MXPA06009239A - Hybrid polypeptides with selectable properties - Google Patents

Abstract

The present invention relates generally to novel, selectable hybrid polypeptides useful as agents for the treatment and prevention of metabolic diseases and disorders which can be alleviated by control plasma glucose levels, insulin levels, and/or insulin secretion, such as diabetes and diabetes-related conditions. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin-resistance, obesity, and diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes.

Description

HYBRID POLYPEPTITS WITH SELECTABLE PROPERTIES RELATED REQUEST The present application claims priority to the provisional application of E.U.A. No. 60 / 543,407, filed February 11, 2004, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to peptide chemistry, and very particularly to hybrid polypeptides with selectable properties.

BACKGROUND OF THE INVENTION Central to many diseases and metabolic disorders are the regulation of insulin levels and blood glucose levels. Insulin secretion is modulated in part by secretagogue hormones, called incretins, which are produced by enteroendocrine cells. The hormone ncretin, glucagon-like peptide-1 ("GLP-1") is a peptide hormone secreted by intestinal cells that has been shown in multiple studies to produce an increasing effect on insulin secretion. GLP-1 is processed from proglucagon in the intestine and increases insulin release induced by nutrients (Krcymann B., et al., Lancet, 2: 1300-1303 (1987)). Several truncated forms of GLP-1 are known to stimulate insulin secretion (insulinotropic action) and cAMP formation [see, e.g. , Mojsov, S., Int. J. Pep. Pro. Res., 40: 333-343 (1992)]. A relationship has been established between several in vitro and mammalian laboratory experiments, especially human, insulinotropic responses to exogenous administration of GLP-1, GLP-1 amide (7-36), and GLP-1 (7-37) acid. (See, e.g., Nauck, MA, et al., Diabetology, 36: 741-744 (1993); Gutniak, M., et al., New Eng. J. of Med., 326 (20): 1316 -1322 (1992); Nauck, MA, et al., J. Clin. Invest., 91: 301-307 (1993); and Thorens, B., et al., Diabetes, 42: 1219-1225 (1993) ). The GLP-1 amide (7-36) exerts a pronounced anti-diabetic effect in insulin-dependent diabetics by stimulating insulin sensitivity and by increasing insulin release induced by glucose at physiological concentrations (Gutniak M., et al., New Eng J. Med., 326: 1316-1322 (1992)). When administered to non-insulin dependent diabetics, the GLP-1 (7-36) amide stimulates insulin release, reduces glucagon secretion, inhibits gastric emptying and increases glucose utilization (Nauck, 1993; Gutniak, 1992; Nauck, 1993). However, the use of GLP-1 type molecules for prolonged therapy of diabetes has been complicated because the half-life of said peptides in the serum is very short. Very particularly, GLP-1 is a peptide of 30 amino acids Proglucagon derivative, a prohormone of 160 amino acids. The actions of different prohormone convertases in the pancreas and intestine result in the production of glucagon and other peptides defined by disease, while the proglucagon digestion results in the production of GLP-1 and GLP-2 as well as two other peptides. . The amino acid sequence of GLP-1 is 100% homologous in all mammals studied so far, implying a critical physiological role. The acid of GLP-1 (7-37) is C-terminally truncated and amidated to form NH2 of GLP-1 (7-36). The biological effects and metabolic turnover of GLP-1 free acid (7-37) OH, and the amide, NH2 of GLP-1 (7-36), are indistinguishable. By convention, amino acid numbering is based on the OH of processed GLP-1 (1-37) from proglucagon. The biologically active GLP-1 is the result of additional processing: NH2 of GLP-1 (7-36). Therefore, the first OH amino acid of GLP-1 (7-37) or NH2 of GLP-1 (7-36) is 7His. In the gastrointestinal tract, GLP-1 is produced by L cells of the intestinal mucosa, colonic and rectal, in response to stimulation by intraluminal glucose. The plasma half-life of active GLP-1 is < 5 minutes, and its metabolic clearance rate is approximately 12-13 minutes (Holst, 1994). The major protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26) that digests the N-terminal His-Ala dipeptide, thus producing metabolites, GLP-1 OH (9-37) or GL2 NH2 -1 (9-36) which are described variously as weak agonists or antagonists, inactive of GLP-1 receptor. The GLP-1 receptor (GLP-1 R) is a G3 protein-coupled receptor of 463 amino acids and is located in pancreatic cells, in the lungs and to a lesser degree in the brain, adipose tissue and kidneys. The stimulation of GLP-1 R by OH of GLP-1 (7-37) or NH2 of GLP-1 (7-36) results in activation of adenylate cyclase, synthesis of cAMP, depolarization of the membrane, increase in calcium intracellular and increased insulin secretion induced by glucose (Holz et al., 1995). GLP-1 is a potent insulin secretagogue that is secreted from the intestinal mucosa in response to food intake. The profound incretin effect of GLP-1 is highlighted by the fact that the "knockout" mice (knocked out) for GLP-1 R are glucose intolerant. The incretin response of GLP-1 infused i.v. It is preserved in diabetic subjects, although the response of incretin to oral glucose in these patients is compromised. The administration of GLP-1 by infusion or subcutaneous injections controls fasting glucose levels in diabetic patients, and maintains the glucose threshold for insulin secretion (Gutniak et al., 1992; Nauck et al., 1986; Nauck et al. al., 1993). GLP-1 has shown tremendous potential as a therapeutic agent capable of increasing insulin secretion in a physiological manner, while avoiding hypoglycemia associated with sulfonylurea drugs. Other important effects of GLP-1 on glucose homeostasis are the suppression of glucagon secretion and inhibition of gastric motility. The inhibitory actions of GLP-1 on the secretion of pancreatic alpha glucagon cells leads to decreases in the production of Hepatic glucose by reduction in gluconeogenesis and glycogenólisís. This antiglucagon effect of GLP-1 is preserved in diabetic patients. The so-called braking effect of GLP-1, in which gastric motility and gastric secretion are inhibited, is effected by vagal efferent receptors or by direct action on intestinal smooth muscle. The reduction of gastric acid secretion by GLP-1 contributes to a lag phase in nutrient availability, thus obviating the need for rapid insulin response. In summary, the gastrointestinal effects of GLP-1 contribute significantly to delayed absorption of glucose and fatty acids and modulate insulin secretion and glucose homeostasis. It has also been shown that GLP-1 induces beta cell-specific genes, such as GLUT-1 transporter, insulin (through the interaction of PDX-1 with insulin gene promoter), and hexokinase-1. Therefore, GLP-1 could potentially reverse the glucose intolerance normally associated with aging, as demonstrated by experiments with rodents. In addition, GLP-1 may contribute to beta cell neurogenesis and increase in beta cell mass, in addition to restoring the function of beta cells during beta cell failure states. The central effects of GLP-1 include increases in satiety coupled with decreases in food intake, effected through the action of hypothalamic GLP-1 R. A 48-hour continuous subcutaneous infusion of GLP-1 in type II diabetic subjects reduced hunger and food intake and increased satiety. These anorexic effects were absent in knockout mice for GLP-1R. The exendins are another family of peptides involved in insulin secretion. The exendins are found in the saliva of the Gila monster, an endogenous lizard from Arizona, and the Mexican pearl lizard. Exendin-3 is present in the saliva of Heloderma horridum, and exendin-4 is present in the saliva of Heloderma suspectum (Eng, J., et al., J Biol. Chem., 265: 20259-62,1990; Eng., J., et al., J. Biol. Chem., 267: 7402-05 (1992)). The exendins have some sequence similarity to several members of the glucagon-like peptide family, with the highest homology, 53%, being to GLP-1 (Goke, et al., J. Biol. Chem., 268: 19650 -55 (1993)). Exendin-4 binds to GLP-1 receptors on insulin-secreting TC1 cells, in scattered acinar cells of pancreas (guinea pig), and in parietal cells of the stomach; the peptide also stimulates the release of somatostatin and inhibits the release of gastrin in isolated stomachs (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993); Schepp, et al., Eur. J. Pharmacol., 69: 183-91 (1994), Eissele, et al., Life Sci., 55: 629-34 (1994)). It was found that exendin-3 and exendin-4 bind to GLP-1 receptors in pancreatic acinar cells, stimulate the production of cAMP in them, and release amylase therefrom (Malhotra, R., et al. ., Relulatory Peptides, 41: 149-56 (1992); Raufman, et al., J. Biol. Chem., 267: 21432-37 (1992); Singh, et al., Regul. Pept, 53: 47- 59 (1994)). The use of the insulinotropic activities of exendin-3 and exendin-4 has been proposed for the treatment of diabetes mellitus and the prevention of hyperglycemia (Eng, U.S. Patent No. 5,424,286). It has been reported that truncated exendin peptides such as exendin [9-39], a carboxyamidated molecule, and fragments 3-39 to 9-39 are potent and selective GLP-1 antagonists (Goke, et al., J. Biol. Chem., 268: 19650-55 (1993); Raufman, JP, et al., J. Biol. Chem., 266: 2897-902 (1991); Schepp, W., et al., Eur. J. Pharm., 269: 183-91 (1994); Montrose-Rafizadeh, et al., Diabetes, 45 (Suppl 2): 152A (1996)). Exendin [9-39] blocks endogenous GLP-1 in vivo, resulting in reduced insulin secretion (Wang, et al., J. Clin. Invest., 95: 417-21 (1995); D'Alessio, et al., J. Clin. Invest., 97: 133-38 (1996)). The receptor apparently responsible for the nonsulinotropic effect of GLP-1 has been cloned from rat pancreatic islet cells (Thorens, B., Proc. Nati, Acad. Sci. USA 89: 8641-8645 (1992)). Exendins and exendin [9-39] bind to the cloned GLP-1 receptor (rat pancreatic-cell GLP-1 receptor, Fehmann HC, et al., Peptides, 15 (3): 453-6 (1994); GLP-1 receptor: Thorens B, et al., Diabetes, 42 (11): 1678-82 (1993)). In cells transfected with the cloned GLP-1 receptor, exendin-4 is an agonist, that is, it increases cAMP, whereas exendin [9-39] is an antagonist, that is, it blocks the stimulatory actions of exendin 4 and GLP-1. Id. Very particularly, exendin-4 is a C-terminal amidated peptide of 39 amino acids found in the saliva of the Gila monster (Heloderma horridum), with 53% amino acid sequence homology to the GLP-1 peptide sequence. See, e.g., Eng, J., et al. "Isolation and Characterization of Exendin-4, and Exendin-3 Analogue from Heloderma suspectum Venom," J. Bio. Chem., 267: 11, p. 7402-7405 (1992), Young, A.A., et al., "Glucose-Lowering and Insulin-Sensitive Actions of Exendin-4," Diabetes, Vol. 48, p. 1026-1034, May 1999. In terms of its activity, exendin-4 is a highly specific agonist for the GLP-1 receptor, and, like GLP-1, is capable of stimulating insulin secretion. Therefore, like GLP-1, exendin-4 is considered a nonsulinotropic peptide. However, unlike GLP-1, exendin-4 has a relatively long half-life in humans, due to its resistance to dipeptidyl peptidase IV which rapidly degrades the GLP-1 sequence in vivo. Furthermore, it has been shown that, in comparison with GLP-1, exendin-4 has a stronger ability to stimulate insulin secretion, and that a lower concentration of exendin-4 can be used to obtain said stimulating activity. See, e.g., US patent. No. 5,424,286, incorporated herein by reference. Thus, the exendin-4 peptides or derivatives thereof (for examples of such derivatives, see, e.g., U.S. Patent No. 6,528,486, incorporated herein by reference, and its corresponding international application WO 01/04156 ) have a greater potential utility for the treatment of conditions that involve the deregulation of insulin levels (e.g., conditions such as diabetes) that either insulin or GLP-1. Another family of peptide hormones involved in metabolic diseases and disorders is the family of amylin peptide hormones, including amylin, calcitonin, calcitonin gene-related peptide, adrenomedullin and intermediate (also known as "AFP-6"). Amylin is a protein hormone of 37 amino acids. It was isolated, purified and chemically characterized as the main component of amyloid deposits in pancreatic islets of human type 2 diabetics (Cooper et al., Proc. Nati, Acad. Sci., USA, 84: 8628-8632 ( 1987)). The amylin molecule has two post-translational modifications: the C-terminus is amidated, and the cysteines at positions 2 and 7 are entangled to form an N-terminal loop. The sequence of the open reading frame of the human amylin gene shows the presence of the proteolytic digestion signal of the dibasic amino acid Lys-Arg, before the N-terminal codon for Lys, and the Gly prior to the proteolytic signal Lys-Arg in the CLAIMS-terminal position, a typical sequence for amidation by protein amidating enzyme, PAM (Cooper et al., Biochem. Biophys. Acta, 1014: 247-258 (1989)). It is believed that amlline regulates gastric emptying, and suppresses glucagon secretion and food intake, thus regulating the rate of appearance of glucose in the circulation. It seems to complement the actions of insulin, which regulates the rate of disappearance of glucose from the circulation and its absorption by peripheral tissues. These actions are supported by experimental findings in rodents and humans, which indicate that amylin It complements the effects of insulin in postprandial glucose control by at least three independent mechanisms, all of which affect the rate of glucose onset. First, amylin suppresses the postprandial secretion of glucagon. Compared with healthy adults, patients with type 1 diabetes do not have circulating amylin and patients with type 2 diabetes have decreased postprandial amylin concentrations. In addition, the infusion of an amylin-specific monoclonal antibody, which binds to the circulating amylin, again resulted in very high glucagon concentrations relative to the controls. These two results indicate a physiological role of endogenous amylin in the regulation of postprandial glucagon secretion. Second, amylin slows gastrointestinal motility and gastric emptying. Finally, it was shown that intrahypothalamic injections of rat amylin reduce feeding in rats and alter the metabolism of neurotransmitters in the hypothalamus. In certain studies, food intake was significantly reduced up to eight hours after the intrahypothalamic injection of rat amylin and rat CGRP. In human trials, an amylin analogue, pramlintide, has been shown to reduce weight or gain in weight. Amylin may be beneficial in treating metabolic conditions such as diabetes and obesity. Amylin can also be used to treat pain, bone disorders, gastritis, to modulate lipids, in particular triglycerides, or to affect body composition such as preferential fat loss and lean tissue reserve.

The hormone calcitonin (CT) was named for secretion in response to induced hypercalcemia and its rapid hypocalcemic effect. It is produced in neuroendocrine cells and is secreted from them in the thyroid which have since been called C cells. The best studied action of CT (1-32) is its effect on osteoclasts. The in vitro effects of CT include rapid loss of curled edges and decreased release of lysozomal enzymes. Finally, the inhibition of osteoclast functions by CT results in a decrease in bone resorption. However, neither a chronic reduction of serum CT in the case of thyroidectomy nor CT in the increased serum found in medullary thyroid cancer seems to be associated with changes in serum calcium or bone mass. Therefore, it is very likely that a primary function of CT (1-32) is to combat acute hypercalcemia in emergency situations and / or protect the skeleton during periods of "calcium stress" such as growth, pregnancy and lactation. (Reviewed in Becker, JCEM, 89 (4): 1512-1525 (2004) and Sexton, Current Medicinal Chemistry 6: 1067-1093 (1999)). Consistent with this are the recent "knockout" mouse data for calcitonin gene, which removes both calcitonin and CGRP-I peptides, which revealed that the mouse had normal levels of basal calcium-related values, but an increased calcemic response (Kurihara H, et al., Hypertens Res. February 2003; 26 Suppl: S 105-8). CT has an effect on plasma calcium levels and It inhibits the function of osteoclasts and is widely used for the treatment of osteoporosis. From the therapeutic point of view, salmon CT (sCT) appears to increase bone density and reduce fracture rates with minimal adverse effects. CT has also been used successfully over the past 25 years as a therapy for Paget's disease of the bones, which is a chronic skeletal disorder that can result in enlarged or deformed bones in one or more regions of the skeleton. CT is also widely used for its analgesic effect on bone pain experienced during osteoporosis, although the mechanism for this effect is not clearly understood. The peptide related to the calcitonin gene (CGRP) is a neuropeptide whose receptors are widely distributed in the body, including the nervous system and the cardiovascular system. This peptide appears to modulate sensory neurotransmission and is one of the most potent endogenous vasodilator peptides discovered to date. The biological effects reported for CGRP include: modulation of substance P in inflammation, nicotinic receptor activity in the neuromuscular junction, stimulation of pancreatic enzyme secretion, a reduction in gastric acid secretion, peripheral vasodilation, cardiac acceleration, neuro-modulation, regulation of calcium metabolism, osteogenic stimulation, insulin secretion, an increase in body temperature and a decrease in food intake. (Wimalawansa, Amylin, calcitonin gene-related peptide, calcitonin and ADM: a peptide superfamily.) Crit Rev Neurobiol. 1997; 11 (2-3): 167-239). An important role of the CGRP is to control blood flow to various organs by its potent vasodilator actions, as evidenced by a decrease in average blood pressure after intravenous administration of a-CGRP. Vasodilatory actions are also supported by recent analysis of homozygous CGRP knockout mice, which showed elevated peripheral vascular resistance and high blood pressure caused by increased peripheral sympathetic activity (Kurihara H, et al., Targeted disruption of ADM and aCGRP genes reveáis their distinct biological roles, Hypertens Res., February 2003; 26 Suppl: S105-8). Therefore, CGRP seems to induce vasodilatory effects, hypotensive effects and an increase in heart rate among other actions. The increased infusion of CGRP in patients with congestive heart failure has shown a sustained beneficial effect on hemodynamic functions without adverse effects, suggesting a use in heart failure. Other indications for the use of CGRP include renal failure, acute and chronic coronary artery ischemia, treatment of cardiac arrhythmia, other peripheral vascular disease such as Raynaud's phenomenon, subarachnoid hemorrhage, hypertension, and pulmonary hypertension. Premetallic toxemia of pregnancy and preterm labor is also potentially treatable. (Wimalawansa, 1997). Recent therapeutic studies include the use of CGRP antagonists for the treatment of migraine headaches. Adrenomedullin (ADM) is expressed almost ubiquitously with many more tissues that contain the peptide than those that do not. A published review of ADM, (Hinson, JP et al., Endocrine Reviews (2000) 21 (2): 138-167) details its effects on the cardiovascular system, cell growth, the central nervous system and the endocrine system, with a Nterval of biological actions that include vasodilation, cell growth, regulation of hormone secretion and natriuresis. Studies in rats, cats, sheep and humans confirm that intravenous infusion of ADM results in potent and sustained hypertension, and is comparable to that of CGRP. However, the hypotensive effect of ADM on mean blood pressure in the anesthetized rat is not inhibited by the CGRP antagonist, CGRPg 37 suggesting that this effect is not mediated by CGRP receptors. The acute or chronic administration of human ADM in rats, anesthetized, conscious or hypertensive, results in a significant decrease in total peripheral resistance accompanied by a fall in blood pressure, with a concomitant increase in heart rate, cardiac output and accident volume cardiovascular. ADM has also been proposed as an important factor in embryogenesis and differentiation and as a survival factor of apoptosis for rat endothelial cells. This is supported by mouse "knockout" studies for ADM, in which mice homozygous for ADM gene loss demonstrated defective vascular formation during embryogenesis and therefore died at mid-gestation. It was reported that mice with ADM +/- heterozygotes had high blood pressure together with susceptibility to tissue injury (Kurihara H, et al., Hypertens Res. February 2003; 26 Suppl: S105-8). ADM affects endocrine organs such as the pituitary, the adrenal glands, reproductive organs and the pancreas. The peptide seems to have a role in the inhibition of ACTH release from the pituitary. In the adrenal glands, it seems to affect the secretory activity of the adrenal cortex in both rat and human and increases adrenal blood flow, which acts as a vasodilator in the adrenal vascular bed in intact rats. It has been shown that ADM is present through the female reproductive tract and plasma levels are elevated in normal pregnancy. Studies in a rat model of preeclampsia show that ADM can reverse hypertension and decrease the mortality of offspring when given to rats during late gestation. Because it did not have a similar effect in animals in early pregnancy or in nonpregnant rats in the preeclampsia model, this suggests that ADM can play an important regulatory role in the utero-placental cardiovascular system. In the pancreas, ADM most likely plays an inhibitory role since it attenuates and delays the insulin response to an oral glucose challenge, resulting in high initial glucose levels. ADM can also affect kidney function. A bolus administered peripherally can significantly reduce average blood pressure and increase renal blood flow, glomerular filtration rate and urine flow. In some cases, there is also an increase in Na + excretion.

ADM also has other peripheral effects on the bones and on the lung. For bones, studies have supported a role beyond the cardiovascular system and fluid homeostasis and have shown that ADM acts on osteoblasts from fetal and adult rodents to increase cell growth comparable to those of known osteoblast growth factors such as transforming growth factor-β. This is clinically important since one of the main challenges in osteoporosis research is to develop a therapy that increases bone mass through osteoblastic stimulation. In the lung, ADM not only causes pulmonary vasodilation, but also inhibits histamine-induced bronchoconstriction or acetylcholine. Recent studies using aerosolized ADM to treat pulmonary hypertension in a rat model indicate that the inhalation treatment of this condition is effective, as evidenced by the fact that average pulmonary artery pressure and total lung resistance were markedly lower in rats treated with ADM than in those treated with saline. This result was achieved without an alteration in systemic blood pressure or heart rate (Nagaya N et al., Am J Physiol Heart Circ Physiol., 2003; 285: H2125-31). In healthy volunteers, it has been shown that the i.v. ADM reduces blood pressure and stimulates heart rate, cardiac output, cAMP levels in plasma, prolactin, norepinephrine and renin. In these patients, there was little or no increase in urine volume or sodium excretion observed. In patients with heart failure or renal failure chronic, ADM i.v. had effects similar to those seen in normal subjects, and also induced diuresis and natriuresis, depending on the dose administered (Nicholls, MG et al., Peptides, 2001; 22: 1745-1752). It has also been shown that treatment with experimental ADM is beneficial in arterial and pulmonary hypertension, septic shock and ischemia / reperfusion injury (Beltowski J., Pol J Pharmacol., 2004; 56: 5-27). Other indications for treatment with ADM include: peripheral vascular disease, subarachnoid hemorrhage, hypertension, preeclamous toxemia of pregnancy and preterm labor, and osteoporosis. The expression of AFP-6 (ie, intermediate) is mainly in the pituitary and gastrointestinal tract. A specific receptor for AFP-6 has not been reported; however, binding studies indicate that AFP-6 binds to all known receptors of the amylin family. It has been shown that AFP-6 increases cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competes with labeled CGRP to bind to their receptors in these cells. In published in vivo studies, the administration of AFP-6 leads to a reduction in blood pressure in both normal and spontaneously hypertensive rats, most likely by interactions with CRLR / RAMP receptors. In vivo administration in mice leads to a suppression of gastric emptying and food intake (Roh et al., J Biol Chem.20 February 2004; 279 (8): 7264-74.) It has been reported that the biological actions of hormones Peptides of the amylin family are generally mediated by binding to two closely related Type II G-protein coupled receptors (GPCRs), the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CRLR). Cloning and functional studies have shown that CGRP, ADM and amylin interact with different combinations of CTR or the CRLR and the receptor activity modifying protein (RAMP). Many cells express multiple RAMPs. It is believed that the co-expression of RAMPs and either CTR or CRLR is required to generate functional receptors for calcitonin, CGRP, ADM and amylin. The RAMP family comprises three members (RAMP1, -2, and -3), which share less than 30% sequence identity, but they have a common topological organization. The co-expression of CRLR and RAMP1 leads to the formation of a receptor for CGRP. The co-expression of CRLR and RAMP2 leads to the formation of a receptor for ADM. The co-expression of CRLR and RAMP3 leads to the formation of a receptor for ADM and CGRP. The co-expression of hCTR2 and RAMP1 leads to the formation of a receptor for amylin and CGRP. The coexpression of hCTR2 and RAMP3 leads to the formation of a receptor for amylin. Another family of additional peptide hormone involved in metabolic diseases and disorders is the leptin family. The mature form of circulating leptin is a protein of 146 amino acids that is normally excluded from the CNS by the hematocerebral barrier (BBB) and the blood-CSF barrier. See, e.g., Weigle et al., 1995. J Clin Invest 96: 2065-2070. Leptin is the afferent signal in a negative feedback loop that regulates food intake and body weight. The leptin receptor is a member of the cytokine receptor family. The anorexigenic effect of leptin depends on the homodimer binding of the Ob-Rb isoform of this receptor that encodes a long intra-cytoplasmic domain that includes several motifs for protein-protein interaction. Ob-Rb is highly expressed in the hypothalamus, suggesting that this region of the brain is an important site of the action of leptin. It has been shown that mutation of the mouse ob gene results in a syndrome that presents pathophysiology that includes: obesity, increased fat deposition in the body, hyperglycemia, hyperinsulinemia, hypothermia, and altered thyroid and reproductive functions in both obese mice ob / ob homozygous males as females (see e.g., Ingalis, et al., 1950. J Hered 41: 317-318) Therapeutic uses for leptin or leptin receptor include (i) diabetes (see, v.gr ., PCT Patent Applications WO 98/55139, WO 98/12224, and WO 97/02004), (ii) hematopoiesis (see, e.g., PCT patent applications WO 97/27286 and WO 98/18486); (iii) infertility (see, e.g., PCT patent applications WO 97/15322 and WO 98/36763) and (iv) tumor suppression (see, e.g., PCT patent applications WO 98). / 48831), each of which is incorporated herein by reference in its entirety The leptin receptor gene (OB-R) has been cloned (Access to GenBank No. AF09) 8792) and locus db has been mapped (see, e.g., Tartaglia, et al., 1995. Cell 83: 1263-1271). Several have also been identified transcriptions of the OB-R, which result from the alternative splicing. Defects in OB-R produce a syndrome in the mutant diabetic ob / ob mouse that is phenotypically identical to the ob / ob mouse (see, e.g., Ghilardi, et al., 1996. Proc. Nati. Acad. Sci. USA 93: 6231-6235). Unlike ob / ob mice, however, administration of recombinant leptin to ob / ob C57BLKS / Jm mice does not result in reduced food intake and body weight (see, e.g., Roberts and Greengerg, 1996. Nutrition Rev. 54: 41-49). Most studies related to leptin are able to report weight loss activity from the administration of recombinant leptin, fragments of leptin and / or leptin receptor variant have administered such constructions directly into the ventricles of the brain. See e.g., Weigle, et al., 1995. J Clin Invest 96: 2065-2070; Barash, et al., 1996. Endocrinology 137: 3144-3147. Other studies have shown significant weight loss activity due to the administration of leptin peptides by intraperitoneal administration (i.p.) to the test subjects. See, Grasso et al., 1997. Endocrinology 138: 1413-1418. In addition, it has been reported that leptin fragments, and most particularly an 18 amino acid fragment comprising residues taken from full length human leptin, function in weight loss, but only under direct administration through a cannula implanted to the cerebral ventricle lateral of rats. See, e.g., PCT patent application WO 97/46585, which is incorporated herein by reference in its entirety.

Another peptide hormone involved in metabolic diseases and disorders is cholecystokinin (CCK). CCK was identified as a report in 1928 from preparations of intestinal extracts for its ability to stimulate gallbladder contraction. Other biological actions of CCK have been reported since then, including stimulation of pancreatic secretion, delay of gastric emptying, stimulation of intestinal motility and stimulation of insulin secretion. See Lieverse et al., Ann. N. Y. Acad. Sci. 713: 268-272 (1994). The actions of CCK, also include as a report effects on cardiovascular function, respiratory function, neurotoxicity and attacks, cancer cell proliferation, analgesia, sleep, sexual and reproductive behaviors, memory, anxiety and behaviors mediated by dopamine. Crawley and Corwin, Pepf / cfes, 15: 731-755 (1994). Other reported effects of CCK include stimulation of pancreatic growth, stimulation of gallbladder contraction, inhibition of gastric acid secretion, release of pancreatic polypeptide and a contractile component of peristalsis. Additional reported effects of CCK include vasodilatation. Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal Tract (3rd ed., 1994); Raven Press, New York). It has been reported that injections of glucagon combinations, CCK and bombesin potentiated the inhibition of intake of condensed milk test food in non-deprived rats on the inhibitions observed with individual compounds. Hinton et al., Brain Res. Bull. 17: 615-619 (1986). It has also been reported that glucagon and CCK synergistically inhibit simulated feeding in rats. LeSauter and Geary, Am. J. Physio. 253: R217-225 (1987); Smith and Gibbs, Annals N. Y. Acad. Sci. 713: 236-241 (1994). It has also been suggested that estradiol and CCK may have a synergistic effect on satiety. Dulawa et al., Peptides 15: 913-918 (1994); Smith and Gibbs, supra. It has also been proposed that signals that are produced from the small intestine in response to nutrients therein can interact synergistically with CCK to reduce food intake. Cox, Behav. Brain Res. 38: 35-44 (1990). In addition, it has been reported that CCK induces satiety in several species. For example, it has been reported that feeding depression was caused by CCK injected intraperitoneally in rats, intraarterially in pigs, intravenously in cats and pigs, in cerebral ventricles in monkeys, rats, dogs and sheep. , and intravenously in obese and non-obese humans. See Lieverse et al., Supra. Studies from several laboratories have reported confirming the behavioral specificity of low doses of CCK on the inhibition in feeding, by comparing the response for food with the response for reinforcers that are not food in both monkeys and rats and showing that CCK induces sequence of behaviors normally observed after ingestion of food (ie, the postprandial satiety sequence). In addition, the comparison of the behavior after CCK with the behavior after the ingestion of food, alone or in combination with CCK has revealed in the form of report similarities behaviors between CCK and the ingestion of food. Crawley and Corwin, supra. It has also been reported that CCK in physiological concentrations in the plasma inhibits food intake and increases satiety in both slender and obese humans. See Lieverse et al., Supra. CCK was characterized in 1966 as a peptide of 33 amino acids. Crawley and Corwin, supra. Specific molecular variants of the CCK amino acid sequence species have been identified. It has been reported that the sequence of 33 amino acids and a truncated peptide, its C-terminal sequence of 8 amino acids (CCK-8) have been identified in pigs, rats, chickens, chinchillas, dogs and humans. It was reported that a sequence of 39 amino acids had been found in pigs, dogs and guinea pigs. It was reported that a sequence of 58 amino acids had been found in cats, dogs and humans. It was reported that frogs and turtles show a sequence of 47 amino acids homologous to both CCK and gastrin. It has been reported that the very recent human intestine contains small amounts of an even larger molecule, called CCK-83. In the rat, it has been reported that a major intermediary form has been identified, and it is called CCK-22. h, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal TraCT (3rd ed., 1994, Raven Press, New York). A non-sulfated CCK-8 and a tetrapeptide (termed CCK-4 (CCK (30-33)) have been reported in rat brain.The C-terminal pentapeptide (termed CCK-4 (CCK (29-33)) retains the structural homology of CCK, and also homology with the neuropeptide, gastrin.

C-terminal octapeptide sulfated, CCK-8, has been reported to be relatively conserved across species. It was reported that the cloning and sequence analysis of a cDNA encoding preprocolecistocinin from rat thyroid carcinoma, porcine brain, and porcine intestine revealed 345 nucleotides encoding a precursor for CCK, which is 115 amino acids and contains all the CCK sequences that were previously reported to have been isolated. Crawley and Corwin, supra. It is said that CCK is distributed throughout the central nervous system and in endocrine cells and enteric nerves of the upper small intestine. CCK agonists include CCK itself (also referred to as CCK-33), CCK-8 (CCK (26-33)), non-sulfated CCK-8, pentagastrin (CCK-5 or CCK (29-33)), and tetrapeptide, CCK-4 (CCK (30-33)). In the pancreatic CCK receptor, it was reported that CCK-8 displaced the binding with a 1000-5000 potency greater than non-sulfated CCK-8 or CCK-4, and it has been reported that CCK-8 is approximately 1000 times more potent than CCK-8 or non-sulfated CCK-4 in the stimulation of pancreatic amylase secretion. Crawley and Corwin, supra. In homogenates of the cerebral cortex, the binding of the CCK receptor is said to be displaced by non-sulfated CCK-8 and by CCK-4 at concentrations that were equimolar, 10 times or 100 times more than sulfated CCK-8. Id. It has been reported that receptors for CCK have been identified in a variety of tissues, and two major subtypes have been described: Type A receptors and type B receptors. Type A receptors have been reported in peripheral tissues including pancreas, gall bladder, pyloric sphincter and vagal afferent fibers, and in discrete areas of the brain. It has been reported that the receptor A subtype (CCKA) is selective for the sulfated octapeptide. The B receptor subtype (CCKs) has been identified throughout the brain and in the stomach, and it has been reported that it does not require sulfation or all eight amino acids. See Reidelberger, J. Nutr. 124 (8 Suppl.) 1327S-1333S (1994); Crawley and Corwin, supra. Yet another family of peptide hormones involved in metabolic diseases and disorders is the pancreatic polypeptide family ("PPF"). Pancreatic polypeptide ("PP") was discovered as a contaminant of insulin extracts and was named by its organ of origin rather than by functional significance (Kimmel et al., Endocrinology 83: 1323-30 (1968)). PP is a peptide of 36 amino acids that contains dictive structural motifs. A related peptide was subsequently discovered in gut extracts and was called peptide YY ("PYY") due to N- and C-terminal tyrosines (Tatemoto, Proc. Nati. Acad. Sci. USA 79: 2514-8 (1982) ). A third related peptide was subsequently found in brain extracts and was termed neuropeptide Y ("NPY") (Tatemoto, Proc. Nati, Acad. Sci. USA 79: 5485-9 (1982), Tatemoto et al., Nature 296: 659-60 (1982)). It has been reported that these three related peptides exert various biological effects. The effects of PP include inhibition of secretion pancreatic and relaxation of the gallbladder. The centrally administered PP produces moderate increases in nutrition that can be mediated by receptors located in the hypothalamus and brainstem (reviewed in Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22 (1998)). The release of PYY occurs after a meal. An alternate molecular form of PYY is PYY (3-36) (Eberlein et al., Peptides 10: 797-803 (1989); Grandt et al., Regul. Pept. 51: 151-9 (1994)). This fragment conutes approximately 40% immunoreactivity similar to total PYY in human and canine inteal extracts and approximately 36% PYY immunoreactivity in total plasma in a fasted state up to slightly above 50% after a food. It is apparently a digee product by dipeptidyl peptidase-IV (DPP4) of PYY. It was reported that PYY (3-36) is a selective ligand at the Y2 and Y5 receptors, which appear to be pharmacologically unique in that they prefer N-terminally truncated NPY analogues (ie, C-terminal fragments). It has been reported that peripheral administration of PYY reduces the secretion of gastric acid, gastric motility, exocrine pancreatic secretion (Yoshinaga et al., Am. J. Physio .263: G695-701 (1992); Guan et al., Endocrinology 128 : 911-6 (1991), Pappas et al., Gastroenterology 91: 1386-9 (1986)), contraction of the gallbladder and inteal motility (Savage et al., Gut 28: 166-70 (1987)). The effects of central injection of PYY on gastric emptying, gastric motility and gastric acid secretion, as seen after direct injection into or around the posterior brain / brainstem (Chen and Rogers, Am. J. Physiol. 269: R787-92 (1995); Chen et al., Regul. Pept. 61: 95-98 (1996); Yang and Tache, Am. J. Physiol. 268: G943-8 (1995); Chen et al., Neurogastroenterol. Motil. 9: 109-16 (1997)), may differ from those effects observed after peripheral injection. For example, centrally administered PYY had some opposite effects to those described here for PYY peripherally injected (3-36) in which the gastric acid secretion was ulated, not inhibited. Gastric motility was suppressed only along with ulation of HRT, but not when it was administered alone, and in fact it was ulating at higher doses through supposed interaction with PP receptors. It has been shown that PYY ulates food and water intake after central administration (Morley et al., Brain Res. 341: 200-3 (1985); Corp et al., Am. J. Physiol. 259: R317-23 (1990)). Metabolic diseases and disorders can take many forms, including obesity, diabetes, dyslipidemia, insulin resistance, cellular apoptosis, etc. Obesity and its associated disorders are common and very serious public health problems in the United States and around the world. Higher body obesity is the strongest known risk factor for type 2 diabetes mellitus, and is a strong risk factor for cardiovascular disease. Obesity is a recognized risk factor for hypertension, atherosclerosis, congestive heart failure, stroke, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovary syndrome, breast, prostate and colon cancers, and incidence increased complications general anesthesia (see, e.g., Kopelman, Nature 404: 635-43 (2000)). Reduces the duration of life and carries a severe risk of previous co-morbidity, as well as disorders such as infections, varicose veins, acanthosis nigricans, eczema, exercise intolerance, insulin resistance, hypercholesterolemia due to hypertension, cholelithiasis, orthopedic injury, and thromboembolic disease (Rissanen et al., Br. Med. J. 301: 835-7 (1990)). Obesity is also a risk factor for the group of conditions called insulin resistance syndrome, or "Syndrome X". A recent estimate of the medical cost of obesity and additional disorders is $ 150 billion worldwide. It is believed that the pathogenesis of obesity is multifactorial but the basic problem is that in obese subjects the availability of nutrients and energy expenditure does not enter the balance until there is excess of adipose tissue. Currently, obesity is an untreatable, chronic, essentially intractable metabolic disorder. A therapeutic drug useful in reducing weight of obese people could have a profound beneficial effect on their health. Diabetes is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria resulting from insufficient production or utilization of insulin. Diabetes severely affects the quality of life of large parts of populations in developed countries. Insufficient insulin production is characterized as type 1 diabetes and insufficient insulin utilization is type 2 diabetes. However, it has now been widely recognized that there are many different diseases related to diabetes that have their onset long before patients have been diagnosed with overt diabetes. Also, the effects of suboptimal control of glucose metabolism in diabetes give rise to a broad spectrum of lipid and cardiovascular related disorders. Dyslipidemia, or abnormal levels of lipoproteins in blood plasma, is a frequent occurrence among diabetics. Dyslipidemia is typically characterized by elevated triglycerides in plasma, low HDL (low density lipoprotein) cholesterol, normal to high LDL (low density lipoprotein) cholesterol levels, and increased levels of LDL (low density lipoprotein) particles. dense, small, in the blood. Dyslipidemia is one of many major contributors to the increased incidence of coronary events and deaths among diabetic subjects. Epidemiological studies have confirmed this by showing a multiple-fold increase in coronary deaths among diabetic subjects when compared to non-diabetic subjects. Several lipoprotein abnormalities have been described among diabetic subjects. Insulin resistance is the decreased ability of insulin to exert its biological action over a wide range of concentrations. In insulin resistance, the body abnormally secretes high amounts of insulin to compensate for this defect and develops a state of impaired glucose tolerance. Failing to compensate for this defective action of insulin, the concentration of glucose in the plasma inevitably increases, resulting in the clinical state of diabetes.

We begin to recognize that insulin resistance and relative hyperinsulinemia have a contributing role in obesity, hypertension, atherosclerosis and type 2 diabetes. The association of insulin resistance with obesity, hypertension and angina has been described as a syndrome, Syndrome X, which has insulin resistance as the common pathogenic link. Apoptosis is an active process of cellular self-destruction that is regulated by extrinsic and intrinsic signals that appear during normal development. It is well documented that apoptosis plays a key role in the regulation of endocrine pancreatic beta cells. There is increasing evidence that in adult mammals the beta cell mass is subject to dynamic changes to adapt insulin production to maintain euglycemia in particular conditions, such as pregnancy and obesity. The control of beta cell mass depends on a sudden balance between cell proliferation, growth and programmed cell death (apoptosis). A disturbance of this balance can lead to alteration of glucose homeostasis. For example, it should be noted that glucose intolerance develops with aging when beta cell replication rates are reduced and human autopsy studies repeatedly showed a 40-60% reduction in beta cell mass in patients with diabetes non-insulin dependent mellitus compared with non-diabetic subjects. There is generally agreement that insulin resistance is an invariable accompaniment of obesity but that normoglycemia is maintained by compensatory hyperinsulinemia until Beta cells became unable to meet the increased demand for insulin, at which point type 2 diabetes begins. Attempts to treat the multiple abnormalities associated with diabetes have prompted the administration of several anti-diabetic drugs in order to face These abnormalities in different patients. Examples of antidiabetic drugs are proteins such as insulin and insulin analogs, and small molecules such as insulin sensitizers, insulin secretagogues and appetite regulating compounds. The need to develop polypeptides to be useful in the above-described metabolic diseases, conditions and disorders persists. Accordingly, an object of the present invention is to provide hybrid polypeptides and methods for producing and using them. All documents referred to herein are incorporated by reference in the present application as set forth fully herein.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates generally to novel, selectable hybrid polypeptides, useful as agents for the treatment and prevention of metabolic diseases and disorders that can be alleviated by controlling plasma glucose levels, insulin levels, and / or insulin secretion. , such as diabetes and conditions related to diabetes. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity and diabetes mellitus of any kind, including type 1, type 2 and gestational diabetes. In one aspect of the invention, hybrid polypeptides having at least one hormonal activity are provided. The hybrid polypeptides of the invention comprise at least two bioactively bound peptide hormone modules covalently linked together, wherein at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity of a component peptide hormone. The bioactive peptide hormone modules are independently selected from: component peptide hormones, component peptide hormone fragments having at least one hormonal activity of the hormone peptide component, analogs and component peptide hormone derivatives having at least one hormonal activity of the component peptide hormones, fragments of analogues and derivatives of peptide hormones components that exhibit at least one hormonal activity of the component peptide hormones, and peptide enhancers. Compound peptide hormones of the invention include: amylin, adrenomedullin (ADM), calcitonin (CT), calcitonin gene-related peptide (CGRP), intermediate, cholecystokinin ("CCK"), leptin, peptide YY (PYY), peptide- 1 similar to glucagon (GLP-1), glucagon-like peptide 2 (GLP-2), oxyntomodulin (OXM), and exendin-4; peptidic increments of The invention includes: structural motifs of component peptide hormones that impart chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition, or other desired pharmacokinetic characteristic to the hybrid polypeptide, and structural motifs of analogs or derivatives of peptide hormones components which impart chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition or other desired pharmacokinetic characteristic to the hybrid polypeptide. In another aspect of the invention, obesity treatment or prevention methods are provided, wherein the method comprises administering a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention to a subject in need thereof. In a preferred embodiment, the subject is an obese or overweight subject. Although "obesity" is generally defined as a body mass index above 30, for purposes of this description, any subject, including those with a body mass index of less than 30, who needs or wishes to reduce body weight is included in the scope of "obese". Subjects who are insulin resistant, glucose intolerant, or have some form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes) may benefit from this method. In yet another aspect of the invention, methods are provided to reduce food intake, reduce nutrient availability, cause weight loss, treating diabetes mellitus or conditions associated with diabetes, and improving the lipid profile (including reducing LDL and triglyceride cholesterol levels and / or changing HDL cholesterol levels), wherein the methods comprise administering to a subject an effective amount of hybrid polypeptide of the invention. In a preferred embodiment, the methods of the invention are used to treat or prevent conditions or disorders that can be alleviated by reducing the availability of nutrients in a subject in need thereof, which comprises administering to said subject a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention. In another embodiment, the methods of the invention are used to treat or prevent conditions or disorders that can be alleviated by levels of plasma glucose control, insulin levels, and / or insulin secretion. In yet another embodiment, the methods of the invention are used to treat diabetes and / or conditions related to diabetes. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and diabetes mellitus of any kind, including type I, type II, and gestational diabetes, complications of diabetes (neuropathy (based on, eg, exendin-4 neurotrophic actions), neuropathic pain (based on, eg, amylin action), retathy, nephropathy, insufficient pancreatic beta-cell mass conditions ( based on, eg, islet neogenesis actions of exendin-4 and GLP-1.) The present invention also relates to compositions pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of at least one hybrid polypeptide of the invention, or a pharmaceutically acceptable salt thereof, together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or vehicles useful in the delivery of the hybrid polypeptides. These and other aspects of the invention will be more clearly understood with reference to the following preferred embodiments and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates the effect of the illustrative compounds of the invention on DIO mouse test. Figure 2 demonstrates the effect of the illustrative compounds of the invention in DIO mouse test. Figures 3A-3B demonstrate the effect of the illustrative compounds of the invention in DIO mouse test. Figure 4A-4B demonstrates the effects of illustrative compounds of the invention on the food intake test, compared to parent peptide compounds.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to novel, selectable hybrid polypeptides, useful as agents for the treatment and prevention of metabolic diseases and disorders that can be alleviated by controlling plasma glucose levels, insulin levels, and / or insulin secretion. , such as diabetes and conditions related to diabetes. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity, and diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes. In one aspect, the invention involves the modular assembly of physiologically, metabolically, and / or pharmacokinetically active peptide modules that can be selected based on "bio-activities," e.g., therapeutic efficacy, range of function, duration of action, physicochemical properties, and / or other pharmacokinetic properties. Without intending to be bound by theory, the present invention refers at least in part to a "tool box" approach, wherein the bioactive peptide hormone modules are linked in binary, tertiary, or other order combinations to create effective, novel therapeutic agents with selectable properties. The "bioactive peptide hormone modules" can be peptide hormones, fragments of peptide with hormonal activity, or structural motifs of peptide hormones that impart chemical, metabolic, and / or other desired pharmacokinetic stability. Peptide hormones may include native peptide hormones, as well as analogs and peptide hormone derivatives, as is known in the art and described herein. In one aspect of the invention, it has been found that the combination of certain physicochemical characteristics of two or more peptide hormones in a single modality can facilitate intervention at several points in a dysfunctional metabolic circuit. As such, in one aspect of the invention, rationally designated hybrid polypeptides that integrate selectable bioactivities into a single polypeptide agent are provided. In one embodiment, the selectable hybrid polypeptides of the invention may involve the use of chemically stable linkers to covalently fix the bioactive modules. In another embodiment, the selectable hybrid polypeptides of the invention may involve the use of digestible linkers, which by themselves may be or form part of a bioactive module. Again, without intending to be bound by any theory, the design of the hybrid polypeptides of the present invention may generally involve: (1) the identification, selection and pairing of bioactive peptide hormone modules for desired therapeutic efficacy and use, and (2) the covalent bond of the bioactive modules (eg, native peptide hormones, analogs or peptide hormone derivatives with hormonal activity, fragments of peptide hormone with hormonal activity, motifs) stabilizers, etc.) either directly or through a linker without loss of bioactivity of the component modules. In certain embodiments, the module selection criteria may include, but are not limited to: (a) desired in vivo efficacy for desired therapeutic or prophylactic indication; (b) optional synergism or dual action of the linked modules for multiple therapeutic or prophylactic indications; and / or (c) chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition, and / or other desired pharmacokinetic characteristics. Section headers are used here for organizational purposes only, and should not be considered as limiting in any way the material described.

Hybrid polypeptides of the invention As mentioned above, the present invention relates in part to hybrid polypeptides comprising at least two bioactive peptide hormone modules selectable from component peptide hormones described herein. The hybrid polypeptides of the present invention will generally be useful in the treatment and prevention of metabolic conditions and disorders. The hybrid polypeptides of the invention will exhibit at least one hormonal activity of a component peptide hormone, and may preferably include at least one additional bioactivity of a second component peptide hormone. In one embodiment, the hybrid polypeptides of the invention they may comprise at least two bioactive peptide hormone modules, wherein each of at least two bioactive peptide hormone modules exhibits at least one hormonal activity of a component peptide hormone. In another embodiment, the hybrid polypeptides of the invention may comprise at least two bioactive peptide hormone modules, wherein at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity of a component peptide hormone and by at least one of the bioactive peptide hormone modules imparts a chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition, and / or other desired pharmacokinetic characteristics to the hybrid polypeptide. In a preferred embodiment, the hybrid polypeptides of the invention may have comparable or superior potency in the treatment and / or prevention of metabolic conditions and disorders, compared to component peptide hormones. In another embodiment, the hybrid polypeptides of the invention may have comparable or superior potency in the treatment and / or prevention of diabetes and / or diabetes-related disorders, as compared to the component peptide hormones. Alternatively, the preferred hybrid polypeptides of the invention may exhibit improved ease of manufacture, stability, and / or ease of formulation, as compared to the component peptide hormones. Very particularly, the hybrid polypeptides of the present invention generally comprise a first module of bioactive peptide hormone covalently linked to at least one additional bioactive peptide hormone module. The bioactive peptide hormone modules can be covalently linked together in any manner known in the art, including but not limited to direct amide linkages or chemical linker groups, as described in more detail below. In one embodiment, chemical linking groups can include peptide mimetics that induce or stabilize the polypeptide conformation. The first bioactive peptide hormone module can be selected from a first component peptide hormone, and can be a peptide hormone (including native peptide hormones as well as analogs and derivatives thereof), a peptide fragment with hormonal activity (including hormone fragments) native peptides as well as analogs and derivatives thereof), or a structural motif of a peptide hormone (including native peptide hormones as well as analogs and derivatives thereof) imparting chemical stability, conformational stability, metabolic stability, receptor interaction, inhibition of protease, and / or other desired pharmacokinetic characteristic to the hybrid polypeptide. Also, the additional bioactive peptide (s) module (s) can be selected from component peptide hormones, and can be a peptide hormone (including native peptide hormones as well as analogues and derivatives thereof) ), a fragment of peptide with activity hormone (including fragments of native peptide hormones as well as analogues and derivatives thereof), or a structural motif of a hormone peptide (including native peptide hormones as well as analogs and derivatives thereof) imparting chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition, and / or other desired pharmacokinetic characteristics to the hybrid polypeptide. The first peptide hormone and the additional peptide hormone can be the same peptide hormone, they can be from the same family of peptide hormones, or they can be different peptide hormones, depending on the desired characteristics of the bioactive peptide hormone modules. As used herein, the term "bioactive" refers to one (1) biological activity in at least one hormonal pathway in vivo, or (2) modulation of therapeutic efficacy, range of function, duration of action, physicochemical properties, and / or other pharmacokinetic properties of said biological activity. Biological activity can be assessed through target hormone receptor binding assays, or through metabolic studies that monitor a physiological indication, as is known in the art and described herein. The modulation of the therapeutic efficacy, extent of function, duration of action, physicochemical properties, and / or other pharmacokinetic properties of said biological activity can be modified through changes in, eg, chemical stability, conformational stability, metabolic stability , receptor interaction, inhibition of protease, and / or other pharmacokinetic characteristics. In one embodiment, the hybrid polypeptides of the invention retain at least about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% by hundred of the biological activity of a component peptide hormone. Preferred hybrid polypeptides are those that have a potency in one of the metabolism related assays known in the art or described herein (eg, receptor binding, food intake, gastric emptying, pancreatic secretion, insulin secretion, reduction of blood glucose, weight reduction, etc.) that is equal to or greater than the power of the peptide hormone component in the same test. Alternatively, the preferred hybrid polypeptides of the invention may have ease of manufactureimproved stability, and / or ease of formulation, compared to component peptide hormones. In another embodiment, the hybrid polypeptides of the invention retain at least about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% by 100% of the biological activity of a peptide hormone native component with respect to the reduction of nutrient availability, the reduction of food intake, the effect of body weight gain, and / or the treatment and prevention of metabolic conditions and disorders . In yet another embodiment, the hybrid polypeptides of the invention exhibit at least about 110%, 125%, 130%, 140%, 150%, 200% or more of the biological activity of a native peptide hormone with respect to the reduction of nutrient availability, the reduction of food intake, the effect of weight gain on the body, and / or the treatment and prevention of metabolic conditions and disorders. In another embodiment, the hybrid polypeptides of the invention exhibit component peptide hormone receptor agonist activity.

Component peptide hormones, analogs and derivatives The component peptide hormones generally include peptide hormones useful in the treatment or prevention of metabolic diseases and disorders including: (a) the family of amylin, including amylin, adrenomedullin ("ADM"), calcitonin ("CT "), peptide related to the calcitonin gene (" CGRP "), intermediate (also known as" AFP-6") and related peptides; (b) cholecystokinin ("CCK"); (c) the leptin family, including leptin and leptin-like peptides; (d) the pancreatic polypeptide family, including pancreatic polypeptide ("PP") and peptide YY ("PYY"); and (e) incretin and incretin mimetics, including: peptide hormones derived from the proglucagon gene such as: glucagon, glucagon-like peptide-1 ("GLP-1"), glucagon-like peptide-2 ("GLP-2" ), and oxintomodulin ("OXM"); and exendins such as: exendin-3 and exendin-4. As described above, the component peptide hormones of the invention also include analogs and derivatives that retain hormonal activity of these native peptide hormones. In one embodiment, said analogs and derivatives are agonists of the target hormone receptor. By "amylin" is meant the human peptide hormone referred to as amylin and secreted from beta cells of the pancreas, and variations of species thereof, as described in the U.S. patent. No. 5,234,906, issued August 10, 1993, for "Hyperglycemic Compositions," the contents of which are incorporated herein by reference. Most particularly, amylin is a polypeptide hormone of 37 amino acids normally co-secreted with insulin by pancreatic beta cells in response to nutrient intake (see, e.g., Koda et al., Lancet 339: 1179-1180, 1992). ). In this sense, "amylin", "wild-type amylin", and "native amylin", i.e., unmodified amylin, are used interchangeably. By "adrenomedullin" or "ADM" is meant the human peptide hormone and variants of the same species. Most particularly, ADM is generated from a preprohormone of 185 amino acids through consecutive enzymatic digestion and amidation. This procedure culminates in the release of a bioactive peptide of 52 amino acids. By "calcitonin" or "CT" is meant the human peptide hormone and variants of species thereof, including salmon calcitonin ("sCT"). Most particularly, CT is a 32 amino acid peptide digested from a larger prohormone. It contains a single disulfide bond, which causes the amino terminal to take the form of a ring. The alternative splicing of the calcitonin pre-mRNA can produce an mRNA which encodes peptide related to the calcitonin gene; that peptide seems to work in the nervous and vascular systems. The calcitonin receptor has been cloned and shown to be a member of the transmembrane G protein-coupled receptor family (seven). By "peptide related to the calcitonin gene" or "CGRP" is meant the human peptide hormone and variants of species thereof, in any physiological form. By "intermediate" or "AFP-6" is meant the human peptide hormone and variants of species thereof, in any physiological form. By "cholecystokinin" or "CCK" is meant the human peptide hormone and variants of species thereof. Most particularly, CCK is a 33 amino acid sequence first identified in humans, and includes an in vivo C-terminal fragment of 8-amino acid ("CCK-8") that has been demonstrated in pigs, rats, chickens, chinchillas, dogs and humans. Therefore, the term CCK-33 will generally refer to human CCK (1-33), while CCK-8 (CCK (26-33)) will refer to the C-terminal octapeptide generically in both sulfated and unsulfated form. unless otherwise specified. In addition, pentagastrin or CCK-5 will refer to the C-terminal peptide CCK (29-33), and CCK-4 will refer to the C-terminal tetrapeptide CCK (30-33). However, as used herein, CCK will generally refer to all variations of the hormone that occur naturally, including CCK-33, CCK-8, CCK-5 and CCK-4, in sulfated form as unsulfated to unless otherwise specified.

By "leptin" is meant leptin that occurs naturally in any species, as well as biologically active isoforms D, or fragments of leptin that occurs naturally and variants of it, and combinations of leptin above. Leptin is the polypeptide product of the ob gene as described in International Patent Publication No. WO 96/05309, which is incorporated herein by reference in its entirety. Analogs and putative leptin fragments are reported in the U.S. patent. 5,521, 283, patent of E.U.A. 5,532, 336, PCT / US96 / 22308 and PCT / US96 / 01471, each of which is incorporated herein by reference in its entirety. By "PP" is meant human pancreatic polypeptide or variants of species thereof, in any physiological form. Therefore, the term "PP" includes both the full length human 36 amino acid peptide as set forth in SEQ ID NO: 1, as variations of PP species, including PP's, e.g., murine, hamster, chicken, bovine, rat and but. In this sense, "PP", "wild type PP", and "native PP", that is, unmodified PP, are used interchangeably. By "PYY" is meant human YY polypeptide or variants of species thereof, in any physiological form. Therefore, the term "PYY" includes both the full length human 36 amino acid peptide, and variations of PYY species, including PPY of eg, murine, hamster, chicken, bovine, rat and but. In this sense, "PYY" and "PYY wild-type" and "PYY native," that is, PYY unmodified, are used interchangeably. In the context of the present invention, all the modifications described with reference to the PYY analog polypeptides of the present invention are based on the native human PYY 36 amino acid sequence. By "GLP-1" is meant peptide-1 similar to human glucagon or variants of species thereof, in any physiological form. The term "GLP-1" includes GLP-1 (1-37), GLP-1 (7-37), and human GLP-1 (7-36) amide, with reference to human GLP-1 (1-37) of full length, and species variations of GLP-1, including PP's, e.g., murine, hamster, chicken, bovine, rat and but. In this sense, "GLP-1," "wild-type GLP-1," and "native GLP-1," ie, unmodified GLP-1, are used interchangeably. By "GLP-2" is meant peptide-2 similar to human glucagon or variants of species thereof, in any physiological form. Most particularly, GLP-2 is a peptide of 33 amino acids, co-secreted together with GLP-1 from the intestinal endocrine cells in the small intestine and the large intestine. By "OXM" is meant human oxintomodulin or species variants thereof in any physiological form. Most particularly, OXM is a 37 amino acid peptide that contains the 29 amino acid sequence of glucagon followed by a carboxy-terminal extension of 8 amino acids. By "exendin" is meant peptide hormone found in the saliva of the monster of Gila, an endogenous lizard of Arizona, and the Mexican pearl lizard, as well as variants of species of the same. Most particularly, exendin-3 is present in the saliva of Heloderma horridum, and exendin-4 is present in the saliva of Heloderma suspectum (Eng, J., et al., J. Biol. Chem., 265: 20259- 62, 1990; Eng., J., et al., J. Biol. Chem., 267: 7402-05 (1992)). The exendins have some sequence similarity with several members of the glucagon-like peptide family, with the highest identity, 53%, being at GLP-1 (Goke, et al., J. Biol. Chem., 268: 19650 -55 (1993)). In this sense, "exendin", "wild type exendin" and "native exendin", that is, unmodified exendin, are used interchangeably. As used herein, an "analogue" refers to a peptide whose sequence was derived from that of a base reference peptide (e.g., PP, PYY, amylin, GLP-1, exendin, etc.), including insertions, substitutions, extensions, and / or deletions of the reference amino acid sequence, which preferably has at least 50 or 55% amino acid sequence identity with the peptide of the peptide base reference, most preferably having at least 70%, 80%, 90% or 95% sequence identity of amino acids with the base peptide. In one embodiment, said analogs may comprise conservative or non-conservative amino acid substitutions (including non-natural amino acids and L and D forms). A "derivative" is defined as a molecule that has the amino acid sequence of a native or analogous reference peptide, but which further has chemical modification of one or more of its amino acid side groups, carbon a atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical portions, creating new bonds, and removing chemical portions. Modifications in the amino acid side groups include, without limitation, acylation of lysine e-amino groups, arginine N-alkylation, histidine or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the amino terminus include, without limitation, the deamino, N-lower alkyl, N-di-lower alkyl, restricted alkyls (eg, branched, cyclic, fused, adamantyl) and N-acyl modifications. Modifications of the terminal carboxyl group include, without limitation, amide, lower alkylamide, restricted alkyls (eg, branched, cyclic, fused, adamantyl), dialkylamide, and modifications of lower alkyl ether. The lower alkyl is C1-C4 alkyl. In addition, one or more side groups, or terminal groups, can be protected by protecting groups known to the peptide expert chemist. The carbon a of an amino acid can be mono- or dimethylated. By "agonist" is meant a compound that induces a biological activity of native human reference peptide, preferably having a better potency than the reference peptide, or within five orders of magnitude (more or less) of potency compared to the peptide of reference, most preferably 4, 3, 2 or 1 order of magnitude, when evaluated by measurements known in the art such as receptor / competition binding studies. In one embodiment, the terms refer to a compound that induces a biological effect similar to that of the native human reference peptide, for example, a compound (1) that has activity in the tests of food intake, gastric emptying, pancreatic secretion , or weight loss similar to the native human reference peptide, or (2) which specifically binds in a reference receptor test or in a competitive binding test with labeled reference peptide. Preferably, the agonists will be joined in such tests with an affinity greater than 1 uM, and most preferably with an affinity greater than 1-5 nM. In another embodiment, the terms refer to a compound that induces a biological effect in the treatment of diabetes or a condition or disorder related to diabetes. Said agonists may comprise a polypeptide comprising an active fragment of a reference peptide or a small chemical molecule. By "amino acid" and "amino acid residue" is meant natural amino acids, non-natural amino acids and modified amino acids. Unless stated otherwise, any reference to an amino acid, generally or specifically by name, includes reference to the stereoisomers D and L if their structure permits such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser) , threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). Non-natural amino acids include, but are not limited to homo-lysine, homo-arginine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, acid 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimetic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, acid 2,3- diaminopropionic, N-ethylglycine, N-ethylaparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methyl isoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline , norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Additional non-natural amino acids include modified amino acid residues that are chemically blocked, reversibly or irreversibly, or chemically modified on their N-terminal amino group or their side chain groups, such as, for example, amino acids or N-methylated D and L residues where the side chain functional groups are chemically modified to another functional group. For example, modified amino acids include methionine sulfoxide; methionine sulfone; aspartic acid- (beta-methyl ester), a modified amino acid of aspartic acid; N-ethylglycine, a glycine modified amino acid; or alanine carboxamide, an amino acid modified alanine. Additional residues that can be incorporated are described in Sandberg et al., J. Med. Chem. 41: 2481-91, 1998. As used herein, "5 Apa" means 5 amino-pentanoyl, "12 Ado" means 12-amino dodecanoyl, "PEG (8)" means 3,6, -dioxioctanoyl, and "PEG (13)" means 1-amino. -4,7,10-thioxa-13-tridecanaminosuccinimoyl. As described above, the native component peptide hormones are known in the art, as are their analogs and derivatives. By reference, the sequences of several native component peptide hormones are provided below in Table 1.

TABLE 1 Illustrative component peptide hormones These peptides are generally C-terminally amidated when expressed physiologically, but need not be for the purposes of the present invention. In other words, the C-terminus of these peptides, as well as the hybrid polypeptides of the present invention, can have a free OH or NH2 group. These peptides may also have other post-translational modifications. One skilled in the art will appreciate that the hybrid polypeptides of the present invention can also be constructed with an N-terminal methionine residue. Analogs of the above component peptide hormones are known in the art, but generally include modifications such as substitutions, deletions and insertions to the amino acid sequence of said component peptide hormones, and any combination thereof. The substitutions, insertions and deletions may be at the N-terminal or C-terminal end, or may be in internal portions of the component peptide hormone. In a preferred aspect, analogs of the component peptide hormones of the invention include one or more modifications of a "non-essential" amino acid residue. In the context of the invention, a "non-essential" amino acid residue is a residue that can be altered, i.e., deleted or substituted, in the native human amino acid sequence of the fragment, e.g., the peptide hormone fragment component, without canceling or substantially reducing the peptide hormone receptor agonist activity component of the resulting analogue. Preferred substitutions include conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain, or physicochemical (e.g., electrostatic, hydrogen bond, isosteric, hydrophobic) characteristics. . Families of amino acid residues having similar side chains are known in the art, these families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g., aspartic acid, acid) glutamic), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), ß-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatics (e.g., tyrosine, phenylalanine, tryptophan, histidine). The present invention also relates to derivatives of the component peptide hormones. Such derivatives include component peptide hormones and analogs thereof conjugated to one or more water soluble polymer molecules, such as polyethylene glycol ("PEG") or fatty acid chains of various lengths (e.g., stearyl, palmitoyl, octanoyl) , etc.), or by the addition of polyamino acids, such as poly-his, poly-arg, poly-lys and poly-ala. Modifications to the component peptide hormones or analogs thereof may also include small molecule substituents, such as short alkyl groups and restricted alkyls (e.g., branched, cyclic, fused), adamantyl) and aromatic groups. Water-soluble polymer molecules will preferably have a molecular weight ranging from about 500 to about 20,000 Daltons. Said polymer conjugations and small molecule substituent modifications can occur individually at the N- or C-terminal or on the side chains of amino acid residues within the sequence of the hybrid polypeptides. Alternatively, there may be multiple derivation sites along the hybrid polypeptide. The replacement of one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine can provide additional sites for referral See, e.g., US patents. Nos. 5,824,784 and 5,824,778. Preferably, the hybrid polypeptides can be conjugated to one, two or three polymer molecules. The water-soluble polymer molecules are preferably coated to an amino, carboxyl or thiol group, and can be linked with N- or C-terminal, or on the side chains of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, the water soluble polymer molecules can be linked with diamine and dicarboxylic groups. In a preferred embodiment, the hybrid polypeptides of the invention are conjugated to one, two or three molecules of PEG through an amino epsilon group on an amino acid lysine. Derivatives of the invention also include component peptide hormones or analogs with chemical alterations to one or more amino acid residues. Such chemical alterations include amidation, glycosylation, acylation, sulfation, phosphorylation, acetylation and cyclization. Chemical alterations can occur individually at the N- or C-terminal or in the side chains of amino acid residues within the sequence of the PPF hybrid polypeptides. In one embodiment, the C-terminus of these peptides may have a free -OH or -NH2 group. In another embodiment, the N-terminus can be blocked with a α-butyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, an octylglycine group (G (Oct )), or an 8-aminoocthanic acid group. In a preferred embodiment, the cyclization It can be through the formation of disulfide bridges. Alternatively, there may be multiple sites of chemical alteration along the hybrid polypeptide.

The amylin family As described above, the component peptide hormones useful in the present invention include peptide hormones of the amylin family, including amylin, adrenomedullin ("ADM"), calcitonin ("CT"), peptide related to calcitonin gene ("CGRP"), intermediate (also known as "AFP-6") and related peptides. Peptide hormones of the native amylin family are known in the art, as are functional analog peptides and derivatives. Certain preferred native peptides, analogs and peptide derivatives are described herein, however, it should be recognized that any peptides of the known amylin family that exhibit hormonal activity known in the art can be used in conjunction with the present invention. Any analog or amylin derivative known in the art can be used in conjunction with the present invention. In one embodiment, the amylin analogs and derivatives have at least one native amylin hormone activity. In certain embodiments, the amylin analogues are agonists of a receptor whose native amylin is capable of specifically binding. Analogs and derivatives of amylin include those described in US 2003/0026812 Al, which is incorporated herein by reference.

Illustrative amylin analogues include: As is known in the art, said amylin analogues are preferably amidated, but within the context of the present invention, they may optionally be in the form of an acid unless otherwise specified. Any analog or derivative of ADM known in the art can be used in conjunction with the present invention. In one embodiment, ADM analogs and derivatives have at least one native ADM hormone activity. In certain embodiments, the ADM analogues are agonists of a receptor whose native ADM is capable of specifically binding. Any analog or derivative of CT known in the art can be used in conjunction with the present invention. In one embodiment, analogs and CT derivatives have at least one native CT hormonal activity. In certain embodiments, CT analogs are agonists of a receptor whose native CT is capable of specifically binding. Analogs and derivatives of Preferred CTs include those described in the US patent. Nos. 4,652,627; 4,606,856; 4,604,238; 4,597,900; 4,537,716; 4,497,731; 4,495,097; 4,444,981; 4,414,149; 4,401, 593; and 4,397,780, which are incorporated herein by reference. Illustrative CT analogues include: As is known in the art, such CT analogues are preferably amidated, but within the context of the present invention, they may optionally be in the acid form unless otherwise specified. Any analog or derivative of CGRP known in the art can be used in conjunction with the present invention. In one embodiment, CGRP analogs and derivatives have at least one native CGRP hormone activity. In certain embodiments, the CGRP analogs are agonists of a receptor whose native CGRP is capable of specifically binding. Preferred CGRP analogs and derivatives include those described in the U.S. Patents. Nos. 4,697,002; and 4,687,839, which are incorporated herein by reference.

Illustrative CGRP analogs include: Any analog or derivative of AFP-6 known in the art can be used in conjunction with the present invention. In one embodiment, analogs and derivatives of AFP-6 have at least one hormonal activity of native AFP-6. In certain embodiments, analogs of AFP-6 are agonists of a receptor whose native AFP-6 is capable of specifically binding. Analogs and derivatives of AFP-6 include those described in WO 2003/022304, which is incorporated herein by reference. Illustrative AFP-6 analogues include: SEC ID: 120 TQAQLLRVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 121 TQAQLLRVGCDTATCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 122 TQAQLLRVGMVLGTMQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 123 TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVEPSSPHSY 124 TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQESAPVEPSSPHSY 125 TQAQLLRVGCVLGTCQVQNLSHRLWQL-RQDSAPVDPSSPHSY 126 TQAQLLRVGCVLGTCQVQNLSHRLWQL-DSAPVDPSSPHSY As is known in the art, such analogues of AFP-6 are preferably amidated, but within the context of the present invention, they may optionally be in the acid form unless otherwise specified.

The CCK family of CCKs, including hCCK and species variants, and various analogs thereof are known in the art. Generally, CCK has a sequence of 33 amino acids first identified in humans, and includes an 8-amino acid C-terminal fragment in vivo ("CCK-8") that has been demonstrated in pigs, rats, chickens, chinchillas, dogs and humans . Other species variants include a sequence of 39 amino acids found in pigs, dogs and guinea pigs, and one of 58 amino acids found in cats, dogs and humans, and a sequence of 47 amino acids homologous to both CCK and gastrin. The C-terminal octapeptide sulphated sequence (CCK-8) is relatively conserved across species, and may be the minimum sequence for biological activity in the periphery of rodents. Therefore, the term CCK-33 will generally refer to human CCK (1-33), while CCK-8 (CCK (26-33)) will refer to the C-terminal octapeptide generically in both the sulfated and unsulfated form unless otherwise specified. In addition, pentagastrin or CCK-5 will refer to the C-terminal peptide CCK (29-33), and CCK-4 will refer to the C-terminal CCK tetrapeptide (30-33). The type A receptor subtype (CCKA) has been reported to be selective for the sulfated octapeptide. The type B receptor subtype (CCKB) has been identified throughout the brain and in the stomach, and it is reported that it does not require sulfation or all eight amino acids. Various methods of selective in vivo and in vitro determination for CCK analogs are known in the art. Examples include in vivo tests involving the contraction of the dog and guinea pig gallbladder after rapid intravenous injection of the compound to be tested for CCK-like activity, and measurements of in vitro tests using rabbit gallbladder strips. See Walsh, "Gastrointestinal Hormones," In Physiology of the Gastrointestinal TraCT (3rd ed., 1994, Raven Press, New York). Certain CCKs and preferred CCK analogs with activity of CCK include: As is known in the art, said CCK peptides are preferably amidated, but within the context of the present invention, they may optionally be in the acid form unless otherwise specified.

The leptin family The peptide hormones component of the leptin family useful in the present invention also include peptide hormones which are components of the leptin family. Peptide hormones component of the native leptin family are known in the art, such as analogs and functional peptide derivatives, certain preferred native peptides, analogs and peptide derivatives are described herein, however it is recognized that any peptides of the known amylin family which exhibit hormonal activity known in the art may be used in conjunction with the present invention. Any analog or derivative of leptin known in the art can be used in conjunction with the present invention. In one embodiment, the leptin analogs and derivatives have at least one native leptin hormone activity. In certain embodiments, the leptin analogues are agonists of a receptor whose native leptin is capable of specifically binding. Preferred analogs and leptin derivatives include those described in, e.g., WO 2004/039832, WO 98/55139, WO 98/12224 and WO 97/02004, all of which are incorporated herein by reference. Illustrative leptin analogs include those wherein the amino acid at position 43 is substituted with Asp or Glu; position 48 is replaced with Ala; position 49 is replaced with Glu, or absent; position 75 is replaced with Ala; position 89 is replaced with Leu; position 93 is replaced with Asp or Glu; position 98 is replaced with Ala; position 117 is replaced with Ser; position 139 is replaced with Leu; position 167 is replaced with Ser, and any combinations thereof. Certain analogs of CCKs and CCK with CCK activity include: The PPF family The component peptide hormones useful in the present invention also include PPF peptide hormones, including PP and PYY. Peptide hormones of native PPF are known in the art, as are analogs and functional peptide derivatives. Certain preferred native peptides, analogs and peptide derivatives are described herein, however it should be recognized that any peptides of the known amylin family that exhibit hormonal activity known in the art can be used in conjunction with the present invention. Any analog or derivative of PPF known in the art can be used in conjunction with the present invention. In one modality, the analogues and PPF derivatives have at least one hormonal activity of a native PPF polypeptide. In certain embodiments, the PPF analogues are agonists of a receptor whose native PPf polypeptide is capable of specifically binding. Preferred analogs and PPF derivatives include those described in WO 03/026591 and WO 03/057235, which are incorporated herein by reference in their entirety. In one embodiment, preferred PPF analogs and derivatives having at least one hormonal PPF activity generally comprise at least two PYY motifs including a poly-proline motif and C-terminal tail motif. Said analogs are generally described in the provisional application of E.U.A. No. 60 / 543,406 filed February 11, 2004, which is incorporated herein by reference. Other preferred PPF analogues are described in PCT / IJS05 / [XXXXX], entitled "Pancreatic Polypeptide Family Motifs and Polypeptides Comprising the Same", Attorney Case 18528. 832, filed concurrently with, the content of which is incorporated herein by reference. By means of antecedents, the investigation has suggested that the differences in receptor affinities of receptor Y correlate with secondary and tertiary structural differences. See, e.g., Keire et al., Biochemistry 2000, 39, 9935-9942. PYY of native swine has been characterized as that which includes two C-terminal helical segments from residues 17 to 22 and 25 to 33 separated by a crepe at residues 23, 24 and 25, a centered round around residues 12-14 and the N-terminal folded close to defects 30 and 31. In addition, the pig PYY Full length has been characterized as that which includes the folding of PP, stabilized by hydrophobic interactions between residues in the N- and C-terminals. See id. A "PYY motif" is generally a structural, primary, secondary or tertiary component of a polypeptide of the native PP family that is critical for biological activity, i.e., the biological activity is substantially decreased in the absence or disturbance of the motif. Preferred PYY motifs include the N-terminal polyproline type II motif of a type II polypeptide of the native PP family, the β-turn motif of the type II polypeptide of the native PP family, the a-helical motif in the C-terminal end of the polypeptide of the native PP family, and the C-terminal tail motif of the polypeptide of the native PP family. Most particularly, in the N-terminal polyproline region, the amino acids corresponding to residues 5 and 8 of a polypeptide of the native PP family are generally conserved as a proline. The ß-type II return motif will generally include amino acids corresponding to residues 12-14 of a polypeptide of the native PP family. The a-helical motif can generally extend from amino acids corresponding to about residue 14 of a polypeptide of the native PP family to any point up to and including the C-terminus, provided that the a-helical motif includes a sufficient number of amino acid residues such that an a-helical turn is formed in solution. The a-helical motif can also include substitutions, insertions and amino acid deletions to the sequence of the family of native PP, provided that the a-helical turn is still formed in solution. The C-terminal tail motif generally includes amino acids corresponding to approximately the last 10 residues of a polypeptide of the native PP family, most preferably the last 7, 6 or 5 residues of a polypeptide of the native PP family, and most preferably the residues of amino acids 32-35. Preferred PYY analogs include those with deletions, insertions and internal substitutions in areas of the molecule of PYY not corresponding to the polyproline motif and / or the C-terminal tail motif. For example, internal deletions are considered in positions 4, 6, 7, 9 0 10.

Incretin and incretin mimetics The component peptide hormones useful in the present invention also include GLP-1 peptide hormones. Native GLP-1 peptide hormones, including GLP-1 (1-37), GLP-1 (7-37) and GLP-1 (7-36) amide, are known in the art, as are the analogues and derivatives of functional peptides. As used herein, GLP-1 refers to all the native forms of GLP-1 peptide hormones. Certain native peptides, analogs and preferred peptide derivatives are described herein, however it should be recognized that any known GLP-1 peptides exhibiting hormonal activity known in the art can be used in conjunction with the present invention.

Any GLP-1 peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, the GLP-1 peptide analogs and derivatives have at least one hormonal activity of a native GLP-1 peptide. In certain embodiments, the GLP-1 peptide analogs are agonists of a receptor to which a native GLP-1 peptide is capable of specifically binding. Preferred GLP-1 peptide analogs and derivatives include those described in, e.g., WO 91/11457, which is incorporated herein by reference. GLP-1 analogs known in the art include: As is known in the art, said GLP-1 analogues can preferably be amidated, but within the context of the present invention, they can be optionally in the acid form unless otherwise specified. Other analogs and derivatives of GLP-1 are described in the patent of E.U.A. No. 5, 545,618 which is incorporated herein by reference. A preferred group of GLP-1 analogs and derivatives include those described in the U.S.A. No. 6,747,006, which is incorporated herein by reference in its entirety. The use in the present invention of a molecule described in the patent of E.U.A. No. 5,188,666, which is expressly incorporated by reference. Another group of molecules for use in the present invention includes compounds described in the U.S.A. No. 5,512,549, which is expressly incorporated herein by reference. Another preferred group of GLP-1 compounds for use in the present invention is described in WO 91/11457, which is incorporated herein by reference. The component peptide hormones useful in the present invention also include GLP-2 peptide hormones. The native GLP-2 peptide hormones, e.g., rat GLP-2 and its homologs including beef GLP-2, porcine GLP-2, degu GLP-2, bovine GLP-2, guinea pig GLP-2 , Hamster GLP-2, human GLP-2, rainbow trout GLP-2 and chicken GLP-2 are known in the art, as are functional peptide analogs and derivatives. Certain native peptides, analogs and derivatives of Preferred peptides are described herein, however, it should be recognized that any known GLP-2 peptides exhibiting hormonal activity known in the art can be used in conjunction with the present invention. Any GLP-2 peptide analog or derivative known in the art can be used in conjunction with the present invention. In one embodiment, the GLP-2 peptide analogs and derivatives have at least one hormonal activity of a native GLP-2 peptide. In certain embodiments, the GLP-2 peptide analogs are agonists of a receptor to which a native GLP-2 peptide is capable of specifically binding. Preferred GLP-2 peptide analogs and derivatives include those described in, e.g., E.U.A. Ser. No. 08 / 669,791 and PCT application: PCT / CA97 / 00252, both of which are incorporated herein by reference. Specific GLP-2 analogs known in the art include: Rat GLP-2 or human altered at position 2 to confer DPP-IV resistance using a Gly instead of an Ala. The component peptide hormones useful in the present invention also include peptide hormones of oxyntomodulin (OXM). Native OXM peptide hormones are known in the art, as are functional analogs and peptide derivatives. Certain preferred native peptides, analogs and peptide derivatives are described herein, however it is recognized that any known OXM peptides that exhibit hormonal activity known in the art can be used in conjunction with the present invention. Any analog or derivative of OXM peptide known in the The technique can be used in conjunction with the present invention. In one embodiment, the OXM peptide analogs and derivatives have at least one hormonal activity of a native OXM peptide. In certain embodiments, the OXM peptide analogs are agonists of a receptor to which a native OXM peptide is capable of specifically binding. The component peptide hormones useful in the present invention also include exendin peptide hormones. Exendin native peptide hormones are known in the art, as are functional peptide analogs and derivatives. Certain native peptides, analogs and preferred peptide derivatives are described herein, however it should be recognized that any known exendin peptides that exhibit hormonal activity known in the art can be used in conjunction with the present invention. Any analogue or derivative of exendin peptide known in the art can be used in conjunction with the present invention. In one embodiment, the exendin peptide analogs and derivatives have at least one hormonal activity of a native exendin peptide. In certain embodiments, the exendin peptide analogs are agonists of a receptor to which a native exendin peptide is capable of specifically binding. Preferred exendin analogs include: As is known in the art, said exendin analogues may preferably be amidated, but within the context of the present invention, they may optionally be in the acid form unless otherwise specified. Exemplary additional illustrative exemplified derivatives and derivatives are disclosed in the PCT application Serial No. PCT / US98 / 16387 filed on August 6, 1998, entitled "Novel Exendin Agonist Compounds", which claims the benefit of the US patent application. Ser. No. 60 / 055,404, filed August 8, 1997, both of which are incorporated herein by reference. Other exendin analogs and derivatives are described in the PCT application Serial No. PCT / US98 / 24210, filed on November 13, 1998, entitled "Novel Exendin Agonist Compounds", which claims the benefit of the provisional application of E.U.A. No. 60 / 065,442 filed on November 14, 1997, both of which are incorporated herein by reference. Other additional exendin analogs and derivatives are disclosed in the PCT application Serial No. PCT / US98 / 24273, filed on November 13, 1998, entitled "Novel Exendin Agonist Compounds", which claims the benefit of the provisional application of E.U.A. No. 60 / 066,029 filed November 14, 1997, both of which are incorporated herein by reference. Other analogues and additional exendin derivatives are described in PCT Application Series No. PCT / US97 / 14199, filed August 8, 1997, entitled "Methods for Regulating Gastrointestinal Activity," which is a continuation in part of the US patent application. Ser. No. 08 / 694,954 filed August 8, 1996, both of which are incorporated herein by reference. Other additional exendin analogs and derivatives are described in the PCT application Serial No. PCT / US98 / 00449, filed on January 7, 1998, entitled "Use of Executives and Agonists Thereof for the Reduction of Food Intake", which claims priority to the provisional US application No. 60 / 034,905 filed January 7, 1997, both of which are incorporated herein by reference. Other analogues and additional exendin derivatives are described in US 2004/0209803 A1, filed December 19, 2003, entitled "Compositions for the Treatment and Prevention of Neuropathy", which is incorporated herein by reference.

Bioactive Peptide Hormone Modules As described above, the hybrid polypeptides of the present invention generally comprise at least two bioactive peptide hormone modules covalently linked together. The bioactive peptide hormone modules can be: (a) native component peptide hormones, (b) analogous or derived from peptide hormones, native components that retain hormonal activity, (c) fragments of peptide hormones, native components that retain hormonal activity, (d) fragments of analogues or derivatives of peptide hormones, native components that retain hormonal activity, (e) structural motifs of peptide hormones, native components that impart chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition, and / or another desired pharmacokinetic characteristic to the hybrid polypeptide; or (f) structural motifs of native component peptide hormone analogues or derivatives that impart chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition, and / or other desired pharmacokinetic characteristics to the hybrid polypeptide. The structural reasons for (e) and (f) will collectively be referred to herein as "peptide enhancers". Preferred bioactive peptide hormone modules include native peptide hormones selected from: amylin, ADM, CT, CGRP, intermediate, CCK (1-33), CCK-8, leptin, PYY (1-36), PYY (3-36) , GLP-1 (1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, exendin-3 and exendin-4. Other preferred bioactive peptide hormone modules include analogs and derivatives of a component peptide hormone selected from: amylin, ADM, CT, CGRP, intermediate, CCK, leptin, PYY (1-36), PYY (3-36), GLP-1 (1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, exendin-3, and exendin-4, wherein the analog or derivative has at least one Hormone activity of the component peptide hormone. The analog may comprise one or more insertions, deletions or substitutions of the amino acid sequence of the component peptide hormone, and the The derivative may comprise one or more chemical modifications of an amino acid residue of a component analog or peptide hormone, as described more fully herein and known in the art. More specifically, the analogs and derivatives may be selected from anyone previously described and / or known in the art. Particularly preferred analogs and derivatives having at least one hormonal activity useful as bioactive peptide hormone modules of the invention include the following: As is known in the art, said peptide compounds may be preferably amidated, but within the context of the present invention, they may optionally be in the acid form unless otherwise specified. Other additional preferred bioactive peptide hormone modules include fragments of a component peptide hormone selected from: amylin, ADM, CT, CGRP, intermediate, CCK, leptin, PYY (1-36), PYY (3-36), GLP-1 (1-37), GLP-1 (7-37), GLP-1 (7 -36), GLP-2, OXM, exendin-3, and exendin-4, wherein the fragment exhibits at least one hormonal activity of the component peptide hormone. Other additional preferred bioactive peptide hormone modules include fragments of analogs or derivatives of a component peptide hormone selected from: amylin, ADM, CT, CGRP, ethermedin, CCK, leptin, PYY (1-36), PYY (3-36) , GLP-1 (1-37), GLP-1 (7-37), GLP-1 (7-36), GLP-2, OXM, exendin-3, and exendin-4, where the fragment presents less a hormonal activity of the component peptide hormone. Again, the analog can comprise one or more insertions, deletions or substitutions of the amino acid sequence of the component peptide hormone, and the derivative can comprise one or more chemical modifications of an amino acid residue of a component analog or peptide hormone, as describes more completely here and known in the art.

Certain preferred fragments that exhibit at least one hormonal activity include the following. However, it is understood that combinations of the above-described analogs and derivatives taken together with fragments known in the art are contemplated, including the preferred fragments described below.

Amiline: amylin (1-36), amylin (1-35), amylin (1-20), amylin (1-18), amylin (1-17), amylin (1-16), amylin (1-15) , amylin (1-7) CT: CT (8-32), CT (8-27), CT (8-26), CT (8-10), CT (18-26), CT (18-27) AFP-6: AFP-6 (18-27) CCK: CCK-8, CCK-5, CCK-4 Leptin: leptin (22-167), leptin (56-73) PYY: PYY (1-35), PYY (1-30), PYY (1-25), PYY (1-15), PYY (1-10), PYY (2-36), PYY (3-36), PYY (4-36), PYY (5-36) GLP-1 GLP-1 (7-37), GLP-1 (7-36), GLP-1 (7-35) Exendin exendin-4 (1-27), exendin-4 (1-28), exendin-4 (1 -29), exendin-4 (1-30) or longer Again, as is known in the art, said peptide compounds may be preferably amidated, but within the context of The present invention may optionally be in the form of unless otherwise specified. In addition, the above preferred fragments may be combined with any of the analogs or derivatives described herein or known in the art. For example, preferred analog fragments may include 5Ala, 14Leu, 25Phe-exendin-4 (1-28), 14Leu, 25Phe-exendin-4 (1-27), 5Ala, 14Leu, 25Phe-exendin-4 (1-28). ), 14Leu, 25Phe- exendin-4 (1-27), or any other combinations of the described fragments, analogs and derivatives. Other preferred bioactive peptide modules include "peptide enhancer", ie, structural motifs of component peptide hormones (including analogs and derivatives thereof) that impart desired chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition. , and / or other pharmacokinetic characteristic to the hybrid polypeptide. Illustrative peptidic enhancers include the following. Again, it is understood that combinations of the above-described analogs and derivatives taken together with the following bioactive peptide modules are contemplated. For example, the last six amino acid residues of analogs and peptide hormone derivatives of the amylin family known in the art and / or described above are also contemplated as preferred bioactive peptide modules.

Exendin-4 exendin-4 (31-39), exendin-4 (32-39), exendin-4 (33-39), exendin-4 (34-39), exendin-4 (35-39), exendin- 4 (36-39), exendin-4 (37-39), exendin-4 (38-39), exendin-4 (39) Considerations for selection of peptide module, separators and linker groups The hybrid polypeptides of the present invention generally comprise at least two bioactive peptide hormone modules of the invention, wherein at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity. The bioactive peptide hormone module having at least one hormonal activity may be located at the N-terminal end of the hybrid polypeptide, the C-terminal end of the hybrid polypeptide, or in the case where the hybrid polypeptide comprises more than two modules of bioactive peptide hormone, may be located in the inner portion of the hybrid polypeptide. In certain embodiments, it may be preferable to locate the bioactive peptide hormone module that exhibits at least one hormonal activity such that the C-terminal end of the bioactive peptide hormone module is aminated. The amidation of the C-terminal end of the bioactive peptide hormone module can be achieved by locating the module at the C-terminal end of the hybrid peptide, or by configuring the module in the C-terminal-to-N-terminal direction at the N-terminus. terminal of the hybrid polypeptide. In both configurations, the C-terminal end of the module Bioactive peptide hormone is available for amidation. The specific component peptide hormones wherein C-terminal amidation may preferably include peptide hormones of the amylin family, CCK, PYY, hGLP-1 (7-36) and hGLP-2. Peptide-specific component hormones wherein C-terminal amidation is not necessarily preferred (indicated otherwise, wherein elongation at the C-terminus of the module is easily tolerated) include exendin-4, exendin-4 (1-28 ), GLP-1 (7-37), frog GLP-1 (7-36), and frog GLP-2. However, if these component peptide hormones are located at the C-terminal end of the hybrid polypeptide, they can still be optionally amidated, and in fact preferably they can be optionally amidated. The bioactive peptide hormone modules can be covalently linked in any manner known in the art. Stable links can be used, or a digestible link can be used. In one embodiment, the carboxy of a first module can be directly linked to the amino of a second module. In another modality, linkers to linked modules can be used. In addition, if desired, back separators or inductors known in the art can be used to stabilize the bond. By way of example, wherein the amidation of the C-terminal end of the N-terminally located bioactive peptide hormone module is not desired, the module can be attached to a second module directly, or using any appropriate linker group known in the art, such as, an alkyl; PEG; amino acid, e.g., Lys, Glu, p-Ala; polyamino acids, eg, poly his, poly-arg, poly-lys, poly-ala, Gly-Lys-Arg (GKR), etc .; bifunctional linker (see, e.g., Pierce catalog, Rockford, II); aminocaproyl ("Acá"), (3-alanyl, 8-amino-3,6-dioxaoctanoyl, or other digestible linker or non-digestible linker known in the art Where amidation of the C-terminus of the hormone module is desired N-terminally located bioactive peptide, the module can again be linked to a second module using any appropriate linker group known in the art More specifically, in the case of a bioactive peptide hormone module having at least one activity Hormone has been configured in the C-terminal- to -N-terminal orientation, resulting in an amino-amino bond, preferred linker groups include dicarboxylic acids, alkyls, PEGs, and amino acids such as Lys, Cys and Glu. mentioned above, the hybrid polypeptides may also preferably include a separator to further stabilize the binding of the bioactive peptide hormone modules. or return inductor known in the art. By way of example, the referred ß-mimetics referred to include mimic A and mimic B illustrated below, also the di-peptides Ala-Aib and Ala-Pro.

A mimic B mimic Illustrative Combinations and Specific Modalities Illustrative combinations of bioactive peptide hormone modules to form the hybrid polypeptides of the invention include combinations of two or more bioactive peptide hormone modules selected from: native peptide hormones, analogs and peptide hormone derivatives exhibiting at least a hormonal activity, fragments of native peptide hormones exhibiting at least one hormonal activity, fragments of analogs and derivatives of hormone peptides exhibiting at least one hormonal activity, and peptidic enhancers, with the proviso that at least one module present at least one hormonal activity. The hybrid polypeptides of the invention will include at least two bioactive peptide hormone modules, wherein each module is composed of component peptide hormones. In the context of the present invention, the peptide hormones component of the hybrid polypeptide may be the same or different, provided that at least two of the component peptide hormones are different. In a preferred embodiment, at least two of the component peptide hormones are from different families of peptide hormone, e.g., the amylin family, CCK, the leptin family, PPF, the proglucagon family, and the family of exendin In certain embodiments, the hybrid polypeptides of the invention may comprise two or more modules having at least one hormonal activity For example, the hybrid polypeptide may comprise a fragment of a first peptide hormone or analog having at least one hormonal activity covalently linked to a fragment of at least one additional peptide hormone analogue. The additional fragment (s) may optionally have at least one hormonal activity. The first peptide hormone may be the same as or different from the additional peptide hormone (s), provided that at least one of the additional peptide hormones is different from the first peptide hormone, and the first Hormone activity may be the same or different from the optional additional hormonal activity. In other embodiments, the hybrid polypeptides of the invention may comprise one or more modules that exhibit at least one hormonal activity in combination with one or more peptidic enhancer modules. For example, a fragment of a first peptide hormone having at least one hormonal activity can be covalently linked to a peptide enhancer, or a fragment of a first peptide hormone having at least one hormonal activity can be covalently linked to a second one. peptide hormone that has at least one hormonal activity, which is in turn linked to a peptidic enhancer. Alternatively, a peptide enhancer may be located between two peptide hormone modules as a stabilizing spacer. Again, the first peptide hormone may be the same or different from the second peptide hormone, and the first hormonal activity it may be the same or different from the second hormonal activity. In another embodiment, the hybrid polypeptides of the invention may comprise two, three, four or more bioactive peptide hormone modules. Illustrative combinations include a module with a hormonal activity in combination with one, two or three peptidic enhancers; two modules with a hormonal activity in combination with one or two peptidic enhancers; three modules with a hormonal activity in combination with a peptidic enhancer, etc. The component peptide hormones are preferably selected from amylin, adrenomedullin, calcitonin, calcitonin gene-related peptide, ethermedin, cholecystokinin, peptide YY leptin, glucagon-like peptide-1, glucagon-like peptide-2, oxintomodulin or exendin-4. Most particularly, combinations of preferred modules include those involving combinations of exendin, amylin and PYY as the component peptide hormones. Particular combinations include exendin-4 / PYY and combinations of PYY / exendin-4, with and without separators or linking groups. Other combinations include exendin / amylin and combinations of amylin / exendin, with and without separators or linking groups. Still other combinations include amylin / PYY and combinations of PYY / amylin, with and without separators or linking groups. In one aspect, preferred module combinations include those that involve a first module comprising exendin-4, a exendin-4 fragment having at least one hormonal activity, an analog or derivative of exendin-4 having at least one hormonal activity, or a fragment of an exendin-4 analogue having at least one hormonal activity in combination with at least one additional module of bioactive peptide hormone. In one embodiment, the first module is linked to one, two or three additional bioactive peptide hormone modules. In preferred embodiments, a first module comprising an exendin-4 peptide is linked to a second bioactive peptide hormone module comprising an amylin peptide having at least one hormonal activity. In another embodiment, the second module is further linked to a third module of bioactive peptide hormone comprising a calcitonin peptide having at least one hormonal activity. In yet another embodiment, the third module can be further linked to a fourth module of bioactive peptide hormone comprising a peptide enhancer selected from amylin peptides. In one embodiment, the first module can be located at the C-terminal end of the hybrid polypeptide. Alternatively, the first module can be located at the N-terminus of the hybrid polypeptide. In certain modalities, separators or linkers such as ßAla can be inserted if desired to link the modules. Preferred exendin-4 peptides include: exendin-4, exendin-4 (1-27), exendin-4 (1-28), 14Leu, 25Phe-exendin-4 (1-28), and 5Ala, 4 Leu, 25Phe-exendin-4 (1-28). Preferred amylin peptides exhibiting at least one hormonal activity include amylin, fragments of amylin such as amylin (1-17), amylin (1-16), amylin (1-15), and amylin (1-7), and amylin analogs such as pramlintide, 2Ala-h-amylan, 27Ala-h-amylin, and fragments thereof. Preferred calcitonin peptides having at least one sCT hormone activity, sCT fragments such as sCT (8-10), sCT (8-27), and calcitonin analogs such as 18Arg-sCT, 14Gln, 18Arg-sCT, 14Gln, 18Arg-sCT, and fragments thereof. Preferred amylin peptidic enhancers include amylin (32-37), amylin (33-37), and amylin (34-37), and analogs thereof. Amylamine / sCT combinations useful in connection with the present invention include those described in PCT / US05 / [XXXXX], Amylin Family Agonist, attorney-in-fact 18528.835, filed concurrently with this, which is incorporated herein by reference. In one aspect, preferred module combinations include those involving a first module comprising exendin-4, a fragment of exendin-4 having at least one hormonal activity, an analog or derivative of exendin-4 having at least one hormonal activity, or a fragment of an exendin-4 analogue having at least one hormonal activity in combination with a peptide enhancer. Preferred exendin-4 compounds include: exendin-4, exendin-4 (1-27), exendin-4 (1-28), 14Leu, 25Phe-exendin-4 (1-28), and 5Ala, 14Leu, 25Phe -exendin-4 (1-28). Preferred peptide enhancers include: PYY (25-36), PYY (30-36) and PYY (31-36). In one embodiment, the first module is located at the C-terminal end of the hybrid polypeptide and the peptide enhancer is located at the N-terminus of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminus of the hybrid polypeptide and the peptide enhancer may be located at the C-terminus of the hybrid polypeptide. In certain modalities, separators or linkers such as ßAla can be inserted if desired to join the modules. In another aspect, preferred module combinations include those involving a first module comprising exendin-4, a fragment of exendin-4 having at least one hormonal activity, an analogue or exendin-4 derivative having at least one a hormonal activity, or a fragment of an exendin-4 analogue having at least one hormonal activity in combination with a second module comprising CCK, a fragment of CCK having at least one hormonal activity, an analog or a derivative of CCK exhibiting at least one hormonal activity, or a fragment of a CCK analog that exhibits at least one hormonal activity. Again, the preferred exendin-4 compounds include: exendin-4, exendin-4 (1-27), exendin-4 (1-28), 14Leu, 25Phe-exendin-4 (1-28), 5Ala, 14Leu, 25Phe-exendin-4 (1-28), and 14Leu-exendin-4 (1-28). Preferred CCK compounds include: CCK-8, and CCK-8 (Phe (CH2SO3)). In one embodiment, the first module is located at the C-terminal end of the hybrid polypeptide and the second module is located at the N-terminal end of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminus of the hybrid polypeptide and the peptide enhancer may be located at the C-terminus of the hybrid polypeptide. In certain modalities, separators or linkers such as ßAla can be inserted if desired to join the modules. In another aspect, combinations of preferred modules include those involving a first module comprising amylin, an amylin fragment having at least one hormonal activity, an analog or amylin derivative having at least one hormonal activity, or a fragment of an amylin analog having at least one hormonal activity in combination with a second module comprising a peptide enhancer, 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 polypeptide and the peptide enhancer is located at the N-terminus of the hybrid polypeptide. Alternatively, the first module may be located at the N-terminus of the hybrid polypeptide and the increase in peptide may be located at the C-terminus of the hybrid polypeptide. In certain modalities, separators or linkers such as ßAla can be inserted if desired to join the modules. Other combinations of preferred moduli include those involving combinations of exendin and CCK or amylin, calcitonin, and CCK as a tertiary combination. Particular combinations include exendin / CCK and CCK / exendin, with and without separators or linkers and linker groups. Other combinations include CCK amylin / calcitonin and CCK / amylin / calcitonin / amylin, with and without separators or linker groups. Each module can independently be a peptide enhancer or can exhibit hormonal activity, depending on the desired properties of the hybrid polypeptide. Other combinations of preferred moduli include those involving combinations of exendin, amylin and calcitonin as ter- and tetra-hybrid molecules. Illustrative combinations include combinations of exendin / amylin / calcitonin; exendin / amylin / calcitonin / amylin; Amyline / calcitonin / exendin; and amylin / calcitonin / amylin / exendin, with and without separators or linker groups. Each module can be independently a peptidic enhancer or it can present a hormonal activity, depending on the desired properties of the hybrid polypeptide. In one embodiment, when one of the bioactive peptide hormone (s) module having at least one hormonal activity is amylin or an analog or fragment thereof, and a second bioactive peptide hormone module comprises CCK, then the hybrid polypeptide should preferably be comprise a third module of bioactive peptide hormone selected from a different component peptide hormone. Third exemplary bioactive peptide hormone modules include calcitonins, most preferably salmon calcitonin, analogues or fragments of the same. In another embodiment, when one of the bioactive peptide hormone (s) module having at least one hormonal activity is amylin or an analog or fragment thereof, and a second bioactive peptide hormone module comprises CT, then the hybrid polypeptide is preferably it must comprise a third module of bioactive peptide hormone selected from a different component peptide hormone. Third exemplary bioactive peptide hormone modules include exendin-4, analogs or fragments thereof. In yet another embodiment, when one of the bioactive peptide hormone module (s) exhibiting at least one hormonal activity is GLP-1 or an analog or fragment thereof, and a second bioactive peptide hormone module is a peptide enhancer comprising a fragment of exendin, then the hybrid polypeptide should preferably comprise a third module of bioactive peptide hormone. Third exemplary bioactive peptide hormone modules include PYY (including analogs, derivatives and fragments thereof) and CCK (including analogs, derivatives and fragments thereof). Within each of the preferred combinations described above, it is understood that reference to a component peptide hormone includes reference to analogs, derivatives, fragments, as well as peptide enhancers related thereto. In a preferred aspect, the hybrid polypeptides include: The hybrid polypeptides of the present invention may also comprise additional modifications including, but not limited to, substitution, deletion and insertion into the amino acid sequence of said hybrid polypeptides and any combination thereof. In a preferred aspect, the hybrid polypeptides of the invention include one or more modifications of a "non-essential" amino acid residue. In the context of the invention, a "non-essential" amino acid residue is a residue that can be altered, i.e., deleted or substituted, in the native human amino acid sequence of the fragment, e.g., the peptide hormone fragment component, without canceling or substantially reducing the agonistic activity of the peptide hormone receptor component of the hybrid polypeptide. Preferred substitutions include conserved amino acid substitutions. A "conserved amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain, or physicochemical characteristics (e.g., electrostatic characteristics, binding bond, isosteric, hydrophobic). Families of amino acid residues having similar side chains are known in the art, these families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g., aspartic acid, acid) glutamic), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline) , phenylalanine, tryptophan), (β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) The present invention also relates to Derivatives of hybrid polypeptides Said derivatives include hybrid polypeptides conjugated to one or more water-soluble polymer molecules, such as polyethylene glycol ("PEG") or fatty acid chains of various lengths (e.g., stearyl, palmitoyl) l, octanoyl, etc.), or by the addition of polyamino acids, such as poly-his, poly-arg, poly-lys and poly-ala. Modifications to the hybrid polypeptides can also include small molecule substituents, such as short alkyls and restricted alkyls (e.g., branched, cyclic, fused, adamantyl), and aromatic groups. Water soluble polymer molecules will preferably have a molecular weight ranging from about 500 to about 20,000.

Daltons. Said polymer conjugations and modifications of small molecule substituents may be presented individually at the N- or C-terminal or on the side chains of amino acid residues within the sequence of the hybrid polypeptides. Alternatively, there may be multiple derivation sites along the hybrid polypeptide. Substitution of one or more amino acids with lysine, aspartic acid, glutamic acid or cysteine may provide additional sites for derivatization. See, e.g., US patents. Nos. 5,824,784 and 5,824,778. Preferably, the hybrid polypeptides can be conjugated to one, two or three polymer molecules.

Water-soluble polymer molecules are preferably coated to an amino, carboxyl, or thiol group, and can be linked by N- or C-terminal, or on the side chains of lysine, aspartic acid, glutamic acid or cysteine. Alternatively, the water soluble polymer molecules can be linked with diamine and dicarboxylic groups. In a preferred embodiment, the hybrid polypeptides of the invention are conjugated to one, two or three molecules of PEG through an epsilon amino group in an amino acid lysine. Derivatives of the hybrid polypeptide of the invention also include hybrid polypeptides with chemical alterations to one or more amino acid residues. These chemical alterations include amidation, glycosylation, acylation, sulphation, phosphorylation, acetylation and cyclization. The chemical alterations can occur singularly in N- or C-terminal or in side chains of amino acid residues within the sequence of PPF hybrid polypeptides. In one embodiment, the C-terminus of these peptides may have a free -OH or -NH2 group. In another embodiment, the N-terminus can be blocked by an isobutyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, an octylglycine group (G (Oct)) ), or an 8-aminooctanoic acid group. In a preferred embodiment, the cyclization can be through the formation of disulfide bridges. Alternatively, there may be multiple sites of chemical alteration along the hybrid polypeptide. Examples of the hybrid polypeptides of the present invention are provided in the sequence listing and are further described in the examples section below.

Use of hybrid polypeptides in the treatment or prevention of metabolic conditions or disorders In another aspect of the invention, obesity treatment or prevention methods are provided, wherein the method comprises administering a therapeutically or prophylactically effective amount of a hybrid polypeptide to a subject who needs the same In a preferred embodiment, the subject is an obese or overweight subject. Although "obesity" generally defined as a body mass index above 30, for purposes of this description, any subject, including those with a body mass index of less than 30, who needs or wishes to reduce body weight is included in the scope of " obese". Subjects who are insulin resistant, glucose intolerant or have any form of diabetes mellitus (eg, type 1, 2 or gestational diabetes) may benefit from this method. In other aspects of the invention, methods to reduce food intake, reduce nutrient availability, cause weight loss, affect body composition, and alter body energy content or increase energy expenditure, treat diabetes mellitus and improving the lipid profile (including reducing the levels of LDL cholesterol and triglycerides and / or changing levels of HDL cholesterol) are provided, wherein the methods comprise administering to a subject an effective amount of a hybrid polypeptide of the invention. In a preferred embodiment, the methods of the invention are used to treat or prevent conditions or disorders that can be alleviated by reducing the availability of nutrients in a subject in need thereof, which comprises administering to said subject a therapeutically or prophylactically effective amount of a hybrid polypeptide of the invention. These conditions and disorders include but are not limited to hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin resistance, obesity and diabetes mellitus of any kind.

Without intending to be limited by theory, it is believed that the effects of peripherally administering hybrid polypeptides of the present invention in the reduction of food intake, in the delay of gastric emptying, in the reduction of nutrient availability, and in the cause of Weight loss is determined by interactions with one or more receptor classes unique to, or similar to, those of the PP family. Most particularly, it appears that a receptor or receptors similar to preferred receptors of PYY (or Y7) are involved. Additional tests useful for the invention include those that can determine the effect of the PPF compounds on the body composition. An illustrative test may be one that involves the use of a diet-induced obese mouse (DIO) model for metabolic disease. Before the treatment period, male C57BI / 6J mice can be fed a high-fat diet (# D12331, 58% of calories from fat; Research Diets, Inc.,) for 6 weeks starting at 4 weeks of age. During the study, mice can continue to eat their high-fat diet. Water ad libitum can be provided throughout the study. A group of non-obese mice of similar age can be fed a low-fat diet (# D12329, 11% of calories from fat) for purposes of comparing metabolic parameters with DIO groups. DIO mice can be implanted with subcutaneous intrascapular (SC) osmotic pumps to deliver either vehicle (50% dimethyl sulfoxide (DMSO) in water) or a compound of the invention. The Pumps of the latter group can be set to supply any amount, e.g., 1000 μg / kg / d of a compound of the invention for 7-28 days. Body weights and food intake can be measured at regular intervals in all study periods. The respiratory quotient (RQ, defined as production of CO H- O2 consumption) and the metabolic rate can be determined using indirect calorimetry of the whole animal (Oxymax, Columbus Instruments, Columbus, OH). Mice can be euthanized by overdose of soflurane, and an adiposity index (bilateral epididymal tier pad weight) is measured. Furthermore, prior to the determination of epididymal weight, body composition (lean mass, fat mass) for each mouse can be analyzed using a dual-energy X-ray absorptiometry instrument (DEXA) per manufacturer's instructions (Lunar Piximus, GE Imaging System). In the methods of the invention, the preferred PPF polypeptide of the invention are those that have a potency in one of the tests described herein (preferably tests of food intake, gastric emptying, pancreatic secretion, weight reduction or body composition) that is greater than the potency of a peptide hormone component in the same test. In addition to relieving hypertension in subjects in need thereof as a result of reducing food intake, weight loss or treating obesity, the compounds of the invention can be used to treat hypotension. The compounds of the invention may also be useful for potentiate, induce, increase or restore the glucose response in pancreatic islets or pancreatic cells. These actions may be useful to treat or prevent conditions associated with metabolic disorders such as those described above and in the patent application of E.U.A. do not. US20040228846. The tests to determine said activity are known in the art. For example, in the patent application of E.U.A. published no. US20040228846 (incorporated herein by reference in its entirety), the tests are described for islet isolation and culture as well as maturation determination of fetal islet. In the examples of patent application US20040228846, hormone peptides derived from intestine including pancreatic polypeptide (PP), neuropeptide Y (NPY), neuropeptide K (NPK), PYY, secretin, peptide-1 similar to giucagon (GLP-1) and bombesin are purchased from Sigma. Type XI collagenase was obtained from Sigma. Means of culture RPMl 1640 and fetal bovine serum were obtained from Gibco. A radioimmunoassay kit containing anti-insulin antibody ([125I] -RIA kit) was purchased from Lineo, St Louis. Post-partum rat islets were obtained from P-02-year-old rats. The islets of adult rats were obtained from rats 6-8 weeks of age. The fetal rat islets were obtained as follows. Pregnant female rats were sacrificed on the e21 days of pregnancy. The fetuses were removed from the uterus. 10-14 pancreas were excised from each litter and washed twice in Hanks pH regulator. The pancreas were stored in the stock, suspended in 6 ml 1 mg / ml of collagenase (Type XI, Sigma) and incubated at 37 ° C for 8-10 minutes with constant agitation. Digestion was stopped by adding 10 volumes of Hanks pH buffer cooled with ice followed by three washings with Hanks pH regulator. The islets were then purified by Ficoll gradient and cultured in 10% fetal bovine serum (FBS) / RPMl medium with or without the addition of 1 μM of IBMX. At the end of five days, the 20 islets were manually collected in each tube and tested for static insulin release. Generally, the islets were first washed with pH regulator KRP and then incubated with 1 ml of KRP pH buffer containing 3 mM (low) glucose for 30 minutes at 37 ° C with constant agitation. After collecting the supernatant, the islets were incubated with 17 mM (high) glucose for one hour at 37 ° C. Insulin released from low or high glucose stimulation was tested by radioimmunoassay (RIA) using the [125I] -RIA equipment. Fetal islets e21 were cultured for 5 days in the presence of 200 ng / ml PYY, PP, CCK, NPK, NPY, secretin, GLP-1 or bombesin. An illustrative in vivo test was also provided using Zucker Diabetic Fatty male rat (ZDF), an inbred rat model (> F30 generations) that spontaneously expresses diabetes in all fa / fa males fed a standard Purina 5008 rodent diet. In fa-fa ZDF males hyperglycemia begins to develop at approximately seven weeks of age and glucose levels (fed) typically reach 500 mg / DL at 10 to 11 weeks of age. The levels of Insulin (fed) are high during the development of diabetes. However, at 19 weeks of age, insulin falls approximately to the level of slender control baits. Triglyceride and cholesterol levels of obese rats are usually higher than those of slender rats. In the test, three groups of 7-week-old ZDF rats, with 6 rats per group, received the infusion treatment per ALZA pump for 14 days: 1) vehicle control, 2 and 3), PYY with two different doses , 100 pmoles / kg / hr and 500 pmoles / kg / hr respectively. Four measurements were taken before the infusion and after the infusion on day 7 and 14: 1) plasma glucose level, 2) plasma insulin level, and 3) plasma triglyceride (TG) level, as well as oral glucose tolerance test (OGTT). Accordingly, these tests can be used with compounds of the invention to test desired activity. Other contemplated uses for the hybrid polypeptides include methods of reducing concentrations of aluminum (Al) in the central nervous system (see US patent 6,734,166, incorporated herein by reference in its entirety) to treat, prevent or retard the onset of Alzheimer's disease. . Tests to determine effects that on Al are known in the art and can be found in the patent of E.U.A. 6,734,166 using diploid and Ts mice. These mice were individually housed in metabolism or polypropylene cages of the Nalgene® brand and given three days to adjust to the cages prior to experimentation. The mice had free access to food (LabDiet® NIH Rat and Moust / Auto 6F5K52, St.

Louis, Mo.) and water during the experiment except for the 16 hours before euthanasia when no food was provided. The mice were given daily subcutaneous injections of either active compound or saline. The mice were sacrificed at the end of day 13 for one experiment and day 3 for another, and the samples were collected. Mouse brain samples were weighed in clean Teflon liners and prepared for analysis by microwave digestion in nitric acid of low trace element grade. The samples were then analyzed for Al content using inductively coupled plasma mass spectrometry (Nuttall et al., Annals of Clinical and Laboratory Science 25, 3, 264-271 (1995)). All tissue handling during the analysis took place in a clean room environment using HEPA air filtration systems to minimize background contamination. The compounds of the invention exhibit a wide range of biological activities, some related to antisecretory and antimotility properties. The compounds can suppress gastrointestinal secretions by direct interaction with epithelial cells or perhaps by inhibition of the secretion of hormones or neurotransmitters that stimulate intestinal secretion. Antisecretory properties include the inhibition of gastric and / or pancreatic secretions that may be useful in the treatment or prevention of diseases and disorders including gastritis, pancreatitis, Barrett's esophagus, and gastroesophageal reflux disease. The compounds of the invention are useful in the treatment of any number of gastrointestinal disorders (see, e.g., Harrison's Principles of Intemal Medicine, McGraw-Hill Inco, New York, 12th Ed.) which are associated with excess intestinal electrolytes and water secretion as well as decreased absorption, .gr., infectious diarrhea, inflammatory diarrhea, short bowel syndrome, or diarrhea that typically occurs after surgical procedures, eg, ileostomy. Examples of infectious diarrhea include without limitation, acute viral diarrhea, acute bacterial diarrhea (eg, Salmonella, Campylobacter and Clostridium or due to protozoal infections), or traveler's diarrhea (eg, Norwalk virus or rotavirus). . Examples of inflammatory diarrhea include, without limitation, malaabsorption syndrome, tropical stomatitis, chronic pancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome. It has also been discovered that the peptides of the invention can be used to treat an emergency or life threatening situation involving a gastrointestinal disorder, e.g., after surgery or due to cholera. The compounds of the invention may also be useful for treating or preventing intestinal damage as opposed to simply treating symptoms associated with intestinal damage (eg, diarrhea). Such damage to the intestine may be, or result from, ulcerative colitis, inflammatory bowel disease, intestinal atrophy, loss of intestinal mucosa and / or loss of intestinal mucosal functions (see WO 03/105763, incorporated herein by reference in its entirety). . Tests for such activity, as described in WO 03/105763, include rats HSD of 11 weeks of age ranging from 250 to 300 grams housed in a light: dark cycle of 12:12 and being allowed ad libitum access to a standard rodent diet (Teklad LM 485, Madison, Wl) and water . The animals were fasted for 24 hours before the experiment. A simple and reproducible rat model of chronic colonic inflammation has been previously described by Morris GP, et al., "Hapten-induced model of chronic inflammation and ulceration in the mouse colon." Gastroenterology. 1989; 96: 795-803. It has a relatively long duration of inflammation and ulceration, giving an opportunity to study the pathophysiology of inflammatory colonic disease in a specifically controlled manner and to evaluate new treatments potentially applicable to inflammatory bowel disease in humans. The rats were anesthetized with 3% isoflurane and placed in a regulated heating plate set at 37 ° C. A forced feeding needle was inserted rectally into the colon 7 cm. Haptentrinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol (v / v) was delivered to the lumen of the colon through the forced feeding needle at a dose of 30 mg / kg, in a total volume of 0.4-0.6. ml, as described in Mazelin, et al., Juton Nerv Syst. 1998; 73: 38 45. The control groups received saline (NaCl 0.9%) intracolonically. Four days after the induction of colitis, the colon was excised from anesthetized rats, which were then euthanized by decapitation. The excised colon and spleen weights were measured and the colons were photographed for gross morphological damage rating. Inflammation was defined as regions of hyperemia and thickening of the intestinal wall. Hybrid polypeptides of the invention can also be used to treat or prevent pancreatic tumors (e.g., inhibit the proliferation of pancreatic tumors). The methods of the invention include reducing the proliferation of tumor cells. The types of benign pancreatic tumor cells that can be treated in accordance with the present invention include adenomas of serous cysts, microcystic tumors and solid cysts tumors. The method is also effective in reducing the proliferation of malignant pancreatic tumor cells such as carcinomas that are produced from the ducts, acini or islets of the pancreas. The patent of E.U.A. 5,574,010 (incorporated by reference in its entirety) provides illustrative tests for testing antiproliferative properties. For example, the '010 patent provides that PANC-1 and MiaPaCa-2 are two human pancreatic adenocarcinoma cancer cell lines that are commercially available from suppliers such as the American Type Culture Deposit, ATCC (Rockville, Md.). The two tumor cells were grown in RPMI-1640 culture medium supplemented with 10% fetal bovine serum, 29.2 mg / l glutamine, 25 μg gentamicin, 5 ml penicillin, streptomycin, and fungizone solution (JRH Biosciences , Lenexa, Kans.) At 37 degrees centigrade in an incubator with 5% CO2 with a NAPCO water blanket. All the cell lines were detached with 0.25% of tripasin (Clonetics, San Diego, Calif.) once or twice a week when a confluent monolayer of tumor cells was obtained. The cells were pelleted for 7 minutes at 500 grams in a centrifuge cooled to 4 degrees Celsius and resuspended in a fortified culture medium RPM) 1640 free of trypsin. Viable cells were counted on a slide with hemocytometer with trypan blue. Ten thousand, 20,000, 40,000 and 80,000 cells of each type were added to 96-well microcropping plates (Costar, Cambridge, Mass.) In a total volume of 200 μl of culture medium per well. The cells were allowed to adhere for 24 hours before the addition of PYY or test peptide. The fresh culture medium was exchanged before the addition of peptides. In vitro incubation of pancreatic tumor cells with either PYY or test compound was continued for 6 hours and 36 hours. PYY was added to cells at doses of 250 pmol, 25 pmol, and 2.5 pmol per well (N = 14). The test compound was added to cell cultures at doses of 400 pmol, 40 pmol, and 4 pmol per well. The control wells received 2 ul of 0.9% saline to simulate volume and physical disturbance with adhered tumor cells. Each 96-well plate contained 18 control wells to allow comparison within each plate during experimentation. Plates of ninety-six (96) wells were repeated 6 times with varying concentrations of PYY and test compound in both PANC-1 and MiaPaCa-2 cells. At the end of the incubation period, 3- (4,5-) bromide was added dimethylthiazolyl-2-yl) -2,5-diphenyl tetrazolium, MTr tetrazolium bromide (Sigma, St. Louis, Mo.) to fresh culture medium at 0.5 mg / ml. The culture media were exchanged and the tumor cells were incubated for 4 hours with MTT tetrazolium bromide at 37 ° C. At the end of the incubation, the culture media were aspirated. Shaped crystal precipitates were dissolved in 200 μl of dimethyl sulfoxide (Sigma, St. Louis, Mo.). Quantitation of solubilized formate was performed by obtaining absorption readings at 500 nm wavelength in an ELISA reader (Molecular Devices, Menlo Park, Calif.). The MTT test measures mitochondrial NADH-dependent dehydrogenase activity, and has been among the most sensitive and reliable method of quantifying in vitro chemotherapy responses of tumor cells. (Alley, M.C., et al., Cancer Res., 48: 589-601, 1988; Carmichael, J., et al., Cancer Res., 47: 936-942, 1987; McHale, AP, et al. ., Cancer Lett., 41: 315-321, 1988, and Saxton, RE, et al., J. Clin. Laser Med. And Surg, 10 (5): 331-336, 1992). The analyzes of absorption readings at 550 nm were analyzed by pooling wells of the same test conditions and verifying differences that occur between the various treatments of peptide concentration or one-way ANOVA. An illustrative in vivo test is also provided. The Mía Paca-2 human pancreatic ductal adenocarcinoma was examined for inhibition of growth in vivo by YY peptide and test compound. Seventy thousand to 100,000 human Mia PaCa-2 cells were orthotopically transplanted in 48 male athymic mice. After a week, the animals were tested with PYY or test compound at 200 pmol / kg / hr using miniosmotic pumps for four weeks. The paired cultures received saline. When the mice were sacrificed, the size and mass of the tumor were measured. The control mice had significant human cancer growth within the pancreas as evidenced by the histological sections. At 9 weeks, ninety percent (90%) of control mice had substantial metastatic disease. The tumor mass was reduced by 60.5% in mice treated with the test compound and 27% in mice treated with PYY. For all indications, in preferred embodiments, the hybrid polypeptide of the invention is peripherally administered at a dose of about 0.5 μg to about 5 mg per day in single or divided doses or controlled continuous release, or at about 0.01 μg / kg to about 500 μg / kg per dose, most preferably about 0.05 μg / kg to about 250 μg / kg, most preferably below about 50 μg / kg. The doses in these ranges will vary with the potency of each analogue or derivative, of course, and can be determined by one skilled in the art. In the methods of the present invention, the hybrid polypeptides of the invention can be administered separately or together with one or more other compounds and compositions that have a long-term or short-term action to reduce the availability of nutrients, including but not limited to other compounds and compositions comprising a amylin or amylin analogue agonist, salmon calcitonin, cholecystokinin (CCK) or CCK agonist, leptin (OB protein) or leptin agonist, exendin or exendin analogue, or GLP-1 or GLP analogue agonist -1. Suitable amylin agonists include, for example, human amylin [25,28,29Pro-] (also known as "pramlintide", and described in U.S. Patent Nos. 5,686,511 and 5,998,367). The CCK used is preferably CCK octapeptide (CCK-8). Leptin is described, for example, in Pelleymounter et al., Science 269: 540-3 (1995); Halaas et al., Science 269: 543-6 (1995); Campfield et al., Science 269: 546-9 (1995). Suitable exendins include exendin-3 and exendin-4, and exendin agonist compounds include, for example, those described in PCT publications WO 99/07404, WO 99/25727, and WO 99/25728.

Production and Polypeptide Purification The hybrid polypeptides described herein can be prepared using standard recombinant techniques or chemical peptide synthesis techniques known in the art, e.g., using an automated or semi-automated peptide synthesizer or both. The hybrid polypeptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques. Several automatic synthesizers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young, Solid Phase Peptide Synthesis, 2a. ed., Pierce Chemical Co. (1984); Tam e al., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232: 341-7 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, 1-284 (1979). Solid phase peptide synthesis can be carried out with an automatic peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc., Foster City, California) using the NMP / HOBt system (Option 1) and the chemistry of tBoc or Fmoc (see, Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B July 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, California) with blocking . Peptides can also be assembled using an advanced chemical technique synthesizer (Model MPS 350, Louisville, Kentucky). The peptides can be purified by CLAR-FI (preparative and analytical) using, e.g., a Waters Delta Prep 3000 system and a preparative C4, C8, or C18 column (10 μ, 2.2x25 cm; Vydac, Hesperia, California). The active peptide can be easily synthesized and then selectively determined in selective determination tests designed to identify reactive peptides. The hybrid polypeptides of the present invention may alternatively be produced by recombinant techniques well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor (1989). These hybrid peptides produced by recombinant technologies can be expressed from a polynucleotide. One of ordinary skill in the art will appreciate that polynucleotides, including DNA and RNA, which encode such diverse fragments of the Hybrid peptides can be obtained from wild-type cDNA, taking into account the degeneracy of codon usage or can be engineered as desired. These polynucleotide sequences can incorporate codons that facilitate the transcription and translation of mRNA in microbial hosts. Said manufacturing sequences can be easily constructed according to methods well known in the art. See, e.g., WO 83/04053. The above polynucleotides can also be optionally encoded as N-terminal methionyl residues. The non-peptidic compounds useful in the present invention can be prepared by methods known in the art. For example, phosphate-containing amino acids and peptides containing said amino acids can be prepared using methods known in the art. See, e.g., Bartlett and Landen, Bioorg Chem. 14: 356-77 (1986). A variety of expression / host vector systems can be used which contain and express a coding sequence for hybrid polypeptides. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti plasmids or pBR322); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary cell lines (CHO), COS cells (such as COS-7), Wl cells 38 , BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293. Illustrative protocols for the recombinant expression of the protein are described below. As such, the polynucleotide sequences provided by the invention are useful for generating new and useful plasmid and viral DNA vectors, new and useful transcaforced and transfected prokaryotic and eukaryotic host cells (including bacterial, yeast and mammalian cells grown in culture). ), and new and useful methods for growth in culture of said host cells capable of expressing the present hybrid polypeptides. The polynucleotide sequences encoding hybrid polypeptides herein may be useful for gene therapy in cases where the overproduction of the peptide hormone (s) component of the chimera is alleviated or the need for increased levels thereof is satisfied. The present invention also provides methods for the production of recombinant DNA of the present hybrid polypeptides. A method is provided for producing hybrid polypeptides from a host cell containing nucleic acids encoding said hybrid polypeptides comprising: (a) culturing host cells containing polynucleotides encoding said hybrid polypeptides under conditions that facilitate the expression of the DNA molecule; and (b) obtaining said hybrid polypeptides. The host cells can be prokaryotic or eukaryotic and include bacteria, mammalian cells (such as Chinese hamster ovary (CHO) cells, monkey cells, newborn hamster kidney cells, cancer cells and other cells), yeast cells and insect cells. Mammalian host systems for the expression of the recombinant protein are also well known to those skilled in the art. The host cell strains can be chosen for a particular ability to process the expressed protein or produce certain post-translational modifications that will be useful for providing protein activity. Such modifications of the polypeptide include, but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translation processing, which differs from a "prepro" form of the protein, may also be important for correct insertion, folding and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38 and the like, have specific cellular machinery and characteristic mechanisms for said post-translation activities, and can be chosen to ensure the correct modification and processing of the introduced foreign protein. Alternatively, a yeast system can be used to generate the hybrid polypeptides of the present invention. The region encoding the hybrid polypeptide cDNA is amplified by PCR. A DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic DNA in a PCR reaction using an initiator containing nucleotides 1-20 of the alpha coupling factor gene and another complementary primer to nucleotides 255-235 of this gene (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)). The pre-pro-alpha leader coding sequence and the hybrid polypeptide coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs the expression of a protein of fusion consisting of the pre-pro-alpha factor fused to the mature hybrid polypeptide. As taught in Rose and Broach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, California (1990), the vector also includes an ADH2 transcription terminator towards the 3 'end of the cloning site, the "2-micron" origin of replication of yeast, the leu-2d gene of yeast, the REP1 and REP2 genes of yeast, the ß-lactamase gene of E.coli, and an origin of replication of E. Coli. The ß-lactamase and leu-2d genes provide selection in bacteria and yeast, respectively. The gene of leu-2d also facilitates the increase in the copy number of the plasmid in yeast to induce higher expression levels. The REP1 and REP2 genes encode proteins involved in the regulation of plasmid copy number. The DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., treatment with lithium acetate (Stearns et al., Meth., 185: 280-97 (1990)). The ADH2 promoter is induced by the depletion of glucose in the growth medium (Price et al., Gene 55: 287 (1987)). The pre-pro-alpha sequence effects the secretion of the fusion protein from the cells. Concomitantly, the yeast KEX2 protein digests the prepro sequence of the mature PYY analogue polypeptides (Bitter et al., Proc. Nati, Acad. Sci. USA 81: 5330-4 (1984)). The hybrid polypeptides of the invention can also be expressed recombinantly in yeast using a commercially available expression system, e.g., the Pichia expression system (Invitrogen, San Diego, California), following the manufacturer's instructions. This system is also based on the sequence of pre-pro-alpha to direct the secretion, but the transcription of the insert is driven by the alcohol oxidase promoter (AOX1) under induction by methanol. The secreted hybrid polypeptide is purified from the yeast growth medium, for example, by the methods used to purify hybrid polypeptide from supernatants of bacterial and mammalian cells. Alternatively, the cDNA encoding hybrid polypeptides can be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, California). This hybrid polypeptide-containing vector is used after in accordance with the manufacturer's instructions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free medium and to produce recombinant protein. The protein is purified and Concentrate medium using a heparin-Sepharose column (Pharmacia, Piscataway, New Jersey) and sequential molecular sizing columns (Amicon, Beverly, Massachusetts), and resuspended in PBS. The SDS-PAGE analysis shows an individual band that confirms the size of the protein and the Edman sequencing in a Proton 2090 peptide sequencer confirms its N-terminal sequence. For example, the DNA sequence encoding the hybrid polypeptide can be cloned into a plasmid containing a desired promoter and optionally a leader sequence (see, e.g., Better et al., Science 240: 1041-3 (1988)). ). The sequence of this construction can be confirmed by automated sequencing. The plasmid is then transformed into E. coli, strain MC1061, using standard procedures using incubation with CaCl2 and heat shock treatment of the bacteria (Sambrook et al., Supra). The transformed bacteria are grown in an LB medium supplemented with carbenicillin, and the production of the expressed protein in growth induced in a suitable medium. If present, the leader sequence will affect the secretion of the hybrid polypeptide and will be digested during secretion. The recombinant protein is purified from the bacterial culture medium by the method described below. Alternatively, the hybrid polypeptides of the invention can be expressed in an insect system. Insect systems for protein expression are well known to those skilled in the art. In such a system, Autographa nuclear polyhedrosis virus Californica (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The hybrid polypeptide coding sequence cloned in a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of hybrid polypeptide will render the polyhedrin gene inactive and produce recombinant virus lacking protein coatings. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the hybrid polypeptide is expressed (Smith et al., J. Virol. 46: 584 (1983); Engelhard et al., Proc. Acad Sci. USA 91: 3224-7 (1994)). In another example, the DNA sequence encoding the hybrid polypeptide can be amplified by PCR and cloned into an appropriate vector, for example pGEX-3X (Pharmacia, Piscataway, New Jersey). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the cloning site of the vector. PCR primers can be generated to include, for example, an appropriate digestion site. The recombinant fusion protein can then be digested from the GST portion of the fusion protein. The pGEX-3X / PYY analog polypeptide construct is transformed into E.coli XL-1 Blue cells (Stratagene, La Jolla, California), and the individual transformants are isolated and grown at 37 ° C in a medium of LB (supplemented with carbenicillin) at an optical density at a wavelength of 600 nm of 0.4, followed by additional incubation for 4 hours in the presence of 0.5 mM isopropyl-β-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis, Missouri). The plasmid DNA of the individual transformants is purified and partially sequenced using automated sequencing to confirm the presence of the PPF hybrid polypeptide encoding gene insert in the proper orientation. The fusion protein, which is expected to be produced as an inclusion body insoluble in bacteria, can be purified as follows. The cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg / ml lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate is clarified by sonication, and cell debris is formed into pellets by centrifugation for 10 minutes at 12,000xg. The pellet containing fusion protein is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, statified on 50% glycerol, and centrifuged for 30 minutes at 6000 xg. The tablet is resuspended in saline regulated at its pH with standard phosphate (PBS) free of Mg ++ and Ca ++. The fusion protein is further purified by fractionating the resuspended pellet on a denaturing SDS polyacrylamide gel (Sambrook et al., Supra). The gel is soaked in 0.4 M KCl to visualize the protein, which is cut and electroeluted in SDS lacking pH regulator that runs on gel. If the GST / PYY analog polypeptide fusion protein is produced in bacteria as a soluble protein, it can be purified using the module of purification of GST (Pharmacia Biotech). The fusion protein can be digested to digest the GST of the PPF hybrid polypeptide. The digestion reaction (20-40 μg of fusion protein, 20-30 units of human thrombin (4000 U / mg (Sigma) in 0.5 ml of PBS) is incubated at 16-48 hours at room temperature and loaded into a denaturing SDS-PAGE gel to fractionate the reaction products The gel is soaked in 0.4 M KCl to visualize the protein strips The identity of the protein strip corresponding to the expected molecular weight of the hybrid polypeptide can be confirmed by sequence analysis of partial amino acids using an automated sequencer (Applied Biosystems Model 473A, Foster City, Calif.) In a particularly preferred method of recombinant expression of the hybrid polypeptides of the present invention, 293 cells can be co-transfected with plasmids containing the cDNA of the hybrid polypeptide in the pCMV vector (5 'CMV promoter, 3' HGH poly A sequence) and pSV2neo (containing the neo resistance gene) by the calcium phosphate method io Preferably, the vectors must be linearized with Seal before transfection. Similarly, an alternative construct using a similar pCMV vector with the incorporated neo gene can be used. The stable cell lines are selected from individual cell clones by limiting the dilution in the growth medium containing 0.5 mg / ml of G418 (antibiotic similar to neomycin) for 10-14 days. The cell lines are selectively determined for polypeptide expression hybrid by ELISA or Western blot, and high expression cell lines expand for large scale growth. It is preferable that the transformed cells are used for long-term high-yield protein production and stable expression is desirable as such. Once said cells are transformed with vectors containing selectable markers together with the desired expression cassette, the cells can be allowed to grow for 1-2 days in an enriched media before they are switched to a selective medium. The selectable marker is designed to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clots of stably transformed cells can proliferate using tissue culture techniques appropriate to the cell. A number of selection systems can be used to recover cells that have been transformed for production of recombinant protein. Said selection systems include, but are not limited to, HSV thymidine kinase genes, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase, in tk-, hgprt- or aprt-, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to aminoglycoside, G418; also, which confers resistance to chlorsulfuron; and hygro, which confers hygromycin resistance. The selectable genes Additional ones that may be useful include trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol in place of histidine. Markers that give a visual indication for identification of transformants include anthocyanins, β-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin. Many of the hybrid polypeptides of the present invention can be produced using a combination of both automated and recombinant peptide synthesis techniques. For example, a hybrid polypeptide of the present invention may contain a combination of modifications including deletion, substitution and insertion by PEGylation. Said hybrid polypeptide can be produced in stages. In the first step, an intermediate polypeptide containing the modifications of deletion, substitution, insertion and any combinations thereof, can be produced by recombinant techniques as described. After an additional purification step as described below, the intermediate polypeptide is PEGylated through chemical modification with an appropriate PEGylation reagent (e.g., from Nectar Transforming Therapeutics, San Carlos, California) to give the hybrid polypeptide wanted. One skilled in the art will appreciate that the above-described process can be generalized to be applied to a hybrid polypeptide that contains a combination of selected modifications of deletion, substitution, insertion, derivatization and other modification means well known in the art and contemplated by the present Nvention It may be desirable to purify the hybrid polypeptides generated by the present invention. Peptide purification techniques are well known to those skilled in the art. These techniques involve, at one level, the crude fractionation of cellular particles into polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Particularly suitable analytical methods for the preparation of a pure peptide are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis and isoelectric focusing. A particularly efficient method of peptide purification is reverse phase HPLC, followed by characterization of purified product by liquid chromatography / mass spectrometry (LC / MS) and mass spectrometry by matrix assisted laser desorption ionization (MALDl). The additional conformation of the purity is obtained by determining the amino acid analysis. Certain aspects of the present invention relate to the purification, and in particular embodiments, to the substantial purification of an encoded protein or peptide. The term "purified peptide" as used herein, refers to a composition, isolable from other components, wherein the peptide is purified to any degree relative to its naturally obtainable state. A purified peptide therefore also refers to a peptide, free from the environment in which it may occur naturally.

Generally, "purified" will refer to a peptide composition that has been subjected to fractionation to remove some other components, and whose composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the peptide forms the main component of the composition, such as constituting approximately 50%, approximately 60%, approximately 70%, approximately 80%, about 90%, about 95% or more of the peptides in the composition. Various techniques suitable for use in peptide purification will be known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like; thermal denaturation, followed by centrifugation; chromatography steps such as ion exchange chromatography, gel filtration, reverse phase, hydroxylapatite and affinity; isoelectric focus; gel electrophoresis; and combinations of those and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps can be changed, or that certain steps can be omitted, and still results in a suitable method for the preparation of a substantially purified protein or peptide. . There is no general requirement that peptides are always provided in their most purified state. In fact, it is contemplated that substantially purified products will have utility in certain modalities. Partial purification can be achieved by using fewer purification steps in combination, or using different forms of the same general purification scheme. For example, it is appreciated that a cation exchange column chromatography performed using a CLAR apparatus will generally result in a greater "number of times" purification than the same technique that uses a low pressure chromatography system. Methods that show a lower degree of relative purification may have advantages in the total recovery of protein product, or in the maintenance of the activity of an expressed protein. It is possible to purify and optionally isolate said hybrid polypeptides from other components obtained in the process. Methods for purifying a polypeptide can be found in the U.S.A. No. 5,849,883. These documents describe specific illustrative methods for the isolation and purification of G-CSF compositions that may be useful in the isolation and purification of the hybrid polypeptides of the present invention. Given the description of these patents, it is clear that one skilled in the art is aware of numerous purification techniques that can be used to purify hybrid polypeptides from a given source. It is also contemplated that a combination of ion exchange chromatography and immunoaffinity can be used to produce purified hybrid polypeptide compositions of the present invention.

Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of at least one hybrid polypeptide of the invention, or a pharmaceutically acceptable salt thereof, together with diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or pharmaceutically acceptable carriers useful in the delivery of hybrid polypeptides. Such compositions may include diluents of varying pH buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives, (e.g., thimerol, benzyl alcohol) and body-forming substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds, such as polylactic acid, polyglycolic acid, etc., or in association with liposomes. Said compositions will influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the present hybrid polypeptides. See, e.g., Remington's Pharmaceutical Sciences 1435-712, 18th ed., Mack Publishing Co., Easton, Pennsylvania (1990). In general, the present hybrid polypeptides will be useful in the same way that the polypeptides of individual components are useful in view of their pharmacological properties. A preferred use is to peripherally administer said hybrid polypeptides for treatment or prevention of metabolic conditions and disorders. In particular, the compounds of the invention possess activity as agents to reduce the availability of nutrients, reduce food intake and effect weight loss. In another embodiment, a preferred use is to administer said hybrid polypeptides for the treatment of diabetes or conditions and disorders related to diabetes. The present hybrid polypeptides can be formulated for peripheral administration, including formulation for injection, oral administration, nasal administration, pulmonary administration, topical administration or other types of administration that will be recognized by one skilled in the art. Very particularly, the administration of pharmaceutical compositions according to the present invention can be by any common route as long as the target tissue is available through that route. In a preferred embodiment, the pharmaceutical compositions can be introduced into the subject by any conventional peripheral method, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g. of term); by oral, sublingual, nasal, anal, vaginal or transdermal administration, or by surgical implantation in a particular site. The treatment may consist of a single dose or a plurality of doses over a period. The controlled continuous release of the compositions of the present invention are also contemplated. The formulation may be liquid or it may be solid, such as lyophilized for reconstitution. The aqueous compositions of the present invention comprise an effective amount of the hybrid polypeptide, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, sotonic and absorption delaying agents, and the like. The use of said media and agents for pharmaceutically active substances is well known in the art. Except that any conventional medium or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Complementary active ingredients can also be incorporated into the compositions. In some cases, it will be convenient to provide a hybrid polypeptide of the invention and another food-reducing, diabetes treatment, plasma glucose-reducing or lipid-altering agent in the plasma, such as an amylin, an agonist analog. of amylin, a CCK or CCK agonist, or a leptin or deleptin agonist, or an exnedin or exendin agonist analog, in a single composition or solution to be administered together. In other cases, it may be more advantageous to administer the additional agent separately from said hybrid polypeptide. The hybrid polypeptide of the invention can be prepared for administration as free base solutions, or pharmacologically salts acceptable in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandélico and similars. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. Such products are easily prepared by methods well known to those skilled in the art. Dispersions in glycerol, liquid polyethylene glycols and mixtures thereof and in oils are also prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In one embodiment, the pharmaceutical compositions of the present invention are formulated to be suitable for parenteral administration, e.g., by injection or infusion. Preferably, the hybrid polypeptide is suspended is an aqueous vehicle, for example, in an isotonic pH buffer at a pH of from about 3.0 to about 8.0, preferably at a pH of from about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to approximately 5.0. Useful pH regulators include sodium citrate-citric acid and phosphate sodium-phosphoric acid, and sodium acetate / acetic acid. A slow release preparation form by replenishment or "deposition" can be used in such a manner that therapeutically effective amounts of the preparation are delivered into the blood stream for many hours or days after injection or transdermal delivery. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that there is an easy syringability. It is also desirable that the hybrid polypeptide of the invention be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the dispersion region and by the use of surfactants. The prevention of the action of microorganisms can be carried out by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents isotonic (for example, sugars or sodium chloride). Prolonged absorption of the injectable compositions can be carried out by using the compositions of agents delaying absorption (for example, aluminum monostearate and gelatin). Sterile injectable solutions can be prepared by incorporating the active compounds in the required amounts in the appropriate solvent with some of the other ingredients listed above, as required, followed by filtered sterilization. In general, the dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques which produce a powder of the active ingredient plus some additional desired ingredient from a previously sterile filtered solution of the same. In general, a therapeutically or prophylactically effective amount of the present hybrid polypeptides will be determined by the age, weight and condition or severity of the diseases, conditions or disorders of the recipient. See, e.g., Remington's Pharmaceutical Sciences 697-773. See also Wang and Hanson, Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S (1988). Typically, a dose of between about 0.001 μg / kg of body weight / day to about 1000 μg / kg of body weight / day can be used, but more or less, as will be recognized by an expert, can be used. The dosage may be one or more times per day, or less frequently, and may be together with other compositions as described herein. It should be noted that the present invention is not limited to the doses mentioned herein. Appropriate doses can be achieved through the use of stabilized tests to determine the level of metabolic conditions or disorders together with pertinent dose response data. The final dose regimen will be determined by the attending physician, considering factors that modify the action of drugs, eg, the specific activity of the drug, severity of the damage and the response of the patient, age, condition, weight body, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, more information will emerge regarding the appropriate dose levels and duration of treatment of specific diseases and conditions. An effective dose will typically be in the range of from about 1 to 30 μg to about 5 mg / day, preferably from about 10 to 30 μg to about 2 mg / day and most preferably from about 5 to 100 μg to about 1 mg / day, most preferably from about 5 μg to about 500 μg / day, for a 50 kg patient, administered in a single dose or dose divided. Preferably, the doses are between about 0.01 to about 100 μg / kg / dose. The exact dose to be administered can be determined by one skilled in the art and depends on the potency of the particular compound, as well as on the age, weight and condition of the individual. The administration should start whenever it is desired suppression of nutrient availability, food intake, weight, modulation of blood glucose or lipids in the plasma, for example, at the first sign of symptoms or shortly after the diagnosis of obesity, diabetes mellitus or insulin resistance syndrome. Administration can be by any route, eg, injection, preferably subcutaneous or intramuscular, oral, nasal, transdermal, etc. Doses for certain routes, for example, oral administration, can be increased taking into account the decreased bioavailability, for example, in approximately 5-100 times. In one embodiment, wherein the pharmaceutical formulation is to be administered parenterally, the composition is formulated to deliver a dose of hybrid polypeptide ranging from 1 μg / kg to 100 mg / kg of body weight / day, preferably at varying doses. 0.1 mg / kg to approximately 50 mg / kg body weight / day. Parenteral administration can be carried out with an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of drug product. Those skilled in the art will readily optimize effective doses and administration regimens as determined by good medical practice and the clinical condition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the routes of administration. The optimum pharmaceutical formulation will be determined by one skilled in the art depending on the route of administration and the desired dose. See, e.g., Remington's Pharmaceutical Sciences, supra, pages 1435-1712. Such formulations can influence the physical state, stability, in vivo release rate and in vivo clearance rate of the agents administered. Depending on the route of administration, an adequate dose can be calculated according to body weight, body surface areas or organ size. Further refinement of the calculations necessary to determine the appropriate treatment dose is routinely made by those skilled in the art without extraordinary experimentation, especially in light of the dose and test information disclosed herein, as well as the pharmacokinetic data observed in clinical trials with animals or humans. It will be appreciated that the pharmaceutical compositions and methods of treatment of the invention may be useful in the fields of human medicine and veterinary medicine. Therefore, the subject to be treated may be a mammal, preferably a human or other animal. For veterinary purposes, subjects include, for example, farm animals including cows, sheep, pigs, horses and goats, companion animals such as dogs and cats, exotic and / or zoo animals, laboratory animals including mice, rats, rabbits. guinea pigs and hamsters, and poultry such as chickens, turkeys, ducks and geese.

In addition, the present invention contemplates a kit comprising a hybrid polypeptide of the invention, suitable components for preparing the hybrid polypeptide of the invention for pharmaceutical application, and instructions for using said hybrid polypeptide and components for pharmaceutical application. To help understand the present invention, the following examples are included. The experiments related to this invention, of course, should not be considered as specifically limiting the invention and said variations of the invention, now known or further developed, which would be within the scope of a person skilled in the art are considered to fall within the scope of the invention. of the invention as described herein and as claimed below.

EXAMPLES The present invention is described in more detail with reference to the following non-limiting examples, which are offered to illustrate the invention more fully, but should not be considered as limiting the scope thereof. The examples illustrate the preparation of the present hybrid polypeptides, and the tests of these hybrid polypeptides of the invention in vitro and in vivo. Those skilled in the art will understand that the techniques described in those examples represent techniques described by the inventors to work well in the practice of the invention, and as such constitute preferred modes for the practice thereof. Nevertheless, it should be appreciated that those skilled in the art in the light of the present disclosure will appreciate that many changes can be made in specific methods that are described and an equal or similar result will be obtained without departing from the spirit and scope of the invention.

EXAMPLE 1 Preparation of hybrid polypeptides The peptides of the invention can be assembled in a Symphony peptide synthesizer (Protein Technologies, Inc.) using Rink amide resin (Novabiochem) with a loading of 0.43-0.49 mmoles / g a 0. 050-0.100 mmoles or a pre-filled Wang resin (Fmoc-Tyr (tBu) - resin from Wang) 0.63 mmoles / g (Novabiochem). The amino acid residues Fmoc (5.0 eq, 0.250-.500 mmol) are dissolved at a concentration of 0.10 M in 1-methyl-2-pyrrolidinone. All other reagents (HBTU, 1-Hydroxybenzotriazole hydrated and N, N-diisopropylethylamine) are prepared as 0.55 M dimethylformamide solutions. The protected Fmoc amino acids are then coupled to the resin bound amino acid using HBTU (2.0 eq, 0.100-0.200 mmoles) , 1-Hydroxybenzotriazole hydrated (1.8 eq, 0.090-0.18 mmol), N, N-diisopropylethylamine (2.4 eq, 0.120-0.240 mmol) for 2 hours. After the last amino acid coupling, the peptide is deprotected using % (v / v) of piperidine in dimethylformamide for 1 hour. Once the peptide sequence is complete, the Symphony peptide synthesizer is programmed to defer the resin. Digestion with trifluoroacetic acid (TFA) of the peptide from the resin is carried out using 93% TFA, 3% phenol, 3% water and 1% triisopropylsilane for 1 hour. The digested peptide is precipitated using tert-butylmethyl ether, pelletized by centrifugation and lyophilized. The pellet is redissolved in water (10.15 ml), filtered and purified by reverse phase HPLC using a C18 column and an acetonitrile / water gradient containing 0.1% TFA. A general procedure for N-blocking the peptides of the invention with fatty acids (e.g., octanoic and stearic acids) is as follows: peptide on rínk amide resin (0.1 mmoles) is suspended in NMP (5 ml) . In a separate vial, HBTU (0.3 mmol), HOBt (0.3 mmol) is dissolved in DMF (5 ml) followed by the addition of DIEA (0.6 mmol). This solution is added to the resin and this suspension is stirred for 2 hours. The solvent is filtered and washed uniformly with NMP (5 ml × 4) and CH 2 Cl 2 (20 ml), dried and subjected to digestion with TFA for 1 hour. The yield of the desired peptide is about 40 mg after digestion and purification. The PEG modification can be carried out in solution on a lysine-free epsilon-amino group or a terminal amino group on a purified peptide using commercially available activated PEG esters. The remaining PEGylated derivatives are purified to homogeneity by reverse phase HPLC and the purity is confirmed by LC / MS and MALDI-MS.

Certain exemplary hybrid polypeptides of the invention are shown below in Table 1-1. Various modifications to the modalized compounds are contemplated, such as chemical modifications such as glycosylation, PEG modifications, etc .; amino acid modifications such as substitutions, insertions and deletions, etc. Furthermore, even when represented as C-terminally amylated, it is understood that the hybrid polypeptides of the invention may alternatively be in the form of the free acid.

TABLE 1-1 Certain illustrative hybrid compounds of the invention SEQ ID: 1 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-ASLRHYLNLVTRQRY-NH2 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NRYYASLRHYLNLVTRQRY- RHYLNLVTRQRY- HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-ßAla-ßAla-ASLRHYLNLVTR HGEGTFTSDLSKQMEEEAVRLFlEWLKNGGPSSGAPPPS-ßAla-ßAla-RHYLNLVTRQRY- HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-ßAla-ßAla-VTRQRY- HGEGTFTSDLSKQMEEEAVRLFIEWLKN-ASLRHYLNLVTRQRY- NH2 NH2 NH2 RHYLNLVTRQRY- HGEGTFTSDLSKQMEEEAVRLFIEWLKN-NH2 10 HGEGTFTSDLSKQMEEEAVRLFIEWLKN-NRYYASLRHYLNLVTRQRY- HGEGTFTSDLSKQMEEEAVRLFIEWLKN-ßAla-ßAla-NH2 11 HGEGTFTSDLSKQMEEEAVRLFIEWLKN-ASLRHYLNLVTRQRY- ßAla-ßAla-RHYLNLVTRQRY - NH2 12 HGEGTFTSDLSKQMEEEAVRLFIEWLKN-ßAla-ßAla-VTRQRY- NH2 13 HGEGAFTSDLSKQLEEEAVRLFIEFLKNNRYYASLRHYLNLVTRQRY- NH2 14 HGEGAFTSDLSKQLEEEAVRLFIEFLKNASLRHYLNLVTRQRY- NH? 15 HGEGAFTSDLSKQLEEEAVRLFIEFLKNRHYLNLVTRQRY- NH, NH HGEGAFTSDLSKQLEEENRYYASLRHYLNLVTRQRY- 16, 17 HGEGAFTSDLSKQLEEEAVRLFlEFLKN-ßAla-ßAla-NH2 18 ASLRHYLNLVTRQRY- HGEGAFTSDLSKQLEEEAVRLFIEFLKN-ßAla-ßAla-NH2 19 RHYLNLVTRQRY- HGEGAFTSDLSKQLEEEAVRLFIEFLKN-ßAla-ßAla-VTRQRY-N NH2 20 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-DY (SO3) MGWMDF- NH2 EXAMPLE 2 Bonding tests The hybrid polypeptides of the invention can be tested in a variety of receptor binding assays using binding test methodologies generally known to those skilled in the art. These tests include those described below. Amylin binding test: Evaluation of the binding of some illustrative compounds of the invention to amylin receptors can be carried out as follows on nuclueus accumbens membranes prepared from brain rat. Male Sprague-Dawley rats (200-250 grams) are sacrificed by decapitation. The brains are removed and placed in saline buffered at pH with cold phosphate (PBS). From the ventral surface, rostral cuts are made to the hypothalamus, laterally joined by the olfactory tracts and extending at an angle of 45 ° medially from these tracts. This basal forebrain tissue, which contains the nucleus accumbens and surrounding regions, is weighed and homogenized in 20 mM of ice-cold pH buffer HEPES (20 mM HEPES acid, pH adjusted to 7.4 with NaOH at 23 ° C) . The membranes are washed three times in fresh pH buffer by centrifugation for 15 minutes at 48,000 x g. The final membrane pellet is resuspended in 20 mM pH buffer HEPES containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF).

Mara measuring the binding of 125 I-amylin (see, Beaumont K et al., Can J Physiol Pharmacol., July 1995; 73 (7): 1025-9), membranes of 4 mg of original wet tissue weight are incubated with 125 I -amiline at 12-16 pM in 20 mM of pH regulator HEPES containing 0.5 mg / ml of bacitracin, 0.5 mg / ml of bovine serum albumin, and 0.2 mM of PMSF. The solutions are incubated for 60 minutes at 2 ° C. Incubations are terminated by filtration through GF / B glass fiber filters (Whatman Inc., Clifton, N.J.) which are pre-soaked for 4 hours in 0.3% poylethyleneimine in order to reduce non-specific binding of radiolabelled peptides. The filters are washed immediately before filtration with 5 ml of cold PBS, and immediately after filtration with 15 ml of cold PBS. The filters are removed and the radioactivity is evaluated in a gamma counter at a counting efficiency of 77%. The competition curves are generated by measuring the binding in the presence of 10"12 to 10" 6 M of an unlabeled test compound and analyzed by non-linear regression using a 4-parameter logistic equation (Inplot program; GraphPAD Software, San Diego ). CGRP receptor binding test: The evaluation of the binding of the compounds of the invention to CGRP receptors are essentially as described for amylin except that membranes prepared from SK-N-MC cells, which are known to express receptors, are used. of CGRP (Muff, R. et al., Ann NY Acad. Sci. 1992: 657, 106-16). Binding tests are performed as described for amylin except that 13,500 cpm of 1251-hCGRP / well or 21.7 pM / well (Amersham) is used.

Adrenomedullin binding test: Adrenomedullin receptor binding can be investigated using HUVECs containing the adrenomedullin receptor (Kato J et al., Eur J Pharmacol, 1995, 289: 383-5) using the Perkin Elmer AlphaScreen test for Cyclic AMP using an optimum of 25-30,000 cells per well. The elevation of cAMP levels is not as great for HUVEC compared to CHO cells. As such, CHO cells can be chosen as a negative control since they do not express the adrenomedullin receptor, if desired. Calcitonin receptor binding assay: Calcitonin receptor binding can be investigated using CHO cells or T47D cells, which also express the calcitonin receptor (Muff R. et al., Ann NY Acad Sci. 1992,657: 106- 16 and Kuestner RE et al., Mol Pharmacol, 1994, 46: 246-55), as is known in the art Leptin binding assay: Two in vitro bioassays are routinely used to evaluate leptin binding and receptor activation (see v. White, et al., 1997. Proc. Nati, Acad. Sci. USA 94: 10657-10662). A fusion protein ("AP-OB") of alkaline phosphatase ("AP") - leptin ("OB") can be used to measure the inhibition of leptin binding in the absence or presence of recombinant mouse leptin (positive control) or peptide, by COS-7 cells transfected with the long form (signaling) of the mouse OB receptor ("OB-RL"). Signal transduction tests can be done on GT1-7 cells co-transfected with AP reporter and OB-RL constructs. The activity of secreted alkaline phosphatase ("SEAP") in response to Stimulation with leptin or mouse peptide can be measured by chemiluminescence. Y1 receptor binding test: Membranes are prepared from confluent cultures of SK-N-MC cells that endogenously express the neuropeptide Y1 receptors. The membranes are incubated with 60 pM of human [125I] peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypeptide for 60 minutes at room temperature in a 96-well polystyrene plate. Then, the contents of the wells are harvested on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried fiberglass plates are combined with scintillant and counted in a Perkin Elmer scintillation counter. Y2 receptor binding test: Membranes are prepared from confluent cultures of SK-N-BE cells that endogenously express the neuropeptide Y2 receptors. The membranes are incubated with 30 pM of human [125I] -peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypeptide for 60 minutes at room temperature in a 96-well polystyrene plate. Then, the contents of the wells are harvested on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried fiberglass plates are combined with scintillant and counted in a Perkin Elmer scintillation counter.

Y4 receptor binding test: K1 cells are transiently transfected with cDNA encoding neuropeptide Y4 gene, and then, forty-eight hours later, membranes are prepared from confluent cell cultures. The membranes are incubated with 18 pM of human [125 I] pancreatic polypeptide (2200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypepide for 60 minutes at room temperature in a 96-well polystyrene plate. Then, the contents of the wells are harvested on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried fiberglass plates are combined with scintillant and counted in a Perkin Elmer scintillation counter. Y5 receptor binding test: K1 cells are transiently transfected with cDNA encoding neuropeptide Y5 gene, and then, forty-eight hours later, membranes are prepared from confluent cell cultures. The membranes are incubated with 44 pM of human [25l] - peptide YY (2200 Ci / mmol, PerkinElmer Life Sciences), and with unlabeled PPF polypepide for 60 minutes at room temperature in a 96-well polystyrene plate. Then, the contents of the wells are harvested on a 96-well glass fiber plate using a Perkin Elmer plate harvester. The dried fiberglass plates are combined with scintillant and counted in a Perkin Elmer scintillation counter.

GLP-1 receptor binding assay: GLP-1 receptor binding activity and affinity can be measured using a binding shift test in which the receptor source is RINmdF cell membranes, and the ligand is [ 125I] GLP-1. Membranes of homogenized RINmdF cells are incubated in 20 mM pH regulator HEPES with 40,000 cpm of [121I] GLP-1 tracer, and variable concentrations of the test compound for 2 hours at 23 ° C with constant mixing. The reaction mixtures are filtered through pre-soaked glass fiber pads with 0.3% solution of PEI and rinsed with pH regulated saline with ice-cold phosphate. The binding counts are determined using a scintillation counter. The binding affinities are calculated using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).

EXAMPLE 3 Food ingestion test in mouse The hybrid polypeptides of the invention can be tested for appetite suppression in the mouse food intake test and for its effect on body weight gain in diet-induced obesity (DIO) mice. The experimental protocols for selective determinations are described below. Female NIH / Swiss mice (8-24 weeks old) are accommodated in a group with a light cycle: darkness of 12:12 hours with lights at 0600. Water and diet of mouse food in standard tablets are available ad libitum, except as indicated. The animals are left fasting starting at approximately 1500 hr, 1 day before the experiment. The morning of the experiment, the animals are divided into experimental groups. In a typical study, n = 4 cages with 3 mice / cage. At time = 0 min, all animals are given an intraperitoneal injection of vehicle or compound in an amount ranging from about 10 nmol / kg to 75 nmol / kg, and a pre-weighed amount is immediately given (10-15 g ) of the standard food. The feed is removed and weighed at 30, 60 and 120 min to determine the amount of food consumed (Morley, Flood et al., Am. J. Physio. 267: R178-R184, 1994). The food intake is calculated by subtracting the weight of the food that remains after v.gr., time point of 30, 60, 120, 180 and / or 240 minutes of the weight of the food initially provided in time = 0. The effects of significant treatment were identified by ANOVA (p <0.05). Where there is a significant difference, the means of the test are compared to the control mean using the Dunnett test (Prism v. 2.01, GraphPad Software Inc., San Diego, California).

EXAMPLE 4 Body weight gain in fattened mice C57B1 / 6 (obesity induced by diet or DIO) Male C57BL / 6 mice (4 weeks old at the beginning of the study) are fed high-fat food (HF, 58% of dietary kcal as fat) or low-fat (LF, 11% of kcal dietary as fat). After 4 weeks with the feed, each mouse was implanted with an osmotic pump (Alzet # 2002) which subcutaneously delivers a predetermined dose of hybrid polypeptide continuously for two weeks. Body weight and food intake are measured every week (Surwit et al., Metabolism-Clinical and Experimental, 44: 645-51, 1995). The effects of the test compound are expressed as the mean +/- d.e. of% change in body weight (i.e.,% change in start weight) of at least 14 mice per treatment group (p <0.05 ANOVA, Dunnett's test, Prism v. 2.01, GraphPad Software Inc. , San Diego, California).

Exendin / PYY hybrids Illustrative hybrid polypeptides of the invention were synthesized using C-terminally truncated exendins (e.g., exendin-4 (1-28) or 5Ala, 14Leu, 25Phe-exendin-4 (1-28)) and an N-terminally truncated PYY spanning 18-36 to 31-36 regions. As such, illustrative hybrid polypeptides generally they comprise two modules, wherein the first module is a fragment of an exendin-4 analog and the second module is a peptide enhancer selected from PYY truncations. For comparison, P-alanine dipeptide spacers were also incorporated between the peptide-binding blocks in various variants (see Table 4-1) - TABLE 4-1 Exendin / PYY hybrids and their effects on the food intake test As shown in Table 4-1, certain compounds illustrative of the invention showed efficacy in the food intake test. Certain peptides were also tested at 75 nmol / kg in the DIO test and proved to be more effective than PYY (figure 1).

Exendin / amylin hybrids Further exemplary hybrid polypeptides of the invention were prepared from C-terminally truncated exendin (1-27), C-terminally truncated amylin peptides (e.g., amylin (1-7), 27Ala- amylin (1-7), and amylin (33-27)), and optional sCT fragments (e.g., sCT (8-10) and 4Gln, 11'18Arg-sCT (8-27)). Although both hybrid polypeptides were very active in the suppression of appetite (see Table 4-2), the onset of action differed from the activity profiles of progenitor molecules (data not shown).

TABLE 4-2 Exendin / amylin hybrids and their effect on the Fl test Both compounds also showed excellent efficacy when they were selectively determined in the DIO test (Figure 2).

Exendin / CCK-8 hybrids Additional illustrative hybrid polypeptides of the invention were prepared from full-length or C-terminally truncated exendin-4 attached to the N-terminus of CCK-8 either directly or through a linker, retaining the N-terminal amide of CCK-8. (Table 4-3). In addition, certain hybrids were prepared incorporating the Tyr (S03) that occurs naturally, although another hybrid was prepared that incorporates the more stable Phe (CH2S03) group. All hybrid polypeptides prepared were active to inhibit food intake (Table 4-3).

TABLE 4-3 Exendin / CCK-8 hybrids and their effect on the food intake test in mice Hybrid polypeptides of exendin / CCK-8 were tested in the DIO test at 25 nmol / kg (Figures 3A and 3B). The data shows an initial weight loss, followed by a rebound effect in all compounds Interestingly, the rebound effect seems to be diminished in hybrids that incorporate the hydropyrically more stable Phe (CH2S0) residue, as well as hybrids that incorporate the 8-amino-3,6-dioxaoctanoyl linker between the exendin and the CCK residues.

Amylin / PYY Hybrid: A hybrid amylin / PYY polypeptide containing truncated segments of each peptide was synthesized. The in vivo activity in the food intake test is shown in Table 4-4.

TABLE 4-4 Hybrid amylin / PYY polypeptide To find out whether the illustrative hybrid polypeptides of the invention are more potent than their progenitor component peptide hormones, illustrative compounds were tested in the food intake test at the minimum effective dose of the most active progenitor molecule. The results are shown below in Figures 4A and 4B, which also compare the effects of parent progenitor peptides (Compounds 1, 11 and 12 are component peptide hormones, analogs or fragments thereof). The data indicate that several peptides are at least as equipotent as parent progenitors in stock. In parallel with in vivo studies, in vitro and functional receptor binding tests (cyclase activity) have been performed for all compounds (data not shown). Although the present invention has been described in terms of examples and preferred embodiments, it is understood that variations and modifications will occur to one skilled in the art. Therefore, it is intended that the appended claims cover all variations that fall within the scope of the invention as claimed.

Claims (25)

NOVELTY OF THE INVENTION CLAIMS

1. - A hybrid polypeptide having at least one hormonal activity, said hybrid polypeptide comprising a first module of bioactive peptide hormone covalently linked to at least one additional module of bioactive peptide hormone; wherein: the bioactive peptide hormone modules are independently selected from the group consisting of: component peptide hormones, component peptide hormone fragments having at least one hormonal activity of the component peptide hormones, analogs and component peptide hormone derivatives that present at least one hormonal activity of the component peptide hormones, fragments of analogs and derivatives of component peptide hormones that exhibit at least one hormonal activity of the component peptide hormones, and peptide enhancers; the component peptide hormones are independently selected from at least two of the group consisting of: amylin, adrenomedullin (ADM), calcitonin (CT), calcitonin gene-related peptide (CGRP), ethermedin, cholecystokinin ("CCK"), leptin, peptide YY (PYY), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), oxyntomodulin (OXM), and exendin-4; Peptide enhancers are independently selected from the group consisting of:

structural motifs of peptide hormones components that impart chemical stability, conformational stability, metabolic stability, receptor interaction, protease inhibition or other desired pharmacokinetic characteristic to the hybrid polypeptide, and structural motifs of analogs or peptide hormone derivatives components that impart chemical stability , conformational stability, metabolic stability, receptor interaction, protease inhibition or other desired pharmacokinetic characteristic to the hybrid polypeptide; and at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity of a component peptide hormone; and further wherein: when at least one bioactive peptide hormone module having at least one hormonal activity of a component peptide hormone is amylin, an amylin fragment having at least one hormonal activity, an analogue or amylin derivative having at least one hormonal activity, or a fragment of an analogue or amylin derivative having at least one hormonal activity, and at least one other bioactive peptide hormone module is CCK, a fragment of CCK exhibiting at least one a hormonal activity, an analog or derivative of CCK having at least one hormonal activity, a fragment of an analogue or derivative of CCK exhibiting at least one hormonal activity, CT, a fragment of CT having at least one activity hormone, an analogue or derivative of CT that exhibits at least one hormonal activity, or a fragment of an analog or derivative of CT that presents at least

a hormonal activity, then the hybrid polypeptide further comprises at least three bioactive peptide hormone modules selected from at least three different component peptide hormones; and when at least one bioactive peptide hormone module having at least one hormonal activity of a component peptide hormone is GLP-1, a fragment of GLP-1 having at least one hormonal activity, an analog or derivative of GLP. -1 presenting at least one hormonal activity, or a fragment of an analog or derivative of GLP-1 having at least one hormonal activity, and at least one other bioactive peptide hormone module is a peptide enhancer comprising a fragment of exendin, then the hybrid polypeptide further comprises at least three bioactive peptide hormone modules.

2. The hybrid polypeptide according to claim 1, further characterized in that the peptidic enhancers are independently selected from the group consisting of: amylin (32-37), amylin (33-37), amylin (34-37), amylin (35-37), amylin (36-37) , amylin (37), ADM (47-52), ADM (48-52), ADM (49-52), ADM (50-52), ADM (51-52), ADM (52), CT (27- 32), CT (27-32), CT (28-32), CT (29-32), CT (30-32), CT (31-32), CT (32), CGRP (32-37), CGRP (33-37), CGRP (34-37), CGRP (35-37), CGRP (36-37), CGRP (37), intermediate (42-47), intermediate (43-47), intermediate (44 -47), intermediate (45-47), intermediate (46-47), intermediate (47), PYY (25-36), PYY (26-36), PYY (27-36), PYY (28-36) , PYY (29-36), PYY (30-36), PYY (31-36), PYY (32-36), PYY (25-35), PYY (26-35), PYY (27-35), PYY (28-35), PYY (29-35), PYY (30-35),

PYY (31-35), PYY (32-35), GLP-1 (29-37) of frog, GLP-1 (30-37) of frog, GLP-2 (24-31) of frog, exendin-4 (31-39), exendin-4 (32-39), exendin-4 (33-39), exendin-4 (34-39), exendin-4 (35-39), exendin-4 (36-39) , exendin-4 (37-39), exendin-4 (38-39), exendin-4 (39), and analogs thereof.

3. The hybrid polypeptide according to claim 1, further characterized in that at least one of the first module of bioactive peptide hormone or at least one additional module of bioactive peptide hormone is a peptide hormone component or fragment of a peptide hormone component presenting at least one hormonal activity of the component peptide hormone.

4. The hybrid polypeptide according to claim 1, further characterized in that at least one of the first module of bioactive peptide hormone or at least one additional module of bioactive peptide hormone is an analog or derivative of a component peptide hormone that presents at least one hormonal activity or a fragment of an analog or derivative of a component peptide hormone exhibiting at least one hormonal activity of the component peptide hormone.

5. The hybrid polypeptide according to claim 1, further characterized in that at least one of the first module of bioactive peptide hormone or at least one additional module of bioactive peptide hormone is peptidic enhancer.

6. The hybrid polypeptide according to claim 1,

further characterized in that the component peptide hormones are independently selected from the group consisting of: amylin, calcitonin, CCK, PYY and exendin-4.

7. The hybrid polypeptide according to claim 1, further characterized in that at least one bioactive peptide hormone module having at least one hormonal activity is located in the N-terminal portion of the hybrid polypeptide.

8. The hybrid polypeptide according to claim 7, further characterized in that at least one bioactive peptide hormone module having at least one hormonal activity located in the N-terminal portion of the hybrid polypeptide is configured in the C-orientation. terminal to N-terminal.

9. The hybrid polypeptide according to claim 8, further characterized in that the N-terminal end of the hybrid polypeptide is amidated.

10. The hybrid polypeptide according to claim 1, further characterized in that at least one bioactive peptide hormone module having at least one hormonal activity is located in the C-terminal portion of the hybrid polypeptide.

11. The hybrid polypeptide according to the claim

10, further characterized in that the C-terminal end of the hybrid polypeptide is amidated.

12. The hybrid polypeptide according to claim 1,

further characterized in that the C-terminal end of a bioactive peptide hormone module is directly attached to the N-terminus of another bioactive peptide hormone module to form the covalent bond.

13. The hybrid polypeptide according to claim 1, further characterized in that the bioactive peptide hormone modules are covalently linked using one or more linker groups independently selected from the group consisting of: alkyls; PEGs of dicarboxylic acids; amino acids; polyamino acids; bifunctional linkers; aminocaproyl (Ac), (3-alanyl, 8-amino-3,6-dioxaoctanoyl, and Gly-Lys-Arg (GKR) 14.- The hybrid polypeptide according to claim 1, further characterized in that the first module of Bioactive peptide hormone is selected from the group consisting of: exendin-4, a fragment of exendin-4 having at least one hormonal activity, an analog or derivative of exendin-4 having at least one hormonal activity, and a fragment of an exendin-4 analogue exhibiting at least one hormonal activity; and at least one additional module of bioactive peptide hormone is independently selected from the group consisting of: amylin, an amylin fragment having at least one hormonal activity, an analogue or amylin derivative having at least one hormonal activity, or a fragment of an amylin analog having at least one hormonal activity, CCK, a fragment of CCK exhibiting at least one hormonal activity, a CCK analog or derivative

having at least one hormonal activity, a fragment of a CCK analog having at least one hormonal activity, CT, a CT fragment exhibiting at least one hormonal activity, an analog or derivative of CT which presents minus a hormonal activity, a fragment of a CT analog having at least one hormonal activity, and a peptide enhancer.

15. The hybrid polypeptide according to claim 14, further characterized in that the first bioactive peptide hormone module is selected from the group consisting of: exendin-4, exendin-4 (1-27), exendin-4 (1- 28), 14Leu, 25Phe-exendin-4 (1-28); 5Ala, 14Leu, 25Phe-exendin-4 (1-28) and 14Leu-exendin-4 (1-28); and at least one additional module of bioactive peptide hormone is independently selected from the group consisting of 25'28, z9Pro-h-amylin, amylin (1-7), 2,7Ala-amylin (1-7), sCT (8 -10), sCT (8-27), 14Gln, 11'18Arg-sCT (8-27), CCK-8, Phe2CCK-8, amylin (33-37), PYY (25-36), PYY (30- 36) and PYY (31 -36).

16. The hybrid polypeptide according to claim 14, further characterized in that the hybrid polypeptide comprises at least three bioactive peptide hormone modules.

17. The hybrid polypeptide according to claim 14, further characterized in that the hybrid polypeptide comprises at least four bioactive peptide hormone modules.

18. The hybrid polypeptide according to claim 14, further characterized in that the first peptide hormone module

bioactive is located at the C-terminal end of the hybrid polypeptide and at least one additional module of bioactive peptide hormone is located at the N-terminus of the hybrid polypeptide.

19. The hybrid polypeptide according to claim 14, further characterized in that the first module of bioactive peptide hormone is located at the N-terminal end of the hybrid polypeptide and at least one additional module of bioactive peptide hormone is located at the end. C-terminal hybrid polypeptide.

20. The hybrid polypeptide according to claim 1, further characterized in that the first bioactive peptide hormone module is selected from the group consisting of: amylin, an amylin fragment having at least one hormonal activity, an analog or derivative of amylin having at least one hormonal activity, and a fragment of an amylin analog having at least one hormonal activity; and at least one additional module of bioactive peptide hormone is a peptide enhancer independently selected from the group consisting of: PYY (25-36), PYY (26-36), PYY (27-36), PYY (28-36) , PYY (29-36), PYY (30-36), PYY (31-36), PYY (32-36), PYY (25-35), PYY (26-35), PYY (27-35), PYY (28-35), PYY (29-35), PYY (30-35), PYY (31-35), PYY (32-35), and analogs thereof.

21. A hybrid polypeptide having at least one hormonal activity, said hybrid polypeptide comprising a first module of bioactive peptide hormone covalently linked to a

second module of bioactive peptide hormone; wherein: the bioactive peptide hormone modules are independently selected from the group consisting of: component peptide hormones, component peptide hormone fragments having at least one hormonal activity of the component peptide hormones, analogues, and component peptide hormone derivatives that present at least one hormonal activity of the component peptide hormones, fragments of analogs and derivatives of component peptide hormones that exhibit at least one hormonal activity of the component peptide hormones, and peptide enhancers; the component peptide hormones are independently selected from at least two of the group consisting of: amylin, PYY, and exendin-4; peptidic enhancers are independently selected from the group consisting of: structural motifs of peptide hormones components that impart chemical stability, conformational stability, metabolic stability, receptor interaction, inhibition of protease or other desired pharmacokinetic characteristic to the hybrid polypeptide, and structural motifs of analogs or peptide hormone derivatives components that impart chemical stability, conformational stability, metabolic stability, receptor interaction, inhibition of protease or other desired pharmacokinetic characteristic to the hybrid polypeptide; and wherein at least one of the bioactive peptide hormone modules exhibits at least one hormonal activity of a component peptide hormone.

22. - The hybrid polypeptide according to claim 21, further characterized in that peptidic enhancers are independently selected from the group consisting of amylin (32-37), amylin (33-37), amylin (34-37), amylin (35-) 37), amylin (36-37), amylin (37), PYY (25-36), PYY (26-36), PYY (27-36), PYY (28-36), PYY (29-36), PYY (30-36), PYY (31-36), PYY (32-36), PYY (25-35), PYY (26-35), PYY (27-35), PYY (28-35), PYY (29-35), PYY (30-35), PYY (31-35), PYY (32-35), exendin-4 (31-39), exendin-4 (32-39), exendin-4 (33 -39), exendin-4 (34-39), exendin-4 (35-39), exendin-4 (36-39), exendin-4 (37-39), exendin-4 (38-39), exendin -4 (39), and analogs thereof.

23. The hybrid polypeptide according to claim 21, further characterized in that the first bioactive peptide hormone module is located at the C-terminal end of the hybrid polypeptide.

24. The hybrid polypeptide according to claim 21, further characterized in that the first bioactive peptide hormone module is located at the N-terminal end of the hybrid polypeptide.

25. The hybrid polypeptide according to claim 21, further characterized in that the hybrid polypeptide comprises combinations of bioactive peptide hormone modules selected from the group consisting of: bioactive peptide hormone modules of exendin-4 / PYY, PYY / exendin 4, exendin / amylin, amylin / exendin, amylin / PYY, and PYY / amylin.