Gln Ser Pro species variants thereof in any physiological form. More Phe Ala Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg Pro Arg particularly, OXM is a 37 amino acid peptide that contains ASn, particularly in the amide form. Pig U25 has the the 29 amino acid sequence of glucagon followed by an 8 sequence: FKVDEEFQGPIVSQNRRYFLFRPRN, particu amino acid carboxyterminal extension. larly its amide form. Other neuromedin U family members 0.091 By “exendin' is meant a peptide hormone found in inlcude the following listed as their SWISS-PROT designa the saliva of the Gila-monster, a lizard endogenous to tions and entry numbers: NEWU CANFA (P34962), NEU Arizona, and the Mexican Beaded Lizard, as well as species U CAVPO (P34966), NEUU CHICK (P34963), NEU variants thereof. More particularly, Exendin-3 is present in U HUMAN (P48645), NEUU LITCE (P81872), the saliva of Heloderma horridum, and exendin-4 is present NEUU MOUSE (Q9QXK8), NEUUPIG (P34964), NEU in the saliva of Heloderma Suspectum (Eng., J., et al., J. Biol. U RABIT (P34965), NEUU RANTE (P20056), and US 2006/0293.232 A1 Dec. 28, 2006

NEW URAT (P12760). Of particular interest are their pro reference peptide, or (2) which binds specifically in a cessed active peptide hormones and analogs, derivatives and reference receptor assay or in a competitive binding assay fragments thereof. Included in the neuromedin Ufamily are with labeled reference peptide. Preferably, the agonists will various truncated or splice varinats, e.g., FLFHYSK bind in Such assays with an affinity of greater than 1 M, and TQKLGKSNVVEELQSPFASQSRGYFLFRPRN. Exem plary of the family is human neuromedin S more preferably with an affinity of greater than 1-5 nM. In with the sequence ILORGSGTAAVDFTKKDHTATWGR another embodiment, the terms refer to a compound which PFFLFRPRN, particularly its amide form. Hybrids of the elicits a biological effect in the treatment of diabetes or a invention having neuromedin module will an anorexigenic diabetes related condition or disorder. Such agonists may effect, and thus have beneficial value in treating obesity, comprise a polypeptide comprising an active fragment of a diabetes, reducing food intake, and other related conditions reference peptide or a small chemical molecule. and disorders as described herein. Of particular interest are neuromedin modules combined with an amylin family pep 0097. By “amino acid” and “amino acid residue' is meant tide, an exendin peptide family or a GLP I peptide family natural amino acids, unnatural amino acids, and modified module. amino acid. Unless Stated to the contrary, any reference to an 0094. As used herein, an “analog refers to a peptide amino acid, generally or specifically by name, includes whose sequence was derived from that of a base reference reference to both the D and the L stereoisomers if their peptide (e.g., PP, PYY. amylin, GLP-1, exendin, etc.), structure allow Such stereoisomeric forms. Natural amino including insertions, Substitutions, extensions, and/or dele acids include alanine (Ala), (Arg), asparagine tions of the reference amino acid sequence, preferably (ASn), aspartic acid (Asp), (CyS), glutamine (Gln), having at least 50 or 55% amino acid sequence identity with glutamic acid (Glu), (Gly), histidine (His), isoleu the base peptide, more preferably having at least 70%, 80%, cine (Ile), (Leu), (LyS), (Met), 90%, or 95% amino acid sequence identity with the base phenylalanine (Phe), proline (Pro), serine (Ser), threonine peptide. In one embodiment, Such analogs may comprise (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). conservative or non-conservative amino acid Substitutions Unnatural amino acids include, but are not limited to homo (including non-natural amino acids and L and D forms). lysine, homo-arginine, aZetidinecarboxylic acid, 2-aminoa 0.095 A "derivative' is defined as a molecule having the dipic acid, 3-aminoadipic acid, beta-alanine, aminopropi amino acid sequence of a native reference peptide or analog, onic acid, 2-aminobutyric acid, 4-aminobutyric acid, but additionally having chemical modification of one or 6-aminocaproic acid, 2-aminoheptanoic acid, 2aminoisobu more of its amino acid side groups, C-carbon atoms, termi tyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, ter nal amino group, or terminal carboxylic acid group. A tiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, chemical modification includes, but is not limited to, adding 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-eth chemical moieties, creating new bonds, and removing ylglycine, N-ethylasparagine, homoproline, hydroxylysine, chemical moieties. Modifications at amino acid side groups allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, include, without limitation, acylation of lysine 8-amino groups, N-alkylation of arginine, histidine, or lysine, alky isodesmosine, allo-isoleucine, N-methylalanine, N-meth lation of glutamic or aspartic carboxylic acid groups, and ylglycine, N-methylisoleucine, N-methylpentylglycine, deamidation of glutamine or asparagine. Modifications of N-methylvaline, naphthalanine, norvaline, norleucine, orni the terminal amino include, without limitation, the desa thine, pentylglycine, pipecolic acid and thioproline. Addi mino, N-lower alkyl, N-di-lower alkyl, constrained alkyls tional unnatural amino acids include modified amino acid (e.g. branched, cyclic, fused, adamantyl) and N-acyl modi residues which are chemically blocked, reversibly or irre fications. Modifications of the terminal carboxy group versibly, or chemically modified on their N-terminal amino include, without limitation, the amide, lower alkyl amide, group or their side chain groups, as for example, N-methy constrained alkyls (e.g. branched, cyclic, fused, adamantyl) lated D and Lamino acids or residues wherein the side chain alkyl, dialkyl amide, and lower alkyl ester modifications. functional groups are chemically modified to another func Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective tional group. For example, modified amino acids include groups known to the ordinarily-skilled peptide chemist. The methionine Sulfoxide; methionine Sulfone; aspartic acid C-carbon of an amino acid may be mono- or dimethylated. (beta-methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a modified amino acid of glycine; or alanine 0096. By “agonist' is meant a compound which elicits a carboxamide, a modified amino acid of alanine. Additional biological activity of native human reference peptide, pref residues that can be incorporated are described in Sandberg erably having a potency better than the reference peptide, or et al., J. Med. Chem. 41: 2481-91, 1998. within five orders of magnitude (plus or minus) of potency compared to the reference peptide, more preferably 4, 3, 2. 0098. As used herein: “5 Apa” means 5 amino-pentanoyl, or 1 order of magnitude, when evaluated by art-known “12 Ado” means 12-amino dodecanoyl, “PEG(8) mean measures such as receptor binding/competition studies. In 3.6,-dioxyoctanoyl, and “PEG (13) means 1-amino-4,7,10 one embodiment, the terms refer to a compound which trioxa-13-tridecanamine Succinimoyl. elicits a biological effect similar to that of native human 0099. As discussed herein native component peptide hor reference peptide, for example a compound (1) having mones are known in the art, as are their analogs and activity in the food intake, gastric emptying, pancreatic derivatives. For reference, the sequences of several native secretion, or weight loss assays similar to native human component peptide hormones are provided in Table 1.

US 2006/0293.232 A1 Dec. 28, 2006 14

TABLE 1-continued Exemplary Component Peptide Hormones Seq ID Description Sequence

SE-20 SFHYLRSRDASSGEEEEGKE-OH (Urocortin 111/Stres scopin)

AI-13 AQAAANAHLMAQI-OH (Urocortin III/Stres scopin)

DA-21 DNPSLSIDLTFHLLRTLLELA-OH (Urocortin)

TL-26 TKFTLSLDWPTNIMNLLFNIAKAKNL-OH (Urocortin III/ Stres scopin)

Human FTLSLDWPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2 Urocortin III

FN-38 LFHYSKTOKLGKSNWWEELOSPFASQSRGYFLFRPRN-NH2 (SLM14) alpha-Atrial SLRRSSCFGGRMDRIGAQSGLGCNSFRY-OH Natriuretic Polypeptide (1-28) human porcine, bovive

Brain c (NSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF)-OH natriuretic peptide, Rat; BNP Rat

Brain SPKMVQGSGGFGRKMDRISSSSGLGCKVLRRH-OH Natriuretic Peptide (BNP) (human)

C-TYPE GLSKGCFGLKLDRIGSMSGLGC-OH Natnuretic peptide, Porcine; Cnp, Porcine

Neuromedin YFLFRPRN-NH2 U-8 (porcine)

Neuromedin YKWNEYQGPWAPSGGFFLFRPRN-NH2 U (rat)

Neuromedin GYFLFRPRN-NH2

Neuromedin RVDEEFOSPFASQSRGYFLFRPRN-NH2 (U25), human US 2006/0293.232 A1 Dec. 28, 2006

0100. These peptides are generally C-terminally ami calcitonin and CGRP: whose C-terminal amide is dated when expressed physiologically, but need not be for generally needed for activity and can tolerate N-terminal the purposes of the instant invention. In other words, the extensions. C-terminus of these peptides, as well as the hybrid polypep tides of the present invention, may have a free —OH or 0.103 Exemplary peptide modules for use in the inven —NH, group. These peptides may also have other post tion further include, C-Terminally extendable peptide mod translational modifications. One skilled in the art will appre ules including, I, II and III: ETI (CSCSSLMD ciate that the hybrid polypeptides of the present invention KECVYFCHLDIIWVNTPEHVVPYGLGSPRS OH: may also be constructed with an N-terminal methionine CSCSSLMDKECVYFCHLDIIW OH), ETII (CSCSS residue. WLDKECVYFCHLDIIWVNTPEQTAPYGLGNPP OH: 0101 Exemplary peptide modules for use in the inven CSCSSWLDKECVYFCHLDIIW OH) and ETIII tion further include, N-terminally extendable peptide mod (CTCFTYKDKECVYYCHLDIIWIN ules (and their analogs and fragments) including , TPEQTVPYGLSNYRGSFR NH2: CTCFTYKD which exists in 2 forms, Apelin 36 and 13, both active at the KECVYYCHLDIIW OH); whose activity lies AJP receptor (LVOPRGSRNGPGPWQGGRRKFRRQR mainly in its first 10 residues (GSSFLSPE PRLSHKGPMPF OH and pERPRLSHKGPMPF OH): HQRVQQRKESKKPPAKLQP OH: GSSFLSPEHQ Prolactin Releasing peptide, which exists in 2 forms, PRP31 OH); , including oxyntomodulin which is a C-ter and PRP20, equally active at GPR10 (SRTHRHSMEIRTP minally extended glucagon with glucagons-like activity DINPAWYASRGIRPVGRF NH2 and TPDINPAWYASR (HSQGTFTSDYSKYLDSRRAQD GIRPVGRF NH2); Gastrin, which exists as big gastrin FVQWLMNTKRNRNNIA-OH: HSQGTFTSDYSKYLD and mini-gastrin, the bulk of activity however residing in SRRAQDFVQWLMNT-OH): GLP-1/GLP-2 whose actici resides in pentagastrin (QLGPQGPPHLVADPSKKQGP ties are retained with or without a C-terminal amide: GIP, WLEEEEEAYGWMDF NH2; pEGPWLEEEEEAYGW which circulates in 2 forms, GIP1-42 and GIP1-30, both MDF NH2; beta-AWMDF NH2); CCK, which exists as fully active at GIP Receptor (YAEGTFISDYSIAMDKI CCK33 or CCK8 (central vs. peripheral; APSGRMSIVKN HQQDFVNWLLAQKGKKNDWKHNITQ-OH: YAEGT LQNLDPSHRISDRDYMGWMDF NH2: DYMGW MDF NH2); Cortistatin, which exists as cortistatin 17 or TISDYSIAMDKIHQQDFVNWLLAQK NH2): C 29 (QEGAPPQQSARRDRMPCRNFFWKTFSSCK OH ropeptide W, which exists as NPW23 and NPW30, equally and DRMPCRNFFWKTFSSCK OH); somatostatin, active at GPR7 and 8 (WYKHVASPRYHTVGRAAGLL which exists as somatostatin 14 or 28 (SANSNPAMAPRE MGLRRSPYLW OH: WYKHVASPRYHTVGRAAGLL RKAGCKNFFWKTFTSC OH: AGCKNFFWKTTTSC MGL-OH); PACAP which exists in 2 forms, PACAP27 and OH); GRP for which a C-terminal 10 amino acid sequence 38 (HSDGIFTDSYSRYRKQMAVKKYLAAV possesses most of the activity (VPLPAGGGTVLTK LGKRYKQRVKNK NH2: HSDGIFTDSYSRYRKQ MYPRGNHWAVGHLM-NH2: GNHWAVGHLM-NH2): MAVKKYLAAVL-NH2): PHI and PHV (HADGVFTSDF for which a C-terminal 10 amino acid region SKLLGQLSAKKYLESLMGKRVSSNISEDPVPV OH: possesses most of the activity (LSWDLPEPRSRASKIRVH HADGVFTSDFSKLLGQLSAKKYLESLM-NH2); GRF, SRGNLWATGHFM-NH2: GNLWATGHFM-NH2); Neuro medin S for which a C-terminal 9 amino acid region which exists in 2 forms GRF29 and GRF40 (YADAIFTN possesses most of the activity (ILORGSGTAAVDFT SYRKVLGQLSARKLLODIMSRQQGESN KKDHTATWGRPFFLFRPRN. NH2: PFFLFRPRN QERGARARL-NH2: YADAIFTNSYRKVLGQL NH2); Neuromedin U for which a C-terminal 9 amino acid SARKLLODIMS OH); PTH 1-34 and 1-37 forms which region possesses most of the activity (FRVDEEFQSP possess activity of full length PTH 1-84 (SVSEIQLM FASQSRGYFLFRPRN. NH2: GYFLFRPRN. NH2): HNLGKHLNSMERVEWLRKKLQDVHN , which exists as long and short forms (KIPY FVALGAPLAPRDAGSQRPRKKED NVLVESHEK ILKRQLYENKPRRPYIL-OH: QLYENKPRRPYIL-OH): SLGEADKADVNVLTKAKSQ; Kiss-1 whose activity lies mainly in its C-terminus (GTSL SVSEIQLMHNLGKHLNSMERVEWL SPPPESSGSPQQPGLSAPHSRQIPAPQ RKKLQDVHNFVAL-OH: SVSEIQLMHNLGKHLNS GAVLVQREKDLPNYNWNSFGLRF NH2: EKDLP NYNWNSFGLRF NH2): RF-amide-3, whose C-terminal MERVEWLRKKLQDVHNF OH) PTH-RP for which fragments possess activity (SAGATANLPLRSGRNMEVS 1-36 possesses activity of full length 1-86 (AVSEHOLLH LVRRVPNLPQRF NH2; VPNLPQRF NH2); Dynor DKGKSIQDLRRRFFLHHLIAEIHTAEI phin, which exists as big dynorphin (A) of dynorphin B RATSEVSPNSKPSPNTKNHPVRFG SDDEGRYLTQET (rimorphin) (YGGFLRRIRPKLKWDNQKRYGGFLR NKVETYKEQPLKTPGKKKKGKP NH2: RQFKVVT-OH: YGGFLRRQFKVVT-OH): AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEI OH) gamma-MSH for which the shorter gamma-MSH1 and 0102 PYY whose C-terminal fragments are active at Y2 the longer gamma-MSH3 have similar activities (YVMGH receptor (YPIKPEAPGEDASPEELNRYYASL RHYLNLVTRQRY-NH2: SLRHYLNLVTRQRY NH2): FRWDRFGRRNSSSSGSSGAGQ-OH: YVMGHFRW AFP-6 whose 7-47 region retains activity (TQAQLL DRF NH2); MSH for which alpha-MSH is an active RVGCVLGTCQVQNLSHRLWQLMG portion of ACTH (SYSMEHFRWGK PAGRQDSAPVDPSSPHSY NH2: VGCVLGTC PVGKKRRPVKVYPNGAEDESAEAFPLEF OH: SYS QVQNLSHRLWOLMGPAGRQDSAPVDPSSPHSY MEHFRWGKPV. NH2); and for which the A, NH2); the amylin family including adrenomdullin, delta, and Y endorphin are active Subpeptides of the larger B US 2006/0293.232 A1 Dec. 28, 2006

endorphin (YGGFMTSEKSQTPLVTLFKNAI with lysine, aspartic acid, glutamic acid, or cysteine may IKNAYKKGE-OH: YGGFMTSEKSQTPLVTLFKNAI provide additional sites for derivatization. See, e.g., U.S. IKNAY OH: YGGFMTSEKSQTPLVTL-OH: YGGFMT Pat. Nos. 5,824,784 and 5,824,778. Preferably, the hybrid SEKSQTPLVT-OH). polypeptides may be conjugated to one, two, or three 0104. The analogs of the above component peptide hor polymer molecules. mones are known in the art, but generally include modifi 0108. The water soluble polymer molecules are prefer cations such as Substitutions, deletions, and insertions to the ably lined to an amino, carboxyl, or thiol group, and may be amino acid sequence of Such component peptide hormones, linked by N or C terminus, or at the side chains of lysine, and any combination thereof. The Substitutions, insertions aspartic acid, glutamic acid, or cysteine. Alternatively, the and deletions may be at the N-terminal or C-terminal end, or may be at internal portions of the component peptide hor water soluble polymer molecules may be linked with mone. In a preferred aspect, analogs of the component diamine and dicarboxylic groups. In a preferred embodi peptide hormones of the invention include one or more ment, the hybrid polypeptides of the invention are conju modifications of a “non-essential amino acid residue. In the gated to one, two, or three PEG molecules through an context of the invention, a “non-essential amino acid resi epsilon amino group on a lysine amino acid. due is a residue that can be altered, i.e., deleted or substi tuted, in the native human amino acid sequence of the 0109) Derivatives of the invention also include compo fragment, e.g., the component peptide hormone fragment, nent peptide hormones or analogs with chemical alterations without abolishing or Substantially reducing the component to one or more amino acid residues. Such chemical alter peptide hormone receptor agonist activity of the resulting ations include amidation, glycosylation, acylation, Sulfation, analog. phosphorylation, acetylation, and cyclization. The chemical alterations may occursingularly at the N- or C-terminus or 0105 Preferred substitutions include conserved amino at the side chains of amino acid residues within the sequence acid substitutions. A "conservative amino acid substitution' of the PPF hybrid polypeptides. In one embodiment, the is one in which the amino acid residue is replaced with an C-terminus of these peptides may have a free —OH or amino acid residue having a similar side chain, or physico NH, group. In another embodiment, the N-terminal end chemical characteristics (e.g., electrostatic, hydrogen bond may be capped with an isobutyloxycarbonyl group, an ing, isosteric, hydrophobic features). Families of amino acid isopropyloxycarbonyl group, an n-butyloxycarbonyl group, residues having similar side chains are known in the art. These families include amino acids with basic side chains an ethoxycarbonyl group, an isocaproyl group (isocap), an (e.g., lysine, arginine, histidine), acidic side chains (e.g., octanyl group, an octyl glycine group (G(Oct)), or an aspartic acid, glutamic acid), uncharged polar side chains 8-aminooctanic acid group. In a preferred embodiment, (e.g., glycine, asparagine, glutamine, serine, threonine, cyclization can be through the formation of disulfide tyrosine, methionine, cysteine), nonpolar side chains (e.g., bridges. Alternatively, there may be multiple sites of chemi alanine, Valine, leucine, isoleucine, proline, phenylalanine, cal alteration along the hybrid polypeptide. tryptophan), B-branched side chains (e.g., threonine, Valine, 3. The Amylin Family isoleucine) and aromatic side chains (e.g., tyrosine, pheny lalanine, tryptophan, histidine). 0110. As discussed herein component peptide hormones 0106 The present invention also relates to derivatives of useful in the present invention include amylin family peptide the component peptide hormones. Such derivatives include hormones including amylin, adrenomedullin (ADM'), cal component peptide hormones and analogs thereof conju citonin (“CT), calcitonin gene related peptide (“CGRP”), gated to one or more water soluble polymer molecules, such intermedin (also known as “AFP-6') and related peptides. as polyethylene glycol (“PEG') or fatty acid chains of Native amylin family peptide hormones are known in art, as various lengths (e.g., Stearyl, palmitoyl, octanoyl, etc.), or by are functional peptide analogs and derivatives. Certain pre the addition of polyamino acids, such as poly-his, poly-arg, ferred native peptides, peptide analogs and derivatives are poly-lys, and poly-ala. Modifications to the component described herein, however it should be recognized that any peptide hormones or analogs thereof can also include Small known amylin family peptides that exhibit hormonal activity molecule Substituents, such as short alkyls and constrained known in the art may be used in conjunction with the present alkyls (e.g., branched, cyclic, fused, adamantyl), and aro invention. matic groups. The water soluble polymer molecules will 0.111) Any amylin analog or derivative known in the art preferably have a molecular weight ranging from about 500 may be used in conjunction with the present invention. In to about 20,000 Daltons. one embodiment, the amylin analogs and derivatives have at 0107 Such polymer-conjugations and small molecule least one 5 hormonal activity of native amylin. In certain Substituent modifications may occursingularly at the N- or embodiments, the amylin analogs are agonists of a receptor C-terminus or at the side chains of amino acid residues which native amylin is capable of specifically binding. within the sequence of the hybrid polypeptides. Alterna Preferred amylin analogs and derivatives include those tively, there may be multiple sites of derivatization along the described in US 2003/0026812 Al, which is hereby incor hybrid polypeptide. Substitution of one or more amino acids porated by reference.

US 2006/0293.232 A1 Dec. 28, 2006

0113 As known in the art, such amylin analogs are 0118. Any CGRP analog or derivative known in the art preferably amidated, but within the context of the present may be used in conjunction with the present invention. In invention, may optionally be in the acid form unless other one embodiment, the CGRP analogs and derivatives have at wise specified. least one hormonal activity of native CGRP. In certain 0114) Any ADM analog or derivative known in the art embodiments, the CGRP analogs are agonists of a receptor may be used in conjunction with the present invention. In which native CGRP is capable of specifically binding. one embodiment, the ADM analogs and derivatives have at Preferred CGRP analogs and derivatives include those least one hormonal activity of native ADM. In certain described in U.S. Patent Nos. 4,697,002; and 4,687,839, embodiments, the ADM analogs are agonists of a receptor which are hereby incorporated by reference. which native ADM is capable of specifically binding. 0115) Any CT analog or derivative known in the art may 0119) Exemplary CGRP analogs include: be 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, SEQ ID: the CT analogs are agonists of a receptor which native CT is capable of specifically binding. Preferred CT analogs and 1 D-Ser-CGRP derivatives include those described in U.S. Pat. Nos. 4,652, 627; 4,606,856; 4,604.238; 4,597,900; 4,537,716; 4,497, 2 D-Thr-CGRP 731: 4,495,097: 4,444,981; 4,414,149; 4,401,593; and 3 D-Asp-CGRP 4.397,780, which are hereby incorporated by reference. 0116 Exemplary CT analogs include: 4 D-Asn-CGRP 5 Ser-CGRP

6 Hse-CGRP SEQ ID: 103 Gly-GT 7 Asp-CGRP 104 ’’Leu-GT 8 Thr-GGRP

105 °Gly, Ser, Gly, ?? des-Tyr-CT 9 Asn-CGRP 106 ''Glu-sCT, 107 "Arg-sCT, 0.120. Any AFP-6 analog or derivative known in the art 108 '''''Arg-sGT, may be used in conjunction with the present invention. In one embodiment, the AFP-6 analogs and derivatives have at 109 'Glu, Arg-sGT, least one hormonal activity of native AFP-6. In certain 110 ''Glu, '''''Arg-scT embodiments, the AFP-6 analogs are agonists of a receptor which native AFP-6 is capable of specifically binding. Preferred AFP-6 analogs and derivatives include those 0117. As known in the art, such CT analogs are prefer described in WO 2003/022304, which is hereby incorpo ably amidated, but within the context of the present inven rated by reference. tion, may optionally be in the acid form unless otherwise specified. 0121 Exemplary AFP-6 analogs include:

SEQ ID: 2O TQAQLLRVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPWDPSSPHSY

21 TQAQLLRVGCDTATCOWONLSHRLWQLMGPAGRODSAPWDPSSPHSY

22 TQAQLLRVGMWLGTMQVQNLSHRLWQLMGPAGRQDSAPWDPSSPHSY

23 TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPWEPSSPHSY

24 TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQESAPWEPSSPHSY

25 TQAQLLRVGCVLGTCQVQNLSHRLWQL----RQDSAPWDPSSPHSY

26 TQAQLLRVGCVLGTCQVQNLSHRLWQL- - - -DSAPWDPSSPHSY

27 RVGCVLGTCQVONLSHRLWOLMGPAGRODSAPWDPSSPHSY US 2006/0293.232 A1 Dec. 28, 2006 19

- continued

SEQ ID: 28 WGCVLGTCOWONLSHRLWQLMGPAGRODSAPWEPSSPHSY

29 WGCVLGTCQVQNLSHRLWQL----RQDSAPWEPSSPHSY

30 GCVLGTCOWONLSHRLWQLMGPAGRODSAPWDPSSPHSY

31 GCNTATCQWQNLSHRLWQL----RQDSAPWDPSSPHSY

32 GGNTATCQWQNLSHRLWQL----RQDSAPWEPSSPHSY

33 GGSNLSTGQWQNLSHRLWQL----RQDSAPWEPSSPHSY

34 GCGNLSTCQWQNLSHRLWQL----RQDSAPWEPSSPHSY

35 GCVLGTCQWQNLSHRLWQL----RQESAPWEPSSPHSY

36 CVLGTGQVONLSHRLWOLMGPAGRQDSAPWDPSSPHSY

37 QVQNLSHRLWQLMGPAGRQDSAPWDPSSPHSY

38 WONLSHRLWQLMGPAGRQDSAPWDPSSPHSY

39 WQNLSHRL----QLMGPAGRQDSAPWDPSSPHSY

40 GTMQVQNLSHRLWQL----RQDSAPWEPSSPHSY

0122). As known in the art, such AFP-6 analogs are tinal Hormones”. In Physiology of the Gastrointestinal Tract preferably amidated, but within the context of the present (3d ed. 1994; Raven Press, New York). invention, may optionally be in the acid form unless other 0.126 Certain exemplary CCKs and CCK analogs with wise specified. CCK activity include: 4. The CCK Family 0123 CCKs, including hCCK and species variants, and various analogs thereof are known in the art. Generally, SEQ ID: CCK has a 33-amino acid sequence first identified in humans, and includes a 8-amino acid in vivo C-terminal 41 DY (SOH) MGWMDF fragment (“CCK-8) that has been reportedly demonstrated 42 DYMGWMDF in pig, rat, chicken, chinchilla, dog and humans. Other species variants include a 39-amino acid sequence found in 43 MGWMDF pig, dog and guinea pig, and a 58-amino acid found in cat, dog and humans, and a 47-amino acid sequences homolo 44 GWMDF gous to both CCK and gastrin. The C-terminal tyrosine 45 WMDF sulfated octapeptide sequence (CCK-8) is relatively con served across species, and may be the minimum sequence 46 KDY (SOH) MGWMDF for biological activity in the periphery of rodents. Thus, the 47 KDYMGWMDF term CCK-33 will generally refer to human CCK(1-33), while CCK-8 (CCK(26-33)) will refer to the C-terminal 48 KMGWMDF octapeptide generically in both the sulfated and unsulfated unless otherwise specified. Further, pentagastrin or CCK-5 49 KGWMDF will refer to the C-terminal peptide CCK(29-33), and the 5 O KWMDF CCK-4 will refer to the C-terminal tetrapeptide CCK(30 33). 0124 The type A receptor subtype (CCKA) has been 0127. As known in the art, such CCK peptides are reported to be selective for the sulfated octapeptide. The preferably amidated, but within the context of the present Type B receptor subtype (CCK) has been identified invention, may optionally be in the acid form unless other throughout the brain and in the stomach, and reportedly does wise specified. not require Sulfation or all eight amino acids. 5. The Leptin Family 0125 Various in vivo and in vitro screening methods for 0.128 Component peptide hormones useful in the present CCK analogs are known in the art. Examples include in vivo invention also include leptin family peptide hormones. assays involving the contraction of the dog or guinea pig Native leptin family peptide hormones are known in art, as gallbladder after rapid intravenous injection of the com are functional peptide analogs and derivatives. Certain pre pound to be tested for CCK-like activity, and in vitro assays ferred native peptides, peptide analogs and derivatives are using strips of rabbit gallbladder. See Walsh, “Gastrointes described herein, however it should be recognized that any US 2006/0293.232 A1 Dec. 28, 2006 20 known amylin family peptides that exhibit hormonal activity ferred native peptides, peptide analogs and derivatives are known in the art may be used in conjunction with the present described herein, however it should be recognized that any invention. known amylin family peptides that exhibit hormonal activity 0129. Any leptin analog or derivative known in the art known in the art may be used in conjunction with the present may be used in conjunction with the present invention. In invention. one embodiment, the leptin analogs and derivatives have at 0.133 Any PPF analog or derivative known in the art may least one hormonal activity of native leptin. In certain embodiments, the leptin analogs are agonists of a receptor be used in conjunction with the present invention. In one which native leptin is capable of specifically binding. Pre embodiment, the PPF analogs and derivatives have at least ferred leptin analogs and derivatives include those described one hormonal activity of a native PPF polypeptide. In certain in, e.g., WO 2004/039832, WO 98/55139, WO 98/12224, embodiments, the PPF analogs are agonists of a receptor and WO97/02004, all of which are hereby incorporated by which native PPF polypeptide is capable of specifically reference. binding. Preferred PPF analogs and derivatives include those described in WO 03/026591 and WO 03/057235, 0130 Exemplary leptin analogs include those where the which are herein incorporated by reference in their entirety. amino acid at position 43 is substituted with Asp or Glu; position 48 is substituted Ala; position 49 is substituted with 0.134. In one embodiment, preferred PPF analogs and Glu, or absent; position 75 is substituted with Ala; position derivatives that exhibit at least one PPF hormonal activity 89 is substituted with Leu; position 93 is substituted with generally comprise at least two PYY motifs including a Asp or Glu; position 98 is substituted with Ala; position 117 polyproline motif and C-terminal tail motif. Such analogs is substituted with Ser, position 139 is substituted with Leu, are generally described in U.S. Provisional Application No. position 167 is substituted with Ser, and any combination 60/543,406 filed Feb. 11, 2004, which is herein incorporated thereof. by reference. Other preferred PPF analogs are disclosed in PCT/US2005/004351, entitled “Pancreatic Polypeptide 0131 Certain exemplary leptin and leptin analogs with Family Motifs and Polypeptides Comprising the Same'. leptin activity include: Attorney Docket 18528.832, the contents of which is hereby incorporated by reference. By way of background, research has suggested that the differences in Y receptor binding affinities are correlated with secondary and tertiary structural SEQ ID: differences. See, e.g., Keire et al., Biochemistry 2000, 39, 51 Asp-leptin 9935-9942. Native porcine PYY has been characterized as including two C-terminal helical segments from residues 17 52 Glu-leptin to 22 and 25 to 33 separated by a kink at residues 23, 24, and 25, a turn centered around residues 12-14, and the N-termi 53 Ala-leptin nus folded near residues 30 and 31. Further, full-length 54 'Glu-leptin porcine PYY has been characterized as including the PP fold, stabilized by hydrophobic interactions among residues 55 Des-AA-leptin in the N- and C-termini. See id. 56 Ala-leptin 0135) A “PYY motif is generally a structural compo 57 Leu-leptin nent, primary, secondary, or tertiary, of a native PP family polypeptide that is critical to biological activity, i.e., bio 58 Asp-leptin logical activity is Substantially decreased in the absence or 59 Glu-leptin disturbance of the motif. Preferred PYY motifs include the N-terminal polyproline type II motif of a native PP family 60 'Ala-leptin polypeptide, the type II P-turn motif of native PP family polypeptide, the a-helical motif at the C-terminal end of 61 '''Ser-leptin native PP family polypeptide, and the C-terminal tail motif 62 '''Leu-leptin of native PP family polypeptide. More particularly, in the N-terminal polyproline region, amino acids corresponding 63 ''Ser-leptin to residues 5 and 8 of a native PP family polypeptide are 64 Asp, 'Glu-leptin generally conserved as a proline. The type II B-turn motif will generally include amino acids corresponding to residues 65 Asp, Ala-leptin 12-14 of a native PP family polypeptide. The O.-helical motif can generally extend from amino acids corresponding to 66 Leu, '''Ser-leptin approximately residue 14 of a native PP family polypeptide 67 Glu, Ser-leptin to any point up to and including the C-terminal end, so long as the C-helical motif includes a sufficient number of amino acid residues such that an O.-helical turn is formed in 6. The PPF Family Solution. The C-helical motif can also include amino acid substitutions, insertions and deletions to the native PP family 0132) Component peptide hormones useful in the present sequence, so long as the C-helical turn is still formed in invention also include PPF peptide hormones, including PP Solution. The C-terminal tail motif generally includes amino and PYY. Native PPF peptide hormones are known in art, as acids corresponding to approximately the last 10 residues of are functional peptide analogs and derivatives. Certain pre a native PP family polypeptide, more preferably the last 7, US 2006/0293.232 A1 Dec. 28, 2006

6, or 5 residues of a native PP family polypeptide, and more 0140. As known in the art, such GLP-1 analogs may preferably amino acid residues 32-35. preferably be amidated, but within the context of the present invention, may optionally be in the acid form unless other 0.136 Preferred PYY analogs include those with internal wise specified. deletions, insertions, and substitutions in areas of the PYY molecule not corresponding to the polyproline motif and/or 0.141. Other GLP-1 analogs and derivatives are disclosed the C-terminal tail motif. For instance, internal deletions at in U.S. Pat. No. 5,545,618 which is incorporated herein by positions 4, 6, 7, 9, or 10 are envisioned. reference. A preferred group of GLP-1 analogs and deriva tives include those disclosed in U.S. Pat. No. 6,747,006, 7. Incretins and Incretin Mimetics which is herein incorporated by reference in its entirety. The 0137 Component peptide hormones useful in the present use in the present invention of a molecule described in U.S. invention also include GLP-1 peptide hormones. Native Pat. No. 5,188,666, which is expressly incorporated by GLP-1 peptide hormones, including GLP-1 (1-37), GLP-1 (7- reference, is also contemplated. Another group of molecules 37), and GLP-1 (7-36)amide, are known in art, as are func for use in the present invention includes compounds tional peptide analogs and derivatives. As used herein, described in U.S. Pat. No. 5,512.549, which is expressly GLP-1 refers to all native forms of GLP-1 peptide hor incorporated herein by reference. Another preferred group of mones. Certain preferred native peptides, peptide analogs GLP-1 compounds for use in the present invention is dis and derivatives are described herein, however it should be closed in WO 91/11457, which is herein incorporated by recognized that any known GLP-1 peptides that exhibit reference. hormonal activity known in the art may be used in conjunc tion with the present invention. 0.142 Component peptide hormones useful in the present 0138 Any GLP-1 peptide analog or derivative known in invention also include GLP-2 peptide hormones. Native the art may be used in conjunction with the present inven GLP-2 peptide hormones, e.g., rat GLP-2 and its homolo tion. In one embodiment, the GLP-1 peptide analogs and gous including ox GLP-2, porcine GLP-2. degu GLP-2, derivatives have at least one hormonal activity of a native bovine GLP-2, guinea pig, GLP-2, hamster GLP-2, human GLP-1 peptide. In certain embodiments, the GLP-1 peptide GLP-2, rainbow trout GLP-2, and chicken GLP-2, are analogs are agonists of a receptor which a native GLP-1 known in art, as are functional peptide analogs and deriva peptide is capable of specifically binding. Preferred GLP-1 tives. Certain preferred native peptides, peptide analogs and peptide analogs and derivatives include those described in, derivatives are described herein, however it should be rec e.g., WO 91/11457, which is hereby incorporated by refer ognized that any known GLP-2 peptides that exhibit hor CCC. monal activity known in the art may be used in conjunction with the present invention. 0139 GLP-1 analogs known in the art include: 0.143 Any GLP-2 peptide analog or derivative known in the art may be used in conjunction with the present inven tion. In one embodiment, the GLP-2 peptide analogs and SEQ ID: derivatives have at least one hormonal activity of a native GLP-2 peptide. In certain embodiments, the GLP-2 peptide 68 'Gln-GLP-1 (7-37) analogs are agonists of a receptor which a native GLP-2 69 D-Gln-GLP-1 (7-37) peptide is capable of specifically binding. Preferred GLP-2 peptide analogs and derivatives include those described in, 70 'Thr-Lys GLP-1 (7–37) e.g., U.S. Ser. No. 08/669,791 and PCT Application PCT/ 71. 'Llys-GLP-1 (7-37) CA97/00252, both of which are hereby incorporated by reference. Specific GLP-2 analogs known in the art include: 72 Gly-GLP-1 (7-36) rat or human GLP-2 altered at position 2 to confer DPP-IV 73 Gln-GLP-1 (7-37) resistance by Substituting a Gly for an Ala. 0.144 Component peptide hormones useful in the present 74 D-Gln-GLP-1 (7-37) invention also include oxyntomodulin (OXM) peptide hor 75 acetyl-Lys-GLP-1 (7-37) mones. Native OXM peptide hormones are known in art, as are functional peptide analogs and derivatives. Certain pre 76 Thr-GLP-1 (7-37) ferred native peptides, peptide analogs and derivatives are 77 D-Thr-GLP-1 (7-37) described herein, however it should be recognized that any known OXM peptides that exhibit hormonal activity known 78 'Asn-GLP-1 (7-37) in the art may be used in conjunction with the present invention. 79 D-Asn-GLP-1 (7-37) 0145 Any OXM peptide analog or derivative known in 8O ??Ser?Arg?' Arg? Gln-GLP-1 (7-37) the art may be used in conjunction with the present inven 81 Thr'Lys-GLP-1 (7–37) tion. In one embodiment, the OXM peptide analogs and derivatives have at least one hormonal activity of a native 82 'Llys-GLP-1 (7-37) OXM peptide. In certain embodiments, the OXM peptide 83 *Arg-GLP-1 (7-37) analogs are agonists of a receptor which a native OXM peptide is capable of specifically binding. 84 *Arg-GLP-1 (7-37) 0146 Component peptide hormones useful in the present invention also include exendin peptide hormones. Native US 2006/0293.232 A1 Dec. 28, 2006 22 exendin peptide hormones are known in art, as are functional 0151. vii. Natriuretic Peptides. peptide analogs and derivatives. Certain preferred native 0152 Natriuretic peptides are a family of hormones that peptides, peptide analogs and derivatives are described consist of atrial natriuretic peptide (ANP), brain natriuretic herein, however it should be recognized that any known peptide (BNP), and C-type natriuretic peptide (CNP). They exendin peptides that exhibit hormonal activity known in the are synthesized and stored as 3 distinct precursor prohor art may be used in conjunction with the present invention. mones, which are the 126 amino acid ANP, 108 amino acid BNP, and 104 amino acid CNP. They are each encoded by 0147 Any exendin peptide analog or derivative known in separate genes and have distinct sites of synthesis and the art may be used in conjunction with the present inven mechanisms of regulation. Parental natriuretic peptide tion. In one embodiment, the exendin peptide analogs and sequences include: derivatives have at least one hormonal activity of a native exendin peptide. In certain embodiments, the exendin pep tide analogs are agonists of a receptor which a native 1881.51 amino acid MSSFSTTTWSFLLLLAFOLLGOTRANIPMYNA exendin peptide is capable of specifically binding. human ANP WSNADLMDFKNLLDHLEEKMPLEDEVWPPQWL preprohormone SDPNEEAGAALSPLPEVPPWTGEVSPAQRDGG ALGRGPWDSSDRSALLKSKLRALLTAPRSLRR 0148 Exemplary exendin analogs include: SSCFGGRMDRIGAQSGLGCNSFRY 189134 amino acid MDPQTAPSRALLLLLFLHLAFLGGRSHPLGSP human BKP GSASDLETSGLQEQRNHLQGKLSELQWEQTSL SEQ ID: preprohormone EPLQESPRPTGVWKSREVATEGTRGHRKMVLY TLRAPRSPKMVQGSGCFGRKMDRISSSSGLGC 185 'Leu, Phe-exendin-4 186 Ala, ''Leu, Phe-exendin-4 190126 amino acid MHLSQLLACALLLTLLSLRPSEAKPGAPPKWP human CNP RTPPAEELAEPQAAGGGQKKGDKAPGGGGANL 187 'Leu, ?? Ala, Phe-exendin-4 preproCNP KGDRSRLLRDLRVDTKSRAAWARLLQEHPNAR KYKGANKKGLSKGCFGLKLDRIGSMSGLGC

0149. As known in the art, such exendin analogs are 0153. The main site of synthesis of the ANP prohormone preferably amidated, but within the context of the present is the atrial myocyte where it is synthesized as a 151-amino acid preprohormone. Removal of a 25-amino acid signal invention, may optionally be in the acid form unless other peptide from its N terminal end occurs in the endoplasmic wise specified. reticulum, leaving a 126-amino acid ANP prohormone 0150. Additional exemplary exendin analogs and deriva (ProANP), the main storage form of ANP within the heart. tives are described in PCT Application Serial No. PCT/ The prohormone consists of 4 biologically active peptide US98/16387 filed Aug. 6, 1998, entitled “Novel Exendin segments: amino acids 1-30 (ProANF 1-30, also known as Agonist Compounds, which claims the benefit of U.S. long acting Na stimulator), 31-67 (ProANF 31-67, also patent application Ser. No. 60/055,404, filed Aug. 8, 1997, known as vessel dilator), 79-98 (ProANF 79-98, also known both of which are herein incorporated by reference. Other as potassium excreter), and 99-126 (ANF, also known as exendin analogs and derivatives are described in PCT Appli atrial natriuretic factor). cation Serial No. PCT/US98/24210, filed Nov. 13, 1998, 0154 BNP was originally isolated from porcine brain but entitled “Novel Exendin Agonist Compounds,” which in humans it is synthesized and secreted from the left claims the benefit of U.S. Provisional Application No. ventricle. Sequence analysis reveals that preproBNP con 60/065,442 filed Nov. 14, 1997, both of which are herein sists of 134 residues and is cleaved to a 108-amino acid incorporated by reference. Still other exendin analogs and ProBNP. Cleavage of a 32-amino acid sequence from the derivatives are described in PCT Application Serial No. C-terminal end of ProBNP results in human BNP (77-108), PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel which is the physiologically active form in plasma. Exendin Agonist Compounds,” which claims the benefit of U.S. Provisional Application No. 60/066,029 filed Nov. 14, O155 CNP is the third member of the natriuretic peptide 1997, both of which are herein incorporated by reference. system and is primarily found in human vascular endothelial Still other exendin analogs and derivatives are described in cells, , and porcine brain. High concentrations of CNP PCT Application Serial No. PCT/US97/14199, filed Aug. 8, are also found in human hypothalamus and midbrain. In 1997, entitled “Methods for Regulating Gastrointestinal humans, preproCNP is a 126-amino acid precursor pro Activity,” which is a continuation-in-part of U.S. patent cessed into proCNP by cleavage of 23 residues from its application Ser. No. 08/694,954 filed Aug. 8, 1996, both of N-terminal end. This 23-amino acid sequence serves as a which are hereby incorporated by reference. Still other . The terminal 22 (105-126) amino acids are exendin analogs and derivatives are described in PCT Appli cleaved from proCNP to yield a biologically active form of cation Serial No. PCT/US98/00449, filed Jan. 7, 1998, CNP. entitled “Use of Exendins and Agonists Thereof for the 0156 Urodilatin is a kidney-derived member of the natri Reduction of Food Intake,” which claims priority to U.S. uretic peptide family and is formed from the same ANP Provisional Application No. 60/034.905 filed Jan. 7, 1997, prohormone and consists of amino acids 95-126. Except for both of which are hereby incorporated by reference. Yet the 4 amino acid N terminal extension, it is identical to ANF other exendin analogs and derivatives are described in US (99-126). Urodilatin appears to be an important regulator of 2004/0209803 A1, filed December 19, 2003, entitled “Com Sodium and water handling in the kidney, as well as a positions for the Treatment and Prevention of Neuropathy.' mediator of sodium excretion in patients with congestive which is hereby incorporated by reference. heart failure (CHF). US 2006/0293.232 A1 Dec. 28, 2006

0157 Natriuretic peptides exert their biologic effects by with ANP, the diuretic and natriuretic effects of BNP are binding to high-affinity receptors on the Surface of target significantly greater. BNP is cleared more slowly than ANP cells. Three subtypes of NPR's NPR-A, NPR-B, and and exerts other effects including Suppressing NPRC have been isolated. Consequently, in one embodi secretion and increasing serum levels of ANP BNP peptides ment is provided a method to screen hybrids for natriuretic can also provide a beneficial decrease in pulmonary capil receptor binding and/or activation. Natriuretic peptides lary wedge pressure, systemic vascular resistance, right including prohormone variants can impart numerous natri atrial pressure and systolic blood pressure, with an increase uretic peptide hormone activities to hybrids of the invention. in cardiac index in patients hospitalized for symptomatic In other embodiments of interest are natriuretic antagonist CHF. In patients with decompensated heart failure, natri hybrids. Natriuresis is the excretion of an excessively large uretic peptide hybrids can provide a beneficial decrease in amount of sodium into the urine. Natriuresis is similar to pulmonary capillary wedge pressure and an improved dys diuresis (the excretion of an unusually large quantity of pnea score. (Dyspnea is an unpleasant sensation of difficulty urine), except that in natriuresis the urine is exceptionally in breathing, typically associated with early stages of cardiac salty. Natriuresis occurs with some diuretics and diseases (as heart failure.) The hybrids containing one, two or three of the adrenal) and can lead to the salt-losing syndrome natriuretic hormone functions provide methods of adminis characterized by dehydration, vomiting, low blood pressure, tration of pharmaceutically active compositions that are and the risk of Sudden death. Exogenous administration of useful for both the prophylactic and therapeutic treatment of the 4 independent circulating peptides of the ANP prohor CHF patients, preferably CHF patients that are decompen mone (1-30, 31-67, 79-98, and 99-126) produce in vivo sated, patients with chronic CHF, and patients with hyper vasodilation, diuresis, Suppression of the -angiotensin tension. The natriuretic portion(s) of a hybrid is sufficient to aldosterone system and enhanced natriuresis and/or kaliure provide a therapeutically effective amount of a natriurertic sis. ProANF 1-30, ProANF 31-67 and ANF 99-126 each peptide to Such patient when administered in a therapeuti have natriuretic, blood pressure lowering and diuretic prop cally effective dose over a therapeutically effective period. erties with ProANF 31-67 and ANF 99-126 having the greatest impact on blood pressure. There are varying effects 0.161. As discussed herein any of the family of therapeu of the ANP peptides on potassium homeostasis: ProANF tically effective natriuretic peptides or their analogs can be 79-98 stimulates potassium excretion, whereas ProANF used. Useful natriuretic peptides include, for example, atrial 31-67 spares potassium loss by inhibiting Na/K ATPase in natriuretic peptide (ANP), brain natriuretic peptide (BNP or the medullary collecting duct cells. Specific to ANF 99-126 B-type natriuretic peptide) and C-type natriuretic peptide is a dose-dependent inhibition of angiotensin II-mediated (CNP). Sequences of useful forms of natriuretic peptides are aldosterone secretion, whereas proANF 31-67 has the prop disclosed in U.S. Patent Publication 20010027181, which is erty of inducing natriuresis through generation of prostag incorporated herein by reference. Examples of ANPs include human ANP (Kangawa et al., BBRC 118:131 (1984)) or that landin. from various species, including pig and rat ANP (Kangawa 0158 BNP produces similar biologic effects as ANF in et al., BBRC 121:585 (1984)). Such ANPs comprise 28 normal humans. Infusions of BNP in normal men produced amino acids. Such ANPs may be administered as a peptide a 2-fold increase in sodium excretion, 50% reduction in having a ring structure of ANP (formation of a disulfide plasma renin, angiotensin II and aldosterone secretion as bond based on CyS), and a C-terminal portion Succeeding the well as a reduction in plasma Volume. ring structure. An example of Such a peptide is a peptide 0159. CNP induces cardiovascular effects similar to the having amino acid residues at the 7-position to the 28-po other natriuretic peptides but does not appear to mediate any sition of ANP is provided in U.S. Patent Application Pub renal effects. When CNP is infused in anesthetized dogs at lication No. 20010027181. Another example is frog ANP. equivalent doses of ANF, plasma coMP rose with a con Specific examples of BNPs that can be used in the methods comitant reduction in mean arterial pressure, right atrial of the invention include human BNP (hBNP). Human BNP pressure and cardiac output, but glomerular filtration rate, comprises 32 amino acids and involves the formation of a renal blood flow and sodium excretion decreased. disulfide bond (Sudoh et al., BBRC 159:1420 (1989)) and U.S. Pat. Nos. 5,114,923, 5,674,710, 5,674,710, and 5,948, 0160 Natriuretic peptides can provide therapeutic benefit 761, each of which is incorporated by reference. Various in heart failure. Congestive heart failure (CHF) is associated BNP's of origin other than human, including as pig BNP and with increases in , endothelin, and with activa rat BNP are also known, and can be used. A further example tion of the renin-angiotensin-aldosterone system, and sym is chicken BNP. Examples of CNPs that can be used in the pathetic nervous systems, mediating vasoconstriction, methods of the invention include pig CNP. Pig CNP com Sodium and water retention, and negative vascular and prises 22 amino acids and involves the formation of a cardiac remodeling. These effects occur despite the elevated disulfide bond, like the above-described ANP and BNP levels of the natriuretic peptides in patients with heart (Sudoh et al., BBRC 168:863 (1990)) (human and rat have failure. In one embodiment of the invention are hybrids that the same amino acid sequence), chicken CNP (Arimura et provide increased or therapeutic serum levels of natriuretic al., BBRC 174: 142 (1991)). Frog CNP (Yoshihara et al., peptide activity for treatment or prevention of cardiac BBRC 173:591 (1990) can also be used. As discussed related diseases and conditions, including CHF. Although herein, one skilled in the art can apply modifications. Such ANF infusion in normal individuals can result in a sustained as a deletion, Substitution, addition or insertion, and/or increase in Sodium excretion and urine flow rates, in the chemical modification to amino acid residues in the amino heart failure patient a marked beneficial reduction in renal acid sequence of a known natriuretic peptide as desired, by response can be obtained. BNP infusion markedly increases known methods. The resulting compound is a compound Sodium excretion in patients with heart failure and exerts which has the activity of acting on a receptor of the starting significant beneficial hemodynamic effects. As compared ANP, BNP or CNP. Analogs having this activity, therefore, US 2006/0293.232 A1 Dec. 28, 2006 24 are included in the hybrids for use in accordance with the ADM, CT, CGRP intermedin, CCK(1-33), CCK-8, leptin, methods of the present invention. PYY(1-36), PYY(3-36), GLP-1(1-37), GLP-1(7-37), GLP 0162. In another embodiment, the hybrids containing one 1 (7-36), GLP-2, OXM, exendin-4, natriuretic peptide hor or more matriuretic functions can be used in treating hyper mones, urocortin family peptides, e.g., Ucn-2 and Ucn-3, tension. In one embodiment a natriuretic hybrid will have no neuromedin family peptides, e.g. neuromedin U25 or splice deleterious effect on heart rate and is not associated with variants, and ANP, BNP, CNP or urodilatin. arrhythmias. In one embodiment the hybrid will comprise at 0166 Other preferred bio-active peptide hormone mod least one, two or three natriuretic peptide functions, for ules include analogs and derivatives of a component peptide example, both ANP and BNP activity. One or more natri hormone selected from: amylin, ADM, CT, CGRP. interme uretic hormone functions can be combined with any other din, CCK, leptin, PYY(1-36), PYY(3-36), GLP-1(1-37), hormone function or peptidic enhancer, as described herein. GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, exendin-3, and In another embodiment the natriuretic portion(s) is a more exendin-4, natriuretic peptide hormones, urocortin family stable analog having an extended in vivo half-life when peptides, e.g., Ucn-2 and Ucn-3, neuromedin family pep compared with that of a native natriuretic peptide. Analogs tides, e.g. neuromedin U25 or splice variants, and ANP, that prevent undesirable cleavage by endogenous enzymes BNP. CNP or urodilatin, wherein the analog or derivative such as NEP are also envisioned. The natriuretic containing exhibits at least one hormonal activity of the component hybrids are also further directed to hypertension reduction, peptide hormone. The analog may comprise one or more diuresis inducement, natriuresis inducement, vascular con insertions, deletions, or Substitutions of the amino acid duct dilatation or relaxation, natriuretic peptide receptors sequence of the component peptide hormone, and the (such as NPR-A) binding, renin secretion Suppression from derivative may comprise one or more chemical modifica the kidney, aldostrerone secretion Suppression from the tions of an amino acid residue of an analog or component adrenal gland, treatment of cardiovascular diseases and peptide hormone, as described more fully herein and known disorders, reducing, stopping or reversing cardiac remodel in the art. ing in congestive heart failure, treatment of renal diseases 0.167 More specifically, analogs and derivatives may be and disorders; treatment or prevention of ischemic stroke, selected from any described above and/or known in the art. and treatment of asthma. Hybrids can be administered to Particularly preferred analogs and derivatives that exhibit at patients that would benefit from inducing natriuresis, diure least one hormonal activity useful as bio-active peptide sis and vasodilatation. Hybrids can be administered alone or hormone modules of the invention include the following: in combination with one or more of the following types of compounds: ACE inhibitors, beta-blockers, diuretics, spironolactone, digoxin, anticoagulation and antiplatelet Amylin: *Ala-h-amylin, ''Ala-h-amylin, Pro-h- amylin, ?, ?Pro-h-amylin, ?, ?, ?Pro-h- agents, and angiotensin receptor blockers. Additional dis amylin, Pro, Val, ?, ?Pro-h-amylin, eases or conditions include renal disorders and diseases, Arg, Pro-h-amylin, Arg, asthma, hypertension and pulmonary hypertension. Hybrids ° 2'2' Pro-h-amylin, 2Pro, 2val, are also useful to treat inflammatory-related diseases, erec ? Pro-h-amylin, Arg, Leu, 2 2'2'Pro-h-amylin, Arg?Leu, tile dysfunction and hypercholesterolemia. * Pro-h-amylin, and ' '-Cyclo-Asp, 0163 Bio-Active Peptide Hormone Modules 'Llys-h-amylin CT: Glu-sCT, Arg-sCT, ''''Arg-sCT, Glu, 0164. As discussed herein the hybrid polypeptides of the "Arg-sCT, 'Glu, '''''Arg-sCT present invention generally comprise at least two bio-active peptide hormone modules covalently linked together. The CGRP D-Ser-CGRP, D-Thr-CGRP, D-Asp-CGRP, bio-active peptide hormone modules may be: (a) native D-Asn-CGRP, Ser-CGRP, Hse-CGRP, component peptide hormones, (b) analogs or derivatives of Asp-CGRP, Thr-CGRP, Asn-CGRP native component peptide hormones that retain hormonal TQAQLLRVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPWDP activity, (c) fragments of native component peptide hor SSPHSY, mones that retain hormonal activity, (d) fragments of ana TQAQLLRVGCDTATCQWQNLSHRLWQLMGPAGRQDSAPWDPS SPHSY, logs or derivatives of native component peptide hormones TQAQLLRVGMWLGTMQVQNLSHRLWQLMGPAGRQDSAPWDPS that retain hormonal activity, (e) structural motifs of native SPHSY, component peptide hormones that impart a desired chemical TQAQLLRVGCVLGTCQWQNLSHRLWQLMGPAGRQDSAPWEPS stability, conformational stability, metabolic stability, bio SPHSY, availability, organ/tissue targeting, receptor interaction, pro TQAQLLRVGCVLGTCQWQNLSHRLWQLMGPAGRQESAPWEPS tease inhibition, plasma protein binding, and/or other phar SPHSY, macokinetic characteristic to the hybrid polypeptide; or (f) CCK DY (OSOH) MGWMDF, DYMGWMDF, MGWMDF, GWMDF, structural motifs of analogs or derivatives of native compo WMDF, KDY (OSOH) MGWMDF, KDYMGWMDF, nent peptide hormones that impart a desired chemical sta KMGWMDF, KGWMDF, KWMDF bility, conformational stability, metabolic stability, bioavail Leptin: Asp-leptin, Glu-leptin, Ala-leptin, ability, organ/tissue targeting, receptor interaction, protease Glu-leptin, Des-AA-leptin, Ala inhibition, plasma protein binding, and/or other pharmaco leptin, Leu-leptin, Asp-leptin, Glu kinetic characteristic to the hybrid polypeptide. The struc leptin, Ala-leptin, ''Leu-leptin, tural motifs of (e) and (f) will collectively be referred to PYY Leu-PYY, Val-PYY, Arg-PYY, Gln-PYY, herein as "peptidic enhancers’. Asn-PYY, Lys-PYY, Pro-PYY, His-PYY, ''Tyr-PYY, 'Pro''Ala-PYY, Leu Pro 0165 Preferred bio-active peptide hormone modules PYY, des-AA-4-PYY include native peptide hormones selected from: amylin, US 2006/0293.232 A1 Dec. 28, 2006

-continued -continued GLP-1 'Gln-GLP-1 (7-37), D-Gln-GLP-1 (7-37), Leptin: leptin (22-167), leptin (56-73) 'Thr-Lys GLP-1 (7-37), Lys-GLP-1 (7-37), Gly-GLP-1 (7-36), Gln-GLP-1 PYY PYY (1-35), PYY (1-30), PYY (1-25), PYY (7-37), D-Gln-GLP-1 (7-37), acetyl-Lys (1-15), PYY (1-10), PYY (2-36), PYY (3- GLP-1 (7-37), Thr-GLP-1 (7-37), D-Thr 36), PYY (4-36), PYY (5-36) GLP-1 (7-37), Asn-GLP-1 (7-37), D-Asn GLP-1 (7-37), ?? Ser?Arg?' Arg? Gln-GLP-1 GLP-1 GLP-1 (7-37), GLP-1 (7-36), GLP-1 (7-35) (7-37), 'Thr, Lys-GLP-1 (7-37), Lys GLP-1 (7-37), Arg-GLP-1 (7-37), Arg Exendin exendin-4 (1-27), exendin-4 (1-28), GLP-1 (7-37) exendin-4 (1–29), exendin-4 (1-30) or longer Exendin ''Leu, Phe-exendin-4, ''Leu, Phe exendin-4, Ala, ''Leu, Phe-exendin 4, and 'Leu, ?? Ala, Phe-exendin-4. 0172 Again, as known in the art, such peptide com pounds may preferably be amidated, but within the context 0168 As known in the art, such peptide compounds may of the present invention, may optionally be in the acid form preferably be amidated, but within the context of the present unless otherwise specified. Further, the above preferred invention, may optionally be in the acid form unless other fragments may be combined with any of the analogs or wise specified. derivatives discussed herein or known in the art. For 0169 Still other preferred bioactive peptide hormone example, preferred analog fragments may include Ala, modules include fragments of a component peptide hormone 'Leu, Phe-exendin-4(1-28), ''Leu, Phe-exendin-4(1- selected from: amylin, ADM, CT, CGRP intermedin, CCK, 27), Ala, ''Leu, Phe-exendin-4(1-28), ''Leu, Phe-exen leptin, PYY(1-36), PYY(3-36), GLP-1(1-37), GLP-1 (7-37), din-4(1-27), or any other combinations of the disclosed GLP-1 (7-36), GLP-2, OXM, a natriuretic peptide, exendin fragments, analogs, and derivatives. 3, and exendin-4, urocortin family peptides, e.g., Ucn-2 and 0173 Yet other preferred bio-active peptide modules Ucn-3, neuromedin family peptides, e.g. neuromedin U25 or include "peptidic enhancer”, i.e., structural motifs of com splice variants, and ANP, BNP. CNP or urodilatin wherein ponent peptide hormones (including analogs and derivatives the fragment exhibits at least one hormonal activity of the thereof) that impart a desired chemical stability, conforma component peptide hormone. tional stability, metabolic stability, bioavailability, organ/ 0170 Yet other preferred bioactive peptide hormone tissue targeting, receptor interaction, protease inhibition, modules include fragments of analogs or derivatives of a plasma protein binding, and/or other pharmacokinetic char component peptide hormone selected from: amylin, ADM, acteristic to the hybrid polypeptide. Exemplary peptidic CT, CGRP intermedin, CCK, leptin, PYY(1-36), PYY(3- enhancers include the following. Again, it should be under 36), GLP-1(1-37), GLP-1 (7-37), GLP-1 (7-37), GLP-1 (7- stood that combinations of the above-described analogs and 36), GLP-2, OXM, ANP BNP. CNP, urodilatin, exendin-3, derivatives taken together with the following bio-active exendin4, a natriuretic peptide hormones, urocortin family peptide modules are contemplated. For example, the last six peptides, e.g., Ucn-2 and Ucn-3, neuromedin family pep amino acid residues of amylin family peptide hormone tides, e.g. neuromedin U25 or splice variants, and ANP, analogs and derivatives known in the art and/or described BNP. CNP or urodilatin, wherein the fragment exhibits at above are also contemplated as preferred bio-active peptide least one hormonal activity of the component peptide hor modules. mone. Again, the analog may comprise one or more inser tions, deletions, or Substitutions of the amino acid sequence Amylin amylin (32-37), amylin (33–37), amylin of the component peptide hormone, and the derivative may Family (34-37), amylin (35-37), amylin (36-37), comprise one or more chemical modifications of an amino amylin (37), ADM (47-52), ADM (48-52) , acid residue of an analog or component peptide hormone, as ADM (49-52), ADM (50-52), ADM (51-52), ADM (52), CT (27-32), CT (27-32), CT described more fully herein and known in the art. (28-32), CT (29-32), CT (30-32), CT 0171 Certain preferred fragments that exhibit at least one (31-32), CT (32), CGRP (32-37), GGRP (33-37), CGRP (34-37), CGRP (35-37), CGRP hormonal activity include the following. However, it should (36-37), CGRP (37), intermedin (42-47), be understood that combinations of the above-described intermedin (43-47) intermedin (44-47), analogs and derivatives taken with fragments known in the intermedin (45-47) intermedin (46-47), art, including the preferred fragments described below, are intermedin (47) contemplated. PYY 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), Amylin: amylin (1-36), amylin (1-35), amylin PYY (26-35), PYY (27-35), PYY (28-35), (1-20), amylin (1-18), amylin (1-17), PYY (29–35), PYY (30–35), PYY (31-35), amylin (1-16), amylin (1-15), amylin (1-7) PYY (32-35) CT: CT (8-32), CT(8-27), CT (8-26), CT(8-10), GLP-1 frog GLP-1 (29-37); frog GLP-1 (30-37); CT (18-26), CT (18-27) and 2 frog GLP-2 (24-31) frog GLP-2 (25-31) AFP-6: AFP-6 (18-27) Exendin-4 exendin-4 (31-39), exendin-4 (32-39), exendin-4 (33-39), exendin-4 (34-39), CGK GCK-8, CCK-5, CCK-4 exendin-4 (35-39), exendin-4 (36-39), US 2006/0293.232 A1 Dec. 28, 2006 26

28)-des-Lys-hAmylin? 1-7)-'Arg-sCt(8-27)-hamy -continued lin(33-37) (see tables herein) its Gly linker species analog is exendin-4 (37-39), exendin-4 (38-39), also specifically intended and disclosed. This species is exendin-4 (39) ‘’GlyGlyGly-Exendin(1-28)-des-Lys-hamylin(1-7)-' 18Arg-SCt(8-27)-hamylin? 33-37), where the three 8. Peptide Module Selection Considerations, Spacers, and are located after the exendin(1-28) sequence. In one embodi Linking Groups ment a linker or spacer is 1 to 30 residues long, in another 0174 The hybrid polypeptides of the present invention embodiment 2 to 30 residues, and in yet another 3-30 generally comprise at least two bio-active peptide hormone residues long, and any integer length from 2 to 30 inclusive; modules of the invention, wherein at least one of the each integer unit is contemplated, e.g. 2, 3, 4, 5, 6, 7, etc. In bio-active peptide hormone modules exhibits at least one one embodiment a Gly linker is used, and in a particular hormonal activity. The bio-active peptide hormone module embodiment a three residue linker Gly-Gly-Gly. that exhibits the at least one hormonal activity may be 0177. Where amidation of the C-terminal end of N-ter located at the N terminal end of the hybrid polypeptide, the minally located bio-active peptide hormone module is C-terminal end of the hybrid polypeptide, or in the event that desired, the module may again be attached to a second the hybrid polypeptide comprises more than two bio-active module using any appropriate linking group known in the peptide hormone modules, may be located in the internal art. More specifically, in the event that a bio-active peptide portion of the hybrid polypeptide. hormone module exhibiting at least one hormonal activity 0175. In certain embodiments, it may be preferable to has been configured in the C-terminal orientation, resulting locate the bio-active peptide hormone module exhibiting the in an amino to amino linkage, preferred linking groups at least one hormonal activity such that the C-terminal end include dicarboxylic acids, alkyls, PEGs, and amino acids of the bio-active peptide hormone module is amidated. Such as Lys, Cys, and Glu. Amidation of the C-terminal end of the bio-active peptide hormone module may be accomplished by locating the 0.178 As mentioned above, the hybrid polypeptides may module at the C-terminal end of the hybrid peptide, or by also preferably include spacer to further stablize the linkage configuring the module in the C-terminal-to-N-terminal of the bio-active peptide hormone modules. Any spacer or direction at the N-terminal end of the hybrid polypeptide. In turn inducer known in the art may be used. By way of both configurations, the C-terminal end of the bio-active example, referred B-turn mimetics include mimic A and peptide hormone module is available for amidation. Specific mimic B illustrated below, also Ala-Aib and Ala-Pro dipep component peptide hormones where C-terminal amidation tides. Their IUPAC names are Mimic A: N-(3S,6S,9S)-2- may be preferable include amylin family peptide hormones, oxo-3-amino-1-azabicyclo4.3.0-nonane-9-carboxylic acid. CCK, PYY. hCLP-1 (7-36), and hCLP-2. Specific compo Mimic B: N-(3S,6S,9R)-2-oxo-3-amino-7-thia-1-azabicyclo nent peptide hormones where C-terminal amidation is not 4.3.0-nonane-9-carboxylic acid. necessarily preferred (stated otherwise, where elongation at the C-terminal end of the module is easily tolerated) include exendin-4, exendin-4(1-28), GLP-1 (7-37), frog GLP-1 (7- mimic A 36), and frog GLP-2. However, if these component peptide hormones are located at the C-terminal end of the hybrid polypeptide, they may still be optionally amidated, and in fact may preferably be optionally amidated. 0176) The bio-active peptide hormone modules may be covalently linked in any manner known in the art. Stable mimic B linkages may be used, or cleavable linkage may be used. In one embodiment, the carboxy of a first module may be directly linked to the amino of a second module. In another embodiment, linking groups may be used to attached mod ules. Further, if desired, spacers or turn inducers known in the art may be employed to stabilize the linkage. By way of / CO example, where amidation of the C-terminal end of the N-terminally located bio-active peptide hormone module is not desired, the module may be attached to a second module 9. Exemplary Combinations and Specific Embodiments directly, or using any appropriate linking group known in the art, Such as, an alkyl, PEG, amino acid, e.g., LyS, Glu, B-Ala; 0.179 Exemplary combinations of bio-active peptide hor polyaminoacids, e.g., poly-his, poly-arg, poly-lys, poly-ala, mone modules to form the hybrid polypeptides of the Gly-Lys-Arg (GKR) etc.; bifunctional linker (see, e.g., invention include combinations of two or more bio-active Pierce catalog, Rockford, Ill.); aminocaproyl (“Aca'), B-ala peptide hormone modules selected from: native peptide nyl, 8-amino-3,6-dioxaoctanoyl, or other cleavable and non hormones, analogs and derivatives of peptide hormones that cleavable linker known in the art. Specifically described exhibit at least one hormonal activity, fragments of native herein, as if each were explicitly drawn, are embodiments of peptide hormones that exhibit at least one hormonal activity, specific hybrids in which the linker in each exemplified fragments of analogs and derivatives of peptides hormones linker-containing hybrid is replaced by a Gly linker, par that exhibit at least one hormonal activity, and peptidic ticularly embodiments where the Gly linker is Gly-Gly-Gly. enhancers, with the proviso that at least one module exhibit As an example, for exemplified species 5 Apa-Exendin(1- at least one hormonal activity. US 2006/0293.232 A1 Dec. 28, 2006 27

0180. The hybrid polypeptides of the invention will (and/or SCT), BNP, and PYY as the component peptide include at least two bio-active peptide hormone modules, hormones. Particular combinations include exendin-4/PYY wherein each module is comprised from component peptide and PYY/exendin-4 combinations, with and without spacers hormones. In the context of the present invention, the or linking groups. Other combinations include exendin/ component peptide hormones of the hybrid polypeptide may amylin and amylin/exendin combinations, with and without be the same or different, with the proviso that at least two of spacers or linking groups. Yet other combinations include the component peptide hormones are different. In a preferred amylin/PYY and PYY/amylin combinations, with and with embodiment, at least two of the component peptide hor out spacers or linking groups. mones are from different peptide hormone families, e.g., the amylin family, CCK, the leptin family, PPF, the proglucagon 0186. In one aspect, preferred module combinations family, the natriuretic peptide family, urocortin family pep include those involving a first module comprising exendin tides, e.g., Ucn-2 and Ucn-3, neuromedin family peptides, 4, a fragment of exendin-4 that exhibits at least one hor e.g. neuromedin U25 or splice variants, and ANP BNP. CNP monal activity, an exendin-4 analog or derivative that exhib or urodilatin and the GLP-1 and exendin family. its at least one hormonal activity, or a fragment of an exendin-4 analog that exhibits at least one hormonal activity 0181. In certain embodiments, the hybrid polypeptides of in combination with at least one additional bio-active pep the invention may comprise two or more modules that tide hormone module. In one embodiment, the first module exhibit at least one hormonal activity. For instance, the is linked to one, two, or three additional bio-active peptide hybrid polypeptide may comprise a fragment of a first hormone modules. peptide hormone or analog that exhibits at least one hor monal activity covalently, linked to a fragment of at least 0187. In preferred embodiments, a first module compris one additional peptide hormone analog. The additional frag ing an exendin-4 peptide is linked to a second bio-active ment(s) may optionally exhibit at least one hormonal activ peptide hormone module comprising an amylin (and/or SCT) ity. The first peptide hormone may be the same or different peptide that exhibits at least one hormonal activity. In from the additional peptide hormone(s), with the proviso another embodiment, the second module is further linked to that at least one of the additional peptide hormones are a third bio-active peptide hormone module comprising a different from the first peptide hormone, and the first hor calcitonin peptide that exhibits at least one hormonal activ monal activity may be the same or different from the ity. In yet another embodiment, the third module may be optional additional hormonal activity. further linked to a fourth bio-active peptide hormone module 0182. In other embodiments, the hybrid polypeptides of comprising a peptidic enhancer selected from amylin pep the invention may comprise one or more modules that tides. In one embodiment, the first module may be located at exhibit at least one hormonal activity in combination with the C-terminal end of the hybrid polypeptide. Alternatively, one or more peptidic enhancer modules. For instance, a the first module may be located at the N terminal end of fragment of a first peptide hormone that exhibits a at least the hybrid polypeptide. In certain embodiments, spacers or one hormonal activity may be covalently linked to a peptidic linkers such as BAla may be inserted if desired to link the enhancer, or a fragment of a first peptide hormone that modules. exhibits at least one hormonal activity may be covalently 0188 Preferred exendin-4 peptides include: exendin-4, linked to a second peptide hormone that exhibits at least one exendin-4(1-27), exendin-4(1-28), ''Leu, Phe-exendin hormonal activity, which is in turn linked to a peptidic 4(1-28), and Ala, '“Leu, Phe-exendin-4(1-28). Also useful enhancer. Alternatively, a peptidic enhancer may be located are exendin(7-15) and its Ser2 analog, HSEGTFTSD (SEQ between two peptide hormone modules as a stabilizing ID NO. ). Preferred amylin peptides that exhibit at spacer. Again, the first peptide hormone may be the same or least one hormonal activity include amylin, amylin frag different from the second peptide hormone, and the first ments such as amylinC1-17), amylin (1-16), amylinC1-15). hormonal activity may be the same or different from the and amylin? 1-7), and amylin analogs such as pramlintide, second hormonal activity. ‘Ala-h-amylin, "Ala-h-amylin, and fragments thereof. Pre 0183 In another embodiment, the hybrid polypeptides of ferred calcitonin peptides that exhibit at least one hormonal the invention may comprise two, three, four, or more bio activity sGT, SCT fragments such as SCT(8-10), SCT(8-27), active peptide hormone modules. Exemplary combinations and, and calcitonin analogs such as ''Ag-SCT, 18Arg include a module with a hormonal activity in combination sCT, 14Glu, Arg-SCT, ''Glu, '''Arg-SCT, and fragments with one, two, or three peptidic enhancers; two modules thereof. Preferred amylin peptidic enhancers include amy with a hormonal activity in combination with one or two lin(32-37), amylin (33-37), and amylin? 34-37), and analogs peptidic enhancers; three modules with a hormonal activity thereof. Amylin/sCT combinations useful in connection with in combination with one peptidic enhancer, etc. the present invention include those disclosed in PCT/ 0184 The component peptide hormones are preferably US2005/004631 Amylin Family Agonist, Attorney Docket selected from amylin, adrenomedullin, calcitonin, calcitonin 18528.835, which is herein incorporated by reference. An gene related peptide, intermedin, cholecystokinin, leptin amylin/sCT chimera particularly useful for creating hybrids peptide YY. glucagon-like peptide-1, glucagon-like peptide of the invention is Compound 10 (described herein and in 2, oxyntomodulin, ANP, BNP. CNP, urodilatin, natriuretic PCT/US2005/004631) and analogs and derivatives thereof. peptide hormones, urocortin family peptides, e.g., Ucn-2 0189 In one aspect, preferred module combinations and Ucn-3, neuromedin family peptides, e.g. neuromedin include those involving a first module comprising exendin U25 or splice variants, and ANP, BNP. CNP or urodilatin or 4, a fragment of exendin-4 that exhibits at least one hor exendin4. monal activity, an exendin-4 analog or derivative that exhib 0185. More particularly, preferred module combinations its at least one hormonal activity, or a fragment of an include those involving combinations of exendin, amylin exendin-4 analog that exhibits at least one hormonal activity US 2006/0293.232 A1 Dec. 28, 2006 28 in combination with a peptidic enhancer. Preferred exen 0193 Yet other preferred module combinations include din-4 compounds include: exendin-4, exendin-4(1-27), those involving combinations of exendin, amylin and calci exendin-4(1-28), ''Leu, Phe-exendin-4(1-28), and Ala, tonin as tertiary and tetra-hybrid molecules. Exemplary '''Leu, Phe-exendin-4(1-28). Preferred peptidic enhancers combinations include exendinfamylin/calcitonin; exendin/ include: PYY(25-36), PYY(30-36) and PYY(31-36). In one amylin/calcitoninfamylin; amylin/calcitonin/exendin; and embodiment, the first module is located at the C-terminal amylin/calcitoninfamylin/exendin combinations, with and end of the hybrid polypeptide and the peptidic enhancer is without spacers or linking groups. Each module may inde located at the N-terminal end of the hybrid polypeptide. pendently be a peptidic enhancer or may exhibit a hormonal Alternatively, the first module may be located at the N-ter activity, depending on the desired properties of the hybrid minal end of the hybrid polypeptide and the peptidic polypeptide. enhance may be located at the C-terminal end of the hybrid polypeptide. In certain embodiments, spacers or linkers such 0194 In one embodiment, when one of the bio-active peptide hormone module(s) that exhibits at least one hor as BAla may be inserted if desired to attach the modules. monal activity is amylin or an analog or fragment thereof, 0190. In another aspect, preferred module combinations and a second bio-active peptide hormone module comprises include those involving a first module comprising exendin CCK, then the hybrid polypeptide should preferably com 4, a fragment of exendin-4 that exhibits at least one hor prise a third bio-active peptide hormone module selected monal activity, an exendin-4 analog or derivative that exhib from a different component peptide hormone. Exemplary its at least one hormonal activity, or a fragment of an third bio-active peptide hormone modules include calci exendin-4 analog that exhibits at least one hormonal activity tonins, more preferably salmon calcitonin, analogs or frag in combination with a second module comprising CCK, a ments thereof. fragment of CCK that exhibits at least one hormonal activity, a CCK analog or derivative that exhibits at least one 0.195. In another embodiment, when one of the bio-active hormonal activity, or a fragment of a CCK analog that peptide hormone module(s) that exhibits at least one hor exhibits at least one hormonal activity. Again, preferred monal activity is amylin or an analog or fragment thereof, exendin-4 compounds include: exendin-4, exendin-4(1-27), and a second bio-active peptide hormone module comprises exendin-4(1-28), ''Leu, Phe-exendin-4(1-28), Ala, '“Leu, CT, then the hybrid polypeptide should preferably comprise ‘Phe-exendin4(1-28), and ''Leu-exendin-4(1-28). Pre a third bio-active peptide hormone module selected from a ferred CCK compounds include: CCK-8, and CCK different component peptide hormone. Exemplary third bio 8(Phe(CHSO)). In one embodiment, the first module is active peptide hormone modules include exendin-4, analogs located at the C-terminal end of the hybrid polypeptide and or fragments thereof. the second module is located at the N-terminal end of the 0196. In yet another embodiment, when one of the bio hybrid polypeptide. Alternatively, the first module may be active peptide hormone module(s) that exhibits at least one located at the N-terminal end of the hybrid polypeptide and hormonal activity is GLP-1 or an analog or fragment thereof, the peptidic enhance may be located at the C-terminal end of and a second bio-active peptide hormone module is a the hybrid polypeptide. In certain embodiments, spacers or peptidic enhancer comprising an exendin fragment, then the linkers such as BAla may be inserted if desired to attach the hybrid polypeptide should preferably comprise a third bio modules. active peptide hormone module. Exemplary third bio-active 0191 In another aspect, preferred module combinations peptide hormone modules include PYY (including analogs, include those involving a first module comprising amylin, a derivatives and fragments thereof) and CCK (including fragment of amylin that exhibits at least one hormonal analogs, derivatives and fragments thereof). activity, an amylin analog or derivative that exhibits at least 0197) Within each of the combinations described herein, one hormonal activity, or a fragment of an amylin analog it is understood that reference to a component peptide that exhibits at least one hormonal activity in combination hormone includes reference to analogs, derivatives, frag with a second module comprising with a peptidic enhancer, ments, as well as peptidic enhancers related thereto. such as PYY(25-36) or PYY(30-36). In one embodiment, the first module is located at the C-terminal end of the hybrid O198) In a preferred aspect, the hybrid polypeptides polypeptide and the peptidic enhancer is located at the include: N-terminal end of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminal end of the hybrid polypeptide and the peptidic enhance may be located at the C-terminal end of the hybrid polypeptide. In certain SEQ ID: embodiments, spacers or linkers such as BAla may be 1 Exendin-4-PYY (22-36) inserted if desired to attach the modules. 2 Exendin-4-PYY (25-36) 0192 Other preferred module combinations include those involving combinations of exendin and CCK or amy 3 Exendin-4-PYY (18-36) lin, calcitonin, and CCK as a tertiary combination. Particular combinations include exendin/CCK and CCK/exendin, with 4 Exendin-4-BAla-BAla-PYY (22-36) and without spacers or linkers and linking groups. Other 5 Exendin-4-BAla-BAla-PYY (25-36) combinations include CCK/amylin/calcitonin and CCK/ amylin/calcitoninfamylin, with and without spacers or link 6 Exendin-4-BAla-BAla-PYY (31-36) ing groups. Each module may independently be a peptidic 7 Exendin-4 (1-28) -PYY (22-36) enhancer or may exhibit a hormonal activity, depending on the desired properties of the hybrid polypeptide.

US 2006/0293.232 A1 Dec. 28, 2006 30

(e.g., lysine, arginine, histidine), acidic side chains (e.g., -continued aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, SEQ ID: tyrosine, methionine, cysteine), nonpolar side chains (e.g., CCK-8-GKR-Glu-hAmylin (1-17) - 'Glu, alanine, Valine, leucine, isoleucine, proline, phenylalanine, "Arg-sCT (18-26)-Amylin (32-37) tryptophan), B-branched side chains (e.g., threonine, Valine, isoleucine) and aromatic side chains (e.g., tyrosine, pheny lalanine, tryptophan, histidine). 0199 Exemplary exendin and neuromedin hybrids 0203 The present invention also relates to derivatives of include the hybrid polypeptides. Such derivatives include hybrid polypeptides conjugated to one or more water Soluble poly Exendin-(1-28)-beta-Ala-beta-Ala-FN-38: mer molecules, such as polyethylene glycol (“PEG”) or fatty HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala acid chains of various lengths (e.g., Stearyl, palmitoyl, FLFHYSKTOKLGKSNWWEELOSPFASQSRGYFLFRPRN-NH2 octanoyl, etc.), or by the addition of polyamino acids, Such as poly-his, poly-arg, poly-lys, and poly-ala. Modifications Exendin- (1-28)-beta-Ala-beta-Ala-Neuromedin (U25 : ) HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala to the hybrid polypeptides can also include Small molecule FRVDEEFOSPFASQSRGYFLFRPRN-NH2 Substituents. Such as short alkyls and constrained alkyls (e.g., branched, cyclic, fused, adamantyl), and aromatic Exendin- (1-28)-beta-Ala-beta-Ala-Neuromedin (U-9) : HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-GYF groups. The water soluble polymer molecules will prefer LFRPRN-NH2 ably have a molecular weight ranging from about 500 to about 20,000 Daltons. The beta-Ala-beta-Ala spacer is optional, and can be 0204 Such polymer-conjugations and small molecule replaced with Gly-Gly-Gly, a mini-PEG group, or other Substituent modifications may occursingularly at the N- or linker known in the art, particularly those described herein. C-terminus or at the side chains of amino acid residues within the sequence of the hybrid polypeptides. Alterna 0200 Exemplary exendin and natriuretic peptide hybrids tively, there may be multiple sites of derivatization along the include exendin-hBNP peptide hybrids, including hybrid polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid, or cysteine may Exendin-(1-28)-beta-Ala-beta-Ala-hBNP: provide additional sites for derivatization. See, e.g., U.S. HGEGTFTSDLSKQMEEEAVRLFLEWLKN-beta-Ala-beta-Ala Pat. Nos. 5,824,784 and 5,824,778. Preferably, the hybrid SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH; polypeptides may be conjugated to one, two, or three and polymer molecules. Exendin-beta-Ala-beta-Ala-hBNP : 0205 The water soluble polymer molecules are prefer HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS -Ala-D- ably linked to an amino, carboxyl, or thiol group, and may Ala-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH. be linked by N or C terminus, or at the side chains of lysine, aspartic acid, glutamic acid, or cysteine. Alternatively, the As in all of the hybrids of the invention, a beta-Ala-beta-Ala water soluble polymer molecules may be linked with spacer is optional, and can be replaced with Gly-Gly-Gly, a diamine and dicarboxylic groups. In a preferred embodi mini-PEG group, or other linker known in the art, particu ment, the hybrid polypeptides of the invention are conju larly those described herein. gated to one, two, or three PEG molecules through an 0201 The hybrid polypeptides of the present invention epsilon amino group on a lysine amino acid. may also comprise further modifications including, but are 0206 Hybrid polypeptide derivatives of the invention not limited to, Substitution, deletion, and insertion to the also include hybrid polypeptides with chemical alterations to amino acid sequence of Such hybrid polypeptides and any one or more amino acid residues. Such chemical alterations combination thereof. In a preferred aspect, the hybrid include amidation, glycosylation, acylation, Sulfation, phos polypeptides of the invention include one or more modifi phorylation, acetylation, and cyclization. The chemical cations of a “non-essential amino acid residue. In the alterations may occursingularly at the N- or C-terminus or context of the invention, a “non-essential amino acid resi at the side chains of amino acid residues within the sequence due is a residue that can be altered, i.e., deleted or substi of the PPF hybrid polypeptides. In one embodiment, the tuted, in the native human amino acid sequence of the C-terminus of these peptides may have a free —OH or fragment, e.g., the component peptide hormone fragment, NH, group. In another embodiment, the N-terminal end without abolishing or Substantially reducing the component may be capped with an isobutyloxycarbonyl group, an peptide hormone receptor agonist activity of the hybrid isopropyloxycarbonyl group, an n-butyloxycarbonyl group, polypeptide. an ethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, an octyl glycine group (G(Oct)), or an 0202 Preferred substitutions include conserved amino 8-aminooctanic acid group. In a preferred embodiment, acid substitutions. A "conservative amino acid substitution' cyclization can be through the formation of disulfide is one in which the amino acid residue is replaced with an bridges. Alternatively, there may be multiple sites of chemi amino acid residue having a similar side chain, or physico cal alteration along the hybrid polypeptide. chemical characteristics (e.g., electrostatic, hydrogen bond ing, isosteric, hydrophobic features). Families of amino acid 0207 Examples of the hybrid polypeptides of the present residues having similar side chains are known in the art. invention are provided in the Sequence Listing and further These families include amino acids with basic side chains discussed in the Examples section herein. US 2006/0293.232 A1 Dec. 28, 2006

10. Use of Hybrid Polypeptides in the Treatment or Preven associated with hypemutrition. In one embodiment Such tion of Metabolic Conditions or Disorders hybrids contain an exendin, GLP 1, amylin and/or sGT portion. 0208 Hybrids of the invention can be useful for reducing food intake, reducing appetite, reducing caloric intake, 0213. In another general aspect, it is now recognized that inducing Satiety, reducing nutrient availability, causing hybrids containing amylin and/or SCT portions can be useful weight loss, affecting body composition, altering body for treating or preventing Barrett's esophagus, Gastroesoph energy content or energy expenditure, improving lipid pro ageal Reflux Disease (GERD) and conditions associated file (including reducing LDL cholesterol and triglyceride therewith. Such conditions can include, but are not limited levels and/or changing HDL cholesterol levels), slowing to, heartburn, heartburn accompanied by regurgitation of gastrointestinal motility, delay gastric emptying, moderating gastric/intestinal contents into the mouth or the lungs, dif the postprandial blood glucose excursions, preventing or ficulty in Swallowing, coughing, intermittent wheezing and inhibiting glucagon secretion, and decreasing blood pres Vocal cord inflammation (conditions associated with Sure. In one embodiment such hybrids contain an exendin, GERD), esophageal erosion, esophageal ulcer, esophageal GLP 1, amylin and/or sGT portion. stricture, Barrett's metaplasia (replacement of normal esophageal epithelium with abnormal epithelium), Barrett's 0209 Thus, in certain embodiments, the hybrids of the adenocarcinoma, and pulmonary aspiration. Such hybrids invention are useful for treating or preventing conditions or have anti-secretory properties, such as inhibition of gastric disorders which can be alleviated by reducing nutrient acids, inhibition of bile acids, and inhibition of pancreatic availability comprising administering to said subject a thera enzymes. Moreover, Such hybrids can have a gastroprotec peutically or prophylactically effective amount of a com tive effect, which renders them particularly useful in the pound of the invention. Such conditions and disorders treatment or prevention of Barrett's esophagus, and/or include, but are not limited to, eating disorders, insulin GERD and related or associated conditions as described resistance, obesity, abnormal postprandial hyperglycemia, herein. diabetes of any kind, including Type I, Type II, and gesta 0214. In another general aspect, hybrids can be further be tional diabetes, Metabolic Syndrome, Dumping Syndrome, useful for treating or preventing pancreatitis, pancreatic hypertension, dyslipidemia, cardiovascular disease, hyper carcinoma, and gastritis, particularly in the treatment and lipidemia, sleep apnea, cancer, pulmonary hypertension, prevention of pancreatitis in patients who have undergone cholecystitis, and osteoarthritis. In one embodiment Such endoscopic retrograde cholangiopancreatography (ERCP). hybrids contain an exendin, GLP 1, amylin and/or SCT Amylin and/or SCT containing hybrid agonists can have a portion. Suprisingly Superior therapeutic effect when combined with 0210 Exemplary peptide module pairings include car Somatostatin. Accordingly, in certain embodiments, methods dioactive/protective peptides, for example a urocortin with a for treating or preventing pancreatitis comprise administer GLP-1 or exendin, an ANP, BNP or CNP with a GLP-1 or ing Such hybrids and administering somatostatin and Soma exendin, and a urocortin with an ANP, BNP or CNP Such to statin agonists to a subject. hybrids will be cardioprotective and particularly useful for 0215. In another general aspect, hybrids are useful for the related diseases and conditions described herein, includ decreasing bone resorption, decreasing plasma calcium, and ing acute or chronic CHF, ischemia reperfusion, myocardial inducing an analgesic effect, particularly to treat bone dis infarction, and for vasodilator actions useful to treat or orders such as osteopenia and osteoporosis. In yet other prevent antihypertensive indications and angina. Ucn 2 and embodiments, hybrids are useful to treat pain and painful 3 are particularly useful in hybrids of the invention. neuropathy. In one embodiment Such hybrids contain an 0211 Non-limiting examples of a cardiovascular condi exendin, GLP1, amylin and/or sGT portion. tion or disease are hypertension, myocardial ischemia, and 0216) In another aspect of the invention, methods for myocardial reperfusion. Compounds of the invention may treating or preventing obesity are provided, wherein the also be useful in treating or preventing other conditions method comprises administering a therapeutically or pro associated with obesity including stroke, cancer (e.g. phylactically effective amount of a hybrid polypeptide to a endometrial, breast, prostate, and colon cancer), gallbladder subject in need thereof. In a preferred embodiment, the disease, sleep apnea, reduced fertility, and osteoarthritis, subject is an obese or overweight subject. While “obesity' is (see Lyznicki et al. Am. Fam. Phys. 63:2185, 2001). In other generally defined as a body mass index over 30; for purposes embodiments, compounds of the invention may be used to of this disclosure, any Subject, including those with a body alter body composition for aesthetic reasons, to enhance mass index of less than 30, who needs or wishes to reduce one's physical capabilities, or to produce a leaner meat body weight is included in the scope of “obese.” Subjects Source. Hybrids are useful to change body composition by who are insulin resistant, glucose intolerant, or have any decreasing fat without significant decrease in muscle mass, form of diabetes mellitus (e.g., type 1, 2 or gestational thus producing a desirable loss of body fat while preserving diabetes) can benefit from these hybrids. In one embodiment lean body mass. In one embodiment Such hybrids contain an such hybrids contain an exendin, PYY. GLP1, amylin and/or exendin, GLP 1, amylin and/or sGT portion. SCT portion. 0212. In another general aspect, hybrids of the invention 0217. In other aspects of the invention, methods of reduc may be used to inhibit the Secretion of ghrelin. Accordingly, ing food intake, reducing nutrient availability, causing compounds of the invention may be utilize this mechanism weight loss, affecting body composition, and altering body to treat or prevent ghrelin related disorders such as Prader energy content or increasing energy expenditure, treating Willi syndrome, diabetes of all types and its complications, diabetes mellitus, and improving lipid profile (including obesity, hyperphagia, hyperlipidemia, or other disorders reducing LDL cholesterol and triglyceride levels and/or US 2006/0293.232 A1 Dec. 28, 2006 32 changing HDL cholesterol levels) are provided, wherein the weight loss, or treating obesity, compounds of the invention methods comprise administering to a subject an effective may be used to treat hypotension. amount of a hybrid polypeptide of the invention. In a 0223 Compounds of the invention may also be useful for preferred embodiment, the methods of the invention are potentiating, inducing, enhancing or restoring glucose used to treat or prevent conditions or disorders which can be responsivity in or cells. These actions may alleviated by reducing nutrient availability in a subject in be useful for treating or preventing conditions associated need thereof, comprising administering to said Subject a with metabolic disorders such as those described above and therapeutically or prophylactically effective amount of a in U.S. patent application no. US20040228846. Assays for hybrid polypeptide of the invention. Such conditions and determining such activity are known in the art. For example, disorders include, but are not limited to, hypertension, in published U.S. patent application no. US20040228846 dyslipidemia, cardiovascular disease, eating disorders, insu (incorporated by reference in its entirety), assays are lin-resistance, obesity, and diabetes mellitus of any kind. In described for islet isolation and culture as well as determin one embodiment such hybrids contain an exendin, PYY. ing fetal islet maturation. In the examples of patent appli GLP1, amylin and/or SCT portion. cation US20040228846, intestine-derived hormone peptides 0218. Without intending to be limited by theory, it is including pancreatic polypeptide (PP), neuropeptide Y believed that the effects of peripherally-administered hybrid (NPY), neuropeptide K (NPK), PYY, , glucagon-like polypeptides of the present invention in the reduction of peptide-1 (GLP-1) and bombesin were purchased from food intake, in the delay of gastric emptying, in the reduction Sigma. Collagenase type XI was obtained from Sigma. of nutrient availability, and in the causation of weight loss RPMI 1640 culture medium and fetal bovine serum were are determined by interactions with one or more unique obtained from Gibco. A radioimmunoassay kit containing receptor classes in, or similar to, those in the PP family. anti-insulin antibody (II-RIA kit) was purchased from More particularly, it appears that a receptor or receptors Linco, St Louis. similar to the PYY-preferring (or Y7) receptors are involved. 0224 Post-partem rat islets were obtained from P-02 year 0219. Additional assays useful to the invention include old rats. Adult rat islets were obtained from 6-8 week old those that can determine the effect of PPF compounds on rats. Fetal rat islets were obtained as follows. Pregnant body composition. An exemplary assay can be one that female rats were sacrificed on pregnancy day E21. Fetuses involves utilization of a diet-induced obese (DIO) mouse were removed from the uterus. 10-14 pancreata were dis model for metabolic disease. Prior to the treatment period, sected from each litter and washed twice in Hanks buffer. male C57BL/6J mice can be fed a high-fat diet (#D12331, The pancreata were pooled, Suspended in 6 ml 1 mg/ml 58% of calories from fat; Research Diets, Inc.) for 6 weeks collagenase (Type XI, Sigma) and incubated at 37° C. for beginning at 4 weeks of age. During the study, the mice can 8-10 minutes with constant shaking. The was continue to eat their high-fat diet. Water can be provided ad stopped by adding 10 volumes of ice-cold Hanks buffer libitum throughout the study. One group of similarly-aged followed by three washes with Hanks buffer. The islets were non-obese mice can be fed a low-fat diet (iD12329, 11% of then purified by Ficoll gradient and cultured in 10% fetal calories from fat) for purposes of comparing metabolic bovine serum (FBS)/RPMI medium with or without addition parameters to DIO groups. of 1 M IBMX. At the end of five days, 20 islets were hand picked into each tube and assayed for static insulin release. 0220 DIO mice can be implanted with subcutaneous Generally, islets were first washed with KRP buffer and then (SC) intrascapular osmotic pumps to deliver either vehicle incubated with 1 ml of KRP buffer containing 3 mM (low) (50% dimethylsulfoxide (DMSO) in water) or a compound glucose for 30 minutes at 37°C. with constant shaking. After of the invention. The pumps of the latter group can be set to collecting the Supernatant, the islets were then incubated deliver any amount, e.g., 1000 g/kg/d of a compound of the with 17 mM (high) glucose for one hour at 37° C. The invention for 7-28 days. insulin released from low or high glucose stimulation were 0221 Body weights and food intake can be measured assayed by radioimmunoassay (RIA) using the 'I-RIA over regular intervals throughout the study periods. Respi kit. E21 fetal islets were cultured for 5 days in the presence ratory quotient (RO, defined as CO production--O con of 200 ng/ml PYY, PP, CCK, NPK, NPY, Secretin, GLP-1 or Sumption) and metabolic rate can be determined using Bombesin. whole-animal indirect calorimetry (Oxymax, Columbus Instruments, Columbus, Ohio). The mice can be euthanized 0225. An exemplary in vivo assay is also provided using by isoflurane overdose, and an index of adiposity (bilateral the Zucker Diabetic Fatty (ZDF) male rat, an inbred (>F30 epididymal fat pad weight) measured. Moreover, prior to Generations) rat model that spontaneously expresses diabe determination of epididymal weight, body composition (lean tes in all faffa males fed a standard rodent diet Purina 5008. mass, fat mass) for each mouse can be analyzed using a Dual In ZDF fa-fa males, hyperglycemia begins to develop at Energy X-ray Absorptiometry (DEXA) instrument per about seven weeks of age and glucose levels (fed) typically manufacturers instructions (Lunar Piximus, GE Imaging reach 500 mg/DL by 10 to 11 weeks of age. Insulin levels System). In the methods of the invention, preferred PPF (fed) are high during the development of diabetes. However, polypeptide of the invention are those having a potency in by 19 weeks of age insulin drops to about the level of lean one of the assays described herein (preferably food intake, control litter mates. Triglyceride and cholesterol levels of gastric emptying, pancreatic secretion, weight reduction or obese rats are normally higher than those of leans. In the body composition assays) which is greater than the potency assay, three groups of 7-week old ZDF rats, with 6 rats per of a component peptide hormone in that same assay. group, received the infusion treatment by ALZA pump for 14 days: 1) vehicle control. 2) and 3), PYY with two 0222. In addition to the amelioration of hypertension in different doses, 100 pmol/kg/hr and 500 pmol/kg/hr respec subjects in need thereof as a result of reduced food intake, tively. Four measurements were taken before the infusion US 2006/0293.232 A1 Dec. 28, 2006

and after the infusion at day 7 and day 14: 1) plasma glucose ischemia. In another embodiment hybrids are useful for level, 2) plasma insulin level, and 3) plasma triglycerides preventing and treating cardiac arrhythmias that reliably (TG) level, as well as oral glucose tolerance (OGTT) test. reduce injury associated with reperfusion and ischemia, and Accordingly, these assays can be used with compounds of enhance patient recovery. In yet a further embodiment the invention to test for desired activity. hybrid treatment after acute stroke or hemorrhage, prefer ably intravenous administration, provides a means for opti 0226. Other uses contemplated for the hybrid polypep mizing insulin secretion, increasing brain anabolism, tides include methods for reducing aluminum (Al) concen enhancing insulin effectiveness by Suppressing glucagon, trations in the central nervous system (see U.S. Pat. No. and maintaining euglycemia or mild hypoglycemia with no 6,734,166, incorporated by reference in its entirety) for risk of severe hypoglycemia or other adverse side effects. In treating, preventing, or delay the onset of Alzheimer's one embodiment such hybrids contain a GLP 1 or exendin disease. Assays for determining effects on Al are known in portion. In a further embodiment a GLP1 or exendin family the art and can be found in U.S. Pat. No. 6,734,166 using module is combined with a natriuretic family peptide, an diploid and Ts mice. These mice were individually housed in amylin family peptide, a urocortin family peptide module to Nalgene(R) brand metabolism or polypropylene cages and obtain enhanced treatment or prevention of cardiovascular given three days to adjust to the cages before experimenta conditions or diseases, including CHF, as described herein. tion. Mice had free access to food (LabCiet(R) NIH Rat and Moust/Auto 6F5K52, St. Louis, Mo.) and water during the 0227. In yet a further embodiment hybrids that are experiment except for the 16 hours prior to euthanasia when capable of lowering insulin resistance or increasing insulin no food was provided. Mice were given daily Subcutaneous sensitivity are useful to treat polycystic syndrome injections of either active compound or saline. Mice were (PCOS). Administering hybrids of the invention can reduce sacrificed at the end of day 13 for one experiment and day or prevent insulin resistance in a Subject suffering from 3 for another, and samples were collected. Mice brain PCOS. In yet another embodiment hybrids prevent the onset samples were weighted in clean teflon liners and prepared of type-2 diabetes in a subject suffering from PCOS. Further for analysis by microwave digestion in low trace element hybrids can restore regular menses, ovulation, or fertility in grade nitric acid. Samples were then analyzed for Al content a subject suffering from PCOS. In one embodiment such using Inductively Coupled Plasma Mass Spectrometry (Nut hybrids contain a GLP1 or an exendin portion for binding tall et al., Annals of Clinical and Laboratory Science 25, 3, and activating a GLP1 receptor. 264-271 (1995)). All tissue handling during analysis took place in a clean room environment utilizing HEPA air 0228. The compounds of the invention exhibit a broad filtration systems to minimize background contamination. range of biological activities, some related to their antisecre Hybrids of the invention are useful for prevention and tory and antimotility properties. The compounds may Sup treatment of nephropathy, including hypertensive and dia press gastrointestinal by direct interaction with betic nephropathy, and nephropathy associated with insulin epithelial cells or, perhaps, by inhibiting secretion of hor resistance and metabolic syndrome. Hybrids achieve these mones or neurotransmitters which stimulate intestinal secre ends by, among other things, improving or preventing wors tion. Anti-secretory properties include inhibition of gastric ening of hypertension, endothelial function, renal function, and/or pancreatic secretions and can be useful in the treat and glomerulosclerosis. In one embodiment, the invention ment or prevention of diseases and disorders including provides a method for preventing or treating nephropathy, gastritis, pancreatitis, Barrett's esophagus, and Gastroesoph including hypertensive and diabetic nephropathy, or that ageal Reflux Disease. related to insulin resistance, comprising administering a 0229 Compounds of the invention are useful in the compound of the invention. Hybrids find further use for treatment of any number of gastrointestinal disorders (see improving endothelial function in a patient having reduced e.g., Harrison's Principles of Internal Medicine, McGraw vasodilatory capacity, or having glomerulosclerosis or any Hill Inco, New York, 12th Ed.) that are associated with other reduction in glomerular flow. Such improvement in excess intestinal electrolyte and water secretion as well as endothelial function serves both to reduce hypertension and decreased absorption, e.g., infectious diarrhea, inflammatory to improve the function of the capillaries of the glomeruli. diarrhea, short bowel syndrome, or the diarrhea which In additional embodiments, the molecules of the invention typically occurs following Surgical procedures, e.g., ileo are useful to prevent progression of nephropathy to ESRD, stomy. Examples of infectious diarrhea include, without to prevent, slow the progression of treat or ameliorate limitation, acute viral diarrhea, acute bacterial diarrhea (e.g., proteinuria and/or glomerulosclerosis. Hybrids are useful for salmonella, campylobacter, and clostridium or due to pro reducing the risk of Suffering from, preventing, or treating tozoal infections), or traveller's diarrhea (e.g., Norwalk cardiac arrhythmias. Hybrids can provide anti-arrhythmic virus or rotavirus). Examples of inflammatory diarrhea effects in patients with cardiac ischemia, cardiac ischemia include, without limitation, malabsorption syndrome, tropi reperfusion, and congestive heart failure. For example, cal sprue, chronic pancreatitis, Crohn's disease, diarrhea, GLP-1 has been found to reduce cardiac injury and enhance and irritable bowel syndrome. It has also been discovered recovery in patients with these disorders. Incretins, includ that the peptides of the invention can be used to treat an ing GLP-1, are glucose-dependent insulinotropic hormones. emergency or life-threatening situation involving a gas GLP-1 and exendin effectively enhance peripheral glucose uptake without inducing dangerous hypoglycemia. They trointestinal disorder, e.g., after Surgery or due to cholera. also strongly suppress glucagon Secretion, independent of its 0230 Compounds of the invention may also be useful for insulinotropic action, and thereby powerfully reduce plasma treating or preventing intestinal damage as opposed to free fatty acid (FFA) levels substantially more than can be merely treating the symptoms associated with the intestinal accomplished with insulin. High FFA levels have been damage (for example, diarrhea). Such damage to the intes implicated as a major toxic mechanism during myocardial tine may be, or a result of ulcerative colitis, inflammatory US 2006/0293.232 A1 Dec. 28, 2006 34 bowel disease, bowel atrophy, loss bowel mucosa, and/or Cambridge, Mass.) in a total volume of 200 ul of culture loss of bowel mucosal function (see WO 03/105763, incor media per well. Cells were allowed to adhere for 24 hours porated herein by reference in its entirety). Assays for such prior to addition of the PYY or test peptide. Fresh culture activity, as described in WO 03/105763, include 11 week old media was exchanged prior to addition of peptides. In vitro male HSD rats, ranging 250-300 grams housed in a 12:12 incubation of pancreatic tumor cells with either PYY or test light:dark cycle, and allowed ad libitum access to a standard compound was continued for 6 hours and 36 hours in length. rodent diet (Teklad LM 485, Madison, Wis.) and water. The PYY was added to cells at doses of 250 pmol, 25 pmol, and animals were fasted for 24 hours before the experiment. A 2.5 pmol per well (N=14). Test compound was added to cells simple and reproducible rat model of chronic colonic cultures at doses of 400 pmol, 40 pmol, and 4 pmol per well. inflammation has been previously described by Morris G. P. et al., “Hapten-induced model of chronic inflammation and Control wells received 2 ul of 0.9% saline to mimic the ulceration in the rat colon.” Gastroenterology. 1989; 96:795 Volume and physical disturbance upon adhered tumor cells. 803. It exhibits a relatively long duration of inflammation Each 96 well plate contained 18 control wells to allow for and ulceration, affording an opportunity to study the patho comparison within each plate during experimentation. physiology of colonic inflammatory disease in a specifically Ninety-six (96) well plates were repeated 6 times with controlled fashion, and to evaluate new treatments poten varying concentrations of PYY and test compound in both tially applicable to inflammatory bowel disease in humans. the PANC-1 and MiaPaca-2 cells. 0231. Rats were anesthetized with 3% isofluorane and 0235. At the end of the incubation period, 3-(4,5-dimeth placed on a regulated heating pad set at 37° C. A gavage ylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide, MTr tet needle was inserted rectally into the colon 7 cm. The hapten razolium bromide (Sigma, St. Louis, Mo.) was added to trinitrobenzenesulfonic acid (TNBS) dissolved in 50% etha fresh culture media at 0.5 mg/ml. Culture media was nol (v/v) was delivered into the lumen of the colon through exchanged and tumor cells were incubated for 4 hours with the gavage needle at a dose of 30 mg/kg, in a total Volume MTT tetrazolium bromide at 37°C. At the end of incubation, of 0 0.4–0.6 mL, as described in Mazelin, et al., Julton Nery culture media was aspirated. Formazon crystal precipitates Syst. 1998:73:38.45. Control groups received saline solution were dissolved in 200 ul of dimethyl sulfoxide (Sigma, St. (NaCl 0.9%) intracolonically. Louis, Mo.). Quantitation of solubilized formazon was per formed by obtaining absorption readings at 500 nm wave 0232 Four days after induction of colitis, the colon was length on an ELISA reader (Molecular Devices, Menlo Park, resected from anesthetized rats, which were then euthanized Calif.). The MTT assay measures mitochondrial NADH by decapitation. Weights of excised colon and spleen were dependent dehydrogenase activity, and it has been among measured, and the colons photographed for scoring of gross the most sensitive and reliable method to quantitative in morphologic damage. Inflammation was defined as regions vitro chemotherapy responses of tumor cells. (Alley, M. C., of hyperemia and bowel wall thickening. et al., Cancer Res., 48:589-601, 1988; Carmichael, J., et al., 0233 Hybrid polypeptides of the invention may also be Cancer Res., 47:936-942, 1987; McHale, A. P., et al., used to treat or prevent pancreatic tumors (e.g., inhibit the Cancer Lett., 41:315-321, 1988; and Saxton, R. E., et al., J. proliferation of pancreatic tumors). Methods of the inven Clin. Laser Med. and Surg., 10(5):331-336, 1992.) Analysis tion include reducing the proliferation of tumor cells. The of absorption readings at 550 nm were analyzed by grouping types of benign pancreatic tumor cells which may be treated wells of the same test conditions and verifying differences in accordance with the present invention include serous cyst occurring between control and the various peptide concen adenomas, microcystic tumors, and solid-cystic tumors. The tration treatments by one-way ANOVA. method is also effective in reducing the proliferation of malignant pancreatic tumor cells Such as carcinomas arising 0236 An exemplary in vivo assay is also provided. The from the ducts, acini, or islets of the pancreas. U.S. Pat. No. human pancreatic ductal adenocarcinoma Mia Paca-2 was 5,574,010 (incorporated by reference in its entirety) pro examined for in vivo growth inhibition by peptide YY and vides exemplary assays for testing anti-proliferative prop test compound. Seventy thousand to 100,000 human Mia erties. For example, the 010 patent provides that PANC-1 PaCa-2 cells were orthotopically transplanted into 48 male and MiaPaca-2 are two human pancreatic adenocarcinoma athymic mice. After one week, the animals were treated with cancer cell lines which are available commercially from either PYY or test compound at 200 pmol/kg/hr via mini suppliers such as American Type Culture Collection, ATCC osmotic pumps for four weeks. The paired cultures received (Rockville, Md.). The two tumor cells were grown in saline. At sacrifice, both tumor size and mass were mea RPMI-1640 culture media supplemented with 10% fetal Sured. Control mice had significant human cancer growth bovine serum, 29.2 mg/L of glutamine, 25 Jug gentamicin, 5 within the pancreas as evidenced by histologic sections. At ml penicillin, Streptomycin, and fungizone solution (JRH 9 weeks, ninety percent (90%) of control mice had substan Biosciences, Lenexa, Kans.) at 37 degrees Celcius in a tial metastatic disease. Tumor mass was decreased by 60.5% NAPCO water jacketed 5% CO, incubator. All cell lines in test treated mice and 27% in PYY treated mice. were detached with 0.25% trypsin (Clonetics, San Diego, 0237 Hybrids are also useful for the therapeutic and Calif.) once to twice a week when a confluent monolayer of prophylactic treatment of neurological and nervous system tumor cells was achieved. Cells were pelleted for 7 minutes disorders associated with neuronal loss or dysfunction, at 500 g in a refrigerated centrifuge at 4 degrees Celcius, and including, but not limited to, Parkinson's Disease, Alzhe resuspended in trypsin free fortified RPMI 1640 culture imer's Disease, Huntington's Disease, ALS, stroke, ADD, media. Viable cells were counted on a hemocytometer slide and neuropsychiatric syndromes, and to enhance or facilitate with trypan blue. learning, memory and cognition in mammals. Particularly 0234 Ten thousand, 20,000, 40,000 and 80,000 cells of useful in this regard are hybrids containing an exendin or each type were added to 96 well microculture plates (Costar, GLP1 active portion, more specifically comprising at least US 2006/0293.232 A1 Dec. 28, 2006

the N-terminal 7-15 amino acids or analog thereof, for 0242. The hybrid polypeptides of the present invention example HSEGTTTSD (SEQ ID NO. ). may alternatively be produced by recombinant techniques well known in the art. See, e.g., Sambrook et al., Molecular 0238 For all indications, in preferred embodiments, the Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor hybrid polypeptide of the invention is administered periph (1989). These hybrid polypeptides produced by recombinant erally at a dose of about 0.5 ug to about 5 mg per day in technologies may be expressed from a polynucleotide. One single or divided doses or controlled continual release, or at skilled in the art will appreciate that the polynucleotides, about 0.01 g/kg to about 500 g/kg per dose, more pref including DNA and RNA, that encode such the various erably about 0.05ug/kg to about 250 ug/kg, most preferably fragments of the hybrid polypeptides may be obtained from below about 50 g/kg. Dosages in these ranges will vary the wild-type cDNA, taking into consideration the degen with the potency of each analog or derivative, of course, and eracy of codon usage, or may be engineered as desired. may be determined by one of skill in the art. These polynucleotide sequences may incorporate codons 0239). In the methods of the present invention, hybrid facilitating transcription and translation of mRNA in micro polypeptides of the invention may be administered sepa bial hosts. Such manufacturing sequences may readily be rately or together with one or more other compounds and constructed according to the methods well known in the art. compositions that exhibit a long term or short-term action to See, e.g., WO 83/04053. The polynucleotides above may reduce nutrient availability, including, but not limited to also optionally encode an N-terminal methionyl residue. other compounds and compositions that comprise an amylin Non-peptide compounds useful in the present invention may or amylin analog agonist, salmon calcitonin, a cholecysto be prepared by art-known methods. For example, phosphate (CCK) or CCK agonist, a leptin (OB protein) or leptin containing amino acids and peptides containing Such amino agonist, an exendin or exendin analog agonist, or a GLP-1 acids may be prepared using methods known in the art. See, or GLP-1 analog agonist. Suitable amylin agonists include, e.g., Bartlett and Landen, Bioorg. Chem. 14: 356-77 (1986). for example, Pro-human amylin (also known as 0243 A variety of expression vector/host systems may be “pramlintide,” and described in U.S. Pat. Nos. 5,686,511 and utilized to contain and express a hybrid polypeptide coding 5.998.367). The CCK used is preferably CCK octapeptide sequence. These include but are not limited to microorgan (CCK-8), more preferably its sulfated form. Leptin is dis isms such as bacteria transformed with recombinant bacte cussed in, for example, (Pelleymounter et al., Science 269: riophage, plasmid or cosmid DNA expression vectors; yeast 540-3 (1995); Halaas et al., Science 269: 543-6 (1995); transformed with yeast expression vectors; insect cell sys Campfield et al., Science 269: 546-9 (1995)). Suitable tems infected with virus expression vectors (e.g., baculovi exendins include exendin-3 and exendin-4, and exendin rus); plant cell systems transfected with virus expression agonist compounds include, for example, those described in vectors (e.g., cauliflower mosaic virus, CaMV; tobacco PCT Publications WO 99/07404, WO 99/25727, and WO mosaic virus, TMV) or transformed with bacterial expres 99/25728. sion vectors (e.g., Ti or pBR322 plasmid); or animal cell 11. Polypeptide Production and Purification systems. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO 0240 The hybrid polypeptides described herein may be cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, prepared using standard recombinant techniques or chemical COS cells (such as COS-7), WI38, BHK, HepG2,3T3, RIN, peptide synthesis techniques known in the art, e.g., using an MDCK, A549, PC12, K562 and 293 cells. Exemplary pro automated or semi-automated peptide synthesizer, or both. tocols for the recombinant expression of the protein are 0241 The hybrid polypeptides of the invention can be described herein. synthesized in Solution or on a solid Support in accordance 0244 As such, polynucleotide sequences provided by the with conventional techniques. Various automatic synthesiz invention are useful in generating new and useful viral and ers are commercially available and can be used in accor plasmid DNA vectors, new and useful transformed and dance with known protocols. See, e.g., Stewart and Young, transfected procaryotic and eucaryotic host cells (including Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. bacterial, yeast, and mammalian cells grown in culture), and (1984); Tam et al., J. Am. Chem. Soc. 105: 6442 (1983); new and useful methods for cultured growth of such host Merrifield, Science 232: 341-7 (1986); and Barany and cells capable of expression of the present hybrid polypep Merrifield, The Peptides, Gross and Meienhofer, eds., Aca tides. The polynucleotide sequences encoding hybrid demic Press, New York, 1-284 (1979). Solid phase peptide polypeptides herein may be useful for gene therapy in synthesis may be carried out with an automatic peptide instances where underproduction of the component peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc., hormone(s) of the chimera would be alleviated, or the need Foster City, Calif.) using the NMP/HOBt (Option 1) system for increased levels of such would be met. and thBoc or Fmoc chemistry (see, Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Ver 0245. The present invention also provides for processes sion 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Bio for recombinant DNA production of the present hybrid systems, Inc., Foster City, Calif.) with capping. Peptides polypeptides. Provided is a process for producing the hybrid may also be assembled using an Advanced Chem Tech polypeptides from a host cell containing nucleic acids Synthesizer (Model MPS 350, Louisville, Ky.). Peptides encoding such hybrid polypeptides comprising: (a) culturing may be purified by RP-HPLC (preparative and analytical) said host cell containing polynucleotides encoding Such using, e.g., a Waters Delta Prep 3000 system and a C4, C8, hybrid polypeptides under conditions facilitating the expres or C18 preparative column (10 L, 2.2x25 cm; Vydac, Hes sion of such DNA molecule; and (b) obtaining such hybrid peria, Calif.). The active peptide can be readily synthesized polypeptides. and then screened in Screening assays designed to identify 0246 Host cells may be prokaryotic or eukaryotic and reactive peptides. include bacteria, mammalian cells (such as Chinese Hamster US 2006/0293.232 A1 Dec. 28, 2006 36

Ovary (CHO) cells, monkey cells, baby hamster kidney tide is purified from the yeast growth medium by, e.g., the cells, cancer cells or other cells), yeast cells, and insect cells. methods used to purify hybrid polypeptide from bacterial and mammalian cell Supernatants. 0247 Mammalian host systems for the expression of the recombinant protein also are well known to those of skill in 0251 Alternatively, the cDNA encoding hybrid polypep the art. Host cell strains may be chosen for a particular tides may be cloned into the baculovirus expression vector ability to process the expressed protein or produce certain pVL1393 (PharMingen, San Diego, Calif.). This hybrid post-translation modifications that will be useful in provid polypeptide-containing vector is then used according to the ing protein activity. Such modifications of the polypeptide manufacturer's directions (PharMingen) to infect include, but are not limited to, acetylation, carboxylation, Spodoptera frugiperda cells in sE9 protein-free media and to glycosylation, phosphorylation, lipidation and acylation. produce recombinant protein. The protein is purified and Post-translational processing, which cleaves a “prepro form concentrated from the media using a heparin-Sepharose of the protein, may also be important for correct insertion, column (Pharmacia, Piscataway, N.J.) and sequential folding and/or function. Different host cells, such as CHO, molecular sizing columns (Amicon, Beverly, Mass.), and HeLa, MDCK, 293, WI38, and the like, have specific resuspended in PBS. SDS-PAGE analysis shows a single cellular machinery and characteristic mechanisms for Such band and confirms the size of the protein, and Edman post-translational activities, and may be chosen to ensure the sequencing on a Proton 2090 Peptide Sequencer confirms its correct modification and processing of the introduced for N-terminal sequence. eign protein. 0252 For example, the DNA sequence encoding the 0248 Alternatively, a yeast system may be employed to hybrid polypeptide may be cloned into a plasmid containing generate the hybrid polypeptides of the present invention. a desired promoter and, optionally, a leader sequence (see, The coding region of the hybrid polypeptide cDNA is e.g., Better et al., Science 240: 1041-3 (1988)). The amplified by PCR. A DNA encoding the yeast pre-pro-alpha sequence of this construct may be confirmed by automated leader sequence is amplified from yeast genomic DNA in a sequencing. The plasmid is then transformed into E. coli, PCR reaction using one primer containing nucleotides 1-20 strain MC1061, using standard procedures employing CaCl2 of the alpha mating factor gene and another primer comple incubation and heat shock treatment of the bacteria (Sam mentary to nucleotides 255-235 of this gene (Kurjan and brook et al., Supra). The transformed bacteria are grown in Herskowitz, Cell, 30: 933-43 (1982)). The pre-pro-alpha LB medium Supplemented with carbenicillin, and produc leader coding sequence and hybrid polypeptide coding tion of the expressed protein is induced by growth in a sequence fragments are ligated into a plasmid containing the suitable medium. If present, the leader sequence will affect yeast alcohol dehydrogenase (ADH2) promoter, Such that secretion of the hybrid polypeptide and be cleaved during the promoter directs expression of a fusion protein consist secretion. The secreted recombinant protein is purified from ing of the pre-pro-alpha factor fused to the mature hybrid the bacterial culture media by the method described herein. polypeptide. As taught by Rose and Broach, Meth. Enz. 185: 234-79. Goeddel ed., Academic Press, Inc., San Diego, 0253 Alternatively, the hybrid polypeptides of the inven Calif. (1990), the vector further includes an ADH2 tran tion may be expressed in an insect system. Insect systems for Scription terminator downstream of the cloning site, the protein expression are well known to those of skill in the art. yeast “2-micron' replication origin, the yeast leu-2d gene, In one Such system, Autographa californica nuclear polyhe the yeast REP1 and REP2 genes, the E. coli B-lactamase drosis virus (AcNPV) is used as a vector to express foreign gene, and an E. coli origin of replication. The B-lactamase genes in Spodoptera frugiperda cells or in Trichoplusia and leu-2d genes provide for selection in bacteria and yeast, larvae. The hybrid polypeptide coding sequence is cloned respectively. The leu-2d gene also facilitates increased copy into a nonessential region of the virus, Such as the polyhe number of the plasmid in yeast to induce higher levels of drin gene, and placed under control of the polyhedrin expression. The REP1 and REP2 genes encode proteins promoter. Successful insertion of hybrid polypeptide will involved in regulation of the plasmid copy number. render the polyhedrin gene inactive and produce recombi nant virus lacking coat protein coat. The recombinant 0249. The DNA construct described in the preceding viruses are then used to infect S. frugiperda cells or Tri paragraph is transformed into yeast cells using a known choplusia larvae in which hybrid polypeptide is expressed method, e.g., lithium acetate treatment (Steams et al., Meth. (Smith et al., J. Virol. 46:584 (1983); Engelhard et al., Proc. Enz. 185: 280-97 (1990)). The ADH2 promoter is induced Natl. Acad. Sci. USA 91: 3224-7 (1994)). upon exhaustion of glucose in the growth media (Price et al., Gene 55: 287 (1987)). The pre-pro-alpha sequence effects 0254. In another example, the DNA sequence encoding secretion of the fusion protein from the cells. Concomitantly, the hybrid polypeptide may be amplified by PCR and cloned the yeast KEX2 protein cleaves the pre-pro sequence from into an appropriate vector, for example, pGEX-3X (Phar macia, Piscataway, N.J.). The pGEX vector is designed to the mature PYY analog polypeptides (Bitter et al., Proc. produce a fusion protein comprising glutathione-S-trans Natl. Acad. Sci. USA 81: 5330-4 (1984)). ferase (GST), encoded by the vector, and a protein encoded 0250 Hybrid polypeptides of the invention may also be by a DNA fragment inserted into the vector's cloning site. recombinantly expressed in yeast using a commercially The primers for the PCR may be generated to include, for available expression system, e.g., the Pichia Expression example, an appropriate cleavage site. The recombinant System (Invitrogen, San Diego, Calif.), following the manu fusion protein may then be cleaved from the GST portion of facturers instructions. This system also relies on the pre the fusion protein. The pGEX-3X/PYY analog polypeptide pro-alpha sequence to direct secretion, but transcription of construct is transformed into E. coli XL-1 Blue cells (Strat the insert is driven by the alcohol oxidase (AOX1) promoter agene, La Jolla, Calif.), and individual transformants are upon induction by methanol. The secreted hybrid polypep isolated and grown at 37° C. in LB medium (supplemented US 2006/0293.232 A1 Dec. 28, 2006 37 with carbenicillin) to an optical density at wavelength 600 is designed to confer resistance to selection, and its presence nm of 0.4, followed by further incubation for 4 hours in the allows growth and recovery of cells that successfully presence of 0.5 mM Isopropyl B-D-Thiogalactopyranoside express the introduced sequences. Resistant clumps of stably (Sigma Chemical Co., St. Louis, Mo.). Plasmid DNA from transformed cells can be proliferated using tissue culture individual transformants is purified and partially sequenced techniques appropriate to the cell. using an automated sequencer to confirm the presence of the 0259. A number of selection systems may be used to desired PPF hybrid polypeptide-encoding gene insert in the recover the cells that have been transformed for recombinant proper orientation. protein production. Such selection systems include, but are 0255 The fusion protein, expected to be produced as an not limited to, HSV thymidine kinase, hypoxanthine-gua insoluble inclusion body in the bacteria, may be purified as nine phosphoribosyltransferase and adenine phosphoribo follows. Cells are harvested by centrifugation; washed in Syltransferase genes, in tk-, hgprt- or aprt-cells, respectively. 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated Also, anti-metabolite resistance can be used as the basis of with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 selection for dhfr, that confers resistance to methotrexate; min. at room temperature. The lysate is cleared by Sonica gpt, that confers resistance to mycophenolic acid; neo, that tion, and cell debris is pelleted by centrifugation for 10 min. confers resistance to the aminoglycoside, G418; also, that at 12,000xg. The fusion protein-containing pellet is resus confers resistance to chlorSulfuron; and hygro, that confers pended in 50 mM Tris, pH 8, and 10 mM EDTA, layered resistance to hygromycin. Additional selectable genes that over 50% glycerol, and centrifuged for 30 min. at 6000xg. may be useful include trpE, which allows cells to utilize The pellet is resuspended in standard phosphate buffered indole in place of tryptophan, or hisD, which allows cells to saline solution (PBS) free of Mg" and Ca". The fusion utilize histinol in place of histidine. Markers that give a protein is further purified by fractionating the resuspended visual indication for identification of transformants include pellet in a denaturing SDS polyacrylamide gel (Sambrook et anthocyanins, B-glucuronidase and its substrate, GUS, and al., supra). The gel is soaked in 0.4 M KCl to visualize the luciferase and its substrate, luciferin. protein, which is excised and electroeluted in gel-running 0260 Many of the hybrid polypeptides of the present buffer lacking SDS. If the GST/PYY analog polypeptide invention may be produced using a combination of both fusion protein is produced in bacteria as a soluble protein, it automated peptide synthesis and recombinant techniques. may be purified using the GST Purification Module (Phar For example, a hybrid polypeptide of the present invention macia Biotech). may contain a combination of modifications including dele 0256 The fusion protein may be subjected to digestion to tion, substitution, and insertion by PEGylation. Such a cleave the GST from the PPF hybrid polypeptide. The hybrid polypeptide may be produced in stages. In the first digestion reaction (20-40 ug fusion protein, 20-30 units stage, an intermediate polypeptide containing the modifica human thrombin (4000 U/mg (Sigma) in 0.5 mL PBS) is tions of deletion, Substitution, insertion, and any combina incubated 16-48 hrs. at room temperature and loaded on a tion thereof, may be produced by recombinant techniques as denaturing SDS-PAGE gel to fractionate the reaction prod described. Then after an optional purification step as ucts. The gel is soaked in 0.4M KCl to visualize the protein described herein, the intermediate polypeptide is PEGylated bands. The identity of the protein band corresponding to the through chemical modification with an appropriate PEGy expected molecular weight of the hybrid polypeptide may be lating reagent (e.g., from Nektar Therapeutics, San Carlos, confirmed by partial amino acid sequence analysis using an Calif.) to yield the desired hybrid polypeptide. One skilled automated sequencer (Applied Biosystems Model 473A, in the art will appreciate that the above-described procedure Foster City, Calif.). may be generalized to apply to a hybrid polypeptide con taining a combination of modifications selected from dele 0257. In a particularly preferred method of recombinant tion, Substitution, insertion, derivation, and other means of expression of the hybrid polypeptides of the present inven modification well known in the art and contemplated by the tion, 293 cells may be co-transfected with plasmids con present invention. taining the hybrid polypeptide cDNA in the pCMV vector (5' CMV promoter, 3' HGH poly A sequence) and pSV2neo 0261. It may be desirable to purify the hybrid polypep (containing the neo resistance gene) by the calcium phos tides generated by the present invention. Peptide purification phate method. Preferably, the vectors should be linearized techniques are well known to those of skill in the art. These with Scal prior to transfection. Similarly, an alternative techniques involve, at one level, the crude fractionation of construct using a similar pCMV vector with the neogene the cellular milieu to polypeptide and non-polypeptide frac incorporated can be used. Stable cell lines are selected from tions. Having separated the polypeptide from other proteins, single cell clones by limiting dilution in growth media the polypeptide of interest may be further purified using containing 0.5 mg/mL G418 (neomycin-like antibiotic) for chromatographic and electrophoretic techniques to achieve 10-14 days. Cell lines are screened for hybrid polypeptide partial or complete purification (or purification to homoge expression by ELISA or Western blot, and high-expressing neity). Analytical methods particularly Suited to the prepa cell lines are expanded for large scale growth. ration of a pure peptide are ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electro 0258. It is preferable that the transformed cells are used phoresis, and isoelectric focusing. A particularly efficient for long-term, high-yield protein production and as Such method of purifying peptides is reverse phase HPLC, fol stable expression is desirable. Once Such cells are trans lowed by characterization of purified product by liquid formed with vectors that contain selectable markers along chromatography/mass spectrometry (LC/MS) and Matrix with the desired expression cassette, the cells may be Assisted Laser Desorption Ionization (MALDI) mass spec allowed to grow for 1-2 days in an enriched media before trometry. Additional confirmation of purity is obtained by they are switched to selective media. The selectable marker determining amino acid analysis. US 2006/0293.232 A1 Dec. 28, 2006

0262 Certain aspects of the present invention concern the 12. Pharmaceutical Compositions purification, and in particular embodiments, the Substantial 0268. The present invention also relates to pharmaceuti purification, of an encoded protein or peptide. The term cal compositions comprising a therapeutically or prophylac “purified peptide' as used herein, is intended to refer to a tically effective amount of at least one hybrid polypeptide of composition, isolatable from other components, wherein the the invention, or a pharmaceutically acceptable salt thereof, peptide is purified to any degree relative to its naturally together with pharmaceutically acceptable diluents, preser obtainable state. A purified peptide therefore also refers to a Vatives, Solubilizers, emulsifiers, adjuvants and/or carriers peptide, free from the environment in which it may naturally useful in the delivery of the hybrid polypeptides. Such OCCU. compositions may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; 0263 Generally, “purified will refer to a peptide com additives such as detergents and solubilizing agents (e.g., position that has been subjected to fractionation to remove Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic various other components, and which composition Substan acid, sodium metabisulfite), preservatives (e.g., thimersol, tially retains its expressed biological activity. Where the benzyl alcohol), and bulking Substances (e.g., lactose, man term “substantially purified” is used, this designation will nitol); incorporation of the material into particulate prepa refer to a composition in which the peptide forms the major rations of polymeric compounds, Such as polylactic acid, component of the composition, such as constituting about polyglycolic acid, etc., or in association with liposomes. 50%, about 60%, about 70%, about 80%, about 90%, about Such compositions will influence the physical state, stability, 95% or more of the peptides in the composition. rate of in vivo release, and rate of in vivo clearance of the present hybrid polypeptides. See, e.g., Remington’s Phar 0264 Various techniques suitable for use in peptide puri maceutical Sciences 1435-712, 18th ed., Mack Publishing fication will be well known to those of skill in the art. These Co., Easton, Pa. (1990). include, for example, precipitation with ammonium Sul phate, PEG, antibodies, and the like; heat denaturation, 0269. In general, the present hybrid polypeptides will be followed by centrifugation; chromatography steps such as useful in the same way that the individual component ion exchange, gel filtration, reverse phase, hydroxylapatite polypeptides are useful in view of their pharmacological and affinity chromatography; isoelectric focusing; gel elec properties. One preferred use is to peripherally administer trophoresis; and combinations of Such and other techniques. such hybrid polypeptides for the treatment or prevention of As is generally known in the art, it is believed that the order metabolic conditions and disorders. In particular, the com of conducting the various purification steps may be changed, pounds of the invention possess activity as agents to reduce or that certain steps may be omitted, and still result in a nutrient availability, reduce food intake, Suppress appetite, suitable method for the preparation of a substantially puri and effect weight loss. In another embodiment, a preferred fied protein or peptide. use is to administer such hybrid polypeptides for the treat ment of diabetes or diabetes related conditions and disor 0265. There is no general requirement that the peptides ders. always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will 0270. The present hybrid polypeptides may be formu have utility in certain embodiments. Partial purification may lated for peripheral administration, including formulation be accomplished by using fewer purification steps in com for injection, oral administration, nasal administration, pull bination, or by utilizing different forms of the same general monary administration, topical administration, or other purification scheme. For example, it is appreciated that a types of administration as one skilled in the art will recog cation-exchange column chromatography performed, utiliz nize. More particularly, administration of the pharmaceuti ing an HPLC apparatus, will generally result in a greater cal compositions according to the present invention may be “-fold' purification than the same technique utilizing a low via any common route so long as the target tissue is available pressure chromatography system. Methods exhibiting a via that route. In a preferred embodiment, the pharmaceu lower degree of relative purification may have advantages in tical compositions may be introduced into the Subject by any total recovery of protein product, or in maintaining the conventional peripheral method, e.g., by intravenous, intra activity of an expressed protein. dermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., term release); 0266 One may optionally purify and isolate such hybrid by oral, Sublingual, nasal, anal, vaginal, or transdermal polypeptides from other components obtained in the pro delivery, or by Surgical implantation at a particular site. The cess. Methods for purifying a polypeptide can be found in treatment may consist of a single dose or a plurality of doses U.S. Pat. No. 5,849,883. These documents describe specific over a period of time. Controlled continual release of the exemplary methods for the isolation and purification of compositions of the present invention is also contemplated. G-CSF compositions that may be useful in isolating and purifying the hybrid polypeptides of the present invention. 0271 The formulation may be liquid or may be solid, Given the disclosure of these patents, it is evident that one Such as lyophilized, for reconstitution. Aqueous composi of skill in the art would be well aware of numerous purifi tions of the present invention comprise an effective amount cation techniques that may be used to purify hybrid polypep of the hybrid polypeptide, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. tides from a given source. The phrase “pharmaceutically or pharmacologically accept 0267 Also it is contemplated that a combination of anion able” refer to molecular entities and compositions that do exchange and immunoaffinity chromatography may be not produce adverse, allergic, or other untoward reactions employed to produce purified hybrid polypeptide composi when administered to an animal or a human. As used herein, tions of the present invention. “pharmaceutically acceptable carrier includes any and all US 2006/0293.232 A1 Dec. 28, 2006 39

Solvents, dispersion media, coatings, antibacterial and anti example, by the use of a coating, such as lecithin, by the fungal agents, isotonic and absorption delaying agents and maintenance of the required particle size in the case of the like. The use of Such media and agents for pharmaceu dispersion and by the use of surfactants. The prevention of tically active substances is well known in the art. Except the action of microorganisms can be brought about by insofar as any conventional media or agent is incompatible various antibacterial an antifungal agents, for example, with the active ingredient, its use in therapeutic composi parabens, chlorobutanol, phenol, Sorbic acid, thimerosal, tions is contemplated. Supplementary active ingredients also and the like. In many cases, it will be preferable to include can be incorporated into the compositions. In some cases, it isotonic agents (for example, Sugars or Sodium chloride). will be convenient to provide a hybrid polypeptide of the Prolonged absorption of the injectable compositions can be invention and another food-intake-reducing, diabetes treat brought about by the use in the compositions of agents ing, plasma glucose-lowering, or plasma lipid-altering delaying absorption (for example, aluminum monostearate agent, such as an amylin, an amylin agonist analog, a CCK and gelatin). or CCK agonist, or a leptin or leptin agonist, or an exendin or exendin agonist analog, in a single composition or solu 0275 Sterile injectable solutions may be prepared by tion for administration together. In other cases, it may be incorporating the active compounds in the required amount more advantageous to administer the additional agent sepa in the appropriate solvent with various of the other ingre rately from said hybrid polypeptide. dients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incor 0272. The hybrid polypeptide of the invention may be porating the various sterilized active ingredients into a prepared for administration as solutions of free base, or sterile vehicle that contains the basic dispersion medium and pharmacologically acceptable salts in water Suitably mixed the required other ingredients from those enumerated above. with a Surfactant, Such as hydroxypropylcellulose. Pharma In the case of sterile powders for the preparation of sterile ceutically-acceptable salts include the acid addition salts injectable solutions, the preferred methods of preparation (formed with the free amino groups of the protein) and are vacuum-drying and freeze-drying techniques that yield a which are formed with inorganic acids such as, for example, powder of the active ingredient plus any additional desired hydrochloric or phosphoric acids, or Such organic acids as ingredient from a previously sterile-filtered solution thereof. acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from 0276 Generally, a therapeutically or prophylactically inorganic bases such as, for example, sodium, potassium, effective amount of the present hybrid polypeptides will be ammonium, calcium, or ferric hydroxides, and Such organic determined by the age, weight, and condition or severity of bases as isopropylamine, trimethylamine, histidine, procaine the diseases, conditions or disorders of the recipient. See, and the like. Such products are readily prepared by proce e.g., Remington’s Pharmaceutical Sciences 697-773. See dures well known to those skilled in the art. Dispersions also also Wang and Hanson, Parenteral Formulations of Proteins can be prepared in glycerol, liquid polyethylene glycols, and and Peptides: Stability and Stabilizers, Journal of Parenteral mixtures thereof and in oils. Under ordinary conditions of Science and Technology, Technical Report No. 10, Supp. storage and use, these preparations contain a preservative to 42:2S (1988). Typically, a dosage of between about 0.001 prevent the growth of microorganisms. ug/kg body weight/day to about 1000 g/kg body weight/ day, may be used, but more or less, as a skilled practitioner 0273. In one embodiment, the pharmaceutical composi will recognize, may be used. Dosing may be one or more tions of the present invention are formulated so as to be times daily, or less frequently, and may be in conjunction Suitable for parenteral administration, e.g., via injection or with other compositions as described herein. It should be infusion. Preferably, the hybrid polypeptide is suspended in noted that the present invention is not limited to the dosages an aqueous carrier, for example, in an isotonic buffer Solu recited herein. tion at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful 0277 Appropriate dosages may be ascertained through buffers include Sodium citrate-citric acid and sodium phos the use of established assays for determining level of meta phate-phosphoric acid, and Sodium acetate/acetic acid buff bolic conditions or disorders in conjunction with relevant ers. A form of repository or “depot' slow release preparation dose-response data. The final dosage regimen will be deter may be used so that therapeutically effective amounts of the mined by the attending physician, considering factors that preparation are delivered into the bloodstream over many modify the action of drugs, e.g., the drugs specific activity, hours or days following transdermal injection or delivery. severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, 0274 The pharmaceutical compositions suitable for the severity of any infection, time of administration and injectable use include sterile aqueous Solutions or disper other clinical factors. As studies are conducted, further sions and sterile powders for the extemporaneous prepara information will emerge regarding appropriate dosage levels tion of sterile injectable solutions or dispersions. In all cases, and duration of treatment for specific diseases and condi the form should be sterile and should be fluid to the extent tions. that easy syringability exists. It is also desirable for the hybrid polypeptide of the invention to be stable under the 0278 An effective dose will typically be in the range of conditions of manufacture and storage and must be pre about 1 to 30 ug to about 5 mg/day, preferably about 10 to served against the contaminating action of microorganisms, 30 ug to about 2 mg/day and more preferably about 5 to 100 Such as bacteria and fungi. The carrier can be a solvent or ug to about 1 mg/day, most preferably about 5 ug to about dispersion medium containing, for example, water, ethanol, 500 g/day, for a 50 kg patient, administered in a single or polyol (for example, glycerol, propylene glycol, and liquid divided doses. Preferably, dosages are between about 0.01 to polyethylene glycol, and the like), Suitable mixtures thereof, about 100 lug/kg/dose. The exact dose to be administered and vegetable oils. The proper fluidity can be maintained, for may be determined by one of skill in the art and is dependent US 2006/0293.232 A1 Dec. 28, 2006 40 upon the potency of the particular compound, as well as EXAMPLES upon the age, weight and condition of the individual. Administration should begin whenever, e.g., Suppression of 0284. The present invention is described in more detail nutrient availability, food intake, weight, blood glucose or with reference to the following non-limiting examples, plasma lipid modulation is desired, for example, at the first which are offered to more fully illustrate the invention, but sign of symptoms or shortly after diagnosis of obesity, are not to be construed as limiting the scope thereof. The diabetes mellitus, or insulin-resistance syndrome. Adminis examples illustrate the preparation of the present hybrid tration may be by any route, e.g., injection, preferably polypeptides, and the testing of these hybrid polypeptides of Subcutaneous or intramuscular, oral, nasal, transdermal, etc. the invention in vitro and/or in vivo. Those of skill in the art Dosages for certain routes, for example oral administration, will understand that the techniques described in these may be increased to account for decreased bioavailablity, for examples represent techniques described by the inventors to example, by about 5-100 fold. function well in the practice of the invention, and as Such constitute preferred modes for the practice thereof. How 0279 Parenteral administration may be carried out with ever, it should be appreciated that those of skill in the art an initial bolus followed by continuous infusion to maintain should in light of the present disclosure, appreciate that therapeutic circulating levels of drug product. Those of many changes can be made in the specific methods that are ordinary skill in the art will readily optimize effective disclosed and still obtain a like or similar result without dosages and administration regimens as determined by good departing from the spirit and Scope of the invention. medical practice and the clinical condition of the individual patient. Example 1 0280 The frequency of dosing will depend on the phar Preparation of Hybrid Polypeptides macokinetic parameters of the agents and the routes of administration. The optimal pharmaceutical formulation will 0285 Peptides of the invention may be assembled on a be determined by one of skill in the art depending on the Symphony peptide synthesizer (Protein Technologies, Inc.) route of administration and the desired dosage. See, e.g., using Rink amide resin (Novabiochem) with a loading of Remington’s Pharmaceutical Sciences, Supra, pages 1435 0.43-0.49 mmol/g at 0.050-0.100 mmol or a pre-loaded 1712. Such formulations may influence the physical state, Wang Resin (Fmoc-Tyr?tBu)-Wang resin) 0.63 mmol/g stability, rate of in vivo release and rate of in vivo clearance (Novabiochem). Fmoc amino acid (5.0 eq, 0.250-0.500 of the administered agents. Depending on the route of mmol) residues are dissolved at a concentration of 0.10 M administration, a Suitable dose may be calculated according in 1-methyl-2-pyrrolidinone. All other reagents (HBTU, to body weight, body Surface areas or organ size. Further 1-Hydroxybenzotriazole hydrate and N,N-Diisopropylethy refinement of the calculations necessary to determine the lamine) are prepared as 0.55 M Dimethylformamide solu appropriate treatment dose is routinely made by those of tions. The Fmoc protected amino acids are then coupled to ordinary skill in the art without undue experimentation, the resin-bound amino acid using, HBTU (2.0 eq, 0.100 especially in light of the dosage information and assays 0.200 mmol), 1-Hydroxybenzotriazole hydrate (1.8 eq, disclosed herein, as well as the pharmacokinetic data 0.090-0.18 mmol), N,N-Diisopropylethylamine (2.4 eq, observed in animals or human clinical trials. 0.120-0.240 mmol) for 2 hours. Following the last amino acid coupling, the peptide is deprotected using 20% (v/v) 0281. It will be appreciated that the pharmaceutical com piperidine in dimethylformamide for 1 hour. Once peptide positions and treatment methods of the invention may be sequence is complete, the Symphony peptide synthesizer is useful in fields of human medicine and veterinary medicine. programmed to cleave the resin. Trifluoroacetic acid (TFA) Thus the subject to be treated may be a mammal, preferably cleavage of the peptide from resin is carried out using 93% human or other animal. For veterinary purposes, Subjects TFA, 3% phenol, 3% water and 1% triisopropylsilane for 1 include for example, farm animals including cows, sheep, hour. The cleaved peptide is precipitated using tert-butyl pigs, horses and goats, companion animals such as dogs and methyl ether, pelleted by centrifugation and lyophilized. The cats, exotic and/or Zoo animals, laboratory animals including pellet is re-dissolved in water (10-15 mL), filtered and mice, rats, rabbits, guinea pigs and hamsters; and poultry purified via reverse phase HPLC using a C18 column and an Such as chickens, turkeys, ducks and geese. acetonitrile/water gradient containing 0.1% TFA. 0282. In addition, the present invention contemplates a 0286 A general procedure for N-capping the peptides of kit comprising a hybrid polypeptide of the invention, com the invention with fatty acids (e.g., octanoic and Stearic ponents Suitable for preparing said hybrid polypeptide of the acids) is as follows: Peptide on rink amide resin (0.1 mmol) invention for pharmaceutical application, and instructions is suspended in NMP (5 mL). In a separate vial, HBTU (0.3 for using said hybrid polypeptide and components for phar mmol), HOBt (0.3 mmol) is dissolved in DMF (5 mL) maceutical application. followed by the addition of DIEA (0.6 mmol). This solution is added to the resin and this Suspension is shaken for 2 hrs. 0283 To assist in understanding the present invention, The solvent is filtered and washed thoroughly with NMP (5 the following examples are included. The experiments relat mLx4) and CHCl (20 mL), dried and is subjected to the ing to this invention should not, of course, be construed as TFA cleavage for 1 hr. The yield of the desired peptide is ca. specifically limiting the invention and Such variations of the 40 mg after cleavage and purification. invention, now known or later developed, which would be within the purview of one skilled in the art are considered to 0287 PEG modification may be carried out in solution on fall within the scope of the invention as described herein and a free epsilon-amino group of lysine or a terminal amino hereinafter claimed. group of a purified peptide using commercially available US 2006/0293.232 A1 Dec. 28, 2006 41 activated PEG esters. The resulting PEGylated derivatives modifications such as glycosylation, PEG modifications, are purified to homogeneity by reverse phase HPLC and the etc.; amino acid modifications such as Substitutions, inser purity is confirmed by LC/MS and MALDI-MS. tions and deletions, etc. Further, even though represented as 0288 Certain exemplary hybrid polypeptides of the C-terminally amidated, it is understood that the hybrid invention are shown in Table 1-1. Various modifications to polypeptides of the invention may alternatively be in the free the embodied compounds are envisioned. Such as chemical acid form.

US 2006/0293.232 A1 Dec. 28, 2006 47

Example 2 T47D cells, which also express the calcitonin receptor (Muff R. etal, Ann NY Acad Sci. 1992, 657:106-16 and Kuestner Binding Assays R. E. et. al. Mol Pharmacol. 1994, 46:246-55), as known in 0289. The hybrid polypeptides of the invention may be the art. tested in a variety of receptor binding assays using binding 0295) Leptin binding assay: Two in vitro bioassays are assay methodologies generally known to those skilled in the routinely used to assess leptin binding and receptor activa art. Such assays include those described herein. tion (see e.g., White, et al., 1997. Proc. Natl. Acad. Sci. 0290 Amylin binding assay: Evaluation of the binding of U.S.A. 94: 10657-10662). An alkaline phosphatase(“AP)- Some exemplary compounds of the invention to amylin leptin (“OER”) fusion protein (“AP-OB') may be used to receptors may be carried out as follows in nuclueus accum measure inhibition of leptin binding in the absence or bens membranes prepared from rat brain. Male Sprague presence of recombinant mouse leptin (positive control) or Dawleys rats (200-250) grams are sacrificed by decapitation. peptide, by COS-7 cells transfected with the long (signaling) Brains are removed and place in cold phosphate-buffered form of the mouse OB receptor (“OE-RL). Signal trans saline (PBS). From the ventral surface, cuts are made rostral duction assays may be done in GT1-7 cells cotransfected to the hypothalamus, bounded laterally by the olfactory with AP reporter and OB-RL constructs. Secreted alkaline tracts and extending at a 45° angle medially from these phosphatase(“SEAP) activity in response to stimulation tracts. This basal forebrain tissue, containing the nucleus with mouse leptin or peptide may be measured by chemi accumbens and Surrounding regions, is weighed and homog luminescence. enized in ice-cold 20 mM HEPES buffer (20 mM HEPES 0296 Y1 receptor binding assay: Membranes are pre acid, pH adjusted to 7.4 with NaOH at 23° C.). Membranes pared from confluent cultures of SK-N-MC cells that endog are washed three times in fresh buffer by centrifugation for enously expresses the neuropeptide Y 1 receptors. Mem 15 minutes at 48,000xg. The final membrane pellet is branes are incubated with 60 pMI-human Peptide YY resuspended in 20 mM HEPES buffer containing 0.2 mM (2200 Ci/mmol, PerkinElmer Life Sciences), and with unla phenylmethylsulfonyl fluoride (PMSF). beled active compound for 60 minutes at ambient tempera 0291) To measure 'I-amylin binding (see, Beaumont K ture in a 96 well polystyrene plate. Then well contents are et al. Can J. Physiol Pharmacol. July 1995; 73(7): 1025-9), harvested onto a 96 well glass fiber plate using a Perkin membranes from 4 mg original wet weight of tissue are Elmer plate harvester. Dried glass fiber plates are combined incubated with 'I-amylin at 12-16 pM in 20 mM HEPES with scintillant and counted on a Perkin Elmer scintillation buffer containing 0.5 mg/ml bacitracin, 0.5 mg/ml bovine COunter. serum albumin, and 0.2 mM PMSF. Solutions are incubated 0297 Y2 receptor binding assay: Membranes are pre for 60 minutes at 2° C. Incubations are terminated by pared from confluent cultures of SK-N-BE cells that endog filtration through GF/B glass fiber filters (Whatman Inc., enously expresses the neuropeptide Y2 receptors. Mem Clifton, N.J.) that are presoaked for 4 hours in 0.3% poyl branes are incubated with 30 pM125I-human Peptide YY ethyleneimine in order to reduce nonspecific binding of (2200 Ci/mmol, PerkinElmer Life Sciences), and with unla radiolabeled peptides. Filters are washed immediately beled active compound for 60 minutes at ambient tempera before filtration with 5 ml cold PBS, and immediately after filtration with 15 ml cold PBS. Filters are removed and ture in a 96 well polystyrene plate. Then well contents are radioactivity assessed in a gamma-counter at a counting harvested onto a 96 well glass fiber plate using a Perkin efficiency of 77%. Competition curves are generated by Elmer plate harvestor. Dried glass fiber plates are combined measuring binding in the presence of 10° to 10 M with scintillant and counted on a Perkin Elmer scintillation unlabeled test compound and are analyzed by nonlinear COunter. regression using a 4-parameter logistic equation (Inplot 0298 Y4 receptor binding assay: CHO-K1 cells are tran program; GraphPAD Software, San Diego). siently transfected with cDNA encoding neuropeptide Y4 gene, and then forty-eight hours later membranes are pre 0292 CGRP receptor binding assay: Evaluation of the pared from confluent cell cultures. Membranes are incubated binding of compounds of the invention to CGRP receptors with 18 pM 125I-human Pancreatic Polypeptide (2200 are essentially as described for amylin except using mem Ci/mmol, PerkinElmer Life Sciences), and with unlabeled branes prepared from SK-N-MC cells, known to express active compound for 60 minutes at ambient temperature in CGRP receptors (Muff, R. et al., Ann NY Acad. Sci. 1992: a 96 well polystyrene plate. Then well contents are harvested 657, 106-16). Binding assays are performed as described for onto a 96 well glass fiber plate using a Perkin Elmer plate amylin except using 13,500 cpm 1251-hCGRP ?well or 21.7 harvestor. Dried glass fiber plates are combined with scin pM/well (Amersham). tillant and counted on a Perkin Elmer scintillation counter. 0293 Adrenomedullin binding assay: Binding to the 0299 Y5 receptor binding assay: CHO-K1 cells are tran adrenomedullin receptor may be investigated using siently transfected with cDNA encoding neuropeptide Y5 HUVECs that contain the adrenomedullin receptor (Kato J gene, and then forty-eight hours later membranes are pre et. al., EurJ Pharmacol. 1995, 289:383-5) using the Perkin pared from confluent cell cultures. Membranes are incubated Elmer AlphaScreenTM assay for cyclic AMP using an opti with 44 pM 125I-human Peptide YY (2200 Ci/mmol, mum of 25-30,000 cells per well. Elevation of cAMP levels PerkinElmer Life Sciences), and with active compound for is not large for HUVEC compared to CHO cells. As such, 60 minutes at ambient temperature in a 96 well polystyrene CHO cells may be chosen as a negative control since they do plate. Then well contents are harvested onto a 96 well glass not express the adrenomedulin receptor if desired. fiber plate using a PerkinElmer plate harvestor. Dried glass 0294 Calcitonin receptor binding assay: Binding to the fiber plates are combined with scintillant and counted on a calcitonin receptor may be investigated using CHO cells or Perkin Elmer scintillation counter. US 2006/0293.232 A1 Dec. 28, 2006 48

0300 GLP-1 receptor binding assay: GLP-1 receptor on at 0600. Water and a standard pelleted mouse chow diet binding activity and affinity may be measured using a are available ad libitum, except as noted. Animals are fasted binding displacement assay in which the receptor source is starting at approximately 1500 hrs, 1 day prior to experi RINm.5F cell membranes, and the ligand is 'IGLP-1. ment. The morning of the experiment, animals are divided Homogenized RINm.5F cell membranes are incubated in 20 into experimental groups. In a typical study, n=4 cages with mM HEPES buffer with 40,000 cpm 'IGLP-1 tracer, and 3 mice/cage. varying concentrations of test compound for 2 hours at 23° 0303 At time=0 min, all animals are given an intraperi C. with constant mixing. Reaction mixtures are filtered toneal injection of vehicle or compound, typically in an through glass filter pads presoaked with 0.3% PEI solution amount ranging from about 10 nmol/kg to 75 nmol/kg, and and rinsed with ice-cold phosphate buffered saline. Bound immediately given a pre-weighed amount (10-15 g) of the counts are determined using a scintillation counter. Binding standard chow. Food is removed and weighed at various affinities are calculated using GraphPad Prism software times, typically 30, 60, and 120 minutes, to determine the (GraphPad Software, Inc., San Diego, Calif.). amount of food consumed (Morley, Flood et al., Am. J. Physiol. 267: R178-R184, 1994). Food intake is calculated Example 3 by Subtracting the weight of the food remaining at the e.g., 30, 60, 120, 180 and/or 240 minute time point, from the Mouse Food Intake Assay weight of the food provided initially at time=0. Significant 0301 The hybrid polypeptides of the invention may be treatment effects are identified by ANOVA (p<0.05). Where tested for appetite Suppression in the mouse food intake a significant difference exists, test means are compared to assay and for their effect on body weight gain in diet the control mean using Dunnett's test (Prism v. 2.01, Graph induced obesity (DIO) mice. The experimental protocols for Pad Software Inc., San Diego, Calif.). the screens are described below. 0304 Activity in the food intake assay and sequence of 0302) Female NIH/Swiss mice (8-24 weeks old) are parent molecules used for the synthesis of hybrids herein group housed with a 12:12 hour light:dark cycle with lights a.

Mouse Food Intake & basal

60 min Cmpd ED50 Description # (nmol/kg) Sequence 30 min 60 min 120 min 180 min Dose

PYY (3–36) 1 3 IKIPEAPGEDASPEELN -31 -38 - 40 -26 10 nmol/Kg RYYASLRHYLNLWTR QRY-NH2

Exendin-4 5 HGEGTFTSDLSKOME - 41 -60 -61 -60 4.8 nmol/Kg EEAWRLFIEWLKINGG PSSGAPPPS-NH2

Exendin-4 11 O3 HGEGTFTSDLSKOME -50 -62 - 49 - 49 16.3 nmol/Kg (1-28) EEAWRLFIEWLKN NH2

Exendin-4 (1-28) 12 13 HGEGAFTSDLSKQLE -53 -61 -50 -53 16.7 nmol/Kg Alas, Leu14, EEAWRLFIEFLKN Phe25 NH2

Rat Amylin 9 KCNTATCATORLANF -58 - 40 -36. -35. 25 nmol/Kg LWRSSNNIGPWLPPTN WGSNTY-NH2

hAmylin (1-7) - 10 26 KCNTATCWLGRLSOE -60 - 47 - 42. -32 25 nmol/Kg Arg-sCT LHRLQTYPRTNTGSN (8-27)-Amylin TY-NH2 (33-37)

CCK-8 26 DY (SO3) MGWMDF- -92 -56 -27 10 nmol/Kg US 2006/0293.232 A1 Dec. 28, 2006 49

Example 4 0307 As shown in Table 4-1, certain exemplary com Body Weight Gain in Fattened C57B1/6 pounds of the invention showed efficacy in the food intake (Diet-induced-obesity, or DIO) Mice assay. Certain peptides were also tested at 75 nmol/kg in the DIO assay and proved to be more efficacious than PYY 0305 Male C57BL/6 mice (4 weeks old at start of study) are fed high fat (HF, 58% of dietary kcal as fat) or low fat (FIG. 1). As observed for other hybrids herein, hybrids can (LF, 11% of dietary kcal as fat) chow. After 4 weeks on retain binding to one, two or more receptors that recognize chow, 5 each mouse is implanted with an osmotic pump the parent molecules. Hybrids were designed that recognize (Alzet # 2002) that subcutaneously delivers a predetermined at least one receptor from each parent or from only one dose of hybrid polypeptide continuously for two weeks. parent, as desired. As observed for other hybrids herein, use Body weight and food intake are measured weekly (Surwit of a linker (which can act as a spacer between each adjacent et al., Metabolism—Clinical and Experimental, 44: 645-51, hormone portion) can provide increased activity, including 1995). Effects of the test compound are expressed as the receptor(s) binding and in vitro and in Vivo activity, such as meantsd of '% body weight change (i.e., '% change from starting weight) of at least 14 mice per treatment group weight loss. The results herein indicate that a C-terminal (p<0.05 ANOVA, Dunnett's test, Prism v. 2.01, GraphPad portion of PYY can modulate activity. Software Inc., San Diego, Calif.). 0308 Exendin/Amylin Hybrids. Further exemplary 0306 Exendin/PYY Hybrids. Exemplary hybrid hybrid polypeptides of the invention were prepared from polypeptides of the invention were synthesized using a C-terminally truncated exendin (1-27), C-terminally trun C-terminally truncated exendins (e.g., exendin-4(1-28) or cated amylin peptides (e.g., amylin(1-7), "Ala-Amylin(1- Ala, ''Leu, Phe-exendin-4(1-28)) and an N-terminally 7), and AmylinC33-27)), and optional SCT fragments (e.g., truncated PYY spanning the 18-36 to 31-36 regions. As Such, the exemplary hybrid polypeptides generally comprise sCT(8-10), '''Arg-SCT(8-27) and ''Glu,'''Arg-SCT(8- two modules, wherein the first module is a fragment of an 27)). Whereas both hybrid polypeptides were very active in exendin-4 analog and the second module is a peptidic appetite Suppression (see Table 4-2), Superior to the same enhancer selected from PYY truncations. For comparison, a dose of rat amylin, the onset of action differed from the B-alanine dipeptide spacers were also incorporated between activity profiles of the parent molecules (data not shown). At the peptide building blocks in several variants (see Table a dose of 1 nmol/kg, Compound 5 was as effective as rat 4-1). amylin.

TABLE 4-1

Exendin/PYY Hybrids, Receptor Binding Data, and Their Effects in the Food Intake Assay Receptor Binding (IC50) nM Mouse Food Intake 96 basal

Description Cmpd # Y2 GLP1R 30 min 60 min 120 min Dose

PYY(3–36) O.04 -31 -38 -40 -26 Ala, 'Leu, Phe-exendin- 1.9 -50 -62 -49 -49 4(1-28) Ala, 'Leu, Phe-exendin- 1.7 1000 -4 -11 -10 0 nmol/Kg 4(1-17)-PYY(18–36) Ala, 'Leu, Phe-exendin- 17 2.1 21 9 -4 0 nmol/Kg 4(1-28)-PYY(22–36) Ala, 'Leu, Phe-exendin- 9 O.81 -3 -5 -22 0 nmol/Kg 4(1-28)-BAla-BAla PYY(22–36) Ala, Leu, Phe-exendin- 2 ind ind 8 -13 -30 0 nmol/Kg 4(1-28)-PYY(25-36) Ala, Leu, Phe-exendin- 3 13 O.22 -9 -25 -42 0 nmol/Kg 4(1-28)-BAla-BAla PYY(25–36) Ala, 'Leu, Phe-exendin- 4 1OOO O.25 - 14 -36 -52 0 nmol/Kg 4(1-28)-BAla-BAla PYY(31–36) exendin-4(1-28)-PYY(25–36) 16 O.29 -30 -37 -45 0 nmol/Kg exendin-4(1-28)-BAla- 7.8 0.16 -24 -40 -52 0 nmol/Kg BAla-PYY(25-36) exendin-4(1-28)-BAla- 1OOO O.19 -49 -56 -61 0 nmol/Kg BAla-PYY(31–36) US 2006/0293.232 A1 Dec. 28, 2006 50

TABLE 4-2 Exendin/Anyin Hybrids and Their Effect in the FI Assay Receptor Binding Assay Mouse Food Intake 96 basal Description Cmpdf GLP-1 Amylin CGRP CT 30 min 60 min 120 min Dose Exendin-4(1-27)- 5 (SEQ -24 -40 -48 25 nmol/Kg Amylin (1–7)- ID No. 11-18Arg 25) sCT(8–27)- Amylin (33–37) Exendin-4(1-27)- 6 (SEQ -59 -66 25 nmol/Kg 2-7Ala ID NO. Amylin (1–7)- 26) sCT(8–10) Exendin-4(1-28)- 14 O.3 O.2 113 O.1 -5 -30 -51 3 nmol/Kg beta-Ala beta-Ala Amylin (1–7)- '''Arg-sCT(8–27)- Amylin (33–37) Exendin-4(1-28)- 15 0.4 O.2 63 O.O3 -8 -36 -51 3 nmol/Kg Gly-Gly 11-18Arg Amylin (33–37) Exendin-4(1-27)- 5 1.7 O.6 178 O.S -20 -10 -26 3 nmol/Kg Amylin (1–7)- 11.18 Arg Amylin (33–37)

0309 Both compounds also showed excellent efficacy compounds are presented in FIG. 5A. Points represent when screened in the DIO assay (FIG. 2). meansd. Peptide was injected intraperitoneally (IP) at t=0 0310. Further exemplary compounds were assayed for immediately following baseline sample into 2-hour fasted effect on blood glucose levels and in a food intake assay. NIH/Swiss mice. Samples were taken at t=30, 60, 120, 180 These tests included compounds 14 and 15. Compound 14, and 240 min. Blood glucose was measured with a One which includes a betaAla linker, is BAla-BAla-Exendin(1- Touch(R) Ultra R. (LifeScan, Inc., a Johnson & Johnson Com 28).hAmylin? 1-7)-'''Arg-sCt(8-27)-hamylin.33–37), pany, Milpitas, Calif.). * p-0.05 vs. Vehicle control; (alternatively written as Exendin(1-28)-BAla-BAla-halmy ANOVA, Dunnett's test. lin(1-7)-'''Arg-SCt(8-27)-hamylinc33–37), while Com 0312. A food intake assay was performed as previously pound 15 contains a Gly linker: ‘’GlyGlyGly-Exendin(1- described herein. The results are presented in FIG. 5B. 28).hAmylin? 1-7)-'''Arg-sCt(8-27)-hamylin.33–37). The Points represent meantsd of n=4 cages (3 mice? cage). longer exendin(1-28) provided increased activity compared Peptide was injected intraperitoneally (IP) at t=0 into over to exendin( 1-27). night-fasted NIH/Swiss mice. Food was introduced imme 0311. A Blood Glucose Assay was performed to test diately after injection and amount consumed measured at effect on lowering blood glucose levels. Female NIH/Swiss t=30, 60, and 120 min. * p-0.05 vs. vehicle control; mice (8-20 weeks old) were group housed with a 12:12 hour ANOVA, Dunnett's test. light:dark cycle with lights on at 0600. Water and a standard 0313 Parental Compound 10 and exendin compounds pelleted mouse chow diet were available ad libitum, except have opposing effects in the Glucose Assay. One would as noted. The morning of the experiment, animals were expect that joining them would result in counteracting divided into experimental groups and fasted Starting at effects or, at best, a dilution of the effect seen with the more approximately 0630hrs. In a typical study, n=2 cages with potent parent compound. However, in the Glucose Assay, 3 mice/cage. At time=0 min, a blood glucose sample was the exemplary hybrid compounds were just as efficacious as taken and immediately followed by an intraperitoneal injec exendin(1-28) and had a longer duration of action. tion of vehicle or compound in an amount ranging from 0314. From the food intake data, the exemplary com about 1 nmol/kg to 25 nmol/kg. Blood glucose was mea pounds were anorexigenic. Activity was generally better sured at 30, 60, 120, 180, and 240 min. Percent pre than the parent compounds dosed individually. Activity was treatment was calculated by dividing the blood glucose at comparable to the parent compounds dosed together but at the e.g., 30, 60, 120, 180 and/or 240 minute time point by the half the concentration of drug (3 nmol/kg hybrid versus 6 blood glucose at time=0 min. Significant treatment effects nmol/kg total for co-dosed parent compounds); thus further were identified by ANOVA (p<0.05). Where a significant demonstrating hybrid superiority. The addition of a linker difference exists, test means were compared to the control increased activity of the hybrids. The Gly-Gly-Gly linker mean using Dunnett's test (Prism v. 4.01, GraphPad Soft was more effective than the betaAla-betaAla linker in this ware Inc., San Diego, Calif.). The results on exemplary CaSC. US 2006/0293.232 A1 Dec. 28, 2006

0315 Exendin/CCK-8 Hybrids. Yet further exemplary 0318. To ascertain if exemplary hybrid polypeptides of hybrid polypeptides of the invention were prepared from full the invention are more potent than their parent component length or C-terminally truncated exendin-4 attached to the peptide hormones, exemplary compounds were tested in the N— terminus of CCK-8 either directly or via a linker, food intake assay at the minimum efficacious dose of the preserving the N-terminal amide of the CCK-8. (Table 4-3). more active parent molecule. The results are shown in FIGS. Further, certain hybrids were prepared incorporating the 4A and 4B, which also compares the effects of pooled parent naturally occurring Tyr(SO), while another hybrid incor peptides (Compounds 1, 11, and 12 are component peptide porating the more stable Phe(CHSO) group was prepared. hormones, analogs or fragments thereof). The data indicate All the prepared hybrid polypeptides were active in inhib that several peptides are at least as equipotent as the pooled iting food intake (Table 4-3). parent peptides. In parallel with the in vivo studies, in vitro

TABLE 4-3 Exendin/CCK-8 Hybrids and Their Effect in the Food Intake Assay Receptor Binding (IC50) nM Mouse Food Intake 96 basal

Description Cmpdf GLP1-R 30 min 60 min 120 min Dose

Exendin-4(1-28) amide 1 O.63 Exendin-4-CCK-8 — (SEQ -12 -28 -28 10 nmol/Kg D NO. 20) Exendin-4(1-28)-CCK-8 7 75 -20 -36 -45 10 nmol/Kg (SEQ D NO. 21) Exendin-4(1-28)-CCK-8 8 31 -24 -47 -66 10 nmol/Kg Phe(CHSO) (SEQ D NO. 22) Exendin-4(1-28)-8- 9 -12 -28 -40 10 nmol/Kg amino-3,6- (SEQ dioxa.octanoyl-CCK-8 D NO. 23)

0316 Exemplary exendin/CCK-8 hybrid polypeptides receptor binding and functional assays (cyclase activity) were tested in the DIO assay at 25 nmol/kg (FIGS. 3A and have been performed for all the compounds (data not 3B). The data shows an initial weight loss, followed by a shown). rebound effect in all compounds. Interestingly, the rebound 0319 Amylin-sCT/leptin hybrids. Further exemplary effect appears to be diminished in hybrids incorporating the hybrids were made which contained a leptin peptide frag more hydrolytically stable Phe(CHSO) residue (compare ment joined to Compound 10, an amylin-SCT-amylin chi FIGS. 3A and 3C), as well as hybrids incorporating a linker, mera described herein. Compound 16 is Ser117, deu119 for example the linker 8-amino-3,6-dioxaoctanoyl, between leptin(116-122)-'''ArgsCT(8-27)-Amylin)33–37). The the exendin and the CCK residues (compare FIGS. 3A and compound bound (RBA=receptor binding assay) the amylin 3B). A ten-fold greater amount of CCK-8 (250 nmol/kg/day) and CT receptors, with some binding to the CGRP receptor. was needed to produce about a -2.8% change at day 2, The compound was also able to activate the CT receptor which rebounded to the HF diet control level at day 7. (CIA assay). 0317 Amylin/PYY Hybrid. An Amylin/PYY hybrid polypeptide was synthesized that contained truncated seg ments of each peptide. In-vivo activity in the food intake Compound Cmpdf# Assay IC50 assay is shown in Table 4-4. Ser117, dLeu119 leptin- 16 amylin RBA O.04 nM '''ArgsCT(8–27)- TABLE 4-4 amylin (33–37) 16 CGRP RBA 81 nM Amylin/PYY Phybrid 16 CT 2.2 nM CYCLASE(C1A) Mouse Food Intake 96 Basal 16 CT RBA (C1A) O.O63 nM 16 GLP RBA (RIN) 1000 nM Description 30 min 60 min 120 min Dose Amylint 1-18)-PYY(19–36) –13 -14 -13 25 nmol/Kg (SEQ ID NO. 38) 0320 This representative molecule was tested for activity in a food intake assay as described herein. Although leptin was not active in this assay, Compound 16 was anorexigenic US 2006/0293.232 A1 Dec. 28, 2006 52 at 1 mg/kg. Compound 16 was also Superior to rat amylin (at at least one of the bio-active peptide hormone modules 25 nmol/kg) in its anorexigenic effect. While Compound 10 exhibits at least one hormonal activity of a component reduced food intake 91-95% compared to controls, much peptide hormone. more effectively; Compound 16 reduced the cumulative 2. The hybrid polypeptide of claim 1, wherein the peptidic intake to 34-38% that of controls. Further exemplary embo enhancers are independently selected from the group con siments include a head-to-headjoining, of the N-terminus of sisting of amylinC32-37), amylin (33-37), amylin(34-37), the leptin peptide with that of the '''ArgsCT(8-27)- amylin(35-37), amylin(36-37), amylinc37), ADM(47-52), amylin? 33-37) compound. ADM(48-52), ADM(49-52), ADM(50-52), ADM(51-52), 0321 CCK/Amylin-sCT Hybrids An exemplary hybrid ADM(52), CT(27-32), CT(27-32), CT(28-32), CT(29-32), of CCK with an amylin-sCT chimera (compound 10) dem CT(30-32), CT(31-32), CT(32), CGRP(32-37), CGRP(33 onstrated relevant receptor specificity and activation. The 37), CGRP(34-37), CGRP(35-37), CGRP(36-37), exemplary compound having sequence DF(P- CGRP(36-37), CGRP(37), intermedin (42-47), intermedin CHSO)MGWMDFGKRKCNTATCATQRLANELVRLQTYP SS site medin (44-47), intermedin (45-47), intermedin demonstrated an IC50 of 0.044 nM in a CT receptor binding 6-47), intermedin (47), PYY(25-36), PYY(26-36), assay, 4.4nM in a CGRP receptor binding assay, 0.083 nM PYY(27-36), PYY(28-36), PYY(29-36), PYY(30-36), in an amylin receptor binding assay, and 1000 nM in a GLP PYY(31-36), PYY(32-36), PYY(25-35), PYY(26-35), Receptor cyclase (RIN) PYY(27-35), PYY(28-35), PYY(29-35), PYY(30-35), PYY(31-35), PYY(32-35), frog GLP-1 (29-37), frog GLP 0322) While the present invention has been described in 1(30-37), frog GLP-2(24-31), exendin-4(31-39), exendin terms of preferred examples and embodiments, it is under 4(32-39), exendin-4 (33-39), exendin-4(34-39), exendin stood that variations and modifications will occur to those 4(35-39), exendin-4 (36-39), exendin-4(37-39), exendin skilled in the art. Therefore, it is intended that the appended 4(38-39), exendin-4(39), and analogs thereof. claims cover all such equivalent variations which come 3. The hybrid polypeptide of claim 1, wherein at least one within the scope of the invention as claimed. of the first bio-active peptide hormone module or the at least one additional bio-active peptide hormone module is a 1. A hybrid polypeptide exhibiting at least one hormonal component peptide hormone or fragment of a component activity, said hybrid polypeptide comprising a first bio peptide hormone that exhibits at least one hormonal activity active peptide hormone module covalently linked to at least of the component peptide hormone. one additional bio-active peptide hormone module; wherein: 4. The hybrid polypeptide of claim 1, wherein at least one the bio-active peptide hormone modules are indepen of the first bio-active peptide hormone module or the at least dently selected from the group consisting of compo one additional bio-active peptide hormone module is an nent peptide hormones, fragments of component pep analog or derivative of a component peptide hormone that tide hormones that exhibit at least one hormonal exhibits at least one hormonal activity or a fragment of an activity of the component peptide hormones, analogs analog or derivative of a component peptide hormone that and derivatives of component peptide hormones that exhibits at least one hormonal activity of the component exhibit at least one hormonal activity of the component peptide hormone. peptide hormones, fragments of analogs and deriva 5. The hybrid polypeptide of claim 1, wherein at least one tives of component peptide hormones that exhibit at of the first bio-active peptide hormone modules or at least least one hormonal activity of the component peptide one additional bio-active peptide hormone module is pep hormones, and peptidic enhancers; tidic enhancer. 6. The hybrid polypeptide of claim 1, wherein the com the component peptide hormones are independently ponent peptide hormones are independently selected from Selected from at least two of the group consisting of: the group consisting of amylin, calcitonin, CCK, PYY, a amylin, adrenomedullin (ADM), calcitonin (CT), cal urocortin family peptide, a neuromedin family peptide, and citonin gene related peptide (CGRP), intermedin, ANP, BNP, CNP or urodilatin and exendin-4. cholecystokinin (“CCK), leptin, peptide YY (PYY), 7. The hybrid polypeptide of claim 1, wherein at least one glucagon-like peptide-1 (GLP-1), glucagon-like pep bio-active peptide hormone module that exhibits at least one tide 2 (GLP-2), oxyntomodulin (OXM), anatriuretic hormonal activity is located at the N-terminal portion of the peptide, aurocortin family peptide, e.g., Ucn-2 and hybrid polypeptide. Ucn-3, a neuromedin family peptide, e.g. neuromedin 8. The hybrid polypeptide of claim 7, wherein the at least U25 or a splice variant, and ANP BNP. CNP or one bio-active peptide hormone module that exhibits at least urodilatin and exendin-4: one hormonal activity located at the N-terminal portion of the peptidic enhancers are independently selected from the hybrid polypeptide is configured in the C-terminal to the group consisting of structural motifs of component N-terminal orientation. peptide hormones that impart a desired chemical sta 9. The hybrid polypeptide of claim 8, wherein the N-ter bility, conformational stability, metabolic stability, bio minal portion of the hybrid polypeptide is amidated. availability, organ/tissue targeting, receptor interaction, 10. The hybrid polypeptide of claim 1, wherein at least protease inhibition, plasma protein binding, or other one bio-active peptide hormone module that exhibits at least pharmacokinetic characteristic to the hybrid polypep one hormonal activity is located at the C-terminal portion of tide, and structural motifs of analogs or derivatives of the hybrid polypeptide. component peptide hormones that impart a desired 11. The hybrid polypeptide of claim 10, wherein the chemical stability, conformational stability, metabolic C-terminal end of the hybrid polypeptide is amidated. stability, bioavailability, organ/tissue characteristic to 12. The hybrid polypeptide of claim 1, wherein the the hybrid polypeptide; and C-terminal end of one bio-active peptide hormone module is US 2006/0293.232 A1 Dec. 28, 2006

directly attached to the N-terminal end of another bio-active derivative that exhibits at least one hormonal activity, and a peptide hormone module to form the covalent attachment. fragment of an amylin analog that exhibits at least one 13. The hybrid polypeptide of claim 1, wherein the hormonal activity; and bio-active peptide hormone modules are covalently attached at least one additional bio-active peptide hormone module using one or more linking groups independently selected is a peptidic enhancer independently selected from the from the group consisting of alkyls; dicarboxylic acids group consisting of: PYY(25-36), PYY(26-36), PEGs, amino acids; polyaminoacids; bifunctional linkers; PYY(27-36), PYY(28-36), PYY(29-36), PYY(30-36), aminocaproyl (Aca), Gly, B-alanyl, 8-amino-3,6-dioxaoc PYY(31-36), PYY(32-36), PYY(25-35), PYY(26-35), tanoyl, and Gly-Lys-Arg (GKR). PYY(27-35), PYY(28-35), PYY(29-35), PYY(30-35), 14. The hybrid polypeptide of claim 1, wherein the first PYY(31-35), PYY(32-35), and analogs thereof. bio-active peptide hormone module is selected from the 21. A hybrid polypeptide exhibiting at least one hormonal group consisting of exendin-4, a fragment of exendin-4 that activity, said hybrid polypeptide comprising a first bio exhibits at least one hormonal activity, an exendin-4 analog active peptide hormone module covalently linked to a sec or derivative that exhibits at least one hormonal activity, and ond bio-active peptide hormone module; wherein: a fragment of an exendin-4 analog that exhibits at least one the bio-active peptide hormone modules are indepen hormonal activity; and dently selected from the group consisting of compo at least one additional bio-active peptide hormone module nent peptide hormones, fragments of component pep is independently selected from the group consisting of: tide hormones that exhibit at least one hormonal amylin, a fragment of amylin that exhibits at least one activity of the component peptide hormones, analogs hormonal activity, an amylin analog or derivative that and derivatives of component peptide hormones that exhibits at least one hormonal activity, or a fragment of exhibit at least one hormonal activity of the component an amylin analog that exhibits at least one hormonal peptide hormones, fragments of analogs and deriva activity, CCK, a fragment of CCK that exhibits at least tives of component peptide hormones that exhibit at one hormonal activity, a CCK analog or derivative that least one hormonal activity of the component peptide exhibits at least one hormonal activity, a fragment of a hormones, and peptidic enhancers; CCK analog that exhibits at least one hormonal activity, the component peptide hormones are independently CT, a fragment of CT that exhibits at least one hor Selected from at least two of the group consisting of: monal activity, a CT analog or derivative that exhibits amylin, PYY, and exendin-4: at least one hormonal activity, a fragment of a CT analog that exhibits at least one hormonal activity, and the peptidic enhancers are independently selected from a peptidic enhancer. the group consisting of structural motifs of component peptide hormones that impart a desired chemical sta 15. The hybrid polypeptide of claim 14, wherein the first bility, conformational stability, metabolic stability, bio bio-active peptide hormone module is selected from the availability, organ/tissue targeting, receptor interaction, group consisting of exendin-4, exendin-4(1-27), exendin protease inhibition, plasma protein binding, or other 4(1-28), ''Leu, Phe-exendin-4(1-28); Ala, ''Leu, Phe pharmacokinetic characteristic to the hybrid polypep exendin-4(1-28) and ''Leu-exendin-4(1-28); and at least tide, and structural motifs of analogs or derivatives of one additional bio-active peptide hormone module is inde component peptide hormones that impart a desired pendently selected from the group consisting of Pro chemical stability, conformational stability, metabolic h-amylin, amylin? 1-7), "Ala-amylin? 1-7), scT(8-10), stability, bioavailability, organ/tissue targeting, recep sCT(8-27), '''Arg-sCT(8-27), ''Glu'''Arg-SCT(8-27), tor interaction, protease inhibition, plasma protein ANP, BNP. CNP urodilatin, CCK-8, Phe’CCK-8, amy binding, or other pharmacokinetic characteristic to the lin(33-37), PYY(25-36), PYY(30-36) and PYY(31-36). hybrid polypeptide; and 16. The hybrid polypeptide of claim 14, wherein the hybrid polypeptide comprises at least three bio-active pep wherein at least one of the bio-active peptide hormone tide hormone modules. modules exhibits at least one hormonal activity of a component peptide hormone. 17. They hybrid polypeptide of claim 14, wherein the 22. The hybrid polypeptide of claim 21, wherein the hybrid polypeptide comprises at least four bio-active peptide peptidic enhancers are independently selected from the hormone modules. group consisting of amylin(32-37), amylin(33-37), amy 18. The hybrid polypeptide of claim 14, wherein the first lin(34-37), amylin (35-37), amylin(36-37), amylinC37), bio-active peptide hormone module is located at the C-ter PYY(25-36), PYY(26-36), PYY(27-36), PYY(28-36), minal end of the hybrid polypeptide and at least one addi PYY(29-36), PYY(30-36), PYY(31-36), PYY(32-36), tional bio-active peptide hormone module is located at the PYY(25-35), PYY(26-35), PYY(27-35), PYY(28-35), N-terminal end of the hybrid polypeptide. PYY(29-35), PYY(30-35), PYY(31-35), PYY(32-35), 19. The hybrid polypeptide of claim 14, wherein the first exendin-4(31-39), exendin-4 (32-39), exendin-4(33-39), bio-active peptide hormone module is located at the N-ter exendin-4 (34-39), exendin-4 (35-39), exendin-4(37-39), minal end of the hybrid polypeptide and at least one addi exendin-4(38-39), exendin-4(39), and analogs thereof. tional bio-active peptide hormone module is located at the 23. The hybrid polypeptide of claim 21, wherein the first C-terminal end of the hybrid polypeptide. bio-active peptide hormone module is located at the C-ter 20. The hybrid polypeptide of claim 1, wherein the first minal end of the hybrid polypeptide. bio-active peptide hormone module is selected from the 24. The hybrid polypeptide of claim 21, wherein the first group consisting of amylin, a fragment of amylin that bio-active peptide hormone module is located at the N-ter exhibits at least one hormonal activity, an amylin analog or minal end of the hybrid polypeptide. US 2006/0293.232 A1 Dec. 28, 2006 54

25. The hybrid polypeptide of claim 21, wherein the position 93 to Asp or Glu, at position 98 to Ala, at position hybrid polypeptide comprises bio-active peptide hormone 117 to Ser, at position 139 to Leu, and at position 167 to Ser. module combinations selected from the group consisting of 32. The hybrid polypeptide of claim 26, wherein the leptin exendin-4/PYY. PYY/exendin-4, exendin/amylin, amylin/ is selected from the group consisting of 43 Asp-leptin, exendin, amylin/PYY, and PYY/amylin bio-active peptide 43Glu-leptin, 48Ala-leptin, 49Glu-leptin, 49Des-AA-leptin, hormone modules. 75Ala-leptin, 89Leu-leptin, 93Asp-leptin, 93Glu-leptin, 26. A hybrid polypeptide exhibiting at least one hormonal 98Ala-leptin, 117Ser-leptin, 139Leu-leptin, 167Ser-leptin, activity, said hybrid polypeptide comprising a first bio 43Asp. 49Glu-leptin, 43 Asp,75Ala-leptin, 89Leu, 117Ser active peptide hormone module covalently linked to at least leptin, 93Glu,167Ser-leptin, and 117Ser, D-119Leu-leptin. one second bio-active peptide hormone module; wherein: 33. The hybrid polypeptide of claim 26, wherein the leptin is a fragment selected from the group consisting of leptin the bio-active peptide hormone modules are indepen (22-167), leptin(116-122), '''Ser, D-Leu-leptin(116-12) dently selected from the group consisting of: and leptin(56-73). component peptide hormones, fragments of component 34. The hybrid polypeptide of claim 26, wherein the leptin peptide hormones that exhibit at least one hormonal peptide hormone module comprises a polypeptide selected activity of the component peptide hormones, analogs from the group consisting of leptin, leptin fragments that and derivatives of component peptide hormones that exhibit at least one hormonal activity, leptin analogs and exhibit at least one hormonal activity of the component derivatives that exhibit at least one hormonal activity, frag peptide hormones, fragments of analogs and deriva ments of leptin analogs and derivatives that exhibit at least tives of component peptide hormones that exhibit at one hormonal activity, and wherein the at least one GLP1 least one hormonal activity of the component peptide peptide hormone module comprises a polypeptide selected hormones, and peptidic enhancers; from the group consisting of glucagon-like peptide-1 (GLP 1), a fragment of GLP 1 that exhibits at least one hormonal the first component peptide hormone comprises a leptin; activity, a GLP1 analog or derivative that exhibits at least the at least one second bio-active peptide hormone mod one hormonal activity, and a fragment of a GLP1 analog that ule comprise a polypeptide independently selected exhibits at least one hormonal activity. from an exendin or GLP1; and 35. The hybrid polypeptide of claim 26, wherein the GLP1 is selected from the group consisting of GLP1(1-37), at least one of the bio-active peptide hormone modules GLP1(1-36), GLP1(7-37), GLP1(7-36), GLP1(7-35) and exhibits at least one hormonal activity of its component analogs or derivatives thereof. peptide hormone. 36. The hybrid polypeptide of claim 26 wherein the GLP1 27. The hybrid polypeptide of claim 26, wherein the leptin is from murine, hamster, chicken, bovine, rat, frog or dog. peptide hormone module comprises a polypeptide selected 37. The hybrid polypeptide of claim 26, wherein the GLP from the group consisting of leptin, leptin fragments that 1 is from human or frog. exhibit at least one hormonal activity, leptin analogs and 38. The hybrid polypeptide of claim 26, wherein the derivatives that exhibit at least one hormonal activity, and GLP1 is selected from the group consisting of HDEFER fragments of leptin analogs and derivatives that exhibit at HAEGTFTSDVSSTLEGQAALEFIAWLVKGRG, HAEG least one hormonal activity. TYTNDVTEYLEEKAAKEFIEWLIKGKPKKIRYS; 28. The hybrid polypeptide of claim 26, wherein the first HAEGTFTSDVTQQLDEKAAKEFIDWLINGGPSKEIIS, and a second bio-active peptide hormone module exhibit at and HAEGTFTSDVSSYLEGQAALEFIAWLVKGR. least one hormonal activity of a component peptide hor 39. The hybrid polypeptide of claim 26, wherein the O. exendin is selected from the group consisting of Gln-GLP 29. The hybrid polypeptide of claim 26, wherein the leptin 1(7-37), D-Gln-GLP-1(7-37), Thr-LysGLP-1-(7-37), comprises the sequence MHWGTLCGFLWLWPYL 'Lys-GLP-1 (7-37), Gly-GLP-1 (7-36), Gln-GLP-1 (7-37), FYVQAVPIQKVQDDTKTLIK D-Gln-GLP-1 (7-37), acetyl-Lys-GLP-1 (7-37), Thr-GLP TIVTRINDISHTQSVSSKQKVTGLDF IPGLHPILTLSK 1(7-37), D-Thr-GLP-1 (7-37), Asn-GLP-1 (7-37), D-- MDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHV Asn-GLP-1 (7-37), Ser Arg-Arg-Gln-GLP-1 (7-37), LAFSKSCHLPWASGLE TLDSLGGVLEASGYSTEV Thr'Lys-GLP-1(7-37), Lys-GLP-1 (7-37), Arg-GLP VALSRLQGSLQDMLWQLDLSPGC, or an active frag 1(7-37), and Arg-GLP-1 (7-37). ment thereof. 40. The hybrid polypeptide of claim 26, wherein the leptin 30. The hybrid polypeptide of claim 26, wherein the leptin peptide hormone module comprises a polypeptide selected comprises the sequence VPIQKVODDTKTLIK from the group consisting of leptin, leptin fragments that TIVTRINDISHTQSVSSKQKVTGLD exhibit at least one hormonal activity, leptin analogs and FIPGLHPILTLSKMDQTLAVYQQIL TSMPSRNVI QISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLG derivatives that exhibit at least one hormonal activity, frag GVLEASGYSTEVVALSR LQGSLQDMLWQLDLSPGC, ments of leptin analogs and derivatives that exhibit at least or an active fragment thereof. one hormonal activity, and 31. The hybrid polypeptide of claim 26, wherein the leptin wherein the at least one exendin peptide hormone module analog comprises one or more amino acid Substitutions at comprises a polypeptide selected from the group con positions 43, 48, 49, 75, 89, 93, 98, 117, 139 or 167, or a sisting of exendin-4, a fragment of exendin-4 that corresponding position in an analog, selected from the group exhibits at least one hormonal activity, an exendin-4 consisting of an amino acid Substitution at position 43 to Asp analog or derivative that exhibits at least one hormonal or Glu, at position 48 to Ala; at position 49 to Glu or is activity, and a fragment of an exendin-4 analog that absent, at position 75 to Ala, at position 89 to Leu, at exhibits at least one hormonal activity. US 2006/0293.232 A1 Dec. 28, 2006

41. The hybrid polypeptide of claim 26, wherein the 55. The hybrid polypeptide of claim 51, wherein the linker exendin is selected from the group consisting of exendin-4, comprises a moiety selected from the group consisting an '''Leu, Phe-exendin-4, Ala, '“Leu, Phe-exendin-4, amino acid Lys, Glu, Gly, Cys, or B-Ala and a polyami '''Leu, Ala, Phe-exendin-4, and active fragments noacid poly-his, poly-arg, poly-lys, poly-ala, betaAla-be thereof. taAla, Gly-Gly-Gly, or Gly-Lys-Arg. 42. The hybrid polypeptide of claim 26, wherein the 56. The hybrid polypeptide of claim 51, wherein the linker exendin comprises the sequence HGEGTFTSDL is 1 to 30 residues long, is 2 to 30 residues, or is 3 to 30 SKQMEEEAVRLFIEWLKNGGPSSGAPPPS or an active residues long. fragment thereof. 57. The hybrid polypeptide of claim 51, wherein the linker 43. The hybrid polypeptide of claim 26, wherein the is 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues long. exendin is selected from the group consisting of exendin(7- 58. The hybrid polypeptide of claim 26 wherein at least 15), Ser-exendin(7-15), exendin-4(1-27), exendin(1-28), one component peptide hormone is produced recombinantly. exendin-4(1-29), exendin-4(1-30), ''Leu, Phe-exendin 59. The hybrid polypeptide of claim 26 wherein the 4(1-27), Ala, ''Leu, Phe-exendin-4(1-27), '“Leu,’Ala, hybrid polypeptide is produced recombinantly. ‘Phe-exendin-4(1-27), ''Leu, Phe-exendin-4(1-28); Ala, 60. The hybrid polypeptide of claim 26 wherein at least '''Leu, Phe-exendin-4(1-28, and ''Leu-exendin-4(1-28). one component peptide hormone is chemically synthesized. 44. The hybrid polypeptide of claim 26, wherein the 61. The hybrid polypeptide of claim 26 wherein the C-terminus is amidated. hybrid polypeptide is chemically synthesized. 45. The hybrid polypeptide of claim 26, wherein any one 62. The hybrid polypeptide of claim 26 wherein the of the component peptide hormone modules has at least at hybrid polypeptide comprises two, three or four exendin least 70% sequence identity to its base peptide. and/or GLP1 peptide hormone modules. 46. The hybrid polypeptide of claim 26, wherein any one 63. A method of treating a patient having a metabolic of the component peptide hormone modules has at least at disease or disorder, comprising administering to a patient in least 80% sequence identity to its base peptide. need thereofatherapeutically effective amount of the hybrid 47. The hybrid polypeptide of claim 26, wherein any one polypeptide of claim 26. of the component peptide hormone modules has at least at least 90% sequence identity to its base peptide. 64. The method of claim 63, wherein the patient is in need 48. The hybrid polypeptide of claim 26, wherein any one of regulating food intake. of the component peptide hormone modules has at least at 65. The method of claim 63, wherein the patient is in need least 95% sequence identity to its base peptide. of regulating body weight. 49. The hybrid polypeptide of claim 26, wherein any one 66. The method of claim 63, wherein the patient is in need of the component peptide hormone modules comprises a of regulating hematopoiesis. D-amino acid. 67. The method of claim 63, wherein the hybrid provides 50. The hybrid polypeptide of claim 26, wherein the leptin a therapeutically effective anorexigenic effect. peptide hormone module is linked at its N-terminus to the 68. The method of claim 63, wherein the hybrid provides C-terminus of the at least one exendin and/or GLP1 peptide a therapeutically effective glucose lowering effect. hormone module. 69. The method of claim 63, wherein the hybrid provides 51. The hybrid polypeptide of claim 26, wherein the leptin a therapeutically effective enhancement of insulin secretion. peptide hormone module is covalently linked through a 70. The method of claim 63, wherein the patient is in need linker to the at least one exendin and/or GLP1 peptide one or more of regulating food intake, regulating body hormone module. weight, hematopoiesis, an anorexigenic effect, glucose low 52. The hybrid polypeptide of claim 51, wherein the linker ering, enhancement of insulin secretion or increase in pan is chemically stable. creatic beta cell mass. 53. The hybrid polypeptide of claim 51, wherein the leptin 71. The method of claim 63, wherein the metabolic peptide hormone module or the at least one exendin and/or disease or disorder is diabetes, type 2 diabetes, type 1 GLP1 peptide hormone module comprises a substitution of diabetes, obesity, hypertension, atherosclerosis, dyslipi one or more amino acids with lysine, aspartic acid, glutamic demia, congestive heart failure, stroke, hypercholester acid, or cysteine to create a linker site. olemia, cardiovascular disease, myocardial ischemia, myo 54. The hybrid polypeptide of claim 51, wherein the linker cardial reperfusion, eating disorders, gestational diabetes, comprises a moiety selected from the group consisting of an diabetic neuropathy, pulmonary hypertension, or associated alkyl, a PEG, an amino acid, a polyaminoacid, a bifunctional with insufficient pancreatic beta cell mass. linker, an aminocaproyl, a 3-alanyl, and an 8-amino-3,6- dioxaoctanoyl. k k k k k